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08 March 2025, Volume 29 Issue 7 Previous Issue   

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Mechanism by which nobiletin inhibits inflammatory response of BV2 microglia Chi Wenxin, Zhang Cunxin, Gao Kai, Lyu Chaoliang, Zhang Kefeng 2025, 29 (7):  1321-1327.  doi: 10.12307/2024.741 Abstract ( 152 )   PDF (1022KB) ( 122 )   Save BACKGROUND: Nobiletin has been found to improve lipopolysaccharide-induced abnormal activation of microglia, excessive release of inflammatory factors and redox imbalance. However, the specific mechanism is not fully understood. OBJECTIVE: To investigate the molecular mechanism by which nobiletin can inhibit lipopolysaccharide-induced inflammation in BV2 microglia.   METHODS: Passage 3 BV2 microglia were divided into three groups: control group was cultured for 24 hours (without any treatment). Lipopolysaccharide group was treated with 10 μg/mL lipopolysaccharide for 24 hours. Lipopolysaccharide + nobiletin group was treated with 20 μmol/L nobiletin for 6 hours and then 10 μg/mL lipopolysaccharide for 24 hours. After the processing, cell proliferation was detected by CCK-8 assay. The level of intracellular reactive oxygen species was detected by fluorescent probe. The mRNA expression levels of nuclear factor κB p65, tumor necrosis factor α, and interleukin-1β were detected by qRT-PCR. The protein expression levels of nuclear factor κB p65, p-nuclear factor κB p65, tumor necrosis factor α, and interleukin-1β were detected by western blot assay. RESULTS AND CONCLUSION: (1) Compared with the control group, the proliferation activity of lipopolysaccharide group was decreased (P < 0.001). Compared with the lipopolysaccharide group, the cell proliferation activity of lipopolysaccharide + nobiletin group was increased (P < 0.001). (2) Compared with the control group, the level of intracellular reactive oxygen species was increased in the lipopolysaccharide group (P < 0.001). Compared with the lipopolysaccharide group, the level of intracellular reactive oxygen species was decreased in the lipopolysaccharide + nobiletin group (P < 0.01). (3) Compared with the control group, the mRNA expression levels of tumor necrosis factor α and interleukin-1β were increased in the lipopolysaccharide group (P < 0.001, P < 0.01). Compared with the lipopolysaccharide group, mRNA expression levels of tumor necrosis factor α and interleukin-1β were decreased in the lipopolysaccharide + nobiletin group (P < 0.01, P < 0.05). (4) Compared with the control group, the protein expression levels of p-nuclear factor κB p65, tumor necrosis factor α, and interleukin-1β in were increased the lipopolysaccharide group (P < 0.001). Compared with the lipopolysaccharide group, the expression of p-nuclear factor κB p65, tumor necrosis factor α, and interleukin-1β was decreased in the lipopolysaccharide + nobiletin group (P < 0.001). (5) These findings suggest that nobiletin attenuates lipopolysaccharide-induced inflammatory response in BV2 microglia by suppressing nuclear factor-κB signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Icariin pretreatment enhances effect of human periodontal stem cells on M1-type macrophages Yu Ting, Lyu Dongmei, Deng Hao, Sun Tao, Cheng Qian 2025, 29 (7):  1328-1335.  doi: 10.12307/2025.014 Abstract ( 172 )   PDF (1526KB) ( 88 )   Save BACKGROUND: Human periodontal stem cells have a certain inhibitory effect on the pro-inflammatory function of M1-type macrophages, and it is not clear whether icariin, which has anti-inflammatory and other pharmacological activities, can enhance the inhibitory effect of human periodontal stem cells on M1-type macrophages. OBJECTIVE: To investigate the effect of icariin on M1 macrophages after pretreatment of human periodontal stem cells.  METHODS: Primary human periodontal stem cells were isolated, cultured and characterized. THP-1 was induced and M1-type macrophages were identified by immunofluorescence staining and PCR. Human periodontal stem cells were cultured with α-MEM complete medium containing concentrations of 10-7, 10-6, 10-5, and 10-4 mol/L icariin, and the cytotoxicity of Icariin on human periodontal stem cells was detected by the CCK-8 assay at 1, 3, 5, and 7 days, respectively. α-MEM complete medium, untreated α-MEM conditioned medium for human periodontal stem cells and α-MEM conditioned medium for human periodontal stem cells pretreated with icariin for 24 hours were conditioned with RPMI-1640 complete medium in a 1:1 ratio for M1-type macrophages in the control, untreated, and pretreated groups, and 24 hours later, the mRNA expression of inflammatory factors in M1 macrophages was detected by RT-PCR. The protein expression of inflammatory factors in M1 macrophages was detected by ELISA. The expression of surface markers and nuclear factor-κB pathway-related proteins in M1/M2 macrophages was detected by western blot assay.  RESULTS AND CONCLUSION: (1) CCK-8 assay results showed that 10-7, 10-6, 10-5, 10-4 mol/L icariin was not cytotoxic to the human periodontal stem cells, and from day 5 onwards, all the concentrations increased the cell viability, and promoted the cell proliferation. 10-4 mol/L icariin was selected for follow-up experiment. (2) RT-PCR and ELISA results showed that compared with the control group , the untreated group and the pretreated group both decreased the expression and secretion of interleukin-1β, interleukin-6, and tumor necrosis factor-α of M1-type macrophages (P < 0.05), and the pretreated group was lower than the untreated group (P < 0.05). (3) Western blot assay results showed that compared with the untreated group, the expression of CD86 was significantly lower in the pretreated group (P < 0.05); compared with the control group, the expression of CD206, a surface marker of M2-type macrophages, was elevated in both the untreated and pretreated groups (P < 0.01), and it was significantly higher in the pretreated group than in the untreated group (P < 0.01). In M1-type macrophages after 24 hours of conditioned culture, compared with the control group, the expression of nuclear factor-κB/P65 was decreased in the untreated group and the pretreated group (P < 0.01), and the expression of p-IκBα was decreased only in the pretreated group (P < 0.01); the expression of both nuclear factor-κB/P65 and p-IκBα was significantly reduced in the pretreated group compared with the untreated group (P < 0.05), while the difference of IκBα in the three groups was not statistically significant. (4) These results indicated that icariin enhanced the inhibitory effect of human periodontal stem cells on M1-type macrophages, and this effect may be related to the inhibition of the nuclear factor-κB signaling pathway of macrophages. Figures and Tables | References | Related Articles | Metrics

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Nerve growth factor promotes chondrogenic differentiation and inhibits hypertrophic differentiation of rabbit bone marrow mesenchymal stem cells Yang Zhihang, Sun Zuyan, Huang Wenliang, Wan Yu, Chen Shida, Deng Jiang 2025, 29 (7):  1336-1342.  doi: 10.12307/2025.005 Abstract ( 93 )   PDF (1559KB) ( 99 )   Save BACKGROUND: Nerve growth factor is a protein that induces nerve growth and regulates biological behaviors such as proliferation and differentiation of mesenchymal stem cells. OBJECTIVE: To investigate the promoting effect of nerve growth factor on chondrogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Rabbit bone marrow mesenchymal stem cells were isolated and cultured, and nerve growth factor was transfected into bone marrow mesenchymal stem cells by lentiviral transfection. The effects of nerve growth factor on the proliferation, migration, hypertrophic differentiation, and chondrogenic differentiation of bone marrow mesenchymal stem cells were detected by CCK-8 assay, cell scratch assay, alizarin red staining, and western blot assay, using the transfected null-loaded virus as control. To further investigate the promoting effect of nerve growth factor on the chondrogenic differentiation of bone marrow mesenchymal stem cells, interleukin 1β was added in bone marrow mesenchymal stem cells transfected with empty virus and nerve growth factor for 14 days. The expression of proteins related to chondrogenic differentiation and hypertrophic differentiation was detected by western blot assay.  RESULTS AND CONCLUSION: (1) CCK-8 assay results showed that nerve growth factor had no significant effect on the proliferation of bone marrow mesenchymal stem cells. (2) Compared with the control group, overexpression of nerve growth factor enhanced the migration ability of the cells, and the expression of cartilage-associated proteins type II collagen and SOX9 was up-regulated (P < 0.05), while the expression of hypertrophic-associated proteins type X collagen and Runx2 was down-regulated (P < 0.05). (3) Compared with the empty virus+interleukin 1β group, the expression of cartilage-associated proteins type II collagen and Sox9 was up-regulated (P < 0.05), and the expression of hypertrophy-associated proteins type X collagen and Runx2 was down-regulated after overexpression of nerve growth factor (P < 0.05). (4) The results indicated that nerve growth factor could promote the chondrogenic differentiation of bone marrow mesenchymal stem cells. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 Figures and Tables | References | Related Articles | Metrics

