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08 November 2024, Volume 28 Issue 31 Previous Issue    Next Issue

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Effect of Pax6 gene expression on hydrogen peroxide-induced aging in bone marrow mesenchymal stem cells Gao Jie, , , Zou Xingxing, Wen Banghong, Li Yuandi, Su Min, , , Hu Rong, , 2024, 28 (31):  4921-4925.  doi: 10.12307/2024.708 Abstract ( 58 )   PDF (1403KB) ( 11 )   Save BACKGROUND: The occurrence and development of various ophthalmic diseases are closely related to excessive oxidative stress, and the inhibition of oxidative stress response may produce preventive and therapeutic effects.  OBJECTIVE: To explore the role of Pax6 gene expression on hydrogen peroxide-induced aging of mouse bone marrow mesenchymal stem cells (BM-MSCs).  METHODS: Resuscitated BM-MSCs, Pax6/BM-MSCs, and shPax6/BM-MSCs were treated with hydrogen peroxide for 24 hours, and then β-galactosidase staining was performed. The proliferation index Ki67 expression and apoptosis were detected by flow cytometry. The expression of senescence-associated molecules (Wnt7a, p21, and p53) was detected by RT-PCR.  RESULTS AND CONCLUSION: (1) After hydrogen peroxide treatment, the cells of the three groups showed senescence phenotype and β-galactosidase staining was positive. Compared with BM -MSCs group, the expression of positive cells in Pax6/BM-MSCs group was less and that in the shPax6/BM-MSCs group was more, and the difference was statistically significant (P < 0.05). (2) The results of flow cytometry showed that compared with BM-MSCs group, the positive expression of Ki67 in the Pax6/BM-MSCs group increased and the level of apoptosis decreased, while the positive expression of Ki67 decreased and the level of apoptosis increased in the shPax6/BM-MSCs group; the difference was significantly different (P < 0.05). (3) RT-PCR showed that compared with the BM-MSCs group, the expression of Wnt7a, p53, and p21 decreased in the Pax6/BM-MSCs  group, while the expression of Wnt7a, p53, and p21 increased in the shPax6/BM-MSCs group; the difference was significantly different (P < 0.05). (4) These findings indicate that overexpression of Pax6 can antagonize the aging progression of BM-MSCs induced by hydrogen peroxide, which may be related to Wnt signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Effects of long-term subculture on biological characteristics of bone marrow mesenchymal stem cells Zhao Wenjing, Liu Baikun, Li Qiulian, Chen Xi 2024, 28 (31):  4926-4930.  doi: 10.12307/2024.702 Abstract ( 64 )   PDF (1789KB) ( 12 )   Save BACKGROUND: There is still controversy whether human bone marrow mesenchymal stem cells can maintain their biological characteristics, energy metabolism patterns, and multidirectional differentiation potential after long-term expression in vitro. Further comprehensive and systematic research is needed.  OBJECTIVE: To evaluate the effects of long-term expansion in vitro on the biological characteristics of mesenchymal stem cells.  METHODS: Human bone marrow mesenchymal stem cells cultured to passage 5, 10, and 15 in vitro. MTT assay was used to detect cell proliferation ability. Flow cytometry was used to detect cell cycle. The multi-differentiation potential of mesenchymal stem cells was detected by inducing to adipogenic, osteogenic and chondrogenic differentiation. Cell migration and invasion abilities were detected by scratch test and Transwell assay. The mitochondrial oxidative phosphorylation and glycolysis function were analyzed using energy metabolism analyzer. The cell senescence was detected by senescence-associated β-galactosidase staining. The expression levels of p21, p16, and p53 proteins were detected by western blot assay.  RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells at passages 5, 10, and 15 grew adherently; the volume of passage 15 mesenchymal stem cells increased and its proliferation ability decreased; the percentage of S-phase cells decreased (P < 0.05). With the increase of culture passages, the migration and invasion abilities decreased gradually (P < 0.05). There was no significant difference in the differentiation potential, demonstrated by adipogenic, osteogenic and chondrogenesis induction. The ability of oxidative phosphorylation of mitochondria and glycolysis decreased gradually (P < 0.05). The number of senescence-associated β-galactosidase-positive cells increased with the increase of passages (P < 0.05), and the expression of senescence protein p21, p16, and p53 increased gradually (P < 0.05). The results indicated that the biological characterization of mesenchymal stem cells changed after long-term in vitro expansion. Mesenchymal stem cells cultured over 10 passages may have a reduced activity due to increasing senescence. Therefore, bone marrow mesenchymal stem cells cultured less than 10 passages are suitable for clinical research/therapy. Figures and Tables | References | Related Articles | Metrics

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Atf7ip is a negative regulator of bone morphogenetic protein 2 promoting osteogenic differentiation in mouse embryonic adult cells Shi Xian, Han Chunqing, Hu Anran, Kuang Shuyun, Ran Yimeng, Wu Yu 2024, 28 (31):  4931-4936.  doi: 10.12307/2024.716 Abstract ( 68 )   PDF (1334KB) ( 12 )   Save BACKGROUND: Whether activating transcription factor 7 interacting protein (Atf7ip) is involved in the regulation in osteogenic differentiation is still controversial, and studying its impact on osteogenic differentiation and its specific mechanisms is of great significance. OBJECTIVE: To investigate the effect of Atf7ip on bone morphogenetic protein 2 promoting osteogenic differentiation of mouse embryonic osteoblast precursor cells (MC3T3-E1). METHODS: MC3T3-E1 cells cultured in vitro were divided into three groups: normal group, interference group (NC-siRNA group, Atf7ip-siRNA group), and high expression group (CMV-VC group and CMV-Atf7ip group), and were transfected for 24 hours, and then treated with 200 ng/mL bone morphogenetic protein 2 for 0, 12, 24, and 48 hours, respectively. qRT-PCR was used to detect the mRNA expression levels of Atf7ip, alkaline phosphatase, osteocalcin, type I collagen α1 in the cells of each group. Western blot assay was used to detect the protein expression of osteogenic differentiation markers Sp7 and Runx2, and the expression of Atf7ip binding molecule SETDB1, histone H3 and H3K9me3. Alkaline phosphatase activity was detected by alkaline phosphatase staining. RESULTS AND CONCLUSION: (1) With the increase of bone morphogenetic protein 2 treatment time, the protein and mRNA expression of Atf7ip decreased, while the protein expression of Sp7, Runx2 and the mRNA expression of osteocalcin and alkaline phosphatase increased significantly (P < 0.05). There was no significant change in the protein expression of Atf7ip binding molecule SETDB1. (2) Compared with the NC-siRNA group, the protein expression of Sp7, Runx2 and the mRNA expression of osteocalcin and type I collagen α1 were significantly up-regulated (P < 0.05), and alkaline phosphatase activity was significantly enhanced; and H3K9 methylation significantly decreased in the Atf7ip-siRNA group (P < 0.05). (3) Compared with the CMV-VC group, the protein expression of Sp7 and Runx, as well as mRNA expression of osteocalcin, alkaline phosphatase, and type I collagen α1 was significantly downregulated (P < 0.05), and the alkaline phosphatase activity was significantly reduced in the CMV-Atf7ip group, while the H3K9 methylation protein in the CMV-Atf7ip group was significantly upregulated compared to the control group (P < 0.05). (4) In conclusion, Atf7ip expression was decreased during bone morphogenetic protein 2-induced osteogenic differentiation of MC3T3-E1, and osteogenic differentiation was significantly increased after knockdown of Atf7ip. Overexpression of Atf7ip significantly weakened osteogenic differentiation, indicating that Atf7ip is a negative regulatory factor of bone morphogenetic protein 2 promoting osteogenic differentiation of MC3T3-E1 cells. Figures and Tables | References | Related Articles | Metrics

