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08 September 2024, Volume 28 Issue 25 Previous Issue    Next Issue

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Primary cilia/intraflagellar transport mediates mechanics-responsive signaling pathway and promotes osteogenic differentiation of bone marrow stromal stem cells Ma Zhanhua, Yan Xu, Jiang Yan, Cao Zhengming, Wang Yongkui, Li Dongzhe, Yang Tengyue, Jin Yikai, Fu Su, Zhang Chunlin 2024, 28 (25):  3937-3941.  doi: 10.12307/2024.192 Abstract ( 161 )   PDF (1375KB) ( 12 )   Save BACKGROUND: Mechanical stimulation has been confirmed to promote osteogenic differentiation of bone marrow stromal stem cells, but the mechanism is unknown. Primary cilia are important mechanoreceptors and regulate various signaling pathways such as TGF-β1/BMP-2/SMAD. They are likely to be important targets for mechanical regulation of bone marrow stromal stem cells.  OBJECTIVE: To investigate the effect and mechanism of fluid shear stress on osteogenic differentiation of bone marrow stromal stem cells.  METHODS: Rat bone marrow stromal stem cells were divided into control group, mechanical stimulation group (fluid shear mechanics intervention by shaking table), mechanical stimulation + IFT88 silencing group (mechanical stimulation + silencing IFT88 expression with siRNA). After 24 hours of intervention, qRT-PCR was utilized to determine the expression of transforming growth factor β1 and bone morphogenetic protein 2. Western blot assay was used to detect the expression of phosphorylated SMAD2/3 protein. Immunofluorescent staining of primary cilia was conducted and morphology was analyzed.  RESULTS AND CONCLUSION: Shear stress stimulation could promote the transcriptional activity of transforming growth factor β1 and bone morphogenetic protein 2 genes, and increase the expression of phosphorylated SMAD2/3 protein. After siRNA interfered with primary cilia, this mechanical response effect was significantly reduced. There was a Spearman correlation between the change ratio of the primary cilium area of bone marrow stromal stem cells and the increased ratio of transforming growth factor β1 and bone morphogenetic protein 2 gene transcription. These findings indicate that primary cilia/intraflagellar transport mediates the activation of fluid shear stress-responsive transforming growth factor β1/bone morphogenetic protein 2/SMAD signaling pathway and promotes osteogenic differentiation of bone marrow stromal stem cells. Figures and Tables | References | Related Articles | Metrics

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Culture, identification, and differentiation of rabbit pituitary stem cells Yue Tingting, Zhu Ting, Mao Jiannan, Li Wei, Hang Chunhua 2024, 28 (25):  3942-3946.  doi: 10.12307/2024.178 Abstract ( 160 )   PDF (1162KB) ( 12 )   Save BACKGROUND: The pituitary gland is an important endocrine organ in the body. Certain diseases can cause damage to the pituitary gland, such as pituitary adenoma and abnormal hormone secretion. Pituitary stem cells, due to their self-renewal and multi-directional differentiation potential, are expected to become a new therapeutic approach for repairing damaged pituitary glands. OBJECTIVE: To isolate and culture pituitary stem cells using the suspension cell ball culture method and identify their proliferation and differentiation ability.  METHODS: Pituitary stem cells were isolated and cultured from the pituitary gland of newborn New Zealand white rabbits using the suspension cell ball culture method, and their morphological characteristics were observed. Immunofluorescence cytochemistry was used to detect the expression of pituitary stem cell markers SOX2 and Nestin. EdU labeling method was utilized to detect the proliferative ability of pituitary stem cells. After adherent and induced differentiation, the hormone levels in the culture medium were detected by ELISA. RESULTS AND CONCLUSION: Pituitary stem cell spheres could be successfully isolated by the suspension cell ball culture method, with strong proliferative ability. Positive expression of stem cell-specific markers SOX2 and Nestin was found in the cultured cells. After induction and differentiation, adrenocorticotropic hormone, thyroid hormone, growth hormone, luteinizing hormone, follicle-stimulating hormone, and prolactin levels significantly increased in the medium (P < 0.001), with strong differentiation ability. Figures and Tables | References | Related Articles | Metrics

