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08 May 2025, Volume 29 Issue 13 Previous Issue    Next Issue

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miR-192-5p targets CKIP-1 to promote osteogenic differentiation of bone marrow mesenchymal stem cells in osteoporosis patients E Zhengkang, Xin Hongwei, Yu Qingbo, Zhang Yunshuai 2025, 29 (13):  2641-2647.  doi: 10.12307/2025.051 Abstract ( 66 )   PDF (1519KB) ( 147 )   Save BACKGROUND: Casein kinase 2 binding protein 1 (CKIP-1) is an important negative control gene in bone formation. After the deletion of this gene, the overall bone of mice was significantly enhanced, and bone formation and bone density were significantly increased. microRNA (miRNA) as the early found small molecular regulator has a regulatory effect on most of the coding genes and plays an important role in osteogenic differentiation.  OBJECTIVE: To investigate the effect and molecular mechanism of miRNA/CKIP-1 axis on osteogenic differentiation of bone marrow mesenchymal stem cells from osteoporosis patients.  METHODS: The miRNA-Seq technology was used to detect the changes of miRNA in bone marrow mesenchymal stem cells in 32 patients with osteoporosis treated in the Department of Orthopedics, Kaifeng Central Hospital from March to June 2022 and healthy people in the physical examination center during the same period. The Targetscan website was used to predict the miRNA targeted to regulate CKIP-1. Luciferase reporter gene assay was used to detect the binding of miRNA and CKIP-1 promoter DNA. miR-192-5p analogs (miR-192-5p mimics)/negative control (NC mimics) or miR-192-5p inhibitors (miR-192-5p inhibitor)/negative control (NC inhibitor) were transfected in bone marrow mesenchymal stem cells. On days 7 and 14 after osteogenic induction, the expression levels of Runt-related transcription factor 2 (Runx2), osteocalcin, anti-osteopontin, bone sialoprotein, and CKIP-1, and the differentiation of bone marrow mesenchymal stem cells into osteoblasts were detected by real-time fluorescence quantitative PCR and alizarin red staining. The regulatory effect of miR-192-5p/CKIP-1/axis on osteogenic differentiation of cells was detected by western blot assay and alizarin red staining. RESULTS AND CONCLUSION: Compared with the healthy group, the expression levels of 16 miRNAs were significantly up-regulated and those of 53 miRNAs were significantly down-regulated in the osteoporosis group (P < 0.05). Using Targetscan website and verified by luciferase reporter gene experiment, it was found that miR-192-5p and CKIP-1 had complementary nucleotide sequences (P < 0.05). Overexpression of miR-192-5p significantly increased the expression levels of Runx2, osteocalcin, osteopontin, and bone sialoprotein (P < 0.05), and inhibition of miR-192-5p significantly decreased the expression levels of Runx2, osteocalcin, osteopontin, and bone sialoprotein (P < 0.05). After silencing the expression of CKIP-1, the protein levels of Runx2, osteocalcin, and osteopontin increased (P < 0.05); the inhibitory effect of knockdown of miR-192-5p on osteogenic differentiation of cells was reversed. Above results confirm that the expression of miR-192-5p is decreased in osteoporosis. miR-192-5p promotes osteogenic differentiation of bone marrow mesenchymal stem cells by targeting the inhibition of CKIP-1 expression. Figures and Tables | References | Related Articles | Metrics

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Small molecule drug TD-198946 enhances osteogenic differentiation of rat bone marrow mesenchymal stem cells Yang Chao, Luo Zongping 2025, 29 (13):  2648-2654.  doi: 10.12307/2025.060 Abstract ( 77 )   PDF (1354KB) ( 102 )   Save BACKGROUND: TD-198946 is a small molecule drug known to induce stem cells to form cartilage; however, its effect on osteogenic differentiation remains unclear.  OBJECTIVE: To investigate the effects of the small molecule drug TD-198946 on promoting osteoblastic differentiation of rat bone marrow mesenchymal stem cells and its mechanism of action.  METHODS: Bone marrow mesenchymal stem cells (BMSCs) were extracted from SD rats. CCK-8 assay was used to evaluate the effect of different concentrations of TD-198946 on the proliferation of bone marrow mesenchymal stem cells to determine the optimal concentration of TD-198946. Then, the optimal concentration of TD-198946 and osteogenic medium were added to induce osteogenic differentiation of bone marrow mesenchymal stem cells. On day 3, qRT-PCR was used to detect the expression of alkaline phosphatase, Runt-related transcription factor 2, osteopontin, osteocalcin, and type I collagen genes. On day 7 of osteogenic induction, alkaline phosphatase staining and western blot assay were performed to detect the expression of Runt-related transcription factor 2, type I collagen, AKT, p-AKT, PI3K, and p-PI3K proteins. On day 21 of osteogenic induction, Alizarin red staining was conducted.  RESULTS AND CONCLUSION: (1) CCK-8 assay results revealed that 100 nmol/L TD-198946 could promote the proliferation of bone marrow mesenchymal stem cells. (2) The results of alkaline phosphatase staining and alizarin red staining showed that TD-198946 could promote osteogenic differentiation of rat bone marrow mesenchymal stem cells. (3) qRT-PCR results showed that TD-198946 could promote the expression of osteogenic genes alkaline phosphatase, Runt-related transcription factor 2, osteopontin, osteocalcin, and type I collagen. (4) Western blot assay results showed that TD-198946 could enhance the expression of Runt-related transcription factor 2, type I collagen, p-PI3K, and p-AKT. The results indicate that the small molecule drug TD-198946 may induce osteogenic differentiation of rat bone marrow mesenchymal stem cells by activating the PI3K/AKT signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Effects of different storage durations on DNA quality of biopsy specimens with novel ultrasound processing Shi Chenxi, Zhu Weidong, Li Sanen, Li Xiuming, Shi Feng, Ding Yayun 2025, 29 (13):  2655-2660.  doi: 10.12307/2025.018 Abstract ( 104 )   PDF (1255KB) ( 251 )   Save BACKGROUND: The technique of ultrasound processing is widely used for molecular biological analysis. It is of great significance to study the DNA quality of tissue with different storage years under new ultrasonic treatment for further specimen quality control of molecular detection. OBJECTIVE: To explore the effects of different storage durations on DNA quality in specimens with ultrasound processing to investigate the optimal storage time for molecular tests. METHODS: Forty specimens of breast biopsy were collected and paraffin specimens were prepared by ultrasonography. These specimens were divided into four groups based on their storage periods: < 1 year, 1-3 years, > 3-5 years, and > 5 years, which contained 10 cases in each group. Paraffin specimens were sliced; each slice was 3 μm thick; 10-15 slices were taken, and DNA was extracted. The mass concentration of DNA was examined by Nanophotometer N60 ultra-micro spectrophotometer and Qubit 4.0 fluorometer. The purity of the DNA was analyzed by the ratio of A260/A280. DNA fragment integrity was measured by capillary electrophoresis (Qsep 100) to evaluate the quality of the DNA fragments.  RESULTS AND CONCLUSION: The mean values of A260/A280 in the four groups were between 1.8 and 2.0, meeting the requirements of tests, without significant differences. The mean values of DNA mass concentration (Qubit concentration) were 30.39, 14.33, 2.52, and 1.95 ng/μL, respectively. The mean values of the N/Q were 6.48, 14.18, 24.56, and 29.86. The mean values of DNA were: 5.64, 1.76, 1.24, and 0.80. The percentage of large DNA fragments averaged 56.08%, 17.72%, 12.68%, and 7.90%. Moreover, the Ct values of the internal control detected by PCR were 15.32, 17.09, 18.39, and 21.24. The three other groups exhibited significantly lower DNA concentration, higher N/Q ratios, decreased DNA quality and percentage of large fragments, and increased values of Ct, compared with the group of within 1 year of storage (P < 0.05). The experimental results suggested that for novel ultrasound processed biopsy specimens, we should prioritize samples stored within 1 year for molecular testing. Samples stored within 3 years can also meet the requirements of second-generation sequencing and other tests. Samples stored within 5 years can only be attempted to carry out PCR. Samples stored for more than 5 years were not recommended to carry out molecular tests. Figures and Tables | References | Related Articles | Metrics