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Erythropoietin-overexpressed umbilical cord mesenchymal stem cells inhibit neuroapoptosis in ischemic-hypoxic SH-SY5Y and its mechanism Li Ruibo, Kong Ning, Sun Lei, Ma Baodong, Jin Ranran, Zhang Wenjin, Yue Han, Zhang Hui 2024, 28 (31):  4937-4944.  doi: 10.12307/2024.704 Abstract ( 59 )   PDF (3448KB) ( 7 )   Save BACKGROUND: Previous studies have successfully constructed erythropoietin-overexpressed umbilical cord mesenchymal stem cells. It was found that the apoptosis of ischemic and hypoxic human neuroblastoma cell line (SH-SY5Y) was significantly reduced by erythropoietin-overexpressed umbilical cord mesenchymal stem cells.  OBJECTIVE: To explore the possible neuroprotective mechanisms of erythropoietin-overexpressed umbilical cord mesenchymal stem cells against ischemic-hypoxic SH-SY5Y and their associated epigenetic mechanisms.  METHODS: Oxygen-glucose deprivation was applied to ischemia-hypoxia-induced SH-SY5Y cell injury, and multifactorial assays were applied to detect the expression levels of inflammatory factors in the cells before and after hypoxia and co-culture, respectively, with mesenchymal stem cells, as well as lentiviral-transfected null-loaded plasmids of the negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells. The expression levels of supernatant inflammatory factors were detected by multifactor assay after co-culture. Proteomics was used to detect the differentially expressed proteins of negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells. Cleavage under targets and tagmentation sequencing was applied to detect genomic H3K4me2 modification, and joint analysis was conducted with RNA-sequencing. Lentiviral vector infection was applied to construct the stable knockdown of REST in SH-SY5Y cells. qRT-PCR and western blot assay were performed to detect the expression level of REST. The apoptosis was detected by flow cytometry after co-culture of oxygen-glucose deprivation treatment with erythropoietin-overexpressed umbilical cord mesenchymal stem cells. The expression difference of H3K36me3 group proteins was detected by western blot assay, and transcriptome sequencing was performed to analyze the differentially expressed genes. RESULTS AND CONCLUSION: (1) Compared with the control group, monocyte chemotactic protein 1, interleukin-6, interleukin-18, and interleukin-1 beta, interferon α2, and interleukin-23 levels significantly increased in the cerebrospinal fluid supernatant of patients with ischemic-hypoxic encephalopathy (P < 0.01). (2) After co-culturing SH-SY5Y cells with erythropoietin-overexpressed umbilical cord mesenchymal stem cells under ischemia and hypoxia, the expression levels of monocyte chemotactic protein 1 and interleukin-6 were significantly reduced. (3) Analysis of protein network interactions revealed significant downregulation of monocyte chemotactic protein 1, interleukin-6 related regulatory proteins CXCL1 and BGN. (4) Transcriptome sequencing analysis found that pro-inflammatory genes were down-regulated, and functional enrichment of histone modifications, and the expression of transcription factors REST and TET3 significantly up-regulated in the erythropoietin-overexpressed umbilical cord mesenchymal stem cell group compared with the negative control mesenchymal stem cell group. (5) Combined analysis of transcriptome sequencing and cleavage under targets and tagmentation revealed changes in epigenetic levels as well as significant activation of the promoter regions of transcription factors REST and TET3. (6) Stable knockdown REST in SH-SY5Y cells was successfully constructed; the transcript levels of REST mRNA and protein expression were both decreased. (7) After the REST knockdown SH-SY5Y cells were co-cultured with erythropoietin-overexpressed umbilical cord mesenchymal stem cells, apoptosis was significantly increased and H3K36me3 expression was significantly decreased. Transcriptome sequencing results showed that the expression of inflammation-related genes Aldh1l2 and Cth, as well as apoptosis-suppressor genes Mapk8ip1 and Sod2 was reduced at mRNA transcription level (P < 0.01). (8) It is concluded that erythropoietin-overexpressed umbilical cord mesenchymal stem cells activated the expression of REST and TET3 by altering the kurtosis of H3K4me2 and upregulated the modification level of H3K36me3, which in turn regulated the expression of inflammation-related genes Aldh1l2 and Cth, as well as apoptosis-suppressor genes Mapk8ip1 and Sod2, and facilitated neuronal survival. Figures and Tables | References | Related Articles | Metrics

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Quercetin targets CCR1 and CXCR4 to promote migration of human bone marrow mesenchymal stem cells Chen Shuang, Xi Zhipeng, Wang Nan, Fang Xiaoyang, Liu Xin, Kang Ran, Xie Lin 2024, 28 (31):  4945-4950.  doi: 10.12307/2024.707 Abstract ( 72 )   PDF (1388KB) ( 12 )   Save BACKGROUND: Quercetin plays an important role in the proliferation and differentiation of bone marrow mesenchymal stem cells, but less research has been done on its mechanism of promoting the migration of bone marrow mesenchymal stem cells.  OBJECTIVE: To study the effect of quercetin on the migration of human bone marrow mesenchymal stem cells through in vitro experiments, and to explore the regulatory role of CCR1 and CXCR4. METHODS: Human bone marrow mesenchymal stem cells were selected as experimental subjects. CCK8 assay was used to detect the effect of quercetin on the proliferative activity of human bone marrow mesenchymal stem cells. Cell scratch assay and Transwell assay were used to detect the in vitro invasive and migratory abilities of human bone marrow mesenchymal stem cells after quercetin treatment, respectively. The role of quercetin in relation to CCR1 and CXCR4 was demonstrated with the help of molecular docking technology. Western blot assay and real-time fluorescence quantitative PCR were used to detect the migration-related chemokine expression after quercetin treatment.  RESULTS AND CONCLUSION: (1) 5 and 10 μmol/L quercetin could significantly promote the proliferation of human bone marrow mesenchymal stem cells, and the drug concentration of 10 μmol/L resulted in the highest cell proliferation efficiency. (2) To better explore the dose-effect relationship of quercetin affecting the migration of human bone marrow mesenchymal stem cells, 5 and 10 μmol/L quercetin were selected for the subsequent experiments, and ligustrazine was used as the positive control drug, and the experiments were divided into blank control group, 5 μmol/L quercetin group, 10 μmol/L quercetin group, and 100 μmol/L ligustrazine group. (3) In vitro migration and invasion ability of human bone marrow mesenchymal stem cells were elevated in a concentration-dependent manner after quercetin treatment, and the migration effect of 10 μmol/L quercetin group was better than that of ligustrazine group. (4) The molecular docking results suggested that there was a strong interaction between quercetin and CCR1 and CXCR4. (5) Quercetin could up-regulate the expression of CCR1 and CXCR4 proteins and mRNA. (6) This study confirmed at the cellular level that quercetin could promote the migration of human bone marrow mesenchymal stem cells by targeting CCR1 and CXCR4. Figures and Tables | References | Related Articles | Metrics

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Notch1-mediated aerobic exercise promotes hippocampal nerve cell proliferation in Alzheimer’s disease mice Li Huijun, Li Chuikun, Wei Cuilan, Zhang Yeting 2024, 28 (31):  4951-4957.  doi: 10.12307/2024.181 Abstract ( 65 )   PDF (2112KB) ( 8 )   Save BACKGROUND: Abnormal Notch1 signaling pathway is mostly found in the brain of Alzheimer’s disease patients, but the role of these signaling pathways in the pathogenesis of Alzheimer’s disease has not been fully clarified. Long-term aerobic exercise can alter the expression of Notch1 by affecting the methylation rate of factors related to the Notch1 signaling pathway. However, it is not clear whether aerobic exercise affects hippocampal nerve cell proliferation and histopathological features of Alzheimer’s disease mice through the Notch1 signaling pathway.  OBJECTIVE: To observe the effects of aerobic exercise on the proliferation and histopathological features of hippocampal nerve cells in Alzheimer’s disease mice after DAPT inhibited the Notch1 signaling pathway. METHODS: APP/PS1 double transgenic Alzheimer’s disease mice aged 3 months were randomly divided into four groups: control group, exercise control group, inhibitor group, and exercise inhibitor group, with 20 mice in each group. The control group was fed naturally, and the exercise group received aerobic exercise intervention. Both natural feeding and exercise intervention lasted for 20 weeks. The mice were injected with solvent or Notch1 inhibitor at week 18. After 20 weeks, the brain tissue was collected, and Aβ1-42, Tau, Ki67, and Notch1 expression levels were detected by real-time PCR, immunofluorescence, and western blot assay.   RESULTS AND CONCLUSION: Compared with the control group, the expressions of Ki67 and Notch1 in the dentate gyrus region of the hippocampus were significantly decreased in the inhibitor group (P < 0.05), but there were no significant differences in Aβ1-42 and Tau. The expression of Ki67 in the dentate gyrus region of the hippocampus in the exercise control group was significantly higher than that in the control group, while the expressions of Aβ1-42, Tau, and Notch1 were significantly lower than those in the control group (P < 0.05). The expressions of Aβ1-42, Tau, Ki67, and Notch1 in the dentate gyrus region of the hippocampus of the exercise inhibitor group were not significantly different from those of the inhibitor group. In conclusion, the Notch1 signaling pathway may mediate exercise to improve the proliferation and histopathological features of hippocampal nerve cells in Alzheimer’s disease mice.   Figures and Tables | References | Related Articles | Metrics