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Detection of immune-related cytokines of bone marrow mesenchymal stem cells in postmenopausal osteoporosis mice by antibody chip and analysis of key differential genes Yang Shanshan, Ouyang Renjun, Tian Jia, Linghu Min, Wang Zhen, Yang Xiaohong 2024, 28 (25):  3947-3954.  doi: 10.12307/2024.182 Abstract ( 143 )   PDF (3442KB) ( 101 )   Save BACKGROUND: In recent years, research on the interaction mechanism between the immune system and the skeleton in postmenopausal osteoporosis has become a hot topic. However, the impact of changes in key immune-related cytokine expression on postmenopausal osteoporosis remains unclear and requires further exploration.  OBJECTIVE: To investigate the differential expression of immune-related cytokines in bone marrow mesenchymal stem cells of mice with postmenopausal osteoporosis by bioinformatics methods.  METHODS: Postmenopausal osteoporosis mouse model was established through ovariectomy. Bone marrow mesenchymal stem cells were obtained by the whole bone marrow adherence method and passaged to passage 2. RayBio L-Series Mouse Antibody Array 308 Glass Slide Kit immune-related factor antibody chip was used to detect the differentially expressed proteins in bone marrow mesenchymal stem cells from ovariectomy and sham-operation mice. Gene ontology, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and protein-protein interaction network analysis were performed to screen common Hub genes by MCC, EPC, and MNC algorithms. RESULTS AND CONCLUSION: This study identified a total of 68 differentially expressed genes. Gene ontology analysis revealed that the differentially expressed genes were enriched in terms including “immune system processes”, “extracellular regions”, and “signal receptor binding”. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the differentially expressed genes were mainly enriched in “cytokine-cytokine receptor interactions”, “tumor necrosis factor signaling pathways”, and “chemokine signaling pathways”. Further screening was performed by constructing a protein-protein interaction network analysis of these 68 differentially expressed genes to identify 8 Hub genes. The violin plot and correlation matrix showed that the expression levels of these 8 Hub genes were significantly down-regulated in the ovariectomy group compared to the sham-operation group. These results demonstrated that there was differential expression of immune-related factors in bone marrow mesenchymal stem cells of postmenopausal osteoporosis mice, and key genes involved in cytokine-cytokine receptor interactions, immune system-related processes, and potential targeted signaling pathways and cellular biological processes were identified, providing new promising targets for the diagnosis, treatment, and prevention of postmenopausal osteoporosis.  Figures and Tables | References | Related Articles | Metrics

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Characteristics of acute graft-versus-host disease of the intestine after unrelated cord blood transplantation Tu Meijuan, Zhang Chunli, Deng Li, Fang Bing, Sun Guangyu, Zhu Xiaoyu, Zhang Xinqiong 2024, 28 (25):  3955-3959.  doi: 10.12307/2024.195 Abstract ( 107 )   PDF (823KB) ( 37 )   Save BACKGROUND: Despite unrelated cord blood transplantation is expected to become an important method for treating malignant hematological diseases, the manifestation and clinical characteristics of acute graft-versus-host disease in the gastrointestinal tract still require further in-depth investigation.  OBJECTIVE: To analyze the clinical characteristics of intestinal acute graft-versus-host disease after unrelated cord blood transplantation.  METHODS: A retrospective analysis was conducted on 668 malignant hematological disease patients after unrelated cord blood transplantation who underwent hematopoietic stem cell transplantation subspecialty in the Department of Hematology, First Affiliated Hospital of University of Science and Technology of China from December 2016 to December 2020. Among them, clinical data of 138 patients with intestinal acute graft-versus-host disease were analyzed, including 76 males and 62 females, with a median age of 13 (1-62) years. All patients were treated with a myeloablative regimen (without antihuman thymocyte globulin) and cyclosporin A combined with mycophenolate mofetil to prevent graft-versus-host disease. RESULTS AND CONCLUSION: (1) The patients with intestinal acute graft-versus-host disease had diarrhea of varying degrees, most of which were yellow-green, yellow-brown watery stools or mucous stools. 