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Human amniotic mesenchymal stem cells overexpressing neuregulin-1 promote skin wound healing in mice Hu Taotao, Liu Bing, Chen Cheng, Yin Zongyin, Kan Daohong, Ni Jie, Ye Lingxiao, Zheng Xiangbing, Yan Min, Zou Yong 2025, 29 (7):  1343-1349.  doi: 10.12307/2025.023 Abstract ( 119 )   PDF (2010KB) ( 90 )   Save BACKGROUND: Neuregulin 1 has been shown to be characterized in cell proliferation, differentiation, and vascular growth. Human amniotic mesenchymal stem cells are important seed cells in the field of tissue engineering, and have been shown to be involved in tissue repair and regeneration. OBJECTIVE: To construct human amniotic mesenchymal stem cells overexpressing neuregulin 1 and investigate their proliferation and migration abilities, as well as their effects on wound healing. METHODS: (1) Human amniotic mesenchymal stem cells were in vitro isolated and cultured and identified. (2) A lentivirus overexpressing neuregulin 1 was constructed. Human amniotic mesenchymal stem cells were divided into empty group, neuregulin 1 group, and control group, and transfected with empty lentivirus and lentivirus overexpressing neuregulin 1, or not transfected, respectively. (3) Edu assay was used to detect the proliferation ability of the cells of each group, and Transwell assay was used to detect the migration ability of the cells. (4) The C57 BL/6 mouse trauma models were constructed and randomly divided into control group, empty group, neuregulin 1 group, with 8 mice in each group. Human amniotic mesenchymal stem cells transfected with empty lentivirus or lentivirus overexpressing neuregulin-1 were uniformly injected with 1 mL at multiple local wound sites. The control group was injected with an equal amount of saline. (5) The healing of the trauma was observed at 1, 7, and 14 days after model establishment. Histological changes of the healing of the trauma were observed by hematoxylin-eosin staining. The expression of CD31 on the trauma was observed by immunohistochemistry.  RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells overexpressing neuregulin-1 were successfully constructed. The mRNA and protein expression of intracellular neuregulin 1 was significantly up-regulated compared with the empty group (P < 0.05). (2) The overexpression of neuregulin 1 promoted the migratory ability (P < 0.01) and proliferative ability of human amniotic mesenchymal stem cells (P < 0.05). (3) Human amniotic mesenchymal stem cells overexpressing neuregulin 1 promoted wound healing in mice (P < 0.05) and wound angiogenesis (P < 0.05). The results showed that overexpression of neuregulin 1 resulted in an increase in the proliferative and migratory capacities of human amniotic mesenchymal stem cells, significantly promoting wound healing and angiogenesis. Related Articles | Metrics

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Effects of conditioned medium and exosomes of human umbilical cord mesenchymal stem cells on proliferation, migration, invasion, and apoptosis of hepatocellular carcinoma cells Jin Kai, Tang Ting, Li Meile, Xie Yuan 2025, 29 (7):  1350-1355.  doi: 10.12307/2025.012 Abstract ( 188 )   PDF (1751KB) ( 189 )   Save BACKGROUND: Mesenchymal stem cells can regulate the tumor microenvironment by secreting extracellular vesicles containing cytokines, growth factors and exosomes for the precise regulation of biological behavior of tumor cells.  OBJECTIVE: To investigate the effects of human umbilical cord-derived mesenchymal stem cell conditioned medium and their released exosomes on the biological properties of hepatocellular carcinoma cells. METHODS: Human umbilical cord mesenchymal stem cell supernatant was collected, centrifuged and filtered at high speed to obtain human umbilical cord mesenchymal stem cell conditioned medium. Human umbilical cord mesenchymal stem cell supernatant was collected and human umbilical cord mesenchymal stem cell exosomes were extracted by ultra-high speed gradient centrifugation. Human umbilical cord mesenchymal stem cell exosomes were labeled with PKH26 and co-cultured with hepatocellular carcinoma cell MHCC97-H. The uptake of exosomes by MHCC97-H cells was observed by fluorescence microscopy. The effects of human umbilical cord mesenchymal stem cell conditioned medium and human umbilical cord mesenchymal stem cell exosomes on biological functions of hepatocellular carcinoma cells were assessed by the CCK-8 proliferation assay, Transwell migration and invasion assay, and the apoptosis assay.   RESULTS AND CONCLUSION: (1) Human umbilical cord mesenchymal stem cell exosomes could be uptaken by MHCC97-H cells and was mainly distributed in the cytoplasm. (2) After treatment with human umbilical cord mesenchymal stem cell conditioned medium, MHCC97-H cells showed a significant increase in proliferation, migration, and invasion (P < 0.001, P < 0.05, P < 0.01), and a significant decrease in apoptosis (P < 0.001), while after treatment with human umbilical cord mesenchymal stem cell exosomes, MHCC97-H cells showed a decrease in proliferation (P < 0.001) and migration, invasion, and apoptosis were significantly enhanced (P < 0.001). (3) The results indicated that human umbilical cord mesenchymal stem cell conditioned medium had the ability to promote the proliferation, migration, invasion, and inhibit apoptosis of MHCC97-H cells, while human umbilical cord mesenchymal stem cell exosomes had the properties of promoting the migration, invasion and apoptosis of MHCC97-H cells, inhibiting the proliferation.  Figures and Tables | References | Related Articles | Metrics

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Three-dimensional gelatin microspheres loaded human umbilical cord mesenchymal stem cells for chronic tendinopathy repair Li Dijun, Jiu Jingwei, Liu Haifeng, Yan Lei, Li Songyan, Wang Bin 2025, 29 (7):  1356-1362.  doi: 10.12307/2025.019 Abstract ( 113 )   PDF (2086KB) ( 120 )   Save BACKGROUND: The absence of blood vessels in tendon tissue makes tendon repair challenging. Therefore, improving tendon healing and raising the efficacy of stem cell and other therapeutic cell transplantation after tendon damage have become hotspots for research in both clinical and scientific contexts. OBJECTIVE: The stem cells and gelatin microcarrier scaffold were joined to form tissue engineered stem cells. Human umbilical cord mesenchymal stem cells cultured in gelatin microcarriers were used to investigate the therapeutic impact and mode of action on tendinopathy healing in rats in vitro and In vivo.  METHODS: (1) In vitro cell experiments: After seeding human umbilical cord mesenchymal stem cells with three-dimensional gelatin microcarriers, the cell vitality and survival were assessed. Human umbilical cord mesenchymal stem cells conventionally cultured were cultured as controls. (2) In vivo experiment: Adult SD rats were randomly assigned to normal group, tendinopathy group, 2D group (tendinopathy + conventional culture of human umbilical cord mesenchymal stem cells), and 3D group (tendinopathy + gelatin microcarrier three-dimensional culture of human umbilical cord mesenchymal stem cells), with 6 rats in each group. Four weeks after therapy, animal behavior tests and histopathologic morphology of the Achilles tendon was examined. RESULTS AND CONCLUSION: (1) In vitro cell experiments: the seeded human umbilical cord mesenchymal stem cells on gelatin microcarriers showed high viability and as time went on, the stem cell proliferation level grew. Compared with the control group, 3D stem cell culture preserved cell viability. (2) In vivo experiment: Following a 4-week treatment, the 3D stem cell culture group showed a significant improvement in both functional recovery of the lower limbs and histopathological scores when compared to the tendinopathy group. The 2D stem cell culture group also showed improvement in tendinopathy injury, but its effect is not as much as the 3D stem cell culture group. (3) The outcomes demonstrate that human umbilical cord mesenchymal stem cells cultured with three-dimensional gelatin microcarrier can promote the repair and regeneration of tendon injury tissue, and the repair effect is better than that of conventional human umbilical cord mesenchymal stem cells. Figures and Tables | References | Related Articles | Metrics

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Exercise preconditioning for eight weeks enhances therapeutic effect of adipose-derived stem cells in rats with myocardial infarction Lou Guo, Zhang Min, Fu Changxi 2025, 29 (7):  1363-1370.  doi: 10.12307/2025.007 Abstract ( 125 )   PDF (1803KB) ( 125 )   Save BACKGROUND: Stem cell transplantation is a novel therapy for myocardial infarction, but the extremely hostile microenvironment in the infarct area results in low survival rate of stem cells and little long-term effect. Exercise preconditioning is a way to induce endogenous protective effects through exercise, which can be used as a new strategy for prevention and treatment of cardiac rehabilitation. OBJECTIVE: To evaluate whether exercise preconditioning potentiates the cardioprotective effects of adipose-derived stem cell transplantation following myocardial infarction in rats and to explore the mechanism of angiogenesis. METHODS: Six-week-old male SD rats were randomly divided into control group, modeling group, stem cell group, and stem cell plus exercise group. Acute myocardial infarction model was made by coronary artery occlusion, and sham operation was performed in control group. The stem cell plus exercise group underwent aerobic exercise for 8 weeks before modeling, and adipose-derived stem cell transplantation was performed 30 minutes after modeling. The stem cell group performed only adipose-derived stem cell transplantation. One and seven days after stem cell transplantation, the expression levels of myocardial total Akt (t-Akt), phosphorylated Akt (p-Akt), vascular endothelial growth factor (VEGF), total endothelial nitric oxide synthase (t-eNOS), and phosphorylated endothelial nitric oxide synthase (p-eNOS) protein were measured by western blotting, and the ratios of p-Akt/t-Akt and p-eNOS/t-eNOS were calculated. At 4 weeks after stem cell transplantation, the heart structure and function as well as myocardial blood flow were detected by color Doppler ultrasound diagnostic system. Myocardial infarction area was measured by TTC staining. Myocardial interstitial collagen deposition was examined by Masson staining. Myocardial capillary density was detected by immunofluorescence staining, and myocardial apoptosis was measured by TUNEL staining.  RESULTS AND CONCLUSION: (1) Four weeks after stem cell transplantation: Compared with control group, left ventricular shortening fraction, left ventricular ejection fraction, myocardial capillary density, and myocardial blood flow decreased (P < 0.05), myocardial infarction area, collagen volume fraction, and apoptosis increased (P < 0.05) in the modeling group. Compared with the modeling group, the above indexes (except for left ventricular fractional shortening and left ventricular ejection fraction) in the stem cell group improved (P < 0.05). Compared with the stem cell group, the above parameters were further improved in the stem cell plus exercise group (P < 0.05). (2) One day after stem cell transplantation: Compared with the control group, the protein expression of t-Akt, p-Akt, VEGF, t-eNOS, p-eNOS and the ratio of p-Akt/t-Akt and p-eNOS/t-eNOS had no significant changes in the modeling group (P > 0.05). Compared with the modeling group, there were no significant changes in the above indexes in the stem cell group (P > 0.05), and p-Akt protein expression and the ratio of p-Akt/t-Akt were up-regulated in the stem cell plus exercise group (P < 0.05). (3) Seven days after stem cell transplantation: Compared with the control group, the protein expression of p-Akt, VEGF, p-eNOS and the ratio of p-Akt/t-Akt and p-eNOS/t-eNOS were decreased in the modeling group (P < 0.05). Compared with the modeling group, there were no significant changes in all parameters in the stem cell group (P > 0.05), and the protein expression of p-Akt, VEGF p-eNOS and the ratio of p-Akt/t-Akt and p-eNOS/t-eNOS were increased in the stem cell plus exercise group (P < 0.05). These findings confirm that exercise preconditioning can potentiate the therapeutic effect of adipose-derived stem cells on cardiac remodeling in rats with myocardial infarction, and its mechanism is associated with the promotion of myocardial angiogenesis and blood perfusion. Figures and Tables | References | Related Articles | Metrics