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Vascular endothelial growth factor combined with basic fibroblast growth factor improves replicative senescence of bone marrow mesenchymal stem cells Shi Weili, Liu Shanshan, Chang Hongbo, Gao Haixia, Wang Xinzhou, Qin Nan, Wu Hong 2024, 28 (31):  4958-4963.  doi: 10.12307/2024.199 Abstract ( 57 )   PDF (1899KB) ( 12 )   Save BACKGROUND: Mesenchymal stem cells are susceptible to senescence during in vitro expansion, which greatly hinders their application in vivo and in vitro. How to improve the replicative senescence of mesenchymal stem cells is an urgent problem to be solved in tissue engineering. OBJECTIVE: To determine whether vascular endothelial growth factor combined with basic fibroblast growth factor can improve the aging of bone marrow mesenchymal stem cells caused by replicative passage. METHODS: Rat bone marrow mesenchymal stem cells were extracted by whole bone marrow adhesion method. Passage 2 cells were selected as normal control group. Passage 7 and later algebraic cells were selected as aging model group. Vascular endothelial growth factor (50 μg/L), basic fibroblast growth factor (10 μg/L), and their combination were administered. Cell proliferation was detected by CCK-8 assay. Cell senescence was observed by β-galactosidase activity staining. Cytoskeleton size and colony formation ability were observed by phalloidine staining and Giemsa staining, respectively, and the levels of senescence-related genes P16, P21, and P53 were detected by qRT-PCR. Gene expression levels of P16, P21, and P53 were tested by qRT-PCR. RESULTS AND CONCLUSION: (1) Vascular endothelial growth factor combined with basic fibroblast growth factor could promote the proliferation of aged bone marrow mesenchymal stem cells, which began to enter the plateau stage on day 9, and the absorbance value of the combined intervention group was significantly higher than that of the model group on day 9 (P < 0.05). (2) The phenotypic markers of the cells in the combined intervention group did not change, and the cell morphology changed from broad to slender. (3) Compared with the model group, the positive rate of β-galactosidase was significantly decreased (P < 0.01); the number of nuclei increased (P < 0.001); the total area of cytoskeleton increased (P < 0.01); colony formation ability was enhanced (P < 0.05); expression level of P16 was decreased (P < 0.01) in the combined intervention group. These results indicate that vascular endothelial growth factor combined with basic fibroblast growth factor can improve the senescence of bone marrow mesenchymal stem cells caused by replicative passage without changing the cell phenotype.  Figures and Tables | References | Related Articles | Metrics

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Overexpression of long non-coding RNA Gm16104 affects osteogenic differentiation of C3H10T1/2 mesenchymal stem cells Lin Zhanying, Lin Ziyun, Huang Liuyan, Zhang Wenxi, Zuo Changqing 2024, 28 (31):  4964-4969.  doi: 10.12307/2024.710 Abstract ( 39 )   PDF (2043KB) ( 15 )   Save BACKGROUND: The osteogenic differentiation of mesenchymal stem cells is regulated by a variety of molecules. Long non-coding RNA (lncRNA) has attracted much attention because they can participate in regulating a variety of biological processes, but the regulatory role of lncRNA on osteogenic differentiation of mesenchymal stem cells has not been fully explored. OBJECTIVE: To explore the effect of lncRNA Gm16104 on osteogenic differentiation of C3H10T1/2 mesenchymal stem cells. METHODS: The C3H10T1/2 mesenchymal stem cells were induced into osteogenic differentiation by bone morphogenetic protein-2. Alkaline phosphatase staining was performed to identify the osteogenic differentiation of the cells 5 days after osteogenic induction. Expression levels of alkaline phosphatase and lncRNA Gm16104 were detected by qRT-PCR after 0, 1, 3, and 5 days of osteogenic differentiation. After transfection of the overexpression plasmid of pcDNA-Gm16104, the osteogenic differentiation was identified by alkaline phosphatase staining and qRT-PCR 4 days after osteogenic induction. The expression levels of osteogenesis-related signalling pathway proteins were detected by western blot assay. RESULTS AND CONCLUSION: (1) After 5 days of osteogenic induction, alkaline phosphatase activity was significantly increased. (2) Compared with 0 days, expression levels of the osteogenic marker gene alkaline phosphatase increased and expression levels of Gm16104 decreased after 1, 3, and 5 days of osteogenic induction. (3) Transfection of C3H10T1/2 cells with pcDNA-Gm16104 plasmid significantly increased the expression level of Gm16104. (4) Overexpression of Gm16104 inhibited alkaline phosphatase activity, the expression levels of the osteogenic marker gene alkaline phosphatase and the osteogenesis-related transcription factor Osterix. (5) Overexpression of Gm16104 inhibited phosphorylated protein expression of PI3K and Akt. (6) The above results suggest that overexpression of Gm16104 may inhibit osteogenic differentiation of C3H10T1/2 mesenchymal stem cells through the PI3K/Akt signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Human placental mesenchymal stem cells inhibit occurrence of pulmonary fibrosis by regulating transforming growth factor-beta 1/Smad3 signaling pathway Cao Jiawei, Ding Shaorui, Tie Hua, Xue Jing, Jia Yuanyuan, Liang Xueyun, Li Feng 2024, 28 (31):  4970-4974.  doi: 10.12307/2024.712 Abstract ( 72 )   PDF (1656KB) ( 10 )   Save BACKGROUND: Human placental mesenchymal stem cells have been shown to be effective in inhibiting the development of pulmonary fibrosis, but the underlying mechanisms remain unclear. OBJECTIVE: To investigate the therapeutic effect and related mechanism of human placental mesenchymal stem cells on silica-induced pulmonary fibrosis in human embryonic lung fibroblasts (MRC-5). METHODS: CCK-8 assay was used to detect the effects of different mass concentrations of silica on the proliferation of MRC-5 at different time points. Immunofluorescence staining was used to screen out the best stimulating mass concentration and time of silica for subsequent experiments. MRC-5 cells were divided into blank group, silica group, and silica + human placental mesenchymal stem cell group. In the blank group, cells were not treated. In the silica group, MRC-5 cells were stimulated with 100 μg/mL silica for 48 hours. In the silica + human placental mesenchymal stem cell group, MRC-5 cells were stimulated with 100 μg/mL silica for 48 hours and then co-cultured with human placental mesenchymal stem cells for 24 hours. Immunofluorescence staining was used to detect the expression of α-smooth muscle actin and collagen type I in cells of each group. Western blot assay was used to detect the expressions of pulmonary fibrosis-related proteins and TGF-β1/Smad 3 signaling pathway-related proteins in cells of each group. RESULTS AND CONCLUSION: (1) CCK-8 assay results suggested that 100 μg/mL silica was the best mass concentration and time to stimulate MRC-5 cells for 48 hours. (2) Immunofluorescence staining results showed that the expression of α-smooth muscle actin and collagen type I in the silica + human placental mesenchymal stem cell group was significantly lower than that in the silica group. (3) Western blot assay results showed that compared with the silica group, the protein expression levels of α-smooth muscle actin, collagen type I, N-cadherin, fibronectin, transforming growth factor-β1, p-Smad3, and Smad3 in the silica + human placental mesenchymal stem cell group were decreased, and the expression of E-cadherin was increased. The difference was statistically significant (P < 0.05). (4) The results showed that human placental mesenchymal stem cells had a significant therapeutic effect on silica-induced pulmonary fibrosis. Human placental mesenchymal stem cells can inhibit the development of pulmonary fibrosis by regulating transforming growth factor-β1/Smad3 signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Strength of association between follicular fluid melatonin levels and pregnancy rates in single-cycle in vitro fertilization-embryo transfer women Liu Shanshan, Wu Juan, Chen Change, Cao Yunxia, Zhang Zhiguo 2024, 28 (31):  4975-4979.  doi: 10.12307/2024.717 Abstract ( 56 )   PDF (874KB) ( 6 )   Save BACKGROUND: In vitro fertilization-embryo transfer is commonly used to solve infertility, but its success rate is not high, the more common reasons are poor endometrial receptivity, poor egg quality, etc. The follicular fluid melatonin can inhibit the aging of the ovary, to a certain extent, can promote the development of embryos, improve the probability of conception, but whether there is a correlation between the two is not known. OBJECTIVE: To explore the correlation between follicular fluid melatonin level and pregnancy rate of single-cycle in vitro fertilization-embryo transfer women.  METHODS: A total of 112 female patients who received in vitro fertilization-embryo transfer treatment in the First Affiliated Hospital of Anhui Medical University from December 2020 to April 2021 were selected as the study subjects. They were divided into quartile array (Q1-Q5) according to the follicular fluid melatonin level from low to high. Among them, the melatonin level of group Q1 was < 6.99 ng/L (n=18), that of group Q2 was 7.00-9.99 ng/L (n=26), that of group Q3 was 10.00-11.99 ng/L (n=27), and that of group Q4 was 12.00-13.99 ng/L (n=18); and melatonin levels in group Q5 were 14.00-19.99 ng/L (n=23). Clinical data characteristics of the five groups were compared. Multi-factor Logistic regression was used to analyze the correlation between follicular fluid melatonin level and pregnancy rate of women with single-cycle in vitro fertilization-embryo transfer and embryo transfer. A restricted cubic spline Logistic regression model was established to analyze the dose-response relationship, and the model was evaluated by clinical decision curve. RESULTS AND CONCLUSION: (1) Compared with the study population with the lowest melatonin quintile (Q1), with the increase of melatonin level (Q2-Q5), the levels of egg harvest and pregnancy success were gradually increased, and the body mass index was gradually decreased, and the differences were significant (P < 0.05). (2) Multivariate Logistic regression analysis showed that after adjusting for confounding factors such as global mass index, number of eggs retrieved, luteinizing hormone, estradiol, progesterone and other confounding factors, follicular fluid melatonin level was still independently correlated with pregnancy rate of single-cycle in vitro fertilization-embryo transfer women (OR=1.538, 95%CI: 1.032-1.837, P < 0.05), and there was significant difference in trend test of follicular fluid melatonin level from low to high quintile array (Ptrend < 0.05). (3) The sensitivity test analysis showed that E value was 2.117. Subgroup analysis showed that the study population with higher levels of luteinizing hormone in follicular fluid had a more significant association between follicular fluid melatonin and pregnancy rate in single-cycle in vitro fertilization-embryo transfer women (P interaction=0.008). (4) The results of restricted cubic spline model analysis showed that there was a nonlinear dose-response relationship between follicular fluid melatonin level and pregnancy rate of single-cycle in vitro fertilization-embryo transfer women (P < 0.05), and there was an overall positive correlation between follicular fluid melatonin level and pregnancy rate of single-cycle in vitro fertilization-embryo transfer women. (5) The results of clinical decision curve analysis showed that the follicular fluid melatonin level had important clinical value in predicting the pregnancy rate of single-cycle in vitro fertilization-embryo transfer women. (6) Follicular fluid melatonin level is closely related to the pregnancy rate of single-cycle in vitro fertilization-embryo transfer women, and with the decrease of follicular fluid melatonin level, the pregnancy rate of single-cycle in vitro fertilization-embryo transfer women also decreases.  Figures and Tables | References | Related Articles | Metrics