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Prediction model and verification of sperm DNA fragments based on traditional Chinese medicine syndrome and semen quality-related parameters Zhou Chao, Yu Guangyu, Yang Shaohua, Gao Leilei, Jin Zhen, Jiang Yueyuan, Li Huan 2025, 29 (13):  2661-2668.  doi: 10.12307/2024.148 Abstract ( 132 )   PDF (1199KB) ( 109 )   Save BACKGROUND: The combination of traditional Chinese medicine syndrome and semen quality-related parameters can jointly predict the occurrence of abnormal increase in sperm DNA fragmentation index (DFI) and draw a column chart, which can significantly improve clinical practicality and application efficiency, provide a basis for comprehensive evaluation of semen quality in clinical practice, take active intervention measures to improve clinical outcomes, and formulate personalized medical plans. OBJECTIVE: To explore the prediction model and verification of sperm DNA fragments based on traditional Chinese medicine syndrome and semen quality-related parameters.   METHODS: Retrospective analysis was made on 420 infertile patients who received traditional Chinese medicine syndrome diagnosis and sperm DNA fragment rate examination in the Department of Traditional Chinese Medicine Andrology, Nanxishan Hospital of Guangxi Zhuang Autonomous Region from July 2019 to July 2021. According to the Manual of Human Semen Examination and Treatment Laboratories (6th Edition), 137 patients with sperm DFI>30% were included in the group of abnormally high sperm DFI, and 283 patients with sperm DFI ≤ 30% were taken as the control group. First, univariate analysis was used to screen the influencing factors of the abnormal increase of sperm DFI. Then, the best matching factor was selected by using the collinearity problem of LASSO correction factors. Then, it was included in the multifactor forward stepwise logistic regression to find out its independent influencing factors and draw a nomogram. Finally, the receiver operating characteristic curve, calibration curve, decision curve analysis and clinical impact curve were used to verify the differentiation and accuracy of the prediction model and its clinical application effectiveness.  RESULTS AND CONCLUSION: (1) The results of the univariate analysis showed that age, body mass index, forward motion rate, total sperm motility, sperm concentration, sperm morphology, kidney yang deficiency syndrome, damp heat downpour syndrome, and kidney sperm deficiency syndrome were the influencing factors for the abnormal increase of sperm DFI (P < 0.05). (2) The best matching factors further screened by LASSO regression were age, body mass index, total sperm motility, sperm concentration, sperm morphology, kidney yang deficiency syndrome, damp heat downpour syndrome, and kidney essence deficiency syndrome (P < 0.05). (3) Multifactor forward stepwise Logistic regression showed that age, body mass index, sperm concentration, total sperm motility, damp heat downpour syndrome, and kidney yang deficiency syndrome were six independent factors that caused the abnormal increase in sperm DFI. (4) Receiver operating characteristic curve showed that the area under the curve of the model group was 0.760(0.713,0.806), and the area under the curve of the validation group was 0.745(0.714,0.776). It showed that the prediction model had good discrimination. (5) The average absolute error of the calibration curve was 0.040, and the Hosmer Lemeshow test (P > 0.05), suggesting that there was no significant statistical difference between the probability of the abnormal increase in DFI of spermatozoa predicted by the model and the probability of the abnormal increase in DFI of spermatozoa actually occurred, which confirmed that the model had good accuracy. (6) Decision curve analysis and clinical impact curve showed that the model group and validation group had the maximum clinical net benefit when the threshold probability values were (0.08-0.84) and (0.09-0.78) respectively, and had good clinical application efficiency within the threshold probability range. (7) These findings conclude that age, body mass index, sperm concentration, total sperm viability, damp heat downpour syndrome and kidney yang deficiency syndrome are independent factors that cause the abnormal increase in sperm DFI. The nomogram of the clinical prediction model constructed by them has good clinical prediction value and clinical application efficiency, and can provide the basis for comprehensive clinical evaluation of semen quality and individualized medical service. Figures and Tables | References | Related Articles | Metrics

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Protective effect of mangiferin on oxidative stress injury in rat bone marrow mesenchymal stem cells Li Xiaofeng, Zhao Duo, Ouyang Qin, Pang Zixiang, Li Yuquan, Chen Qianfen 2025, 29 (13):  2669-2674.  doi: 10.12307/2025.059 Abstract ( 83 )   PDF (1073KB) ( 73 )   Save BACKGROUND: Mesenchymal stem cell transplantation has a certain effect on spinal cord injury, but there are still some problems such as low survival rate and poor efficiency of cell transplantation caused by local oxidative stress environment. OBJECTIVE: To explore the protective effect of mangiferin on oxidative stress of bone marrow mesenchymal stem cells. METHODS: Rat bone marrow mesenchymal stem cells at passage 3 were added to mangiferin and incubated for 2 hours according to the gradient of concentration (0, 20, 40, 80, and 160 µmol/L). A serum-free medium containing 400 µmol/L H2O2 was added, and a gradient concentration of mangiferin was added again and cultured for 12 and 24 hours. Rat bone marrow mesenchymal stem cells were cultured as normal control group. Cell survival was detected by MTT assay in each group. The superoxide dismutase, malondialdehyde, and catalase in the culture medium were detected in accordance with the instruction of the kit. RESULTS AND CONCLUSION: Compared with normal control group, cell viability in the H2O2 group was significantly decreased (P < 0.01), and the levels of superoxide dismutase and catalase were significantly decreased (P < 0.01), while the level of malondialdehyde was significantly increased (P < 0.01). Compared with H2O2 group, the survival rate of cells was significantly increased (P < 0.01), and the levels of superoxide dismutase and catalase were significantly increased (P < 0.01), while the level of malondialdehyde was significantly decreased (P < 0.01) in the mangiferin group. Mangiferin showed concentration-dependent antioxidant stress protective activity above 20 µmol/L, and no cytotoxicity below 160 µmol/L. These findings indicate that antioxidant mangiferin can increase the antioxidant activity of bone marrow mesenchymal stem cells and provide a new preconditioning strategy for bone marrow mesenchymal stem cell transplantation. Figures and Tables | References | Related Articles | Metrics

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Acellular cartilage extracellular matrix homogenate combined with oxymatrine on treatment of osteoarthritis in rats Wu Fuzhang, Zhang Pengli, Zhang Zhenhua, He Yongbing, Sun Heyan 2025, 29 (13):  2675-2682.  doi: 10.12307/2025.042 Abstract ( 92 )   PDF (1235KB) ( 43 )   Save BACKGROUND: Acellular cartilage extracellular matrix is effective in the treatment of osteoarthritis, but its efficacy is limited when applied alone. Oxymatrine alleviates interleukin-1β-induced chondrocyte apoptosis and extracellular matrix degradation. OBJECTIVE: To observe the effect of different doses of oxymatrine combined with acellular cartilage extracellular matrix injection in knee cavity on osteoarthritis in rats. METHODS: The femoral cartilage of SD rats was obtained. The acellular cartilage extracellular matrix was prepared by physical, chemical and enzyme methods. Fifty SD rats were selected and divided into sham operation group, osteoarthritis group, simple material group, low-dose drug group, and high-dose drug group by random number table method, with 10 rats in each group. The latter four groups were treated with modified Hulth method to establish the rat model of osteoarthritis. After successful modeling, acellular cartilage extracellular matrix homogenate was injected into the knee cavity of rats in the simple material group. Acellular cartilage extracellular matrix homogenate containing 50, 100 µg oxymatrine was injected into the knee cavity of rats in the low-dose drug group and the high-dose drug group, respectively. Samples were taken 4 weeks after injection for relevant detection. RESULTS AND CONCLUSION: (1) Compared with the sham operation group, the concentrations of interleukin 1β, tumor necrosis factor ɑ, and malondialdehyde in the joint effusion were increased (P < 0.05), and the concentration of superoxide dismutase in the joint effusion was decreased (P < 0.05) in the osteoarthritis group. Compared with osteoarthritis group, the levels of interleukin 1β, tumor necrosis factor ɑ, and malondialdehyde in joint effusion were decreased (P < 0.05), while the level of superoxide dismutase was increased (P < 0.05) in the low-dose drug group and high-dose drug group, and the changes were more significant in high-dose drug group. (2) TUNEL staining showed that compared with sham operation group, apoptotic chondrocytes increased in osteoarthritis group. Compared with the osteoarthritis group, the apoptotic chondrocytes decreased in the simple material group, the low-dose drug group, and the high-dose drug group, and the decrease was more significant in the high-dose drug group. (3) Hematoxylin-eosin staining and immunohistochemical staining showed that the knee cartilage was seriously degraded, the expression of type II collagen was decreased (P < 0.05), and the expression of matrix metalloproteinase-9 was increased (P < 0.05) in the osteoarthritis group. Compared with the osteoarthritis group, the knee cartilage degeneration of rats in the simple material group, the low-dose drug group, and the high-dose drug group was significantly improved; the expression of type II collagen was increased (P < 0.05) and the expression of matrix metalloproteinase 9 was decreased (P < 0.05), and the improvement was most significant in the high-dose drug group. (4) Western blot assay showed that compared with sham operation group, the expression of Nrf2, HO-1, and Bcl-2 protein in cartilage tissue decreased (P < 0.05); expression levels of Cleaved-caspase-3, Cleaved-caspase-9, and Bax were increased (P < 0.05) in the osteoarthritis group. Compared with the osteoarthritis group, the protein expression levels of Nrf2, HO-1, and Bcl-2 were increased (P < 0.05), and the protein expressions of Cleaved-caspase-3, Cleaved-caspase-9, and Bax were decreased (P < 0.05) in the low-dose and high-dose drug groups. The improvement was more significant in the high-dose drug group. (5) In conclusion, intracavicular injection of acellular cartilage extracellular matrix and oxymatrine can promote the synthesis of extracellular matrix, inhibit inflammation and oxidative stress, and suppress chondrocyte apoptosis, and play a therapeutic role in osteoarthritis, which may be related to the activation of Nrf2/HO-1 pathway.  Figures and Tables | References | Related Articles | Metrics