53 patients (38.4%) had blood stools, 82 patients (57.9%) had skin involvement, 18 patients (13.0%) had a secondary intestinal bacterial infection, and 90 patients (65.2%) had cytomegaloviremia. (2) The clinical characteristics of patients (70 cases, 50.7%) with grade 1-2 intestinal acute graft-versus-host disease were compared with those (68 cases, 49.3%) with grade 3-4 intestinal acute graft-versus-host disease. It was found that the age of grade 3-4 intestinal acute graft-versus-host disease patients was higher than that of grade 1-2 intestinal acute graft-versus-host disease patients (P < 0.001), and they were complicated with cytomegaloviremia probably (P=0.035). Diarrhea lasted longer (P=0.00) and the length of hospital stay increased substantially (P < 0.001). However, there were no significant differences in recipient gender, pre-transplant disease status, HLA matching, diagnosis, combined skin graft-versus-host disease, and secondary intestinal infection rate in patients of the two groups. (3) These findings conclude that the clinical characteristics of intestinal acute graft-versus-host disease after unrelated cord blood transplantation are complex, which affects the prognosis and quality of life of patients seriously and requires early identification and precise treatment. Figures and Tables | References | Related Articles | Metrics

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Interleukin-4 regulates macrophage polarization and osteogenic differentiation of bone marrow mesenchymal stem cells Zhang Jie, Xiao Tianjiao, Li Li, Kang Jiabing, Zhan Jifan, Wei Yan, Tian Ai 2024, 28 (25):  3960-3966.  doi: 10.12307/2024.189 Abstract ( 130 )   PDF (1344KB) ( 89 )   Save BACKGROUND: Interleukin-4 can promote the osteogenic effect of bone substitute materials, but its molecular mechanism is not yet clear. Further elucidating the mechanism of interleukin-4 promoting osteogenic effect can help find safe, economical, and effective methods for the regeneration treatment of alveolar bone defects in patients. OBJECTIVE: To explore the effect of interleukin-4 intervention on polarization transformation of macrophages and osteogenic differentiation of bone marrow mesenchymal stem cells and its possible mechanism. METHODS: RAW264.7 cells in the M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours. RAW264.7 cells in the interleukin-4+M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours and then interleukin-4 was added for 24 hours. RAW264.7 cells in the interleukin-4+AG+M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours, and then interleukin-4 and AG-490, a JAK/STAT pathway inhibitor, were added for 24 hours. After intervention, immunofluorescence staining was used to analyze the expression of inducible nitric oxide synthase and CD206, the phenotypic marker protein of macrophages. ELISA kit was used to detect the expression of interleukin-10 and tumor necrosis factor-α in the supernatant of cell culture. The gene expressions of nodular receptor protein-3 (NLRP3), interleukin-1β, and caspase-1 were detected by RT-qPCR. The expression levels of tyrosine protein kinase 1 (JAK1)/ phosphorylated tyrosine protein kinase 1 (p-JAK1), signal transduction and transcription activator 6 (STAT6)/phosphorylated signal transduction and transcription activator 6 (p-STAT6), NLRP3, pro-interleukin-1β and pro-caspase-1 were detected by western blot assay. Then, RAW264.7 cells in the above four groups were indirectly co-cultured with bone marrow mesenchymal stem cells by transwell for 24 hours, followed by alkaline phosphatase staining and alizarin red staining. The mRNA expressions of alkaline phosphatase, collagen type I, and osteocalcin were detected by RT-qPCR. RESULTS AND CONCLUSION: (1) Immunofluorescence and ELISA results showed that interleukin-4 intervention could promote the expression of CD206 and interleukin-10 in M2 macrophages, and inhibit the secretion of inducible nitric oxide synthase and tumor necrosis factor-α. (2) RT-qPCR results showed that interleukin-4 could suppress the expression of NLRP3, interleukin-1β, and caspase-1 mRNAs. (3) Western blot assay showed that interleukin-4 could promote the expression of JAK1/p-JAK1, STAT6/p-STAT6 and NLRP3 proteins. (4) The alkaline phosphatase staining and alizarin red staining of bone marrow mesenchymal stem cells co-cultured with the interleukin-4+M1 group were significantly enhanced, and the mRNA expressions of alkaline phosphatase, collagen type I, and osteocalcin were significantly increased. It is concluded that interleukin-4 may inhibit the activation of NLRP3 by up-regulating JAK1/STAT6 pathway, thus promoting the transformation of macrophages from M1 polarization to M2 polarization, and finally enhancing the osteogenic differentiation ability of bone marrow mesenchymal stem cells.  Figures and Tables | References | Related Articles | Metrics