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Icariin-containing serum promotes chondrocyte proliferation and chondrogenic differentiation of stem cells in the co-culture system of three kinds of cells Liu Qi, Li Linzhen, Li Yusheng, Jiao Hongzhuo, Yang Cheng, Zhang Juntao 2025, 29 (7):  1371-1379.  doi: 10.12307/2025.013 Abstract ( 105 )   PDF (2684KB) ( 138 )   Save BACKGROUND: The capability of repairing articular cartilage damage is very limited, and tissue engineering technology provides new therapeutic options for repairing damaged cartilage, in which the interaction and induction between chondrocytes, bone marrow mesenchymal stem cells, and synovial mesenchymal stem cells is the basis of autologous healing of cartilage damage. OBJECTIVE: To construct the chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell co-culture system to simulate the in vitro microenvironment of chondrocytes, and to explore the optimal cell inoculation ratio, meanwhile to observe the effects of icariin-containing serum on the proliferation of chondrocytes and the chondrogenic differentiation of stem cells in the system. METHODS: Rat knee chondrocytes, bone marrow mesenchymal stem cells and synovial mesenchymal stem cells were extracted, cultured and identified, and a chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell non-contact co-culture system was constructed according to different cell inoculation ratios. After 72 hours of co-culturing, the chondrocyte proliferative activity and phenotypic ability were observed, and the co-culture system with the best overall effect was selected. New Zealand white rabbits were gavaged with icariin solution (0.25 mg/mL) to prepare icariin-containing serum, and cultured in conventional complete medium (high sugar DMEM culture medium containing 10% fetal bovine serum and 1% double antibody by volume) as the control group, while the experimental group was intervened by adding 10% icariin-containing serum by volume on the basis of above. The proliferative activity of chondrocytes and the expression of collagen type II were tested for the two groups after 24 and 48 hours. The differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells into chondrocytes in the co-culture system was tested by immunofluorescence staining after 14 days.  RESULTS AND CONCLUSION: (1) The three kinds of cells grew normally adherently to the wall in different ratios of co-culture, where chondrocytes showed the best proliferative activity and phenotypic ability in the co-culture system when chondrocytes: bone marrow mesenchymal stem cells: synovial mesenchymal stem cells = 2:1:1. (2) Compared with the control group, the proliferative activity and type II collagen expression of chondrocytes in the experimental group were significantly increased after 24 hours (P < 0.01), and the two groups still had difference after 48 hours (P < 0.05). The two groups showed obvious chondrogenic differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells after 14 days (P < 0.01), and some of the cells appeared round or oval, and the cytoplasmic type II collagen immunofluorescence staining was positive. The fluorescence intensity of the experimental group was significantly higher than that of the control group (P < 0.01). (3) The results showed that the chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell co-culture system could be successfully established by the non-contact co-culture method, and the best chondrocyte proliferative activity and phenotypic ability could be obtained when the cell ratio was 2:1:1. Icariin-containing serum had the promoting effect on chondrocyte proliferation, and chondrogenic differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells in the system.  Figures and Tables | References | Related Articles | Metrics

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Effects of macrophage migration inhibitory factor on survival, proliferation, and differentiation of human embryonic stem cells Huang Ting, Zheng Xiaohan, Zhong Yuanji, Wei Yanzhao, Wei Xufang, Cao Xudong, Feng Xiaoli, Zhao Zhenqiang 2025, 29 (7):  1380-1387.  doi: 10.12307/2025.020 Abstract ( 163 )   PDF (1341KB) ( 141 )   Save BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which is secreted in different types of stem cells and can regulate the proliferation, differentiation and migration of various types of stem cells. Our previous research has confirmed that human embryonic stem cells secrete MIF and that its concentration in the culture medium is relatively stable. However, whether MIF is involved in the survival, proliferation and differentiation of human embryonic stem cells remains unclear. OBJECTIVE: To investigate the effects of MIF on survival, proliferation, and differentiation of human embryonic stem cells.  METHODS: (1) Human embryonic stem cells H9 were cultured. The growth curve of cells was detected and plotted by CCK-8 assay. Enzyme-linked immunosorbent assay was used to determine the level of MIF in the medium. (2) To determine the effects of exogenous MIF on the survival and proliferation of human embryonic stem cells, different groups were established: the control group, which was cultured in stem cell medium without any modifications; the exogenous MIF group, which was treated with different concentrations (30, 100, 300 ng/mL) of MIF in the stem cell medium; the MIF inhibitor ISO-1 group, which was treated with different concentrations (2, 7, 21 μmol/L) of ISO-1 in the stem cell medium; and the MIF+ISO-1 group, which was treated with different concentrations of ISO-1 along with 100 ng/mL of MIF. Cell viability was assessed using the CCK-8 assay. (3) To further elucidate the effect of MIF gene on survival and proliferation of human embryonic stem cell, the MIF knockout H9 cell line was constructed by CRISPR-Cas 9 technology to observe the lineage establishment. (4) To determine the effect of high concentrations of MIF on human embryonic stem cell differentiation, 100 ng/mL MIF and 100 ng/mL of CXCR4 neutralizing antibody were separately added to the normal stem cell culture medium. The expression levels of self-renewal factors (KLF4, c-MYC, NANOG, OCT4, and SOX2) and differentiation transcription factors (FOXA2, OTX2) were measured using real-time quantitative polymerase chain reaction, immunofluorescence staining, and western blot analysis. RESULTS AND CONCLUSION: (1) The logarithmic growth phase of H9 cells was between 3-6 days. Under normal growth conditions, human embryonic stem cells secreted MIF at a concentration of approximately 20 ng/mL, independent of cell quantity. (2) Compared to the control group, the addition of different concentrations of MIF had no effect on the proliferation of human embryonic stem cells (P > 0.05). ISO-1 significantly inhibited the proliferation of human embryonic stem cells, with a stronger inhibition observed at higher concentrations of ISO-1 (P < 0.05). The addition of MIF in the presence of ISO-1 reduced the inhibitory effect of ISO-1 (P < 0.05). (3) Real-time quantitative polymerase chain reaction showed that knocking out 50% of the MIF gene resulted in a significant decrease in the growth vitality of human embryonic stem cells and failure to establish cell lines. (4) Adding 100 ng/mL exogenous MIF to the culture medium resulted in a decrease in the mRNA, protein, and fluorescence expression levels of the self-renewal transcription factor KLF4, while the mRNA, protein, and fluorescence expression levels of the differentiation factor FOXA2 increased. (5) When 100 ng/mL CXCR4 neutralizing antibody was added to the culture medium, the mRNA and protein expression levels of KLF4 increased, while the mRNA and protein expression levels of FOXA2 decreased, contrary to the expression trend observed in the MIF group. In conclusion, the endogenous secretion of MIF by human embryonic stem cells is essential for their survival. The addition of MIF to the culture medium does not promote the proliferation of human embryonic stem cells. However, it can lead to a decrease in the expression of the self-renewal factor KLF4 and an increase in the expression of the transcription factor FOXA2. This provides a clue for further investigation into the effects and mechanisms of MIF on the differentiation of human embryonic stem cells. The MIF-CXCR4 axis plays a regulatory role in this process. Figures and Tables | References | Related Articles | Metrics