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Comparative proteomic analysis of rat adipose-derived mesenchymal stem cells and their exosomes Zhang Xinrui, Han Yue, Lei Lei, Liu Jianyu, Geng Chengkui 2025, 29 (13):  2683-2689.  doi: 10.12307/2025.046 Abstract ( 98 )   PDF (1814KB) ( 117 )   Save BACKGROUND: Research on adipose-derived mesenchymal stem cells and their exosomes often uses proteomics to analyze their roles in tissue repair, but comparative proteomic analyses between the two are scarce. OBJECTIVE: To analyze adipose-derived mesenchymal stem cells and their exosomes using proteomic analysis.  METHODS: Rat adipose-derived mesenchymal stem cells were isolated, cultured, and identified. Exosomes were then extracted from cell supernatant and identified. Differentially expressed proteins of adipose-derived mesenchymal stem cells and their exosomes were analyzed using DIA proteomics. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed on these differentially expressed proteins.  RESULTS AND CONCLUSION: (1) Adipose-derived mesenchymal stem cells displayed a mainly spindle-shaped, fibroblast-like morphology. (2) The exosome suspension protein concentration was 7.66 mg/mL, as determined by the BCA method. (3) Exosomes exhibited a characteristic teacup shape with a visible double-layer membrane vesicle structure. The center presented a low electron density component. The exosomes showed a peak particle size distribution of 112.2 nm, a concentration of 7.5×1011 particles/mL, and an average diameter ranging from 70 to 140 nm. (4) Exosomes expressed high levels of surface marker proteins CD9 and TSG101. (5) Gene Ontology analysis of differentially expressed proteins revealed enrichment in the extracellular matrix and synapses, with functions related to ion binding, ribosome binding, and particularly cell adhesion and translation. (6) Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the differentially expressed proteins were primarily involved in extracellular matrix receptor interaction, ribosome, and cytokine receptor interaction, and also associated with various metabolic diseases like cholesterol and thyroid disorders.  Figures and Tables | References | Related Articles | Metrics

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miR-212-3p regulates senescence of bone marrow mesenchymal stem cells by targeting MAPK3 Zhong Liying, Li Shundong, Wang Cong 2025, 29 (13):  2690-2697.  doi: 10.12307/2025.039 Abstract ( 92 )   PDF (1931KB) ( 161 )   Save BACKGROUND: Bone marrow mesenchymal stem cells in patients with osteoporosis show significant senescence and decreased activity and osteogenic differentiation. miR-212-3p inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells. However, its regulation of senescence of bone marrow mesenchymal stem cells and its mechanism remain unclear. OBJECTIVE: To investigate the effect of miR-212-3p on senescence of bone marrow mesenchymal stem cells by targeting mitogen-activated protein kinase 3 (MAPK3) and its mechanism.  METHODS: Rat bone marrow mesenchymal stem cells were isolated and cultured in vitro, and the third generation was collected for the following experiments: (1) Cultured in two groups: The control group was added with complete culture medium, and the model group was added with complete culture medium containing H2O2. After 72 hours of culture, β-galactosidase activity, miR-212-3p and MAPK3 mRNA expression, as well as MAPK3, p16, and p21 protein expression were detected. (2) Cultured in three groups: control group, inhibitor control group, and miR-212-3p inhibitor group. After transfection for 24 hours, miR-212-3p, mRNA and protein expression of MAPK3 were detected. (3) Dual luciferase reporter gene combined with qRT-PCR and western blot assay were used to verify the targeting regulation of miR-212-3p and MAPK3. (4) Cultured in different groups: control inhibitor group, miR-212-3p inhibitor group, miR-212-3p inhibitor+interference control group, and miR-212-3p inhibitor+MAPK3 interference group. After transfection for 24 hours, MAPK protein and mRNA expression levels in cells were detected. They were divided into control group, H2O2 group, H2O2+ control inhibitor group, H2O2+miR-212-3p inhibitor group, H2O2+miR-212-3p inhibitor+interference control group, and H2O2+miR-212-3p inhibitor+MAPK3 interference group. Cells were transfected for 24 hours and then cultured with H2O2 for 72 hours. Aging-related β-galactosidase activity and p16 and p21 protein expression were detected. RESULTS AND CONCLUSION: (1) Compared with the control group, β-galactosidase activity, miR-212-3p mRNA expression and p16, p21 protein expression were increased in the model group (P < 0.05), while MAPK3 mRNA and protein expression levels were decreased (P < 0.05). (2) Compared with the control group, the mRNA expression of miR-212-3p was decreased (P < 0.05), and the mRNA and protein expression levels of MAPK3 were increased (P < 0.05) in miR-212-3p inhibitor group. (3) Double luciferase reporter gene experiment confirmed that MAPK3 was the downstream target gene of miR-212-3p. (4) Compared with the control inhibitor group, the mRNA and protein expression levels of MAPK3 were increased in miR-212-3p inhibitor group (P < 0.05). Compared with the miR-212-3p inhibitor group, the mRNA and protein expression levels of MAPK3 in the miR-212-3p inhibitor+MAPK3 interference group were decreased (P < 0.05). Compared with H2O2+control inhibitor group, β-galactosidase activity in H2O2+miR-212-3p inhibitor group was decreased (P < 0.05). Compared with H2O2+miR-212-3p inhibitor group, β-galactosidase activity in H2O2+miR-212-3p inhibitor+MAPK3 interference group was higher than that in H2O2+miR-212-3p inhibitor group (P < 0.05). Compared with the H2O2+control inhibitor group, the protein expression levels of p16 and p21 in the H2O2+miR-212-3p inhibitor group were decreased (P < 0.05). Compared with H2O2+miR-212-3p inhibitor group, the protein expression levels of p16 and p21 in H2O2+miR-212-3p inhibitor+MAPK3 interference group were increased (P < 0.05). (5) To conclude, downregulation of miR-212-3p inhibits the senescence of rat bone marrow mesenchymal stem cells, and its mechanism of action may be achieved by targeting up-regulation of MAPK3 expression. Figures and Tables | References | Related Articles | Metrics

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Effect of rotating cell culture system on proliferation and stemness maintenance of epidermal stem cells in the elderly Zhang Min, Dong Xixi, Huang Meng, Wang Chaoxiang, Zhang Luyue, Cao Junkai 2025, 29 (13):  2698-2705.  doi: 10.12307/2025.079 Abstract ( 104 )   PDF (2130KB) ( 110 )   Save BACKGROUND: Due to the aging population and the increase in elderly diseases, research and utilization of epidermal stem cells in the elderly have become increasingly important. However, due to the weak proliferation and drying ability of epidermal stem cells in the elderly, their application in biomedical and clinical research is limited. OBJECTIVE: To investigate the ability of rotating cell culture system to promote the proliferation and maintenance of epidermal stem cells in elderly individuals.  METHODS: Epidermal stem cells were obtained from children (7-12 years old) and the elderly (over 60 years old) using traditional planar culture methods. After identification by cell morphology and cell immunofluorescence, the differences in proliferation ability of epidermal stem cells between the children and the elderly were analyzed by flow cytometry, CCK-8 assay, and cell clone formation assay. Elderly epidermal stem cells were cultured using a rotating cell culture system and 3D TableTrix® microcarriers. The cell proliferation and expression of stemness markers were detected by Live/Dead staining, cell doubling efficiency, cell doubling time, and real-time quantitative polymerase chain reaction. RESULTS AND CONCLUSION: (1) Under planar culture conditions, the children group had stronger proliferative ability than the elderly group. (2) Compared with planar culture, the dynamic cultivation of elderly epidermal stem cells using a rotating cell culture system had a higher population doubling (P < 0.01) and a shorter population doubling time (P < 0.01). (3) The dynamic cultivation of rotating cell culture system promoted the self-assembly of elderly epidermal stem cells to form 3D human epidermal stem cell spheres based on microcarrier scaffolds, which had similar epidermal stem cell proliferation as the children group. (4) After dynamic culture, the expression of stemness marker genes (ITGA6 and ITGB1), basal cell marker genes (K5) and differentiation marker genes (K10 and K1) of elderly epidermal stem cells reached the same level as that of children. The results showed that the dynamic cultivation of rotating cell culture system can not only improve the efficiency of elderly epidermal stem cell culture, promote cell proliferation and self-assembly into 3D cell spheres, but also maintain a certain stemness of elderly epidermal stem cells without complete terminal differentiation. Figures and Tables | References | Related Articles | Metrics