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Isolation, purification and identification of rat annulus fibrosus-derived stem cells by adherent method combined with fibronectin differential adhesion method Ma Dong, Chen Qiqing, Zhao Jirong, Huang Junkai, Yang Yunyun, Zhu Bao, Zhao Ning, Ma Tong 2024, 28 (31):  4980-4986.  doi: 10.12307/2023.771 Abstract ( 52 )   PDF (1963KB) ( 6 )   Save BACKGROUND: At present, bone marrow mesenchymal stem cells are the main seed cells used in cell therapy for the treatment of intervertebral disc degeneration. However, the use of bone marrow mesenchymal stem cells as seed cells for the regeneration of fibrous rings is at risk of heterotopic ossification and teratoma at the repair site. Therefore, it is of great economic and social significance to find a new kind of seed cells for tissue engineering of annulus fibrosus for the treatment of intervertebral disc degeneration. OBJECTIVE: To isolate and purify rat annulus fibrosus-derived stem cells by adherent method combined with fibronectin differential adhesion screening method, and to observe its purification effect and biological characteristics.  METHODS: Annulus fibrosus tissue was obtained from a SD rat intervertebral disc. Primary annulus fibrosus-derived stem cells were obtained by the mechanical-enzymatic digestion method. Annulus fibronectin differential adhesion method was used to purify annulus fibrosus-derived stem cells. Morphological changes and proliferation of cells were observed through a microscope. The expression levels of stem cell markers were detected by immunofluorescence technique and qPCR. The screened cells were subjected to multi-lineage cell differentiation and characteristic gene detection. RESULTS AND CONCLUSION: (1) The purified cells grew well, and most of them were angular and star-shaped multi-process cells, which had good proliferation ability. (2) Cells were positive for cell membrane surface antigens CD73, CD90 and CD105, while negative for CD45 and CD34. (3) After specific induction, cells could successfully differentiate into osteoblasts, chondroblasts and lipoblasts. (4) Collagen-I, Runx-2 after osteogenic induction, Collagen II, Sox-9 after chondrogenic induction, and PPAR -γ and LPL after lipogenic induction were highly expressed in cells, and the difference was significant compared with that before induction (P < 0.05). (5) These findings confirm that the adherent method combined with fibronectin differential adhesion method is effective enough to screen, isolate and purify rat annulus fibrosus-derived stem cells, and has good cell biological properties, good proliferation ability and multiple differentiation potential. Figures and Tables | References | Related Articles | Metrics

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Granulocyte colony-stimulating factor combined with high-intensity intermittent exercise preconditioning improved cardiac remodeling in rats with acute myocardial infarction Sun Yuma, Ma Wenchao, Fu Changxi 2024, 28 (31):  4987-4994.  doi: 10.12307/2024.703 Abstract ( 67 )   PDF (1547KB) ( 55 )   Save BACKGROUND: Exercise training is an important non-drug rehabilitation method for many heart diseases, and it can also enhance the heart’s tolerance to adverse stressors (such as myocardial ischemia), that is, exercise preconditioning. Granulocyte colony-stimulating factor can effectively mobilize stem cell homing and differentiation and promote the repair of damaged myocardium. However, the effect of the combination of the two treatments is not yet clear. OBJECTIVE: To explore the effect of granulocyte colony-stimulating factor supplementation combined with high-intensity intermittent exercise preconditioning on cardiac remodeling in rats with acute myocardial infarction and investigate its possible mechanism. METHODS: Totally 58 male Wistar rats were divided into sham group (n=10), model group (n=12), model drug group (n=12), model exercise group (n=12) and model combined treatment group (n=12). The myocardial infarction rat model was made by coronary artery ligation. The model exercise group and the model combined treatment group were pretreated with 8 weeks of high-intensity intermittent exercise on an electric treadmill before modeling. The model drug group and the model combined treatment group were subcutaneously injected with human recombinant granulocyte colony-stimulating factor 3 hours after modeling for 5 days (10 μg/kg per day). At 8 weeks after administration, echocardiography was used to detect heart structure and function; heart was stained with 2,3,5-triphenyltetrazolium chloride and Masson staining to obtain myocardial infarct area and collagen volume fraction, respectively. Vessel density and cell apoptosis rate were detected by immunofluorescence. Real-time fluorescent quantitative PCR was utilized to detect the mRNA expression of embryonic genes (brain natriuretic peptide, β-myosin heavy chain) and myocardial contraction regulatory factors (α-myosin heavy chain, sarcoplasmic reticulum Ca2+-ATPase). Western blot assay was used to detect the protein expression of cardiac stromal cell-derived factor 1, CXC chemokine receptor protein 4, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), Bcl2, Bax, and cleaved caspase-3. RESULTS AND CONCLUSION: (1) Compared with sham group, myocardial infarct size, collagen volume fraction, and apoptosis rate increased (P < 0.05); vessel density, left ventricular fractional shortening, and left ventricular ejection fraction decreased (P < 0.05); brain natriuretic peptide and β-myosin heavy chain mRNA increased (P < 0.05), α-myosin heavy chain and sarcoplasmic reticulum Ca2+-ATPase mRNA and α-myosin heavy chain/β-myosin heavy chain ratio decreased (P < 0.05); stromal cell-derived factor 1, CXC chemokine receptor protein 4, Bax, cleaved caspase-3 protein expression increased (P < 0.05); p-JAK2, p-STAT3, and Bcl-2 protein expression decreased (P < 0.05) in the model group. (2) Compared with the model group, the above indexes in the model drug and model exercise groups were significantly improved (P < 0.05). Compared with the model drug and model exercise groups, the above parameters were further ameliorated (P < 0.05) in the model combined treatment group. (3) The results showed that supplementation of granulocyte colony-stimulating factor or high-intensity intermittent exercise preconditioning alone can improve cardiac remodeling in rats with acute myocardial infarction, and the combined therapy has a better effect, which may be related to the induction of stem cell homing and the activation of JAK2/STAT3 signaling pathway to inhibit cardiomyocyte apoptosis. Figures and Tables | References | Related Articles | Metrics

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Effects of conditioned medium of acute myeloid leukemia on biology of mesenchymal stem cells Zhang Chike, Wang Feiqing, Wu Dan, Yang Bo, Cheng Jinyang, Chen Juan, Tang Dongxin, Liu Yang, Li Yanju 2024, 28 (31):  4995-5002.  doi: 10.12307/2024.705 Abstract ( 53 )   PDF (3872KB) ( 15 )   Save BACKGROUND: At present, the biological functions and molecular changes of bone marrow mesenchymal stem cells in the tumor microenvironment of acute myeloid leukemia are still unclear. OBJECTIVE: To explore the changes in the biological function of bone marrow mesenchymal stem cells in acute myeloid leukemia and the role of acute myeloid leukemia conditioned medium by bioinformatics and experiment. METHODS: Differential genes were screened from GEO data sets, and enrichment analysis was performed. The protein-protein interaction network was constructed and the Hub gene was obtained. Bone marrow mesenchymal stem cells from patients with acute myeloid leukemia and healthy donors were cultured. Bone marrow mesenchymal stem cells from healthy donors were treated with acute myeloid leukemia conditioned culture solution. Each group was subjected to the adipogenic differentiation, osteogenic differentiation, staining of β-galactosidase, detection of the cell cycle, and validation of Hub genes. RESULTS AND CONCLUSION: (1) Gene expression data of bone marrow mesenchymal stem cells from acute myeloid leukemia patients and healthy donors were obtained from GSE84881, and 184 up-regulated genes and 140 down-regulated genes were screened. (2) The biological functions of enrichment mainly include cell cycle, adipocyte differentiation, cell metabolism, and MYC pathway. According to the Degree algorithm, 10 up-regulated Hub genes and 10 down-regulated Hub genes were selected. (3) The cell in vitro experiment found that: compared with the control group, the surface antigen of acute myeloid leukemia mesenchymal stem cells did not change, but it showed enhanced lipid differentiation ability, weakened osteogenic differentiation ability, increased β-galactosidase positive cell number, altered cell morphology, arrested cell cycle, increased LGALS3 expression, and decreased MYC expression. Mesenchymal stem cells from healthy donors showed similar changes after being cultured in acute myeloid leukemia conditioned medium. (4) The results show that biological function of mesenchymal stem cells is altered in the acute myeloid leukemia microenvironment, which provides new insights into the interaction between mesenchymal stem cells and tumor cells.  Figures and Tables | References | Related Articles | Metrics