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Osteogenesis-induced exosomes derived from human periodontal ligament stem cells promote osteogenic differentiation of human periodontal ligament stem cells in an inflammatory microenvironment Aikepaer · Aierken, Chen Xiaotao, Wufanbieke · Baheti 2025, 29 (7):  1388-1394.  doi: 10.12307/2025.024 Abstract ( 157 )   PDF (1939KB) ( 25 )   Save BACKGROUND: The osteogenic differentiation ability of exosomes derived from osteogenic mesenchymal stem cells is well established. However, their impact on the osteogenic differentiation of human periodontal ligament stem cells in an inflammatory microenvironment remains unclear. OBJECTIVE: To examine the impact of exosomes derived from osteogenesis-induced human periodontal ligament stem cells on the osteogenic differentiation of human periodontal ligament stem cells within an inflammatory microenvironment. METHODS: Human periodontal ligament stem cells were isolated and cultured. After 3 days of osteogenic induction, exosomes were extracted. Human periodontal ligament stem cells were divided into four groups. Control group was treated with osteogenesis-induced medium. The exosome group was treated with osteogenesis-induced medium containing 5 μg/mL exosomes. Inflammatory model and inflammatory model + exosome groups were treated with 1 μg/mL lipopolysaccharide for 24 hours to construct a cellular inflammatory microenvironment. The inflammatory model group was treated with osteogenesis-induced medium after lipopolysaccharide intervention. The inflammatory model + exosome group was treated with osteogenesis-induced medium containing 5 μg/mL exosome. The osteogenic differentiation ability of human periodontal ligament stem cells was assessed using alkaline phosphatase staining and alizarin red staining. The expressions of Runt-related transcription factor 2, osteopontin, osteoblast-specific transcription factor Osterix (OSX) and wnt pathway-related protein β-catenin were detected by real-time fluorescence quantitative PCR and western blot assay.  RESULTS AND CONCLUSION: (1) Compared with the control group, the relative area stained by alkaline phosphatase, the relative area stained by mineralized nodules and the expression levels of Runx2, osteopontin, and OSX were significantly decreased in the inflammatory model group (P < 0.05). (2) Compared with the inflammatory model group, the expression of Runx2, osteopontin, and OSX in the inflammatory model + exosome group was significantly increased in the relative area of alkaline phosphatase staining, the relative area of mineralized nodules staining (P < 0.05). (3) Compared with the control group, the expression of wnt pathway-related protein β-catenin was significantly increased in the inflammatory model group (P < 0.05). Compared with the inflammatory model group, the expression of β-catenin in the inflammatory model + exosome group was significantly decreased (P < 0.05). These findings indicate that exosomes derived from human periodontal ligament stem cells induced by bone formation can enhance the osteogenic differentiation of human periodontal ligament stem cells within an inflammatory microenvironment, and the mechanism may be related to wnt/β-catenin signaling pathway. Figures and Tables | References | Related Articles | Metrics

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An experimental method for simultaneous extraction and culture of primary cortical neurons and microglial cells from SD rats He Longcai, Song Wenxue, Ming Jiang, Chen Guangtang, Wang Junhao, Liao Yidong, Cui Junshuan, Xu Kaya 2025, 29 (7):  1395-1400.  doi: 10.12307/2025.011 Abstract ( 178 )   PDF (1270KB) ( 137 )   Save BACKGROUND: Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders, and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction. It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously. OBJECTIVE: To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells.  METHODS: Newborn suckling SD rats were taken within 24 hours. The brain was removed and placed in a dish with DMEM, and the pia mater was removed for later use. Primary neurons were extracted from the same brain tissue, and then the remaining brain tissue was used to extract microglial cells. The whole process was performed on ice. Extraction and culture steps of primary cortical neurons: The cerebral cortex was taken 2.0-3.0 mm with forceps, and the tissue was digested with papain for 20 minutes. After aborting digestion, the blown tissue presented an adherent tissue suspension. The supernatant cell suspension was obtained, filtered, and dispensed into 15 mL centrifuge tubes. After centrifugation and re-suspension, the cells were inoculated onto 6-well plate crawls coated with L-polylysine. Neuronal morphology was observed at 1-day intervals, and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7. Microglia extraction and culture steps: The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction, digested by trypsin for 20 minutes. After digestion was stopped, the tissue was blown to a homogenate, and then the homogenate was transferred to the culture bottle for culture. On day 14, the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours. Microglial cells were shed in the supernatant. Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining. RESULTS AND CONCLUSION: (1) After 24 hours of culture, the neurons were adherent to the wall, the cytosol was enlarged, and some neurons developed synapses. After 3 and 5 days of culture, the cytosol was further enlarged, and most of the neurons were in the form of synapses, and some neurons were growing in clusters. On day 7, neuronal synapses were prolonged and thickened, and they were connected with each other to form a network. The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining. (2) The cells grew close to the wall on day 1 of culture. On days 3, 5, and 7, the density of microglial cells was small, and the cell morphology was bright oval or round, but the cells basically grew in clumps on the upper layer of other cells. On day 10, the density of microglial cells increased significantly. On day 14, microglial cells grew in dense clumps on the upper layer of other cells, and then they could be isolated and purified. The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence; their purity was greater than 95%. (3) The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity, good morphology, and high viability. Figures and Tables | References | Related Articles | Metrics

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Changes in ferroptosis in hippocampal neurons of vascular dementia model rats treated with Tongmai Kaiqiao Pill Zhao Nannan, Li Yanjie, Qin Hewei, Zhu Bochao, Ding Huimin, Xu Zhenhua 2025, 29 (7):  1401-1407.  doi: 10.12307/2024.740 Abstract ( 139 )   PDF (2005KB) ( 183 )   Save BACKGROUND: Research has demonstrated a close association between ferroptosis and vascular dementia. Tongmai Kaiqiao Pill has a certain effect on improving the cognitive function of vascular dementia patients, but its mechanism is unclear. OBJECTIVE: To explore the interventional effects and molecular mechanisms of Tongmai Kaiqiao Pill for vascular dementia based on the regulation of ferroptosis by the nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/glutathione peroxidase 4 (GPX4) signaling pathway.  METHODS: Among eighty-four SD male rats, 12 rats were used as the sham-operated group, and the rest of them were prepared as a model of vascular dementia by the modified 2-VO method, and then randomly divided into the model group, the Tongmai Kaiqiao Pills high-, moderate-, and low-dosage (27.6, 13.8, and 6.9 g/kg) groups, the combined group (Tongmai Kaiqiao Pill high-dosage+ML385, 20 mg/kg), and the donepezil hydrochloride group (0.45 mg/kg). The drug was given once a day by intragastric administration. The combined group was also intraperitoneally injected Nrf2 inhibitor ML385, once a day, for 4 weeks. Morris water maze was used to detect the learning memory ability of rats. Hematoxylin-eosin staining was used to observe the histopathological changes in the hippocampus of rats in each group. Colorimetric assay was used to detect the content of reduced glutathione, ferrous ion (Fe2+), and malondialdehyde in the serum of rats. Prussian blue staining was used to detect the iron deposition in the hippocampal tissue of rats. Transmission electron microscopy was used to observe the ultrastructural changes of mitochondria in rat hippocampal tissues. Western blot assay was used to detect the protein expression levels of Nrf2, HO-1, GPX4, XCT, and ferritin heavy chain 1 (FTH1) in rat hippocampal tissues.  RESULTS AND CONCLUSION: (1) In comparison to the sham operation, rats in the model group exhibited a significantly prolonged latency period (P < 0.05) and a reduced number of platform crossings (P < 0.05). Additionally, the hippocampal tissues of these rats displayed loosely organized structure, deeply stained cell nuclei, and solidified or lysed chromatin. Ferri ions aggregated in CA1 region. There were atrophied mitochondria with dissolved cristae and thickened mitochondrial membranes. Fe2+, malondialdehyde, and reduced glutathione levels in rat serum were found to be elevated (P < 0.05). A significant reduction in the expression of GPX4, HO-1, XCT, Nrf2, and FTH1 proteins was detected in the hippocampus (P < 0.05). (2) Compared to the model group, the average escape latency of the rats was significantly reduced following intervention with Tongmai Kaiqiao Pills and donepezil hydrochloride (P < 0.05), with an increased number of platform crossings (P < 0.05). Hippocampal neurons showed significant recovery. Notably, iron aggregation in the CA1 region was significantly reduced, and mitochondrial structure and function were improved. There were significant reductions in Fe2+ and malondialdehyde levels, while the levels of GPX4, HO-1, XCT, Nrf2, and FTH1 in rat hippocampal tissues, and reduced glutathione in serum were significantly increased (P < 0.05). (3) The high-dose Tongmai Kaiqiao Pills exhibited a treatment effect comparable to that of donepezil hydrochloride (P > 0.05), with a significant prolongation of water maze escape latency (P < 0.05), a reduced number of platform crossings (P < 0.05), and insignificant neuronal pathological changes in the CA1 area. However, the combined group showed increased iron deposition, elevated malondialdehyde and Fe2+ levels in blood serum (P < 0.05), reduced glutathione content (P < 0.05), hippocampal tissue mitochondrial atrophy, and reduced expression of Nrf2, XCT, HO-1, GPX4, and FTH1 proteins (P < 0.05). Within a certain range, higher doses of Tongmai Kaiqiao Pills demonstrated a more pronounced effect, comparable to the efficacy of high-dose donepezil hydrochloride. (4) It is concluded that Tongmai Kaiqiao Pills have been shown to mitigate histopathological changes in the rat hippocampus and enhance cognitive function in rats with vascular dementia. The mechanism of action is likely associated with the suppression of ferroptosis through the activation of the Nrf2/HO-1/GPX4 signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Verbascoside inhibits Erastin-induced ferroptosis of dopaminergic nerve cell line MN9D cells Zhang Mingyang, Yang Xinling 2025, 29 (7):  1408-1413.  doi: 10.12307/2025.008 Abstract ( 128 )   PDF (1573KB) ( 88 )   Save BACKGROUND: In recent years, more and more studies have confirmed that ferroptosis of dopaminergic neurons is involved in the pathogenesis of Parkinson’s disease, and verbascoside has been confirmed to have antioxidant, anti-inflammatory and neuroprotective effects. OBJECTIVE: To investigate the protective effect of verbascoside on Erastin-induced ferroptosis of MN9D cells and its action mechanism. METHODS: MN9D cells were divided into control group, model group (20 μmol/L Erastin group), Erastin+1 μg/mL verbascoside group, Erastin+5 μg/mL verbascoside group, and Erastin+10 μg/mL verbascoside group. MN9D cells were cultured in a CO2 incubator for 24 hours, then pretreated with different mass concentrations of verbascoside for 8 hours, and induced with 20 μmol/L Erastin for 24 hours. The levels of reduced glutathione, superoxide dismutase, total iron ion, and malondialdehyde were detected by ELISA. The expression of tyrosine hydroxylase was detected by immunohistochemistry. The expressions of tyrosine hydroxylase, nuclear factor erythrocyte-2-associated factor 2, heme oxygenase-1, and glutathione peroxidase 4 were detected by western blot assay.  RESULTS AND CONCLUSION: (1) Compared with the control group, the levels of reduced glutathione and superoxide dismutase were significantly decreased (P < 0.05), and the levels of malondialdehyde and total iron ion were significantly increased in the model group (P < 0.05). Compared with the model group, the levels of reduced glutathione and superoxide dismutase were significantly increased (P < 0.05), and the levels of malondialdehyde and total iron ionized water were decreased in 1, 5, 10 μg/mL verbascoside groups (P < 0.05). (2) Compared with the control group, the area of tyrosine hydroxylase positive cells in the model group was significantly reduced (P < 0.05). Compared with the model group, the area of tyrosine hydroxylase positive cells was significantly increased in 1, 5, 10 μg/mL verbascoside groups (P < 0.05). (3) Compared with the control group, the protein expressions of tyrosine hydroxylase, nuclear factor erythrocyte-2-associated factor 2, heme oxygenase-1 and glutathione peroxidase 4 were significantly decreased in the model group (P < 0.05). Compared with the model group, the protein expression levels of tyrosine hydroxylase, nuclear factor erythrocyte-2-associated factor 2, heme oxygenase-1, and glutathione peroxidase 4 were significantly increased in 1, 5, 10 μg/mL verbascoside groups (P < 0.05). The results suggested that verbascoside could inhibit Erastin-induced ferroptosis in MN9D cells, possibly by activating nuclear factor erythrocyte-2-associated factor 2/ heme oxygenase-1/glutathione peroxidase 4 pathway. Figures and Tables | References | Related Articles | Metrics