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Effect of apoptosis-inducing factor gene knockdown on bone marrow mesenchymal stem cell transplantation for myocardial infarction Han Dunzheng, Qin Xiaozhou, Pan Xiudi, Lu Waner, Dai Ying, Chen Yanxun, Cheng Xianfei, Tang Muhan 2024, 28 (25):  3967-3973.  doi: 10.12307/2024.197 Abstract ( 111 )   PDF (1210KB) ( 73 )   Save BACKGROUND: Numerous basic and clinical trials have confirmed that the low survival rate after bone marrow mesenchymal stem cell transplantation is a serious constraint on its long-term therapeutic effect. Previous studies have shown that apoptosis-related factors play an important role in the apoptosis of bone marrow mesenchymal stem cells, of which apoptosis-inducing factor may be a key factor. OBJECTIVE: Bone marrow mesenchymal stem cells, of which apoptosis-inducing factor was knocked down, were transplanted into infarcted myocardium of mice, aiming to certify the importance of apoptosis-inducing factor in the survival of bone marrow mesenchymal stem cells to further recover cardiac function after infarction. METHODS: Firstly, bone marrow mesenchymal stem cells were infected with LV-AIF-shRNA lentivirus to down-regulate the expression of apoptosis-inducing factor protein. Flow cytometry, western blot assay, and RT-qPCR were used to detect the infection efficiency of lentivirus. CCK-8 assay was used to detect the cell viability of bone marrow mesenchymal stem cells with apoptosis-inducing factor knockdown under hypoxic and ischemic conditions. Then, with the mouse model of acute myocardial infarction constructed, the normal bone marrow mesenchymal stem cells and bone marrow mesenchymal stem cells with apoptosis-inducing factor gene knockdown were transplanted into the model, respectively. The expression of apoptosis-inducing factor was examined by fluorescence immunoassay. Serum brain natriuretic peptide levels were detected by ELISA. Cardiac ultrasound was used to detect cardiac function. Myocardial fibrosis was observed by Masson staining. The expression of SRY gene was detected by RT-qPCR in apoptosis-inducing factor-knocked bone marrow mesenchymal stem cells after transplantation, reflecting cell survival. RESULTS AND CONCLUSION: (1) Bone marrow mesenchymal stem cells with apoptosis-inducing factor gene knockdown were successfully established by LV-AIF-shRNA lentivirus infection, following 97.7% of infection efficiency, and notably decline of the expression of apoptosis-inducing factor (P < 0.001). (2) Under ischemia and hypoxia, the cell viability of apoptosis-inducing factor knockdown bone marrow mesenchymal stem cells was significantly increased compared with normal bone marrow mesenchymal stem cells. (3) Compared with normal bone marrow mesenchymal stem cells after transplantation, the survival number of bone marrow mesenchymal stem cells in the infarcted myocardium after apoptosis-inducing factor gene knockdown was significantly increased to 3.71 times (P < 0.001), and the apoptosis-inducing factor protein expression and myocardial fibrosis degree in the infarcted area were significantly reduced. (4) Compared with normal bone marrow mesenchymal stem cells, the serum brain natriuretic peptide level of bone marrow stem cells with apoptosis-inducing factor gene knockdown after transplantation was significantly decreased (P < 0.05), and left ventricular ejection fraction and left ventricular shortening fraction were significantly improved (P < 0.05). (5) These findings confirm that apoptosis-inducing factor gene knockdown can reduce myocardial fibrosis and improve cardiac function after acute myocardial infarction via enhancing the bone marrow mesenchymal stem cell viability and increasing the bone marrow mesenchymal stem cell survival after transplantation in the donor. Figures and Tables | References | Related Articles | Metrics