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Effect of cell mechanics on morphogenesis of MDCK lobular organoid Cao Yu, Wu Dang, Ouyang Mingxing, Deng Linhong 2024, 28 (31):  5003-5009.  doi: 10.12307/2024.725 Abstract ( 84 )   PDF (1757KB) ( 25 )   Save BACKGROUND: The development of tissues and organs in the body is a precise and autonomously regulated process, and the function of biomechanical factors at this macroscale is a basic scientific question worth exploring.  OBJECTIVE: To investigate the roles of cell mechanics in morphogenesis of the lobular organoid of 3D Madin-Darby canine kidney (MDCK). METHODS:  The formation of MDCK lobular organoid was visualized by fluorescence resonance energy transfer technology, and the influence of different cellular mechanical signals and extracellular matrix environment on lobular organoid formation and corresponding changes in extracellular regulated protein kinases (ERK) activity were examined.  RESULTS AND CONCLUSION: (1) Inhibition of ERK signaling pathway can inhibit the growth of MDCK lobular organoid. (2) Inhibition of cell contractile force signals such as ROCK pathway and Myosin II activity, reduced ERK activity and lobular organoid size. (3) Selective inhibition of calcium channels in plasma membrane and endoplasmic reticulum led to reduced ERK activity and lobular organoid growth. (4) By inhibiting the mechanically-sensitive receptor Piezo ion channel or integrin signal on the cell membrane, the lobular organoid became smaller or MDCK cells could not generate tissue morphology. (5) Extracellular matrix compositions affected the morphogenesis of lobular organoid. The addition of type I collagen in Matrigel changed the lobular organoid to elongated shape. (6) The results of this study preliminarily show that mechanical signals in the cells and extracellular matrix environment play an important role in culturing MDCK lobular organoid, and provides certain molecular mechanisms.  Figures and Tables | References | Related Articles | Metrics

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Indolepropionic acid inhibition of microglial cell M1 polarization for treatment of spinal cord injury Teng Yilin, Xi Deshuang, Feng Yanbin, Liang Yu, Deng Hao, Zeng Gaofeng, Zong Shaohui 2024, 28 (31):  5010-5016.  doi: 10.12307/2024.713 Abstract ( 64 )   PDF (1385KB) ( 16 )   Save BACKGROUND: Indolepropionic acid has been shown to reduce diabetes-induced central nervous system inflammation. However, there is a lack of research on whether to inhibit microglia M1 polarization for the treatment of spinal cord injury. OBJECTIVE: To investigate the mechanism of indolepropionic acid inhibition of microglial cell M1 polarization for the treatment of spinal cord injury through cell and animal experiments.  METHODS: (1) In vitro experiments: BV2 cell viability was assessed using the CCK-8 assay to determine optimal concentrations of indolepropionic acid. Subsequently, BV2 cells were categorized into control group, administration group (50 μmol/L indolepropionic acid), lipopolysaccharide group (100 ng/mL lipopolysaccharide), and treatment group (100 ng/mL lipopolysaccharide + 50 μmol/L indolepropionic acid). Nitric oxide content was quantified using the Griess method. Real-time quantitative PCR and western blot assay were employed to measure mRNA and protein levels of pro-inflammatory factors. Cell immunofluorescence staining was conducted to assess inducible nitric oxide synthase expression. The Seahorse assay was employed to assess glycolytic stress levels in BV2 cells. (2) In vivo experiments: 30 SD rats were randomly divided into three groups: sham surgery group, spinal cord injury group, and indolepropionic acid group. Motor function recovery in rats after spinal cord injury was assessed using BBB scoring and the inclined plane test. Immunofluorescence staining of spinal cord tissue was conducted to evaluate the expression of inducible nitric oxide synthase in microglial cells. ELISA was employed to measure protein expression levels of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in spinal cord tissue. RESULTS AND CONCLUSION: (1) In vitro experiments: Indolepropionic acid exhibited significant suppression of BV2 cell viability when its concentration exceeded 50 μmol/L. Indolepropionic acid achieved this by inhibiting the activation of the nuclear factor κB signaling pathway, thereby suppressing the mRNA and protein expression levels of pro-inflammatory cytokines (interleukin-1β and tumor necrosis factor-α), as well as the M1 polarization marker, inducible nitric oxide synthase, in BV2 cells. Additionally, indolepropionic acid notably reduced the glycolytic level in BV2 cells induced by lipopolysaccharides. (2) In vivo experiments: Following indolepropionic acid intervention in spinal cord injury rats, there was a noticeable increase in BBB scores and the inclined plane test angle. There was also a significant decrease in the number of M1-polarized microglial cells in spinal cord tissue, accompanied by a marked reduction in the protein expression levels of pro-inflammatory cytokines (interleukin-1β and tumor necrosis factor-α). (3) These results conclude that indolepropionic acid promotes functional recovery after spinal cord injury by improving the inflammatory microenvironment through inhibition of microglia M1 polarization. Figures and Tables | References | Related Articles | Metrics

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In vitro human lymphocyte proliferation assay under different extraction conditions and doses of two types of test samples Xu Jianxia, Fu Haiyang, Qu Shoufang 2024, 28 (31):  5017-5021.  doi: 10.12307/2024.718 Abstract ( 60 )   PDF (765KB) ( 5 )   Save BACKGROUND: In vitro lymphocyte proliferation test is often used to detect the potential immunogenicity of medical devices, but no detailed extraction conditions and dose are given in the relevant standards.  OBJECTIVE: To investigate the effects of different extraction conditions of the test product and different doses of the extract on in vitro human lymphocyte proliferation, and to consider the factors that need to be considered when selecting test conditions for in vitro lymphocyte proliferation test.  METHODS: In the experiment, the homogenous bone repair material and heparin-modified intraocular lens were divided into the following 12 groups: (1) Experimental group 1: 24-hour complete medium (RPMI modified medium containing 10% fetal bovine serum) extract of 200 µL + lymphocyte suspension of 50 µL; (2) negative control group 1: 24-hour complete medium 200 µL + lymphocyte suspension 50 µL; (3) experimental group 2: 24-hour complete medium extract 100 µL + lymphocyte suspension 100 µL; (4) negative control group 2: 24-hour complete medium 100 µL + lymphocyte suspension 100 µL; (5) experimental group 3: 72-hour RPMI modified medium extract (addition of 10% fetal bovine serum before experiment) 200 µL + lymphocyte suspension 50 µL; (6) negative control group 3: 72-hour RPMI modified medium (addition of 10% fetal bovine serum before experiment) 200 µL + lymphocyte suspension 50 µL; (7) experimental group 4: 72-hour RPMI modified medium extract (addition of 10% fetal bovine serum before experiment) 100 µL + lymphocyte suspension 100 µL; (8) negative control group 4: 72-hour RPMI modified medium (addition of 10% fetal bovine serum before experiment) 100 µL + lymphocyte suspension 100 µL; (9) positive control group 1: complete medium containing 10 μg/mL plant hemagglutinin-M 200 µL + lymphocyte suspension 50 µL; (10) positive control group 2: complete medium containing 10 μg/mL plant hemagglutinin-M 100 µL + lymphocyte suspension 100 µL; (11) blank control group 1: 250 µL complete medium; (12) control group 2: 200 µL complete medium. After 3 days of culture, the proliferation of lymphocytes was detected by CCK-8 assay. RESULTS AND CONCLUSION: (1) Under different test conditions, the extracts of the allogeneic bone repair material could enhance the activity of human lymphocytes. Under the condition of 72-hour leaching in RPMI modified medium and the volume ratio of leaching solution and lymphocyte suspension was 4:1, the most significant effect was observed. Heparin-modified intraocular lens extract also had obvious inhibitory effect on lymphocyte activity under this condition; its inhibitory effect on lymphocyte activity may be related to the heparin in the extract. However, the activity of lymphocytes was slightly enhanced by heparin-modified intraocular lens extract under the experimental conditions of complete medium extraction for 24 hours and the volume ratio of extract to lymphocyte suspension was 4:1. (2) Under different extraction conditions and doses, the results of in vitro lymphocyte proliferation test may be quite different. The selection of test conditions should be combined with the clinical application of the product, and the inherent characteristics of the product should also be considered. Figures and Tables | References | Related Articles | Metrics