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Construction of lentiviral vectors for solute carrier family 1 member 5  overexpression and knockdown and stably transfected RAW264.7 cell line Guo Daxin, Fan Susu, Zhu Zhendong, Hou Jianhong, Zhang Xuan 2025, 29 (7):  1414-1421.  doi: 10.12307/2025.025 Abstract ( 227 )   PDF (4012KB) ( 163 )   Save BACKGROUND: Solute carrier family 1 member 5 (SLC1A5) plays a potential role in a variety of diseases, but the exact mechanism of action is unclear. The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets. OBJECTIVE: To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines, so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation.  METHODS: Primers were designed and synthesized based on the SLC1A5 gene sequence, and the gene segment was amplified using polymerase chain reaction. Subsequently, the target gene segment was directionally inserted into the GV492 vector plasmid, which had been digested with AgeI/NheI enzymes, to construct recombinant lentiviral plasmids. Positive clones were further selected, and their sequences were confirmed. The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector, along with the target plasmid vector, was co-cultured with 293T cells for transfection, resulting in the production and titration of lentiviral stocks. Furthermore, RAW264.7 cells were cultured in vitro, and the working concentration of puromycin was determined. Lentiviruses were separately co-cultured with RAW264.7 cells, and transfection efficiency was determined by measuring fluorescence intensity. Stable transfected cells were selected using puromycin, and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines.  RESULTS AND CONCLUSION: (1) Sequencing results indicated a perfect match between the sequencing and target sequences, confirming the successful construction of recombinant lentiviral vectors. (2) The titer for the overexpression SLC1A5 lentivirus was 1×109 TU/mL, while the titer for the knockdown SLC1A5 lentivirus was 3×109 TU/mL. (3) The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL. (4) The optimal conditions for transfecting RAW264.7 cells with overexpression/knockdown expression of SLC1A5 lentivirus involved the use of HiTransG P transfection enhancer with a multiplicity of infection value of 50. (5) A significant upregulation of the gene and protein expression levels of SLC1A5 was detected in cell lines stably overexpressing SLC1A5, while gene and protein expression levels of SLC1A5 were significantly decreased in the knockdown stable cell lines. These findings indicate that lentiviral vectors for mouse SLC1A5 overexpression and knockdown have been successfully constructed and a stably transfected RAW264.7 cell line has been obtained. Figures and Tables | References | Related Articles | Metrics

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Inflammatory factor interferon-gamma affects migration and apoptosis of human vascular smooth muscle cells through pyroptosis pathway Wan Lingling, Wu Mengying, Zhang Yujiao, Luo Qingqing 2025, 29 (7):  1422-1428.  doi: 10.12307/2025.004 Abstract ( 124 )   PDF (1294KB) ( 84 )   Save BACKGROUND: Successful uterine spiral artery remodeling is necessary for normal pregnancy, in which vascular smooth muscle cells are important cells. Interferon-γ is associated with the loss of vascular smooth muscle cells during early pregnancy. However, the specific mechanism is not fully understood.   OBJECTIVE: To investigate the effects of interferon-γ on migration and apoptosis of vascular smooth muscle cells through NLRP3/caspase-1/GSDMD pyroptosis pathway. METHODS: Human vascular smooth muscle cells were divided into control group and interferon-γ group. The control group was cultured normally, and the interferon-γ group was treated with 10 ng/mL interferon-γ for 24 hours. The migration ability of vascular smooth muscle cells was detected by Transwell assay. The apoptosis of vascular smooth muscle cells was detected by TUNEL assay and flow cytometry. The mRNA expression levels of NLRP3 and caspase-1 were detected by qPCR. Western blot assay was utilized to detect NLRP3, caspase-1, and cleaved N-terminal GSDMD protein expression levels. RESULTS AND CONCLUSION: Compared with the control group, the migration ability and apoptosis rate of vascular smooth muscle cells in interferon-γ group were significantly increased (P < 0.05). Compared with the control group, the mRNA expression levels of NLRP3 and caspase-1 in vascular smooth muscle cells of interferon-γ group were significantly increased (P < 0.05). Compared with control group, the expression levels of NLRP3, caspase-1, and cleaved N-terminal GSDMD protein in vascular smooth muscle cells in the interferon-γ group were significantly increased (P < 0.05). The results suggest that interferon-γ may regulate the migration and apoptosis of vascular smooth muscle cells through NLRP3/caspase-1/GSDMD pyroptosis pathway.  Figures and Tables | References | Related Articles | Metrics

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Comprehensive analysis of genes related to endometrial receptivity and alternative splicing events in northwest Tibetan cashmere goats De Ji, Suo Langda,  Wei Yuchen,  Wang Bin, Awangcuoji, Renqingcuomu, Cui Jiuzeng, Zhang Lei, Ba Gui 2025, 29 (7):  1429-1436.  doi: 10.12307/2025.010 Abstract ( 90 )   PDF (2544KB) ( 126 )   Save BACKGROUND: Endometrial receptivity is a key factor in embryo implantation in northwest Tibetan cashmere goats, and the expression of genes related to endometrial receptivity and their variable splicing are still unclear. OBJECTIVE: To analyze and explore genes and variable splicing events related to endometrial receptivity in northwest Tibetan cashmere goats.  METHODS: On days 5 and 15 of pregnancy (representing pre receptive endometrium group and receptive endometrium group), three northwest Tibetan cashmere goats were randomly selected. Endometrial tissue was collected and stained with hematoxylin and eosin to observe tissue morphology. Immunohistochemical staining was used to detect the expression of endometrial receptive marker proteins leukemia inhibitory factor and vascular endothelial growth factor. After the total RNA was extracted and the quality test was qualified, transcriptome sequencing was performed to search differentially expressed mRNAs, lncRNAs, circRNAs, and miRNAs, perform functional prediction, and analyze alternative splicing mRNAs and lncRNAs related to endometrial receptivity.  RESULTS AND CONCLUSION: (1) Compared with the pre receptive endometrium group, the expression levels of leukemia inhibitory factor and vascular endothelial growth factor proteins in the endometrial tissue of the receptive endometrium group were significantly increased. (2) The sequencing results showed that the differentially expressed genes were mostly mRNA and lncRNA genes, including 250 upregulated mRNAs, 193 upregulated lncRNAs, 135 downregulated mRNAs, and 123 downregulated lncRNAs, which were significantly enriched in the Wnt, Hedgehog, and Hippo signaling pathways. (3) Alternative splicing event analysis uncovered 8 differentially expressed variable splicing transcripts, which were all mRNA transcripts, including 2 downregulated and 6 upregulated, and were significantly associated with vascular endothelial growth factor receptor signaling, cell motility, and embryonic development.  Figures and Tables | References | Related Articles | Metrics