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Mechanism of human umbilical cord mesenchymal stem cell-derived exosomes against mouse renal ischemia/reperfusion injury Li Lingyu, Wei Huafeng, Luo Hao, Wang Hao, He Jiahui, Yao Yawei, Lyu Xinghua 2025, 29 (13):  2706-2712.  doi: 10.12307/2025.048 Abstract ( 138 )   PDF (1940KB) ( 189 )   Save BACKGROUND: Human umbilical cord mesenchymal stem cell-derived exosomes are involved in multiple injury repair processes, and the effects and the specific mechanisms of renal ischemia/reperfusion injury have not been fully elucidated. OBJECTIVE: To investigate the molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes in the treatment of renal ischemia/reperfusion injury. METHODS: (1) Human umbilical cord mesenchymal stem cells were cultured and exosomes were obtained and identified using an exosome extraction kit. (2) The distribution of exosomes in the kidney of mice with renal ischemia/reperfusion injury was examined by intravital fluorescence imaging. (3) Thirty C57/BL6 male mice were divided into five groups according to the random number table method: sham operation group, renal ischemia/reperfusion group, sham operation group+Compound C group, renal ischemia/reperfusion+exosome group (exosome group), and renal ischemia/reperfusion+exosome+Compound C group (exosome+Compound C group), with 6 mice in each group. Except the sham operation group, bilateral renal pedicles were clamped for 45 minutes and a mouse model of renal ischemia/reperfusion injury was established after 24 hours of reperfusion. In sham operation+Compound C group and exosome+Compound C group, AMPK inhibitor Compound C was intraperitoneally injected 30 minutes before model establishment. In the exosome group and exosome+Copmpound C group, exosomes were injected through the tail vein 15 minutes before renal pedicle clipping. The levels of serum creatinine and urea nitrogen, interleukin 6, and tumor necrosis factor α in renal tissue, and the expression of apoptosis-related factors in renal tissue were detected after 24 hours of reperfusion in each group.  RESULTS AND CONCLUSION: (1) Human umbilical cord mesenchymal stem cell exosomes had the typical tea tray morphology, with the diameter distribution in the range of 40-160 nm, and expressed the specific marker membrane protein of exosome surface. (2) Murine kidneys after renal ischemia/reperfusion injury were more likely to gather human umbilical cord mesenchymal stem cell exosomes compared with the sham operation group. (3) Exosome pretreatment reduced renal injury and the level of renal cell apoptosis in mice treated with renal ischemia/reperfusion injury. Moreover, this protective effect could be reversed by AMPK inhibitors. These findings verify that human umbilical cord mesenchymal stem cell-derived exosomes exerting a protective effect on renal ischemia/reperfusion injury may be related to the activation of the AMPK/YAP1 pathway to antiapoptosis. Figures and Tables | References | Related Articles | Metrics

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Beta-hydroxybutyric acid improves energy dysfunction of mouse hippocampal neuron HT22 cells induced by amyloid-β protein 1-42 Ye Yucai, Fu Chaojing, Li Yan, Li Xinru, Chai Shifan, Cai Hongyan, Wang Zhaojun 2025, 29 (13):  2713-2719.  doi: 10.12307/2025.074 Abstract ( 88 )   PDF (1321KB) ( 279 )   Save BACKGROUND: Patients with Alzheimer’s disease have severe brain energy disorders. In recent years, brain energy rescue strategies based on ketone body intervention have attracted more and more attention in the treatment of Alzheimer’s disease. OBJECTIVE: To investigate whether β-hydroxybutyric acid can improve energy dysfunction by improving mitochondrial bioenergy function in HT22 cells of mouse hippocampal neurons induced by amyloid-β protein 1-42 (Aβ1-42).  METHODS: HT22 cells were divided into four groups: Control, β-hydroxybutyric acid, Aβ1-42, Aβ1-42+ β-hydroxybutyric acid. Related detection kits were respectively used to detect HT22 cell survival rate, adenosine triphosphate level, α-ketoglutarate dehydrogenase activity, Na+K+-ATPase activity, mitochondrial membrane potential, and reactive oxygen species levels.  RESULTS AND CONCLUSION: Compared with the control group, the survival rate, adenosine triphosphate level, α-ketoglutarate dehydrogenase activity, Na+K+-ATPase activity, and mitochondrial membrane potential of HT22 cells were significantly decreased (P < 0.05), and the level of reactive oxygen species was significantly increased (P < 0.05) in the Aβ1-42 group. Compared with the Aβ1-42 group, the survival rate, adenosine triphosphate level, α-ketoglutarate dehydrogenase activity, Na+K+-ATPase activity, and mitochondrial membrane potential of HT22 cells were significantly increased (P < 0.05), and the reactive oxygen species level was significantly decreased (P < 0.05) in the Aβ1-42+β-hydroxybutyric acid group. These results showed that β-hydroxybutyric acid improved mitochondrial bioenergetic function and ultimately improved Aβ1-42-induced energy impairment and survival rate in HT22 cells.  Figures and Tables | References | Related Articles | Metrics

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Lineage tracing for mammary stem cells using Hopx reporter mice Shi Jianyun, Li Wenjing, Peng Ying, Jia Zhenhua, Zhang Shujin, Tan Lulu, Yuan Yitong, Du Ruochen 2025, 29 (13):  2720-2727.  doi: 10.12307/2025.088 Abstract ( 63 )   PDF (1665KB) ( 216 )   Save BACKGROUND: Mammary stem cells are vital for the development and homeostasis of mammary gland tissue. The occurrence of breast cancer has a close relationship with the mammary stem cells. Recent studies have shown that Hopx, as an important transcriptional regulator of morphogenesis and cell differentiation, has been confirmed to be expressed in a variety of adult stem cells such as nerves, intestines, hair follicles and lungs. However, its role in mammary stem cells has not been reported so far.   OBJECTIVE: To investigate whether Hopx expression marks mammary stem cells.  METHODS: (1) Female Hopx-LacZ transgenic mice aged 8 weeks were selected to detect the background expression of Hopx in breast tissue by β-galactosidase staining. (2) Female wild-type mice at 4, 6, and 8 weeks of age and 14.5 days of gestation were selected for whole-tissue magenta staining and K14 and K8 immunofluorescence staining, respectively. (3) Female Hopx-CreERT2; Rosa26LacZ transgenic mice aged 8 weeks and 17.5 days of gestation were selected and stained with breast β-galactosidase. (4) The 4-week-old female Hopx-CreERT2;Rosa26LacZ transgenic mice were selected. The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day, three times), and breast β-galactosidase staining was performed 4 weeks after injection. The 8-week-old female Hopx-CreERT2;Rosa26LacZ transgenic mice were selected. The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day, three times), and breast β-galactosidase staining was performed 4 and 10 weeks after the last injection. (5) Female Hopx-CreERT2; Rosa26LacZ transgenic mice aged 8 weeks were selected. The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day, three times). Hopx-CreERT2;Rosa26LacZ transgenic mice were pregnant 2 weeks after injection. The mammary tissue of mice at 17.5 days of the first pregnancy and 17.5 days of the third pregnancy was stained with β-galactosidase.  RESULTS AND CONCLUSION: (1) The results of β-galactosidase staining showed that the mammary ducts of Hopx-LacZ transgenic mice at 8 weeks of age did contain Hopx-positive cells and were located in the basal epithelia, with a small number. (2) Whole-mount staining of mammary glands and immunofluorescence staining results exhibited that the mammary glands of mice had different characteristics with corresponding developmental stages such as puberty, maturity, and pregnancy, and underwent a series of complex epithelial remodeling processes. (3) The results of β-galactosylase staining showed that Hopx-labeled positive cells in the mammary duct of Hopx-CreERT2; Rosa26LacZ transgenic mice at 17.5 days of gestation increased compared with female Hopx-CreERT2; Rosa26LacZ transgenic mice at 8 weeks of age. (4) The results of β-galactosylase staining showed that the Hopx-labeled positive cells in the mammary glands of 4- and 8-week-old female Hopx-CreERT2; Rosa26LacZ transgenic mice after tamoxifen injection were located in the basal epithelium with a small number. (5) The results of β-galactosidase staining showed that Hopx-labeled positive cells in the mammary glands of mice at 17.5 days of the first and third gestation were located in the basal epithelia around the alveoli, and the number of Hopx-labeled positive cells at 17.5 days of the third gestation was more. (6) In conclusion, Hopx reporter-marked basal epithelial cells belong to dormant mammary stem cells, which are responsible for the growth of the mammary glands during pregnancy and contribute to acinar formation. Figures and Tables | References | Related Articles | Metrics