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Action mechanism of Bushenhuoxue decoction on promoting nucleus pulposus-like differentiation of adipose-derived stem cells Guo Zehua, Li Zhaoyong, Chen Long, Duan Jiahao, Jiang Haobo, Chen Guangxue, Su Youxian, Liu Enxu, Yang Shaofeng 2024, 28 (25):  3974-3980.  doi: 10.12307/2024.188 Abstract ( 114 )   PDF (1538KB) ( 44 )   Save BACKGROUND: Stem cell transplantation is a new way to prevent and cure intervertebral disc degeneration. However, whether the transplanted stem cells can survive, proliferate, differentiate, and restore the function of nucleus pulposus cells after transplantation, is the key and difficult point to overcome.  OBJECTIVE: To explore the effects of Bushenhuoxue decoction on survival, proliferation, and nucleus pulposus-like differentiation of adipose-derived stem cells. METHODS: A Transwell chamber was used to construct a co-culture model of human adipose-derived stem cells and human degenerative nucleus pulposus cells. The experiment was divided into control group, model group, drug-containing serum group, and drug-free serum group. Except for the control group, the co-culture system of other groups was treated with 50 μmol/L tert-butyl hydrogen peroxide for 24 hours. The drug-containing serum group and drug-free serum group were treated with DMEM low-glucose complete culture medium containing drug-containing serum of Bushenhuoxue decoction or drug-free serum with 20% volume fraction for 48 hours. The sublayer adipose-derived stem cells were taken. Toluidine blue staining was used to detect proteoglycan synthesis levels. Real-time PCR method was used to detect mRNA expression of type II collagen, proteoglycan and SRY-box transcription factor 9. The protein expression of SOX9 was detected by western blot assay. Lactate dehydrogenase assay was used to detect cytotoxicity. Flow cytometry was used to detect reactive oxygen species, and β-galactosidase staining was used to detect cell senescence.  RESULTS AND CONCLUSION: (1) Compared with the control group, the proportion of necrotic cells in the model group increased; toluidine blue staining became lighter, and the expression levels of type II collagen, proteoglycan, SOX9 mRNA and SOX9 protein decreased (P < 0.05). Compared with the model group, the drug-containing serum of Bushenhuoxue decoction could significantly reduce cell injury and promote the expression of type II collagen, proteoglycan, SOX9 mRNA, and SOX9 protein (P < 0.05), but the improvement in the drug-free serum group was not significant (P > 0.05). (2) Compared with the control group, the contents of cytotoxicity, reactive oxygen species, and cell senescence in the model group were significantly increased. Compared with the model group, the microenvironment of the coculture system was significantly improved by drug-containing serum of Bushenhuoxue decoction (P < 0.05), while drug-free serum had no significant effect on the microenvironment of the co-culture system (P > 0.05). (3) The results show that Bushenhuoxue decoction can promote the survival, proliferation, and nucleus pulposus-like differentiation of adipose-derived stem cells. Figures and Tables | References | Related Articles | Metrics

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Comparison of small extracellular vesicles derived from stem cells and tissue on de novo adipose regeneration Yang Baohua, Zhou Xiaojie, Jing Wei, Tian Weidong, Yu Mei 2024, 28 (25):  3981-3987.  doi: 10.12307/2024.171 Abstract ( 125 )   PDF (1887KB) ( 17 )   Save BACKGROUND: De novo adipose regeneration induced by small extracellular vesicles has become a promising method for repairing soft tissue defects. However, due to different animal models and small extracellular vesicle application dosages, it is difficult to quantitatively compare the therapeutic effect of small extracellular vesicles from various sources on adipose regeneration.   