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Target of neohesperidin in treatment of osteoporosis and its effect on osteogenic differentiation of bone marrow mesenchymal stem cells Zhang Zhenyu, Liang Qiujian, Yang Jun, Wei Xiangyu, Jiang Jie, Huang Linke, Tan Zhen 2025, 29 (7):  1437-1447.  doi: 10.12307/2025.030 Abstract ( 110 )   PDF (3446KB) ( 126 )   Save BACKGROUND: Previous studies have found that neohesperidin can delay bone loss in ovariectomized mice and has the potential to treat osteoporosis, but its specific mechanism of action remains to be explored. OBJECTIVE: To explore the key targets and possible mechanisms of neohesperidin in the treatment of osteoporosis based on bioinformatics and cell experiments in vitro. METHODS: The gene expression dataset related to osteoporosis was obtained from GEO database, and the differentially expressed genes were screened and analyzed in R language. The osteoporosis-related targets were screened from GeneCards and DisGeNET databases, and the neohesperidin-related targets were screened from ChEMBL and PubChem databases, and the common targets were obtained by intersection of the three. The String database was used to construct the PPI network of intersection genes, and the key targets were screened. The DAVID database was used for GO and KEGG enrichment analysis. The AutoDock software was used to verify the molecular docking between the neohesperidin and the target protein. The effect of neohesperidin on osteogenic differentiation of C57 mouse bone marrow mesenchymal stem cells was detected. Complete medium was used as blank control group; osteogenic induction medium was used as the control group; and osteogenic induction medium containing different concentrations of neohesperidin (25, 50 μmol/L) was used as experimental group. The expression of alkaline phosphatase, the degree of mineralization, the expression of osteogenic-related genes and target genes during osteogenic differentiation of cells were measured at corresponding time points. RESULTS AND CONCLUSION: (1) 9 253 differentially expressed genes, 2 161 osteoporosis-related targets, and 326 neohesperidin-related targets were screened. There were 53 common targets among the three. All 53 genes were up-regulated in osteoporosis samples. The PPI network screened the target gene PRKACA of research significance. GO function and KEGG pathway enrichment analysis showed that neohesperidin’s treatment of osteoporosis through PRKACA target mainly depended on biological processes such as protein phosphorylation and protein autophosphorylation, acting on endocrine resistance, proteoglycan in cancer, and estrogen signaling pathway to play a therapeutic role. Molecular docking results showed that neohesperidin had a certain binding ability to the protein corresponding to the target PRKACA. (2) The results of alkaline phosphatase staining showed that neohesperidin could promote the expression of alkaline phosphatase in the early stage of osteogenic differentiation of mesenchymal stem cells. Alizarin red staining showed that neohesperidin could promote the mineralization of osteogenic differentiation of mesenchymal stem cells. RT-qPCR results showed that neohesperidin could increase the mRNA expression of alkaline phosphatase, PRKACA, and osteocalcin. (3) These results indicate that neohesperidin may promote osteogenic differentiation through PRKACA target on the estrogen signaling pathway to prevent and treat osteoporosis. Figures and Tables | References | Related Articles | Metrics

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Identification and drug sensitivity analysis of key molecular markers in mesenchymal cell-derived osteosarcoma Zhang Haojun, Li Hongyi, Zhang Hui, Chen Haoran, Zhang Lizhong, Geng Jie, Hou Chuandong, Yu Qi, He Peifeng, Jia Jinpeng, Lu Xuechun 2025, 29 (7):  1448-1456.  doi: 10.12307/2025.009 Abstract ( 125 )   PDF (5122KB) ( 181 )   Save BACKGROUND: Osteosarcoma has a complex pathogenesis and a poor prognosis. While advancements in medical technology have led to some improvements in the 5-year survival rate, substantial progress in its treatment has not yet been achieved. OBJECTIVE: To screen key molecular markers in osteosarcoma, analyze their relationship with osteosarcoma treatment drugs, and explore the potential disease mechanisms of osteosarcoma at the molecular level.  METHODS: GSE99671 and GSE284259 (miRNA) datasets were obtained from the Gene Expression Omnibus database. Differential gene expression analysis and Weighted Gene Co-expression Network Analysis (WGCNA) on GSE99671 were performed. Functional enrichment analysis was conducted using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes separately for the differentially expressed genes and the module genes with the highest positive correlation to the disease. The intersection of these module genes and differentially expressed genes was taken as key genes. A Protein-Protein Interaction network was constructed, and correlation analysis on the key genes was performed using CytoScape software, and hub genes were identified. Hub genes were externally validated using the GSE28425 dataset and text validation was conducted. The drug sensitivity of hub genes was analyzed using the CellMiner database, with a threshold of absolute value of correlation coefficient |R| > 0.3 and P < 0.05.  RESULTS AND CONCLUSION: (1) Differential gene expression analysis identified 529 differentially expressed genes, comprising 177 upregulated and 352 downregulated genes. WGCNA analysis yielded a total of 592 genes with the highest correlation to osteosarcoma. (2) Gene Ontology enrichment results indicated that the development of osteosarcoma may be associated with extracellular matrix, bone cell differentiation and development, human immune regulation, and collagen synthesis and degradation. Kyoto Encyclopedia of Genes and Genomes enrichment results showed the involvement of pathways such as PI3K-Akt signaling pathway, focal adhesion signaling pathway, and immune response in the onset of osteosarcoma. (3) The intersection analysis revealed a total of 59 key genes. Through Protein-Protein Interaction network analysis, 8 hub genes were selected, which were LUM, PLOD1, PLOD2, MMP14, COL11A1, THBS2, LEPRE1, and TGFB1, all of which were upregulated. (4) External validation revealed significantly downregulated miRNAs that regulate the hub genes, with hsa-miR-144-3p and hsa-miR-150-5p showing the most significant downregulation. Text validation results demonstrated that the expression of hub genes was consistent with previous research. (5) Drug sensitivity analysis indicated a negative correlation between the activity of methotrexate, 6-mercaptopurine, and pazopanib with the mRNA expression of PLOD1, PLOD2, and MMP14. Moreover, zoledronic acid and lapatinib showed a positive correlation with the mRNA expression of PLOD1, LUM, MMP14, PLOD2, and TGFB1. This suggests that zoledronic acid and lapatinib may be potential therapeutic drugs for osteosarcoma, but further validation is required through additional basic experiments and clinical studies. Figures and Tables | References | Related Articles | Metrics

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Bibliometric analysis of research process and current situation of brain aging and exosomes Lyu Liting, Yu Xia, Zhang Jinmei, Gao Qiaojing, Liu Renfan, Li Meng, Wang Lu 2025, 29 (7):  1457-1465.  doi: 10.12307/2025.015 Abstract ( 174 )   PDF (4310KB) ( 244 )   Save BACKGROUND: In recent years, with the rapid development of biomedicine, the study of brain aging and exosomes has attracted more and more attention, but there is no bibliometrics analysis in this field.  OBJECTIVE: To objectively analyze domestic and foreign literature on brain aging and exosomes in the past 15 years, to summarize the research status, hot spots, and development trends in this field.  METHODS: Using the core database of Web of Science as a search platform, we downloaded the literature on brain aging and exosomes published from the establishment of the database to December 28, 2022, and analyzed the data from the aspects of country or region, institution, author, keywords, and co-cited literature using CiteSpace 6.1.R6 visualization software. RESULTS AND CONCLUSION: A total of 1 045 research articles were included, and the number of publications on brain aging and exosomes research both domestically and internationally was showing an increasing trend year by year. The United States ranked first with 429 papers, and China ranked second with 277 papers. Louisiana State University ranked first with 16 articles. Professor Lukiw Walter J from Louisiana State University in the United States was the author with the highest number of publications, and Professor Bartel DP from the Massachusetts Institute of Technology was the author with the most citations. The most prolific Journal was the International Journal of Molecular Sciences. Alzheimer’s disease, microRNA, gene expression, extracellular vesicles, exosomes, oxidative stress, and biomarkers are the most relevant terms. According to the research on hot topics, biomarkers have become a new research hotspot. The above results indicate that the research on brain aging and exosomes has gradually increased in the past 15 years. The research direction has gradually shifted from the initial exploration of the expression of miRNAs in central nervous system diseases related to brain aging to the search for biomarkers that can identify and diagnose neurodegenerative diseases. The study of exocrine miRNAs to protect central nervous system from damage has emerged as promising therapeutic strategy. Figures and Tables | References | Related Articles | Metrics