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Isolation and culture of adult mouse cerebrospinal fluid-contacting neurons in vitro and characterization of self-renewal capacity Shangguan Zeyu, , Chen Chanjuan, Li Qizhe, Tan Wei, Yan Haijian, Wang Chunqing, Dou Xiaowei, Li Qing 2025, 29 (13):  2728-2735.  doi: 10.12307/2025.028 Abstract ( 82 )   PDF (1496KB) ( 83 )   Save BACKGROUND: We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro, but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons. There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.  OBJECTIVE: To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro, and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.   METHODS: Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes. High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium. The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons, and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed. An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5% CO2, 37°C thermostat, respectively. The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation, CCK8 assay, qPCR, and western blot assay. RESULTS AND CONCLUSION: (1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse, which expressed Nestin and SOX2 in vitro, and were able to form neurospheres and pass on continuously. (2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse, and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter, whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P < 0.000 1). (3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P < 0.000 1). (4) qPCR and western blot assay revealed that the mRNA (P < 0.000 1) and protein expression levels (P < 0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse. (5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.  Figures and Tables | References | Related Articles | Metrics

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Lycium barbarum polysaccharide regulates mitochondrial dynamics to improve H2O2-induced apoptosis of SH-SY5Y cells Wang Jiwei, Li Yanbing, Guo Minfang, Meng Tao, Yu Jingwen, Liu Xiaoqin, Mu Bingtao, Jia Siwei, Ma Cungen, Yu Jiezhong 2025, 29 (13):  2736-2743.  doi: 10.12307/2025.049 Abstract ( 116 )   PDF (2571KB) ( 110 )   Save BACKGROUND: A large number of studies have shown that neurodegenerative diseases are closely related to oxidative stress injury and the imbalance of mitochondrial dynamics. Lycium barbarum polysaccharides have a neuroprotective effect. However, it is not clear whether lycium barbarum polysaccharides can ameliorate apoptosis induced by oxidative stress injury by regulating abnormal mitochondrial dynamics. OBJECTIVE: To study the effect of lycium barbarum polysaccharides on apoptosis induced by H2O2 in SH-SY5Y human neuroblastoma cells.  METHODS: SH-SY5Y cells were cultured in three groups. The control group was cultured for 24 hours. The hydrogen peroxide group was treated with H2O2 for 24 hours, and the lycium barbarum polysaccharide group was treated with lycium barbarum polysaccharide for 2 hours and then treated with H2O2 for 24 hours. After treatment, the levels of malondialdehyde, glutathione, and superoxide dismutase in the precipitation of the cells were detected by kit. Mitochondrial membrane potential was detected by JC-1 kit. Cell viability was detected by MTT assay. Apoptosis was detected by TUNEL. The expression levels of mitochondrial dynamics-related proteins (phosphorylated promoter protein 1, mitochondrial fission protein 1, mitochondrial fusion protein 1, mitochondrial fusion protein 2, and optic atrophy protein 1) and apoptotic proteins (Bax, Bcl-2, and Caspase-3) were detected by immunofluorescence staining and western blot assay. RESULTS AND CONCLUSION: (1) Compared with the control group, the levels of malondialdehyde were increased (P < 0.05), and the levels of superoxide dismutase and glutathione were decreased (P < 0.05) in the H2O2 group. Compared with the H2O2 group, the malondialdehyde level was decreased (P < 0.05), and the superoxide dismutase and glutathione levels were increased (P < 0.05) in the lycium barbarum polysaccharide group. (2) The mitochondrial membrane potential in the H2O2 group was lower than that in the control group (P < 0.05), and that of lycium barbarum polysaccharide group was higher than that of the H2O2 group (P < 0.05). (3) Compared with the control group, the apoptosis rate and the expression of Bax and Caspase-3 protein were increased (P < 0.05), while the cell viability and the expression of Bcl-2 protein were decreased (P < 0.05) in the H2O2 group. Compared with the H2O2 group, the apoptosis rate and the expression of Bax and Caspase-3 protein were decreased (P < 0.05), while the cell viability and the expression of Bcl-2 protein were increased (P < 0.05) in the lycium barbarum polysaccharide group. (4) Compared with the control group, the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were increased (P < 0.05), and the protein expression levels of mitochondrial fusion protein 1, mitochondrial fusion protein 2, and optic atrophy protein 1 were decreased (P < 0.05) in the H2O2 group. Compared with the H2O2 group, the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were decreased (P < 0.05), and the protein expression levels of mitochondrial fusion protein 1, mitochondrial fusion protein 2, and optic atrophy protein 1 were increased (P < 0.05) in the lycium barbarum polysaccharide group. (5) These results indicate that lycium barbarum polysaccharide can improve SH-SY5Y cell apoptosis caused by oxidative stress damage by regulating mitochondrial dynamics. Figures and Tables | References | Related Articles | Metrics

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Effects of periodontal ligament stem cells-derived exosomes on biological characteristics of periodontal ligament stem cells in an inflammatory environment Jiang Zhiliang, Luo Yaxin, Hu Zhengqi, Yang Li, Yang Chanchan, Chen Hong, Liu Xiaoyi, Huang Yan, Yang Kun 2025, 29 (13):  2744-2752.  doi: 10.12307/2025.038 Abstract ( 72 )   PDF (2861KB) ( 72 )   Save BACKGROUND: In recent years, the application of exosomes of periodontal ligament stem cells in periodontal tissue regeneration engineering has been widely studied, but the effect of exosomes on periodontal ligament stem cells derived from inflammatory environment is still unclear.  OBJECTIVE: To investigate the effects of exosomes secreted by periodontal ligament stem cells from healthy and inflammatory environments on the proliferation and differentiation of periodontal ligament stem cells from inflammatory environments.  METHODS: Human periodontal ligament stem cells from healthy and inflammatory tissues were isolated and cultured by enzyme digestion method. Exosomes were extracted from two kinds of periodontal ligament stem cells using ultracentrifugation. Passage 3 periodontal ligament stem cells derived from inflammatory tissue were selected and cultured in three groups. Cells in the blank group were cultured routinely. The healthy exosome group was added with exosomes secreted by peripheral ligament stem cells derived from healthy tissue. The inflammatory exosome group was added with exosomes secreted by human periodontal ligament stem cells derived from inflammatory tissue. Cell proliferation and cloning were detected. The expression of alkaline phosphatase, the formation of mineralized nodules, and the expression of mRNA and protein of genes related to osteogenesis were detected under osteogenic differentiation.  RESULTS AND CONCLUSION: (1) CCK-8 assay and clonal formation test showed that compared with the blank group, two kinds of exosomes could promote the proliferation and colony formation of periodontal ligament stem cells from inflammatory tissue (P < 0.05), and the effect of the healthy exosome group was stronger than that of the inflammatory exosome group (P < 0.05). (2) Alkaline phosphatase and alizarin red staining showed that compared with the blank group, the two kinds of exosomes could promote the expression of alkaline phosphatase and the formation of mineralized nodules in periodontal ligament stem cells from inflammatory tissue, and the promoting effect of the healthy exosome group was stronger than that of the inflammatory exosome group. RT-PCR and western blot assay showed that compared with the blank group, the two kinds of exosomes could promote the expression of alkaline phosphatase, RUNX2, and type I collagen mRNA and protein in periodontal ligament stem cells from inflammatory tissue (P < 0.05). The promoting effect of the healthy exosome group was stronger than that of the inflammatory exosome group (P < 0.05). (3) The results showed that exosomes secreted by human periodontal ligament stem cells could promote the proliferation and osteogenic differentiation of periodontal ligament stem cells derived from inflammatory environments, and the promoting effect of exosomes secreted by human periodontal ligament stem cells derived from healthy tissues was better than that from human periodontal ligament stem cells derived from inflammatory tissues. Figures and Tables | References | Related Articles | Metrics