OBJECTIVE: To compare the regenerative effects of small extracellular vesicles derived from stem cells and small extracellular vesicles from tissue. METHODS: Small extracellular vesicles derived from adipose-derived stem cells and from adipose tissue were isolated by ultracentrifugation. The particle number, particle size, morphology, and protein expression of small extracellular vesicles were identified by nanoparticle tracking analysis, transmission electron microscopy and western blot assay. A quantitative and evaluative subcutaneous model for adipose regeneration in C57 mice was established using a customized silicone tube. The regenerative effects of induced de novo adipose were compared by cell counting and hematoxylin-eosin staining. RESULTS AND CONCLUSION: (1) Small extracellular vesicles derived from adipose-derived stem cells and from adipose tissue were isolated by ultracentrifugation. Both small extracellular vesicles were round-shape in transmission electron microscopy with particle size between 50-200 nm, and abundant with the small extracellular vesicles marker protein CD81, CD63 and TSG101. (2) An equal number of small extracellular vesicles were mixed with matrigel in customized silicone tubes, implanted subcutaneously in the back of mice to establish a cell-free and quantifiable adipose regeneration model. (3) On days 3 and 7 after implantation, the results of cell counting and hematoxylin-eosin staining showed that both small extracellular vesicle groups recruited more host cells than the blank group, and the small extracellular vesicles derived from adipose tissue group were superior to the small extracellular vesicles derived from adipose-derived stem cell group. (4) 4 weeks after implantation, hematoxylin-eosin staining of the contents in silicone tubes showed that small extracellular vesicles induced de novo adipose regeneration in vivo, and the small extracellular vesicles derived from adipose tissue group were superior to the small extracellular vesicles derived from adipose-derived stem cell group. The above results indicated that small extracellular vesicles derived from tissues have a superior effect on inducing de novo adipose regeneration compared to small extracellular vesicles derived from stem cells.  Figures and Tables | References | Related Articles | Metrics

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Aerobic exercise preconditioning improves therapeutic effect of bone marrow mesenchymal stem cells on acute myocardial infarction Zhang Min, Lou Guo, Fu Changxi 2024, 28 (25):  3988-3993.  doi: 10.12307/2024.187 Abstract ( 118 )   PDF (1554KB) ( 29 )   Save BACKGROUND: Stem cell therapy is an alternative treatment strategy for restoring damaged myocardial tissue after acute myocardial infarction. Exercise preconditioning can induce endogenous cardioprotective effects in the body. However, the efficacy and mechanism of the combined application are still unclear.  OBJECTIVE: To explore the effect and possible mechanism of exercise preconditioning combined with bone marrow mesenchymal stem cells on the therapeutic effect in rats with acute myocardial infarction. METHODS: Seventy male SD rats were randomly divided into sham operation group, model group, stem cell therapy group, exercise preconditioning group, and combined intervention group. Rats in the exercise preconditioning group and combined intervention group underwent 8-week aerobic exercise on the treadmill before modeling. The animal model of acute myocardial infarction was made by ligating the anterior descending coronary artery. The stem cell therapy group and the combined intervention group were injected with bone marrow mesenchymal stem cells (1×109 L-1, 1 mL) through the tail vein the next day after modeling. After 4 weeks of treatment, the exercise performance was evaluated by a graded treadmill exercise test. The cardiac structure and function were detected by echocardiography. The left ventricle was isolated. 2,3,5-Triphenyltetrazolium chloride staining was used to evaluate myocardial infarct size. Masson staining was used to obtain collagen volume fraction. CD31 immunohistochemical staining was used to detect myocardial capillary density. TUNEL staining was used to detect myocardial cell apoptosis. Immunoblotting was used to detect protein expression levels of stromal cell-derived factor 1, CXC chemokine receptor protein 4, tumor necrosis factor-α, interleukin-10, and vascular endothelial growth factor.  