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Identification and validation of characterized gene NFE2L2 for ferroptosis in ischemic stroke Wang Mi, Ma Shujie, Liu Yang, Qi Rui 2025, 29 (7):  1466-1474.  doi: 10.12307/2025.027 Abstract ( 147 )   PDF (3982KB) ( 259 )   Save BACKGROUND: Ferroptosis is closely associated with the pathogenesis of ischemic stroke, and targeting ferroptosis is a promising regimen for the treatment of ischemic stroke, but the specific regulatory targets are unclear. OBJECTIVE: To screen ferroptosis-related characterized genes in ischemic stroke by bioinformatics and machine learning methods and validate them by cellular experiments to investigate the role of ferroptosis in ischemic stroke.  METHODS: Eligible ischemic stroke-related datasets and ferroptosis expression datasets were selected based on GEO database and FerrDb database, and ferroptosis-related differential genes were screened by t-test. GO functional enrichment analysis with KEGG signaling pathway enrichment analysis was performed for ferroptosis-related differential genes. Characterized genes for ferroptosis in ischemic stroke were screened by PPI network analysis and machine learning. The reliability and biological functions of the characterized genes were explored using ROC analysis and GSEA analysis, followed by cell experiment. HT22 cells were divided into control and ischemic stroke groups. No intervention was made in the control group, and 0.1 mM H2O2 was added to the ischemic stroke group for 24 hours to simulate cellular oxidative stress injury and ferroptosis. The ferroptosis and the expression of characterized genes were verified by real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and western blot assay. RESULTS AND CONCLUSION: (1) Forty-five ferroptosis-associated differential genes were obtained, and GO and KEGG enrichment analyses revealed that the differential genes were closely associated with oxidative stress, autophagy, ferroptosis, adipocytokine signaling pathway, and mitochondrial metabolism. (2) A total of one ferroptosis characterized gene, nuclear factor erythroid 2-related factor 2 (NFE2L2), was identified by the MCODE plugin and cytoHubba plugin in the PPI network with the LASSO algorithm and SVM-RFE algorithm in machine learning. (3) Receiver operating characteristic curve analysis of NFE2L2 revealed that the diagnostic prediction models constructed in the training and validation sets had good accuracy and specificity. GSEA analysis of NFE2L2 revealed that the characterized gene was involved in the regulation of ischemic stroke pathogenesis through immunity, inflammatory response, amino acid metabolism, and neurofactor regulation. (4) RT-PCR and western blot analyses showed that the acyl coenzyme A synthetase long chain family, member 4 (ACSL4) mRNA and protein expression levels were significantly higher in the ischemic stroke group compared with the control group (P < 0.05), and the glutathione peroxidase 4 (GPX4) mRNA and protein expression levels were significantly lower in the ischemic stroke group (P < 0.05). Compared with the control group, the mRNA and protein expression levels of the characterized gene NFE2L2 were significantly higher in the ischemic stroke group (P < 0.05). (5) It suggests that ischemic stroke is closely related to ferroptosis, and targeting the characterized gene NFE2L2 may provide certain ideas and directions for the study and treatment of ischemic stroke.  Figures and Tables | References | Related Articles | Metrics

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Research hotspots and frontiers of stem cells for Alzheimer’s disease Xie Liugang, Cui Shuke, Guo Nannan, Li Aoyu, Zhang Jingrui 2025, 29 (7):  1475-1485.  doi: 10.12307/2025.026 Abstract ( 236 )   PDF (3812KB) ( 285 )   Save BACKGROUND: Stem cells can promote nerve regeneration, repair damaged nerves, inhibit inflammation and apoptosis of nerve cells, and provide a new way for the treatment of Alzheimer’s disease.  OBJECTIVE: To make a bibliometrical analysis of the articles on stem cell therapy for Alzheimer’s disease published internationally from 2004 to 2023, in order to reveal the research hotspot and trend of stem cell therapy for Alzheimer’s disease.  METHODS: From the Web of Science Core Collection database, by using Excel, VOSviewer, and Citespace software, the annual number of publications, countries, institutions, journals/co-cited journals, authors, and keywords of articles related to stem cells and Alzheimer’s disease published from January 1, 2004 to October 31, 2023 were visually analyzed.  RESULTS AND CONCLUSION: A total of 3 521 core papers were included, and the number of published papers increased year by year. The United States is the country with the most papers. Harvard Medical School is the most prolific institution. Maiese kenneth is the author with the most papers. International Journal of Molecular Sciences has the most papers in this field. The journal PLoS One published the most citations. At present, the field of stem cell therapy for Alzheimer’s disease focuses on pathophysiological mechanism and animal experimental research, and “neurogenesis”, “oxidative stress”, “extracellular vesicles”, and “mesenchymal stem cells” are the research trends in this field. Stem cell therapy for Alzheimer’s disease has broad prospects. In the future, exchanges and cooperation between institutions and authors should be strengthened to further explore the main mechanism of stem cell therapy for Alzheimer’s disease, solve possible clinical problems such as immune rejection, effectiveness, and safety, and further tap the potential of stem cells in the treatment of Alzheimer’s disease. Figures and Tables | References | Related Articles | Metrics

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Visualization analysis of macrophage polarization in tissue repair process Chang Jinxia, Liu Yufei, Niu Shaohui, Wang Chang, Cao Jianchun 2025, 29 (7):  1486-1496.  doi: 10.12307/2025.032 Abstract ( 193 )   PDF (4978KB) ( 204 )   Save BACKGROUND: During tissue repair and regeneration, macrophages exhibit multiple activities such as promoting inflammation, anti-inflammation, fibrosis, and wound healing at various stages of tissue damage. The heterogeneity and balanced polarization of macrophages are decisive in organ repair.   OBJECTIVE: To explore the research hotspots and development trends in the field of macrophage polarization in tissue repair through visualization analysis methods, as well as the research level of global scientific and clinical workers in this field.  METHODS: Using bibliometric analysis methods, this study employed Citespace literature visualization analysis software and VOSviewer tools, retrieving related literature from 2013 to 2023 in the Web of Science Core Collection’s Science Citation Index Expanded (SCI-Expanded) and Social Sciences Citation Index Expanded (SSCI-Expanded) databases. The analysis results were presented in a dynamic map format, revealing the main trends and focuses of the research. RESULTS AND CONCLUSION: The number of publications in this field had dramatically increased from 2013 to 2023, with a significant rise starting in 2017. Chinese researchers had the highest number of publications, with 642 papers, while American researchers began focusing on this field early on. Professor Elisseeff Hennifer H had made a substantial contribution to the research in this area. Shanghai Jiao Tong University had produced the most publications. In recent years, keywords such as “hyaluronic acid” and “regulation” had been prevalent. Macrophage polarization research in tissue repair primarily concentrates on its multifunctional regulatory mechanisms, interactions with other cell types, and its behavior under specific pathological conditions. The main research areas include the role of macrophages in wound healing, cardiovascular diseases, chronic inflammation, tumor microenvironments, and regenerative medicine. A deeper understanding of the multifunctionality and polarization mechanisms of macrophages can lead to the development of new therapeutic strategies to enhance tissue repair and regeneration, thereby improving patient treatment outcomes. Figures and Tables | References | Related Articles | Metrics

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Role and mechanism of platelet-derived growth factor BB in repair of growth plate injury Peng Hongcheng, Peng Guoxuan, Lei Anyi, Lin Yuan, Sun Hong, Ning Xu, Shang Xianwen, Deng Jin, Huang Mingzhi 2025, 29 (7):  1497-1503.  doi: 10.12307/2024.742 Abstract ( 125 )   PDF (985KB) ( 119 )   Save BACKGROUND: In the initial stage of growth plate injury inflammation, platelet-derived growth factor BB promotes the repair of growth plate injury by promoting mesenchymal progenitor cell infiltration, chondrogenesis, osteogenic response, and regulating bone remodeling. OBJECTIVE: To elucidate the action mechanism of platelet-derived growth factor BB after growth plate injury. METHODS: PubMed, VIP, WanFang, and CNKI databases were used as the literature sources. The search terms were “growth plate injury, bone bridge, platelet-derived growth factor BB, repair” in English and Chinese. Finally, 66 articles were screened for this review. RESULTS AND CONCLUSION: Growth plate injury experienced early inflammation, vascular reconstruction, fibroossification, structural remodeling and other pathological processes, accompanied by the crosstalk of chondrocytes, vascular endothelial cells, stem cells, osteoblasts, osteoclasts and other cells. Platelet-derived growth factor BB, as an important factor in the early inflammatory response of injury, regulates the injury repair process by mediating a variety of cellular inflammatory responses. Targeting the inflammatory stimulation mediated by platelet-derived growth factor BB may delay the bone bridge formation process by improving the functional activities of osteoclasts, osteoblasts, and chondrocytes, so as to achieve the injury repair of growth plate. Platelet-derived growth factor BB plays an important role in angiogenesis and bone repair tissue formation at the injured site of growth plate and intrachondral bone lengthening function of uninjured growth plate. Inhibition of the coupling effect between angiogenesis initiated by platelet-derived growth factor BB and intrachondral bone formation may achieve the repair of growth plate injury. Figures and Tables | References | Related Articles | Metrics