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Astragaloside inhibits astrocyte activation and inflammatory response induced by inflammation Yu Jingwen, Guo Minfang, Zhang Bingxin, Mu Bingtao, Meng Tao, Zhang Huiyu, Ma Cungen, Yin Jinzhu, Song Lijuan, Yu Jiezhong 2024, 28 (31):  5022-5028.  doi: 10.12307/2024.726 Abstract ( 72 )   PDF (2853KB) ( 17 )   Save BACKGROUND: Astrocytes play an important role in the pathology of central nervous system diseases. The phenotypic and functional changes in astrocytes suggest that it may be an effective therapeutic target for central nervous system diseases. Our previous studies have confirmed that astragaloside can inhibit the lipopolysaccharide-induced astrocyte inflammatory response. Whether astragaloside can regulate the phenotype and function of astrocytes through Notch-1 and its downstream signaling pathway remains unclear. OBJECTIVE: To explore the effect of astragaloside on astrocyte activation and inflammatory response induced by inflammation and its possible mechanism. METHODS: Cerebral cortex astrocytes derived from neonatal C57BL/6 mouse were cultured in vitro. CCK-8 assay was used to determine the optimum concentration of astragaloside and Notch active inhibitor DAPT. The astrocytes were divided into five groups: PBS group, lipopolysaccharide group, lipopolysaccharide + astragaloside group, lipopolysaccharide + DAPT group and lipopolysaccharide + DAPT + astragaloside group. The secretion level of inflammatory factors was detected by ELISA, and the level of nitric oxide was detected by Griess method. The astrocytes and splenic mononuclear cells were co-cultured in Transwell chamber to observe the migration of CD4T cells. The expression of astrocyte activation marker GFAP, A1 marker C3 and A2 marker S100A10 as well as Notch 1 and Jag-1 was detected by immunofluorescence staining. The expressions of CFB, C3, S100A10, PTX3, Notch-1, Jag-1, and Hes were detected by western blot assay. RESULTS AND CONCLUSION: (1) According to the results of CCK8 assay, the final concentration of astragaloside was selected as 25 μmol/L and the final concentration of DAPT was 50 μmol/L for follow-up experiments. (2) Compared with PBS group, interleukin-6, interleukin-12 and nitric oxide secretion levels in the lipopolysaccharide group were significantly increased (P < 0.05, P < 0.05, P < 0.01). Compared with the lipopolysaccharide group, interleukin-6 (all P < 0.05), interleukin-12 (P > 0.05, P < 0.05, P < 0.05) and nitric oxide (P < 0.05, P < 0.01, P < 0.01) secretion significantly reduced in the lipopolysaccharide + astragaloside group, lipopolysaccharide +DAPT group, lipopolysaccharide + DAPT + astragaloside group. (3) Compared with the PBS group, the expression of GFAP that is the marker of activated astrocytes and the migration of CD4 T cells were significantly increased in the lipopolysaccharide group (P < 0.01). Compared with the lipopolysaccharide group, astrocyte activation was significantly inhibited and CD4 T cell migration was significantly reduced in the lipopolysaccharide + astragaloside, lipopolysaccharide +DAPT, lipopolysaccharide + DAPT + astragaloside group (P < 0.05, P < 0.05, P < 0.01). (4) Compared with the PBS group, the expressions of A1 markers C3 and CFB in the lipopolysaccharide group were increased (P < 0.01, P < 0.05), and the expressions of A2 markers S100A10 and PTX3 were decreased (P < 0.01, P < 0.05). Compared with the lipopolysaccharide group, C3 (all P < 0.01) and CFB (both P < 0.05) were significantly reduced and S100A10 (all P < 0.01) and PTX3 (P < 0.05, P < 0.05 and P > 0.05) were increased in the lipopolysaccharide + astragaloside, lipopolysaccharide +DAPT, lipopolysaccharide + DAPT + astragaloside group. (5) Compared with the PBS group, the expressions of Jag-1, Notch-1 and Hes in the lipopolysaccharide group were significantly increased (all P < 0.01). Compared with the lipopolysaccharide group, the expressions of Jag-1 (all P < 0.01), Notch-1 (all P < 0.01) and Hes (P < 0.05, P < 0.01 and P < 0.01) were significantly reduced in the lipopolysaccharide + astragaloside, lipopolysaccharide +DAPT, lipopolysaccharide + DAPT + astragaloside group. (6) The results indicate that astragaloside can promote the transformation of astrocytes from A1 to A2 by regulating Notch-1 signaling pathway, reduce the secretion of inflammatory factors and the migration of CD4 T cells, and thus inhibit astrocyte activation and inflammatory response.  Figures and Tables | References | Related Articles | Metrics

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Circ0005512 promotes microglia/macrophage pyroptosis after spinal cord injury in female rats Zhang Yan, Zhang Wenkai, Zhang Wenxiu, Liu Tao, Ma Ziqian, Chen Xueming 2024, 28 (31):  5029-5035.  doi: 10.12307/2024.711 Abstract ( 50 )   PDF (1424KB) ( 7 )   Save BACKGROUND: Neuroinflammation is an important factor leading to secondary spinal cord injury, and microglia/macrophage pyroptosis is a significant part of post-spinal cord injury neuroinflammation. Studies have shown that microglia/macrophage undergoes pyroptosis after spinal cord injury, but the regulatory mechanism of circular RNA (circRNA) in microglia/macrophage pyroptosis after spinal cord injury remains unclear.  OBJECTIVE: To investigate the role and mechanism of circRNA0005512 in regulating microglia/macrophage pyroptosis after spinal cord injury.  METHODS: Female Wistar rats were divided into sham group and spinal cord injury group. Motor function was evaluated using the Basso, Beattie, and Bresnahan (BBB) scale. Cavity volume was assessed by hematoxylin-eosin staining. Differential expression of circRNA in spinal cord tissue was screened using RNA-sequencing and circ0005512 was validated by real-time PCR. Immunofluorescence, western blot assay, ELISA, and real-time PCR were performed to detect cell pyroptosis in the rats and lipopolysaccharide-induced microglial cell line HAPI cell models. Gene knockdown was used to confirm the regulatory role of circRNA0005512 in microglia/macrophage pyroptosis.   RESULTS AND CONCLUSION: (1) Seven days after spinal cord injury, evident cavities were observed at the injury site. Immediately after spinal cord injury, the motor function of rats was completely lost. Over time, the motor function of rats in the spinal cord injury group gradually partially recovered, and there was a significant difference in BBB scores compared to the sham group. (2) Circ0005512 was significantly upregulated according to the results of the RNA-sequencing and confirmed in both the animal and cell models. (3) Immunofluorescence, western blot assay, real-time PCR, and ELISA confirmed the significant upregulation of cell pyroptosis markers (NLRP3, GSDMD, and caspase-1) in spinal cord injury tissue and lipopolysaccharide-induced HAPI cells. (4) In the cell model, knockdown of circ0005512 resulted in significantly decreased levels of cell pyroptosis marker-NLRP3. (5) The results above indicate that circ0005512 promotes pyroptosis in microglia/macrophages after spinal cord injury.  Figures and Tables | References | Related Articles | Metrics