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Preclinical study of platelet-rich plasma combined with adipose stem cell transplantation in accelerating wound healing: a systematic evaluation and meta-analysis Lin Li, Jiao Linxi, Yu Fangning, Ma Yichao, Zhang Bo, Xu Xuying 2025, 29 (13):  2753-2763.  doi: 10.12307/2025.041 Abstract ( 91 )   PDF (2192KB) ( 75 )   Save OBJECTIVE: Researches show that a combination of platelet-rich plasma and adipose-derived stem cells can accelerate the healing of skin lesions. However, systematic evidence for the combination of the two is still lacking. The purpose of this study was to assess the efficacy of a combination of two interventions in a clinical rodent skin wound model.  METHODS: We searched PubMed, Embase, Cochrane, and CNKI and selected the studies of platelet-rich plasma, adipose-derived stem cell transplantation, or their combination on skin wounds in experimental animals published until July 2023. Wound healing and wound transformation growth factor β, CD31, type I collagen, and vascular endothelial growth factor were used as indicators. RevMan 5.3 and Stata 15.0 were used to analyze the data.  RESULTS: A total of 12 studies were included, of which 8 studies used rats as experimental subjects and 4 studies used mice as experimental subjects. The experimental group was treated with platelet-rich plasma combined with adipose-stem cell transplantation, and the control group was treated with platelet-rich plasma alone. The results of meta-analysis showed that the wound healing rate of the experimental group at 3, 7, and 10 days after treatment was greater than that of the control group [SMD=2.65, 95%CI(1.29, 4.01), Z=3.81, P=0.000 1; SMD=3.38, 95%CI(2.47, 4.30), Z=7.24, P < 0.000 01; SMD=2.62, 95%CI(1.50, 3.73), Z=4.61, P < 0.000 01]. The wound healing time of the experimental group was shorter than that of the control group [SMD=-2.12, 95%CI(-3.5,-0.74), P=0.003]. The expression of transforming growth factor β, positive rate of CD31, expression of type I collagen, and vascular endothelial growth factor in wound of experimental group were higher than those of control group [SMD=5.65, 95%CI(1.22, 10.08), Z=2.50, P=0.01; SMD=2.49, 95%CI(1.96, 3.02), Z=9.28, P < 0.000 01; SMD=3.44, 95%CI(0.72, 6.17), Z=2.48, P=0.01; SMD=2.38, 95%CI(0.97, 3.79), Z=3.30, P=0.001 0].  CONCLUSION: Our results show that platelet-rich plasma + adipose-derived stem cells combined treatment can improve the wound healing rate, shorten the wound healing time, and at the same time increase the expression of transforming growth factor β, CD31, type I collagen, and vascular endothelial growth factor to accelerate healing. Due to the limitations of the model, more animal testing and clinical trials are needed. Figures and Tables | References | Related Articles | Metrics

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Mechanism of different sources of mesenchymal stem cells in treatment of premature ovarian failure Liu Yanyan, Ma Yuanyuan, Huang Xianghua, Zhang Jingkun 2025, 29 (13):  2764-2773.  doi: 10.12307/2025.017 Abstract ( 216 )   PDF (1338KB) ( 264 )   Save BACKGROUND: A large number of cell and animal tests have confirmed the effect of mesenchymal stem cells on improving ovarian function, and some clinical trials have been completed and their effectiveness has been preliminarily confirmed, bringing hope to women with premature ovarian failure. OBJECTIVE: To summarize and analyze the mechanism, research progress, and related clinical trials of mesenchymal stem cells from different sources in the treatment of premature ovarian failure in recent years, so as to provide a theoretical basis for further research and clinical application of mesenchymal stem cell therapy for premature ovarian failure. METHODS: Using “mesenchymal stem cells, premature ovarian failure” as keywords in Chinese and English, the relevant literature was searched in CNKI, WanFang Data, Chinese Medical Database, and PubMed database, and finally 72 articles that met the requirements were included for review. RESULTS AND CONCLUSION: Currently, there are seven types of commonly used mesenchymal stem cells for premature ovarian failure, which are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, menstrual blood mesenchymal stem cells, amniotic mesenchymal stem cells, amniotic fluid mesenchymal stem cells, and adipose mesenchymal stem cells. The mechanisms include inhibiting apoptosis and promoting proliferation, anti-inflammatory and inhibiting oxidative stress, homing, promoting angiogenesis, anti-fibrosis, parasecretory, immune regulation, autophagy, and improving microenvironment. Cell and animal experiments have proven that different sources of mesenchymal stem cells can have better intervention effect on premature ovarian failure through various mechanisms, and delay the progress of premature ovarian failure to a certain extent. If it can be successfully applied to the clinic in the future, it can alleviate the psychological and physical pain of patients to a great extent. However, due to the lack of comprehensive and accurate evidence in clinical studies such as stem cell source, administration mode and dose, adverse reactions, etc., further studies are still needed to confirm it in the future, and its long-term safety needs to be further observed. Figures and Tables | References | Related Articles | Metrics

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Construction strategy for vascularization of organoids Liu Mingyu, Fan Wenjuan 2025, 29 (13):  2774-2783.  doi: 10.12307/2025.052 Abstract ( 233 )   PDF (1161KB) ( 166 )   Save BACKGROUND: The effective promotion of internal angiogenesis in organoids is a current focal issue in organoid culture. Vascularized organoids, as a newly developed bioculture technology, have significant research and application value in the study of living tissue development, disease formation mechanisms, tissue replacement therapy, and drug screening. OBJECTIVE: To summarize the methods or strategies for vascularization of organoids in recent years, analyze the formation mechanism of vascularization of organoids and the construction strategies, with a view to providing reliable ideas for more in-depth study of the mechanism of organoid genesis and for clinical translation. METHODS: The authors utilized the PubMed and CNKI databases for related formation collection. The keywords were “organoids, vascularization, vascular, vascular development, vessel” in Chinese and English. Finally, 77 papers were included for summarization. RESULTS AND CONCLUSION: (1) The mechanism of vascularization formation in organoid organs involves three key factors, namely seed cells, cytokines, and extracellular matrix. Seed cells provide the essential cell source for vascularized organoids; cytokines play an important role in guiding angiogenesis within organoids, and the extracellular matrix provides an external growth environment for vascular cells, promoting the occurrence of vascularized organoids. (2) The construction strategies of vascularized organoids include cell self-reorganization, microvascular fragment infiltration, transplantation into host, and microfluidic chip. In vitro induction of pluripotent stem cells to differentiate into endothelial progenitor cells can integrate with adjacent tissues and have the potential for angiogenesis, so pluripotent stem cells can be used to construct vascularized organoids by self-reorganization. Microvascular fragments retain their cellular complexity, natural structure, and phenotypic plasticity, which is more conducive to simulating natural microvessels and promoting vascularization of organoids. Transplantation into host is currently the best method to achieve complete blood perfusion in organoids, while microfluidic chip provides a solution for achieving extracorporeal blood supply in organoids. (3) Multiple construction strategies of organoid such as co-differentiation of multiple stem cell types, precise regulation of signaling molecules, microvascular infiltration, and in vivo host transplantation, have introduced vascular components into organoids to some extent, making them closer to the corresponding tissues in terms of function and maturity. However, the challenge of achieving perfusion remains, and so far, only in vivo transplantation in hosts has enabled effective perfusion in organoids. Therefore, organoids still face numerous challenges in terms of vascularization. Figures and Tables | References | Related Articles | Metrics

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Role and clinical application progress of exosome-derived non-coding RNA in microenvironment of osteoarthritis Li Zhichao, Yang Zhenguo, Wang Lei, Wang Wenbo, Xue Jingcai, Liu Wenbin, Cao Hui 2025, 29 (13):  2784-2792.  doi: 10.12307/2025.033 Abstract ( 118 )   PDF (2183KB) ( 154 )   Save BACKGROUND: Osteoarthritis is a common degenerative joint disease, and the etiology and development of its pathogenesis are still unclear. Timely diagnosis and treatment of early osteoarthritis are crucial, and there is currently no definite and effective method. Extracellular vesicles come from a wide range of sources, including non-coding RNAs such as small RNAs, circular RNAs, and long chain non-coding RNAs. Extracellular vesicles non-coding RNAs can be directly delivered from primitive cells to neighboring or remote cells, regulating cell activity through intercellular communication and playing an important regulatory role in reshaping the bone and joint microenvironment.  OBJECTIVE: To summarize the intervention effects of exosome-derived non-coding RNAs on the joint microenvironment of osteoarthritis and the progress made in clinical application, and to clarify the potential of exosome-derived non-coding RNAs in the diagnosis and treatment of osteoarthritis.  METHODS: Search terms “exosomes, non-coding RNA, osteoarthritis, application, signal pathway, synovial fluid, cartilage cells, cartilage matrix, subchondral, mechanism” were used for the search on PubMed database. Finally, 66 related articles were included for review analysis. RESULTS AND CONCLUSION: (1) Exosome-derived non-coding RNAs play an important regulatory role in the joint microenvironment during the pathogenesis of osteoarthritis, mainly reflected in: exosome non-coding RNAs regulating the inflammatory response in the joint, degeneration of chondrocytes and cartilage matrix, subchondral bone remodeling, and intercellular communication. (2) The non-coding RNAs in exosomes can serve as biomarkers for osteoarthritis, aiding in the early diagnosis and monitoring of disease progression and prognosis. (3) Exosome non-coding RNAs serve as therapeutic targets for osteoarthritis.  Exosomes carry miRNAs to the articular chondrocytes and cartilage matrix to play a regulatory role. (4) Exosomes non-coding RNAs can improve the effect of cartilage tissue engineering by regulating gene expression and promoting intercellular communication to repair or regenerate damaged cartilage. (5) In future research, researchers should continue to explore the intervention mechanism of non-coding RNAs derived from exosomes on osteoarthritis, and apply them to clinical practice in combination with the latest research outcomes in cartilage tissue engineering, which will effectively help solve the pain of osteoarthritis patients.  Figures and Tables | References | Related Articles | Metrics