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Mechanism of exosomal miRNA involved in tumor chemotherapy resistance Weng Zongqin, Zhao Hailong 2025, 29 (7):  1504-1511.  doi: 10.12307/2025.016 Abstract ( 181 )   PDF (1401KB) ( 169 )   Save ACKGROUND: As a common method of cancer treatment, chemotherapy has a certain therapeutic effect in the early stage. However, as the frequency of chemotherapy increases, the resistance of tumor cells to drugs gradually increases, that is, chemotherapy resistance. At present, the main mechanisms of chemotherapy resistance include the failure of chemotherapy drugs to reach the tumor site, the loss of drug activity leading to a decrease in drug efficacy, and tumor cells evading drug attack by reshaping their metabolic functions, gene phenotypes, and microenvironment. However, the specific mechanism remains to be discussed in depth.  OBJECTIVE: To summarize the mechanisms by which exosomal miRNA is involved in drug resistance in different tumors, and to propose potential targets. METHODS: The PubMed database was searched using the English search terms “hematological malignancies, liver cancer, lung cancer, breast cancer, melanoma, colorectal cancer, drug resistance, exosomal miRNAs.” The search time range was from January 2013 to August 2023. Preliminary screening was carried out by reading the titles and abstracts, and the exclusion criteria were for literature with poor relevance to the research content or repetitive content. Finally, 60 articles were included for review. RESULTS AND CONCLUSION: (1) Exosomal miRNA enters the intercellular matrix and circulation, and participates in the formation and development of different tumor biological processes, including tumor microenvironment remodeling, angiogenesis, and drug resistance, by exerting local paracrine or distal effects. (2) Exosomal miRNA can penetrate the cell membrane into the cytoplasm of another cell, activate or inhibit the signaling pathway in the recipient cell by delivering miRNA, and maintain cell survival, proliferation, and metastasis during chemotherapy, thus leading to drug resistance. (3) Many exosomal miRNAs related to chemotherapy resistance have been identified and studied as common tumor treatment targets. (4) Although exosomal miRNA holds great promise as potential biomarkers and therapeutic targets, there are still some research deficiencies that need to be addressed: Firstly, there is a lack of standardized protocols for exosome isolation and characterization, resulting in differences in the methods used in different studies; secondly, although the presence and abundance of miRNAs in exosomes have been widely studied, their functional roles and mechanisms are not completely clear. Elucidating the specific targets and pathways regulated by exosomal can provide valuable insights into their biological significance and potential therapeutic applications. Addressing these research gaps will contribute to a more comprehensive understanding of exosomal miRNAs and their potential applications in various fields, including diagnosis, therapy, and personalized medicine. Figures and Tables | References | Related Articles | Metrics

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Molecular mechanisms underlying role of mesenchymal stem cell secretome Li Jialin, Zhang Yaodong, Lou Yanru, Yu Yang, Yang Rui 2025, 29 (7):  1512-1522.  doi: 10.12307/2025.022 Abstract ( 270 )   PDF (1279KB) ( 263 )   Save BACKGROUND: A large number of studies have confirmed that the therapeutic effectiveness of mesenchymal stem cell secretome is comparable to that of mesenchymal stem cells, but the mechanism of its action is still unclear. OBJECTIVE: To summarize the research progress of mesenchymal stem cell secretome in recent years, to investigate the molecular mechanism of its therapeutic effect, to analyze the current problems and to look forward to the future development. METHODS: The terms “exosomes, mesenchymal stem cells secrete, extracellular vesicles, mesenchymal stem cells, mechanism” were used as English search terms in the PubMed database. Articles that were not related to the research purpose of the article and duplicated articles were excluded. At the same time, we combined the method of literature tracking. Finally, 109 articles that met the criteria were incuded for the review. RESULTS AND CONCLUSION: (1) The mesenchymal stem cell secretome promotes tissue repair and regeneration through delivering genetic material, immunomodulatory factors, growth factors, etc. to target cells, by activating anti-apoptotic, regulating angiogenesis, modulating fibrosis and pro-survival pathways in target cells. (2) The potential of mesenchymal stem cell secretome in disease therapy has also been confirmed. Numerous research results have shown that mesenchymal stem cell secretome can be used as a new cell-free treatment for inflammatory and degenerative diseases. (3) Mesenchymal stem cell secretome has been engineered to have more efficient therapeutic effects in recent years. However, due to the heterogeneity of the mesenchymal stem cell secretome and the complexity of its components, the exact mechanism of its therapeutic effect is still unclear. (4) At present, further research is needed to identify the key targets of mesenchymal stem cell secretome, and innovative specific and enhanced mesenchymal stem cell secretome should be developed by combining with engineering and genetic engineering technologies in the future. Figures and Tables | References | Related Articles | Metrics

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Effect of extracellular vesicles for diagnosis and therapy of oral squamous cell carcinoma Cao Yue, Ye Xinjian, Li Biyao, Zhang Yining, Feng Jianying 2025, 29 (7):  1523-1530.  doi: 10.12307/2025.029 Abstract ( 93 )   PDF (958KB) ( 176 )   Save BACKGROUND: Extracellular vesicles are secreted into the extracellular milieu by a wide range of cell types, including tumor cells, under different physiological and pathophysiological conditions, where a wide range of biological signals and cell-to-cell signaling exists. Tumor-derived extracellular vesicles may exacerbate cancer progression, survival, invasion, and promote angiogenesis.  OBJECTIVE: To review the research progress of extracellular vesicles in the diagnosis and treatment of oral squamous cell carcinoma. METHODS: Literature search was performed by the first author in PubMed, WanFang, CNKI and other databases with the keywords “EVs, oral squamous cell carcinoma, diagnosis and treatment, biopsy, tissue engineering” in Chinese and English. Finally, 63 articles were included for analysis.  RESULTS AND CONCLUSION: (1) In oral squamous carcinoma saliva biopsies, extracellular vesicles play a crucial role in the progression of oral squamous cell carcinoma by acting as an information transfer tool between tumor cells and the surrounding microenvironment, carrying a wide range of biomolecules including soluble proteins, lipids, DNA, and RNA. These tiny vesicles not only play a key role in tumor growth and spread, but also provide important information about the biological properties of the tumor. (2) Saliva biopsy, as a non-invasive diagnostic method, can open up new possibilities for early diagnosis and targeted treatment of oral squamous cell carcinoma by analyzing the extracellular vesicles therein. (3) It has been found that bioactive molecules, such as microRNAs (miRNAs) and specific proteins, contained within extracellular vesicles can serve as biomarkers for oral squamous carcinoma and improve the accuracy of early diagnosis. Specific proteins in extracellular vesicles such as EHD2, CAVIN1, PF4V1, and CXCL7 show potential as novel predictive biomarkers. (4) In addition, this paper highlights the potential application of extracellular vesicles in the treatment of oral squamous carcinoma. Through engineering modifications, extracellular vesicles can serve as a new generation of nanoscale drug delivery systems to enhance the efficiency and specificity of targeted tumor therapy. (5) Future studies will further explore the effect and mechanism of extracellular vesicles in oral squamous cell carcinoma, which is expected to improve patients’ survival and quality of life. Figures and Tables | References | Related Articles | Metrics

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Physical factors and action mechanisms affecting osteogenic/odontogenic differentiation of dental pulp stem cells Sun Yuting, Wu Jiayuan, Zhang Jian 2025, 29 (7):  1531-1540.  doi: 10.12307/2024.743 Abstract ( 258 )   PDF (1061KB) ( 239 )   Save BACKGROUND: Dental pulp stem cells are one of the stem cells with great potential in oral and maxillofacial tissue engineering. Compared with mesenchymal stem cells, dental pulp stem cells have the advantages of convenient collection, less ethical problems and higher potential of proliferation and differentiation. Currently, except for biochemical factors, physical stimulation also plays a critical role in the osteogenic/odontogenic differentiation of dental pulp stem cells. OBJECTIVE: To review the relevant physical factors and the possible signaling pathway affecting the osteogenic/odontogenic differentiation of dental pulp stem cells to find the optimal induction conditions affecting their differentiation. METHODS: PubMed and CNKI databases were searched for relevant articles using “dental pulp stem cells (DPSCs), osteogenesis differentiation, odontoblastic differentiation, hypoxia, mechanical force, laser therapy, magnetic fields, microgravity” as English and Chinese search terms. Seventy-nine articles regarding physical factors affecting osteogenic/odontogenic differentiation of dental pulp stem cells were selected for the review. RESULTS AND CONCLUSION: (1) Direct or indirect physical signals in the microenvironment have shown broad application prospects in regulating the directed differentiation of stem cells. Many related physical factors, for example, hypoxia, mechanical stimulation (dynamic hydrostatic pressure, mechanical tension, shear force, etc.), laser, microgravity, and magnetic field, have positive influences on the osteogenic/odontogenic differentiation of dental pulp stem cells. Owing to the complex mechanical environment of stomatognathic system, mechanical stimulation is a key physical factor in changing cellular environment and is also a frontier in tissue engineering. It will provide new ideas for investigating the response of dental pulp stem cells to the mechanical environment in the diagnosis and treatment of oral diseases. (2) Because this field is relatively “young”, the parameters of equipment have not been unified and the relevant results are not consistent. The optimal induction parameters and conditions of related physical factors should be further explored and optimized. (3) Scaffold material, one of the three elements of tissue engineering, plays a role in promoting the osteogenic/odontogenic differentiation of dental pulp stem cells, and promotes the development of materials science and clinical technology. (4) The signaling pathways involve Notch, Wnt, MAPK, etc. The biological basis of regulating the behavior of dental pulp stem cells is not clear. The specific mechanism will be further explored in the future to provide new ideas for dental pulp regeneration and bone tissue engineering under the influence of physical factors. Figures and Tables | References | Related Articles | Metrics