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Intracranial transplantation of human bone marrow mesenchymal stem cells alleviates rat brain ischemia-reperfusion injury Song Wenxue, Liao Yidong, Ming Jiang, He Longcai, Chen Guangtang, Chen Chen, Wang Zili, Xiong Mingsong, Cui Junshuan, Xu Kaya 2024, 28 (31):  5036-5041.  doi: 10.12307/2024.709 Abstract ( 67 )   PDF (1549KB) ( 6 )   Save BACKGROUND: Studies have found that activation of nuclear factor-erythroid 2-related factor 2/heme oxidase-1 (Nrf2/HO-1) pathway can alleviate oxidative stress caused by cerebral ischemia-reperfusion injury, but whether human bone marrow mesenchymal stem cells (hBMSC) can activate Nrf2/HO-1 pathway to alleviate cerebral ischemia-reperfusion injury is still lacking relevant studies. OBJECTIVE: To investigate whether intracranial transplantation of hBMSC alleviates oxidative stress injury in cerebral ischemia-reperfusion animal models by activating Nrf2/HO-1 pathway. METHODS: Totally 40 male SPF SD rats were randomly divided into sham operation group, model group, hBMSC transplantation group, hBMSC+solvent group and hBMSC+Nrf2 inhibitor group. Each group consisted of eight animals. In the model group and the hBMSC transplantation group, middle cerebral artery occlusion model was prepared by thread embolization method. The thread embolization was removed 1 hour later, and 30 μL PBS or hBMSC cultured to at least passage 5 was injected into the right cortex and striatum of rats. In the hBMSC+Nrf2 inhibitor group and hBMSC+solvent group, the left ventricle was injected with Nrf2 inhibitor Brusatol and its solvent dimethyl sulfoxide respectively 24 hours before model establishment, then the middle cerebral artery occlusion model was prepared, and hBMSC was injected. Relevant indexes were detected 3 days after transplantation. RESULTS AND CONCLUSION: (1) CT and TTC staining showed the same area and volume of cerebral infarction: model group > hBMSC+Nrf2 inhibitor group > hBMSC+solvent group > hBMSC transplantation group > sham operation group. (2) Hematoxylin-eosin staining and Nissl’s staining showed that the ischemic brain tissue was intact and the neurons were normal in the sham operation group. Compared with the model group, the pathological morphology and neuronal injury of the hBMSC transplantation group and the hBMSC+solvent group were significantly improved. Compared with the hBMSC+solvent group, the hBMSC+Nrf2 inhibitor group had more serious pathological morphology and neuronal damage. (3) Western blot assay and oxidative stress index detection results showed that compared with the sham operation group, Nrf2 and HO-1 proteins were decreased (all P < 0.05), malondialdehyde was increased and superoxide dismutase was decreased (all P < 0.05) in the model group. Compared with the model group, the expression levels of Nrf2 and HO-1 proteins were increased (all P < 0.05), malondialdehyde was decreased and superoxide dismutase was increased (all P < 0.05) in the hBMSC transplantation group and the hBMSC+solvent group. Compared with the hBMSC+solvent group, the expression levels of Nrf2 and HO-1 proteins were simultaneously decreased (all P < 0.05), and malondialdehyde was increased and superoxide dismutase was decreased (all P < 0.05) in the hBMSC+Nrf2 inhibitor group. (4) These results indicate that hBMSC can alleviate cerebral ischemia-reperfusion injury possibly by activating Nrf2/HO-1 pathway. Figures and Tables | References | Related Articles | Metrics

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miRNA derived from mesenchymal stem cells and its derivatives in treatment of pathological scar Li Yue, Qiao Hua 2024, 28 (31):  5042-5047.  doi: 10.12307/2024.701 Abstract ( 55 )   PDF (1070KB) ( 16 )   Save BACKGROUND: Stem cell therapy has become an emerging method of treating pathological scars. miRNAs are involved in scarring mechanisms, and the targeted action of some stem cell sources of miRNA is mediated by exosomes. OBJECTIVE: To review the biological properties of miRNA derived from mesenchymal stem cells and its derivatives, the mechanism of treatment of scarring through anti-inflammation, suppression of excessive tissue reconstruction and antioxidation. METHODS: The first author used a computer in September 2023 to retrieve the relevant literature published from January 2000 to September 2023, searching for “stem cell, exosome, miRNA, keloid” in English, and “stem cells, exosome, keloid” in Chinese, eventually incorporating 74 papers for analysis. RESULTS AND CONCLUSION: (1) miRNAs with high expression of exosomes derived from mesenchymal stem cells increase the proportion of M2-type macrophages by promoting the polarization of macrophages, target the regulation of transforming growth factor β, transforming growth factor β receptors or related signaling pathways, inhibit the expression of pro-inflammatory factors, promote the expression of anti-inflammatory factors and other mechanisms to inhibit inflammation and thus suppress scar lesions. (2) miRNAs with high expression of exosomes derived from mesenchymal stem cells can reduce the secretion of matrix metalloproteinases, regulate the balance between matrix metalloproteinase inhibitors and matrix metalloproteinases, inhibit the proliferation and migration of fibroblasts and myofibroblasts, directly reduce the production of collagen and other mechanisms, and ultimately lead to the normal degradation of extracellular matrix, thereby inhibiting excessive tissue remodeling and cicatricial lesions. (3) miRNAs with high expression of exosomes from mesenchymal stem cells can improve the resistance of scar fibroblasts to oxidative stress by regulating reactive oxygen species and hypoxia-inducing factors, and then regulate the proliferation and apoptosis of scar fibroblasts to inhibit scar lesions. (4) Exosomes derived from mesenchymal stem cells have good prospects for scar treatment. Studies on this aspect can find mirnas that regulate inflammatory cells, inflammatory factors, signaling pathways, matrix metalloproteinases, fibroblasts, reactive oxygen species, hypoxia-inducing factors and other key factors from the three aspects of inflammation, tissue remodeling and oxidative stress. Then, by inducing mesenchymal stem cells with high expression of the above miRNA, exosomes were extracted, and finally verified and clinical trials were carried out. Figures and Tables | References | Related Articles | Metrics

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Interleukin-1beta enhances migration and adhesion of mesenchymal stem cells in inflammatory environments Wu Qixiang, Fang Chenyu, Zhang Lei 2024, 28 (31):  5048-5054.  doi: 10.12307/2024.715 Abstract ( 64 )   PDF (1266KB) ( 17 )   Save BACKGROUND: Mesenchymal stem cells possess characteristics such as rapid renewal, targeted homing, tissue repair, and immune regulation, which provide potential for the treatment of inflammatory diseases. In most inflammatory diseases, interleukin-1β is highly expressed. Both exogenous and endogenous mesenchymal stem cells unavoidably exist in an environment with high interleukin -1β concentration.  OBJECTIVE: To study the interaction of interleukin-1β with mesenchymal stem cells in inflammatory environment and the mechanism of its influence on the migration and adhesion of mesenchymal stem cells to provide a theoretical basis for adjusting stem cell therapy strategies. METHODS: The first author searched for studies involving interleukin-1β enhancing migration and adhesion of mesenchymal stem cells by computer on CNKI, WanFang, VIP, PubMed, and Web of Science using search terms “interleukin-1β, mesenchymal stem cell, nuclear factor-κB, MAPK, ERK, p38, migration, adhesion” in Chinese and English. The literature tracing method was also used to search for some of the literature. Finally, 65 articles were included in the review analysis. RESULTS AND CONCLUSION: (1) In the inflammatory environment, interleukin-1β can regulate the migration and adhesion ability of mesenchymal stem cells. This effect may be achieved by recruiting IRAK1 through interleukin-1RI and then activating TAK1 and IKK in turn. After IKK phosphorylation, nuclear factor-κB and ERK signaling pathways are activated or CXCR expression is upregulated through the p38 pathway to promote mesenchymal stem cell migration and adhesion. However, further standardized research needs to be carried out based on the genetic background of mesenchymal stem cells, the dose and processing time of interleukin-1β. (2) In vitro experiments using pre-stimulated mesenchymal stem cells with interleukin-1β can change the survival environment of mesenchymal stem cells and alter their secretion factors to make them develop towards a more anti-inflammatory direction. On the other hand, under the premise of producing higher levels of anti-inflammatory and pro-nutrient factors, extracted mesenchymal stem cell exosomes can exert anti-inflammatory effects. (3) It has been observed in various animal disease models that pre-stimulating mesenchymal stem cells with interleukin-1β regulates their immune regulation ability, thereby affecting the development and outcome of inflammation. However, this is limited to preclinical basic research only; further verification on efficacy and safety of stem cell therapy with interleukin-1β pre-treated mesenchymal stem cells is required in clinical settings.  Figures and Tables | References | Related Articles | Metrics

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Co-culture technology of mesenchymal stem cells and macrophages Yang Xiaoqian, Song Aimei, Song Hui 2024, 28 (31):  5055-5062.  doi: 10.12307/2024.706 Abstract ( 68 )   PDF (1248KB) ( 14 )   Save BACKGROUND: In the co-culture environment of mesenchymal stem cells and macrophages, mesenchymal stem cells can promote the polarization of macrophages into anti-inflammatory macrophages to reduce inflammation, and macrophages can promote the osteogenic differentiation of mesenchymal stem cells. The co-culture of both plays an important role in regulating the immune system and promoting tissue regeneration.  OBJECTIVE: To summarize the methods, influencing factors and possible mechanisms of co-culture between mesenchymal stem cells and macrophages, and to provide theoretical basis and experimental methods for the application of co-culture of mesenchymal stem cells and macrophages in tissue engineering.  METHODS: The first author searched the relevant articles published from January 1970 to September 2023 in PubMed and CNK by computer from January to September 2023. The Chinese and English key words were “mesenchymal stem cells, macrophages, co-culture”. Finally, 63 articles were included and analyzed.  RESULTS AND CONCLUSION: (1) In vitro co-culture of mesenchymal stem cells and macrophages can be divided into direct contact co-culture and indirect contact co-culture according to the model, and two-dimensional cell co-culture and three-dimensional cell co-culture according to the dimension. (2) The co-culture of mesenchymal stem cells and macrophages can promote the polarization of macrophages towards M2 type and enhance the osteogenic effect of mesenchymal stem cells. (3) In the co-culture model, the methods of co-culture, the proportion and time of co-culture, the phenotype of macrophages, and the cell source and conditions all affected the immune regulation of macrophages and the osteogenesis of mesenchymal stem cells. (4) Cell interaction in co-culture may regulate the immune function of macrophages, proliferation, migration and osteogenesis of mesenchymal stem cells through cell-secreted soluble factors, extracellular vesicles, cell-cell contact, and metabolic pathways. (5) Mesenchymal stem cells and macrophages can enhance cardiac function after acute myocardial infarction, promote epithelial wound healing, reduce lung inflammation, improve renal function, and accelerate bone repair. (6) There are still some problems in co-culture of mesenchymal stem cells and macrophages, such as the selection of co-culture conditions, the maintenance of good cell state and interaction of co-cultured cells. (7) The co-culture of mesenchymal stem cells and macrophages can improve the local inflammatory microenvironment and promote tissue regeneration and repair, so it will have a broad application prospect in tissue engineering. Figures and Tables | References | Related Articles | Metrics