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Cellular and molecular mechanisms of platelet-rich plasma in promoting wound healing Long Chenyan, Cheng Biao, Tian Ju 2025, 29 (13):  2793-2801.  doi: 10.12307/2025.058 Abstract ( 172 )   PDF (1080KB) ( 123 )   Save BACKGROUND: Although platelet-rich plasma has shown potential in promoting wound healing, further research and validation are still needed for its clinical application. Additionally, there are still some controversies and uncertainties regarding the action mechanisms of platelet-rich plasma. OBJECTIVE: To provide an overview and analysis of the mechanisms of platelet-rich plasma in promoting wound healing, in order to enhance understanding of this treatment method and provide useful guidance for future basic research and clinical applications. METHODS: Relevant literature published from January 2000 to December 2023 was retrieved from the China National Knowledge Infrastructure (CNKI) and PubMed databases. The English and Chinese search terms included “platelet rich plasma, PRP, wound, wound healing, refractory wounds, chronic wounds, cellular and molecular mechanisms, signaling pathways, regenerative medicine.” A total of 80 articles were reviewed based on inclusion and exclusion criteria. RESULTS AND CONCLUSION: (1) Platelet-rich plasma has many potential advantages in wound healing as it can provide abundant bioactive substances such as growth factors and cytokines. (2) Platelet-rich plasma regulates the function of various cell types involved in wound healing, including epidermal cells, vascular endothelial cells, fibroblasts, stem cells, and inflammatory cells. (3) In platelet-rich plasma therapy, extracellular vesicles and mitochondria may play important roles in wound healing. (4) Platelet-rich plasma hydrogels can be widely used to enhance tissue regeneration and serve as functional carriers for drug delivery. (5) Platelet-rich plasma regulates the synthesis and degradation of the extracellular matrix, affecting matrix remodeling during the wound healing process. (6) Platelet-rich plasma may influence tissue repair and regeneration by dynamically regulating multiple signaling pathways and the epithelial-mesenchymal transition, providing a new perspective for research in this field. Figures and Tables | References | Related Articles | Metrics

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Platelet-rich plasma intervenes in chondrocyte autophagy and apoptosis for treatment of osteoarthritis Wang Yaomin, Zhang Kefan, Wang Dening, Ren Qiang, Li Jian, Shi Hui 2025, 29 (13):  2802-2811.  doi: 10.12307/2025.099 Abstract ( 138 )   PDF (1171KB) ( 178 )   Save BACKGROUND: In the process of intervening in the development of osteoarthritis, platelet-rich plasma plays an important role by intervening in autophagy, apoptotic cytokines and signal transduction pathways.  OBJECTIVE: To summarize the structure of cytokines and signaling pathways involved in the diagnosis of osteoarthritis by platelet-rich plasma in recent years, as well as its correlation with chondrocyte apoptosis and autophagy, in order to provide effective targets for the future treatment of osteoarthritis.  METHODS: Literature search was conducted in CNKI, WanFang Data, VIP, PubMed, Web of Science, and Medline databases using “platelet-rich plasma, chondrocyte, apoptosis, autophagy, osteoarthritis, cytokines, signaling pathway” as Chinese and English search terms. A systematic summary and induction were made for the 66 included articles. RESULTS AND CONCLUSION: Current research has shown that platelet-rich plasma can promote cartilage repair and assist bone tissue healing through various pathways, mainly divided into three aspects: (1) Platelet-rich plasma regulates the extension, closure, and maturation of microautophagosomes, promotes chondrocyte megaautophagy and molecular chaperone-mediated cell autophagy under specific conditions, enhances the expression of autophagy-related factors such as LC3II/I and Beclin1, inhibits the expression of P62/SQSTM1. Currently, there is no clear research directly exploring the specific effect of platelet-rich plasma on heat shock proteins, and further research is needed in this field in the future. (2) The various growth factors released by platelet-rich plasma inhibit the expression of pro-apoptotic factors Caspase, interleukin-1β, and tumor necrosis factor alpha, promote the expression of anti-apoptotic factor Bcl-2, and prevent chondrocyte apoptosis and degeneration. (3) By activating the PI3K/AKT/mTOR signaling pathway, NF-κB signal transduction pathway, death receptor pathway, mitochondrial stress pathway and other pathways, platelet-rich plasma inhibits the expression of Bax and Caspase, and prevents the release of cytochrome c, thereby inhibiting the death and necrotic apoptosis of chondrocytes. In general, platelet-rich plasma promotes cartilage repair, supports cartilage regeneration, and plays an anti-inflammatory role, and its biological effect in chondrocytes usually depends on the regulation of autophagy and apoptosis-related cytokines and signaling pathways. Figures and Tables | References | Related Articles | Metrics

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Applications and advances of single-cell transcriptome sequencing technology in osteoporosis research Sun Chengtao, Sun Guangjiang , Qi Xiaonan , Cheng Ming , Yao Xiaosheng 2025, 29 (13):  2812-2821.  doi: 10.12307/2025.034 Abstract ( 244 )   PDF (1288KB) ( 361 )   Save BACKGROUND: The incidence of fragility fractures caused by osteoporosis is increasing year by year, and it is urgent to explore its pathophysiology, potential biomarkers, therapeutic targets and effective drugs. There are multiple cells in the bone microenvironment, which are crucial for maintaining bone metabolism. In recent years, single-cell RNA sequencing technology has been developed to characterize the transcriptome of single cells, and has been gradually applied to the study of osteoporosis prevention and treatment.  OBJECTIVE: To review the application and progress of single-cell transcriptome sequencing technology in osteoporosis research.  METHODS: The first author used a computer search of Web of Science, PubMed database, and CNKI from 2009 to 2023. Chinese and English search terms were “single-cell RNA sequencing, bone marrow derived stromal cells, osteoblasts, osteocytes, osteoclasts, immune cells, bone marrow vascular endothelial cells.” Through reading and screening of relevant literature, 89 articles were finally included in the review analysis. RESULTS AND CONCLUSION: (1) Single-cell transcriptome sequencing technology can gain an in-depth understanding of the trajectory and regulatory mechanism of cell development, proliferation, differentiation. (2) Candidate genes affecting bone mineral density are mainly concentrated in bone marrow mesenchymal stem cell subpopulation, in which Sox9 is a key transcription factor. (3) The differential expression of Runx2 in different subsets of osteoblasts controls the differentiation of bone marrow mesenchymal stem cells into osteoblasts. (4) RAB38 is related to histone modification and transcriptional regulation during osteoclast differentiation. (5) Studies at the single-cell level have confirmed that there are many kinds of intercellular communication in the bone microenvironment, and osteoclast may conduct intercellular communication through CD160-TNFRSF14 ligand-receptor binding. The neutrocyte/monocyte may interact with osteoblasts through the RESISTIN pathway. Plasma dendritic cells communicate with osteoblast cell lines through the epidermal growth factor pathway. Both can provide directions for target prediction and drug development. (6) An unrecognized capillary subset is called S-type endothelial cells, which originates entirely from the secondary ossification center of the epiphysis, and is even more malleable than H-type blood vessels. (7) This paper reviews the progress of single-cell transcriptome sequencing in characterizing the differentiation fate and differentiation trajectory of different bone tissue cells, identifying new cell subsets, identifying cell regulatory factors, and clarifying intercellular communication from a cellular perspective, so as to provide cell-level information for exploring the pathogenesis of osteoporosis, screening potential diagnostic markers, predicting therapeutic targets, and exploring the mechanism of drug action. (8) In the future, single-cell sequencing technology will provide more valuable information for changes in the bone microenvironment in the direction of single-cell multiomics (genomics, proteomics, and metabolomics) and other technologies such as spatial transcriptome technology.  Figures and Tables | References | Related Articles | Metrics