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Exosome-derived microRNA with bone and joint diseases: role and mechanism Yuan Ping, Wang Zhihua, Wang Weizhou, Wang Wentong, He Fei 2024, 28 (31):  5063-5069.  doi: 10.12307/2024.723 Abstract ( 56 )   PDF (1123KB) ( 16 )   Save BACKGROUND: Exosomes are vesicle-like structures secreted by cells into extracellular compartments in the form of cytosol, which contain a large amount of microRNAs with important intercellular communication roles. MicroRNAs in exosomes rely on exosome transport and are able to enter target cells to exert important biological regulatory effects. In common bone and joint diseases, abnormal or damaged bone metabolism releases a large number of exosomes, while some exosome-derived microRNAs also promote the progression of osteoarthritis. Therefore, exosome-derived microRNAs are closely related to the skeletal system and are important for the development as well as diagnosis and treatment of many osteoarticular diseases.  OBJECTIVE: To review the research progress of exosome-derived microRNAs in bone metabolism and bone and joint diseases. METHODS: Using “exosomes, extracellular vesicle, microRNA, miRNA, bone, bone diseases, bone formation, bone regeneration, bone resorption, bone destruction” as Chinese and English search terms, articles were searched on CNKI, Metasys, and PubMed databases. Finally, 86 articles were included for summarization.  RESULTS AND CONCLUSION: Exosome-derived microRNAs can regulate bone metabolism by affecting bone formation and bone resorption, and are closely related to the development of bone and joint diseases such as fracture healing, osteoporosis, osteoarthritis, rheumatoid arthritis, osteonecrosis of the femoral head, and osteosarcoma. Exosome-derived microRNAs will be an effective means of diagnosis and treatment of certain bone and joint diseases in the future. However, the current research on exosome-derived microRNAs in osteoarthritic diseases is limited, and more explorations and researches are still needed to diagnose and treat osteoarthritic diseases using exosome-derived microRNAs. Figures and Tables | References | Related Articles | Metrics

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Role of non-coding RNA and exosomes in pathogenesis of gestational diabetes mellitus and their early diagnostic value Hu Lingli, Li Na, Li Jingyang, Zhang Eryun, Chen Yu, Gu Ying 2024, 28 (31):  5070-5077.  doi: 10.12307/2024.714 Abstract ( 32 )   PDF (1106KB) ( 11 )   Save BACKGROUND: In recent years, there have been many studies on the mechanism of exosomal non-coding RNA in gestational diabetes mellitus, but there is a lack of the latest systematic review of exosomes from different sources, especially placental sources.  OBJECTIVE: To summarize the changes and potential roles of microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), and exosomes in gestational diabetes mellitus to provide potential targets for early screening and treatment of clinical gestational diabetes mellitus. METHODS: A literature search was conducted on PubMed, Web of Science, China National Knowledge Infrastructure, WanFang Data, and VIP databases to retrieve relevant articles on non-coding RNA or exosomal non-coding RNA in relation to gestational diabetes mellitus. A total of 74 articles were included for review.  RESULTS AND CONCLUSION: (1) Non-coding RNAs play important pathological and physiological roles in the lifecycle activities, and increasing evidences suggest that non-coding RNAs are involved in the occurrence and development of gestational diabetes mellitus by regulating various physiological functions. This provides a new direction for the research of gestational diabetes mellitus. (2) Exosomes are widely present in the human body. Various cells can secrete exosomes, such as red blood cells, epithelial cells, and placental cells. Non-coding RNAs found in exosomes from different sources have been demonstrated to play a role in the pathogenesis, diagnosis, and treatment of gestational diabetes mellitus. (3) MiRNA and gestational diabetes mellitus: The role of peripheral blood miRNA in gestational diabetes mellitus is mainly to affect the functions of trophoblast cells, pancreatic beta cells and blood glucose levels in gestational diabetes mellitus; placental miRNA can reflect the severity of gestational diabetes and impair the function of trophoblast cells. (4) LncRNA and gestational diabetes mellitus: Peripheral blood lncRNA can induce insulin resistance through the phosphatidylinositol 3-kinase/protein kinase B pathway and may provide new insights for the diagnosis and treatment of gestational diabetes mellitus; placental lncRNA can regulate proliferation and migration of placental trophoblast cells, promoting the occurrence and development of gestational diabetes mellitus. (5) CircRNA and gestational diabetes mellitus: Peripheral blood and placental circRNA can induce the occurrence and development of gestational diabetes mellitus by impairing the proliferation, migration and metabolism of placental trophoblast cells. (6) Non-coding RNA in exosomes and gestational diabetes mellitus: Peripheral blood non-coding RNA in exosomes can affect gestational diabetes mellitus blood glucose levels and glucose homeostasis, and participate in the occurrence and development of gestational diabetes mellitus by influencing placental function. (7) Non-coding RNA has the potential to serve as biomarkers for early diagnosis of gestational diabetes mellitus. Additionally, engineered exosomes can better achieve targeted therapy for gestational diabetes mellitus. These latest findings provide a reference for both basic research and clinical translation of gestational diabetes mellitus. (8) In the future, improvements in the extraction and purification methods of peripheral blood exosomes should be improved, and factors such as race, diet and physical activity should be excluded to improve the reproducibility of results. Further prospective clinical studies are required to explore the clinical application of circulating non-coding RNA and exosomes in the prediction and diagnosis of gestational diabetes mellitus. Figures and Tables | References | Related Articles | Metrics

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Research, development and advance in precise screening of lung cancer drugs with tumor organoids Liu Jiwei, Liu Weici, Mao Wenjun 2024, 28 (31):  5078-5084.  doi: 10.12307/2024.719 Abstract ( 68 )   PDF (1153KB) ( 26 )   Save BACKGROUND: Lung cancer is one of the most common malignant tumors with the worst prognosis worldwide. Its incidence rate and mortality rate have long been among the top of malignant tumors. The heterogeneity and drug resistance are among the reasons contributing to its poor prognosis. Lung cancer organoid, which is a 3D in vitro model cultured from patient-derived tumor cells recapitulating the biological characteristics of the primary tumor, can be used for various researches of lung cancer. OBJECTIVE: To review the application and research progress of lung cancer organoids in chemotherapy, targeted therapy, and immunotherapy drug sensitivity screening, analyze its limitations, aiming to provide new strategies for personalized and precision medicine of lung cancer. METHODS: The first author searched relevant articles published from 2013 to 2023 in CNKI and PubMed in July 2023. The search terms were “organoid, lung cancer organoid, lung cancer experimental model, precision medicine, drug sensitivity test, chemotherapy, targeted therapy, immunotherapy” in Chinese and English. Finally, a total of 84 articles were incorporated. RESULTS AND CONCLUSION: (1) Compared with traditional lung cancer research models, which can only demonstrate two-dimensional cell activities, lack cell-to-cell interactions, and suffer from species differences, lung cancer organoids offer a diverse cell source and continuously optimized culture conditions. They can simulate cellular interactions in a three-dimensional context while retaining the biological characteristics of the original tumor. These organoids represent a promising research model with significant potential, providing a solid foundation for their use in cancer drug screening. (2) Lung cancer organoids have shown preliminary significance in guiding anticancer drug selection. Their predictive outcomes align closely with actual clinical outcomes, marking a pivotal step towards precision in lung cancer treatment. By assessing the efficacy of common chemotherapy, targeted therapy, and immunotherapy drugs, these organoids enable the customization of individualized treatment strategies, reducing unnecessary drug trials and toxic and side reaction while exploring possible alternative drugs or combination regimens for drug resistance issues so as to guide the precision treatment of rare mutated lung cancer and fill the clinical gap. A more comprehensive drug evaluation is provided by comparing the activity of different drugs. Additionally, it is essential to consider the internal heterogeneity of organoids, emphasizing the importance of multiple sampling to enhance result accuracy. (3) Lung cancer organoids have limitations in practical applications such as inconsistent success rates and purity in cultivation and the lack of vascular tissue. To address these shortcomings, improvements are needed in cultivation conditions, expedited testing processes, and the development of multi-organ systems to simulate the overall effects of drugs in multiple organs. These enhancements will contribute to a more accurate assessment of drug efficacy and toxicity, thereby enhancing the precision of lung cancer treatment. Figures and Tables | References | Related Articles | Metrics