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Application of single-cell RNA sequencing in spinal cord injury Wang Peigeng, Ye Dongping 2025, 29 (13):  2822-2831.  doi: 10.12307/2025.044 Abstract ( 238 )   PDF (1074KB) ( 360 )   Save BACKGROUND: In recent years, the study of single-cell RNA sequencing technology in spinal cord injury has provided new insights into cellular and molecular heterogeneity as well as structural changes in the central nervous system after trauma. OBJECTIVE: To review the research progress of single-cell RNA sequencing technology in spinal cord injury, comprehensively and deeply expound the application of single-cell RNA sequencing technology in spinal cord injury.  METHODS: A computerized system was used to search the articles published from 2009 to 2023 in PubMed, Web of Science, CNKI, and WanFang databases with the Chinese and English search terms of “single-cell RNA sequencing, spinal cord injury, sequencing technology.” Articles with poor quality, repetitive content, and non-relevance were excluded, and 57 articles were finally included for review and analysis. RESULTS AND CONCLUSION: At present, the research of single-cell RNA sequencing technology in spinal cord injury can be summarized as follows: (1) Cell subsets such as microglia, astrocytes, oligodendrocytes, macrophages, B cells, neurons, and neural stem cells were identified, and specific marker genes of these subsets were identified. (2) Microglia remain permanently active after spinal cord injury and coordinate the early stages after spinal cord injury through proliferation, immunity, and homeostatic function. Astrocytes play many important functions in spinal cord injury in an activated manner, including maintaining microenvironment balance, removing necrotic tissue, forming a protective barrier, and glial scars. Both macrophages and microglia play an important role in chronic neuroinflammation following spinal cord injury. (3) Neural stem cells and neuronal subsets can self-renew after spinal cord injury. Newly discovered neuronal subsets such as SCVsx2:: Hoxa7: Zfhx3 → lumbar and SCVsx2: Hoxa10 can regenerate to natural targets and facilitate the recovery of motor function. (4) The discovery of dynamic changes in cell subsets improves our understanding of the course of spinal cord injury lesions and provides new insights into the treatment of spinal cord injury at different time points. Up to now, more basic research and sufficient clinical experiments are needed to validate the results of single-cell RNA sequencing in these studies. In the future, single-cell RNA sequencing technology is expected to open a new window for the diagnosis and treatment of spinal cord injury by interdisciplinary collaboration with bioinformatics, computer science, tissue engineering, and clinical medicine. Figures and Tables | References | Related Articles | Metrics

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Relationship between macrophage subtypes in obese adipose tissue and metabolic diseases Zhao Yuqing, Wang Wei, You Huijuan, Chen Liyuan, Chen Yan, Wang Qinglu, Yang Fengying 2025, 29 (13):  2832-2841.  doi: 10.12307/2025.506 Abstract ( 135 )   PDF (1411KB) ( 260 )   Save BACKGROUND: Macrophage subtypes exhibit tissue heterogeneity, and the adipose tissue macrophage phenotype is largely influenced by obesity. Local and systemic inflammatory responses caused by obese adipose tissue macrophages are considered a vital pathological mechanism of obesity-associated metabolic diseases. OBJECTIVE: To summarize the inflammatory characteristics of different macrophage subtypes in adipose tissue and their relationship with obesity-associated metabolic diseases, aiming to provide a reference basis for targeting specific macrophage subtypes to explore preventive and treatment strategies for obesity-associated metabolic diseases. METHODS: Literature retrieval was conducted in CNKI and PubMed using Chinese and English search terms “obesity, adipose tissue, adipose tissue macrophage, macrophage polarisation, metabolic diseases.” The search results were accepted or excluded according to the inclusion criteria. Ninety-one papers that met the criteria were finally included for review. RESULTS AND CONCLUSION: (1) Macrophages have tissue heterogeneity. Under normal conditions, adipose tissue macrophages are mainly composed of anti-inflammatory M2 resident macrophages, which maintain tissue inflammation homeostasis. Under obese conditions, a large number of foreign infiltrating macrophages surround hypertrophic adipocytes, and most of them exhibit pro-inflammatory characteristics. Therefore, it is believed that adipose tissue macrophages of pro-inflammatory M1 type may actually be a collection of multiple pro-inflammatory subtypes. Further understanding of the characteristics of various pro-inflammatory subtypes helps us to gain a deeper understanding of the mechanisms underlying inflammatory disorders in obese adipose tissue. (2) In obesity, foreign infiltrating macrophages form crown-like structures around hypertrophic adipocytes. Currently, six different subtypes of the crown-like structure have been identified, most of which exhibit pro-inflammatory properties and a few of which possess anti-inflammatory characteristics. Thus, taking full advantage of the anti-inflammatory subtypes while inhibiting the differentiation of the pro-inflammatory subtypes may be a new target for alleviating inflammatory damage in obese adipose tissue. (3) M3, MMe, CD9+ and LAM adipose tissue macrophage subtypes have been found to be involved in the occurrence and development of metabolic diseases such as atherosclerosis, diabetes, insulin resistance, and cancer. DARC+ and MFehi adipose tissue macrophage subtypes play a vital role in the treatment of non-alcoholic fatty liver disease, obesity insulin resistance, iron death, and other related metabolic diseases. The above studies further suggest that inflammatory disorders caused by externally infiltrated macrophages in obese adipose tissue are an important pathological basis for obesity-induced metabolic diseases. Further in-depth research on the characteristics of various subtypes has important theoretical and practical significance. Figures and Tables | References | Related Articles | Metrics

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Potential of mitochondrial transplantation in treatment of sarcopenia Li Wei, Yin Hongtao, Sun Yongchen, Xu Weijuan, Sun Jinling, Jin Xiaodong 2025, 29 (13):  2842-2848.  doi: 10.12307/2025.053 Abstract ( 156 )   PDF (975KB) ( 138 )   Save BACKGROUND: Sarcopenia is a comprehensive condition of aging induced decline in skeletal muscle mass and strength and represents a major health challenge for the elderly. Accumulating evidence suggests that mitochondrial dysfunction plays a key role in the pathogenesis of sarcopenia. OBJECTIVE: To summarize the mechanisms by which dysregulation of mitochondrial quality control leads to sarcopenia and to explore whether mitochondrial transplantation may be a potential target for the treatment of sarcopenia. METHODS: We searched PubMed and CNKI databases for relevant articles published from 2009 to 2023 using the keywords “sarcopenia, mitochondrial dysfunction, mitochondrial quality control, mitochondrial transplantation, limitations.” RESULTS AND CONCLUSION: Given the key role of mitochondrial dysfunction in the pathogenesis of sarcopenia, mitochondrial transplantation may serve as a possible strategy for the treatment of sarcopenia by improving mitochondrial bioenergetics and modulating mitochondria related signaling pathways. Although some preclinical and clinical studies have confirmed the potential of mitochondrial transplantation for the treatment of various diseases, there are still some urgent questions regarding the specific details of mitochondrial transfer. Figures and Tables | References | Related Articles | Metrics

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Effect of NLRP3 inflammasome in microglia after spinal cord injury Zhou Wenyang, Liao Yehui, Tian Minghao, He Baoqiang, Zhong Dejun 2025, 29 (13):  2849-2860.  doi: 10.12307/2025.035 Abstract ( 193 )   PDF (2146KB) ( 480 )   Save BACKGROUND: NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is closely related to neuroinflammation after spinal cord injury, in which microglial polarization and pyroptosis play a key role. Targeted regulation of NLRP3 can induce microglial polarization from M1 proinflammatory phenotype to M2 anti-inflammatory phenotype and regulate microglial pyroptosis, which is a promising therapeutic strategy. OBJECTIVE: To summarize the molecular mechanism and therapeutic strategies of NLRP3 inflammasome in microglia after spinal cord injury. METHODS: Databases of PubMed, Web of Science, and CNKI were searched for the articles with search terms “spinal cord injury, NLRP3, microglia, polarization, pyroptosis” in English and “spinal cord injury, NLRP3, microglia, polarization, pyroptosis, inflammation” in Chinese. Finally, a total of 79 articles were included according to the inclusion and exclusion criteria. RESULTS AND CONCLUSION: (1) Currently, there is no consensus on the complex pathogenesis of spinal cord injury. A large number of studies have shown that spinal cord injury is closely related to inflammatory factors and signaling pathways. The NLRP3 inflammasome is a hot topic in current research as a mechanism of disease and a breakthrough in treatment. (2) The NLRP3 inflammasome plays a key role in the inflammatory response, oxidative stress, and neuronal recovery after spinal cord injury. (3) Microglia are immune cells in the brain and spinal cord and are the most important regulatory factors in secondary spinal cord injury. After spinal cord injury, microglia adjust the internal environment, mainly manifested as polarization and necrosis, produce a large number of inflammatory factors, hinder the nerve regeneration and functional recovery of spinal cord injury, and regulating the phenotype change of microglia is another key factor in the treatment of spinal cord injury. (4) The NLRP3 inflammasome is closely related to microglia. After spinal cord injury, NLRP3 is mainly expressed in microglia, which promotes the polarization of microglia to M1 and accelerates the production of cleavage proteins, further disrupting the microenvironment and aggravating the progression of spinal cord injury. (5) Many molecules participate in the regulation of NLRP3 inflammasomes in microglia, involving signaling pathways. Among them, nuclear factor-κB and MAPK signaling pathways promote NLRP3 inflammasome, while the rest inhibit this inflammasome. (6) At present, a large number of exogenous molecules and drugs regulate NLRP3 inflammasomes, with a wide range of clinical application prospects. Relevant drugs are in the clinical trial stage and obtain good effects, such as the NLRP3-specific inhibitor MCC950. However, key issues such as how to precisely control targeted delivery and the impact on other tissues and organs urgently need to be resolved. With the deepening of research, it is expected to make new breakthroughs in delaying the treatment of spinal cord injury in the future. Figures and Tables | References | Related Articles | Metrics