RESULTS AND CONCLUSION:  (1) Intervention efficacy: Compared with the sham operation group, exercise performance, left ventricular ejection fraction, left ventricular fractional shortening, and CD31 positive cell rate decreased (P < 0.05); myocardial infarct size, collagen volume fraction, and myocardial apoptotic rate increased (P < 0.05) in the model group. Compared with the model group, exercise performance was not statistically significant (P > 0.05) in the stem cell therapy group, and the exercise performance improved (P < 0.05) in the exercise preconditioning and combined intervention groups; left ventricular ejection fraction, left ventricular fractional shortening, and CD31 positive cell rate increased (P < 0.05), and the myocardial infarct size, collagen volume fraction, and cardiomyocyte apoptosis rate decreased (P < 0.05) in the stem cell therapy, exercise preconditioning, and combined intervention groups. Compared with the stem cell therapy group, exercise performance, left ventricular ejection fraction, left ventricular shortening fraction, and CD31 positive cell rate increased (P < 0.05), myocardial infarct size, collagen volume fraction, and myocardial cell apoptosis rate decreased (P < 0.05) in the combined intervention group. (2) Protein expression: Compared with the sham operation group, the expression of tumor necrosis factor-α increased (P < 0.05), while interleukin-10 and vascular endothelial growth factor expression decreased (P < 0.05) in the model group. Compared with the model group, the expression of CXC chemokine receptor protein 4 increased (P < 0.05) in the stem cell therapy group and combined intervention group, and the expression of tumor necrosis factor-α decreased (P < 0.05) while interleukin-10 and vascular endothelial growth factor increased (P < 0.05) in the stem cell therapy group, exercise preconditioning group, and combined intervention group. Compared with the stem cell therapy group, the expression of tumor necrosis factor-α decreased (P < 0.05), while CXC chemokine receptor protein 4, interleukin-10, and vascular endothelial growth factor increased (P < 0.05) in the combined intervention group. To conclude, exercise preconditioning can enhance the therapeutic effect of bone marrow mesenchymal stem cells in rats with acute myocardial infarction, which can inhibit cardiac remodeling, improve cardiac function, and delay the progress of heart failure. Its mechanism is related to the promotion of stem cell homing, inhibition of inflammatory response, and promotion of angiogenesis. Figures and Tables | References | Related Articles | Metrics

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Neuron-derived extracellular vesicles promote neurogenesis of neural stem cells Li Zhen, Sun Xiao, Xie Yongpeng, Rong Wang, Sun Haitao 2024, 28 (25):  3994-3999.  doi: 10.12307/2024.186 Abstract ( 130 )   PDF (1827KB) ( 32 )   Save BACKGROUND: It has been shown that neural stem cells can differentiate into neurons, astrocytes, and oligodendrocytes. Mesenchymal stem cells-derived extracellular vesicles have also been shown to cross the blood-brain barrier to reach sites of central nervous injury and promote neural repair. However, it is not clear whether neuron-derived extracellular vesicles promote the differentiation of neural stem cells in a direction that is beneficial for neurogenesis. OBJECTIVE: To investigate whether neuron-derived extracellular vesicles facilitate neural stem cell differentiation towards neurogenesis.  METHODS: Neurons and neural stem cells were extracted from neonatal SD rat cerebral cortex by trypsin digestion. Cell supernatants of neurons were collected. Neuron-derived extracellular vesicles were extracted. Neural stem cells cultured for 10 days were co-cultured with neuron-derived extracellular vesicles or PBS for 7 days. Immunoblotting, immunofluorescence, and RT-qPCR were used to detect proteins specifically expressed by neurons, neural stem cells, oligodendrocytes, and astrocytes. RESULTS AND CONCLUSION: The neural stem cells co-cultured with neuron-derived extracellular vesicles showed high expression of neuron-specific proteins and oligodendrocyte-specific proteins including β3-tubulin, neurofilament 200 and myelin basic protein, and low expression of astrocyte-specific protein glial fibrillary acidic protein. These results suggest that neuron-derived extracellular vesicles can promote the differentiation of neural stem cells into neurons and oligodendrocytes and prevent the differentiation of neural stem cells into astrocytes. Figures and Tables | References | Related Articles | Metrics

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Targeted induction of human umbilical cord mesenchymal stem cells cultured with human peripheral blood serum into neural stem cells Han Xia, Zhao Ruidong, Yang Junli 2024, 28 (25):  4000-4004.  doi: 10.12307/2024.194 Abstract ( 106 )   PDF (1590KB) ( 34 )   Save BACKGROUND: There are many kinds of cell media with different components, which have a great influence on cell growth. Several studies in and outside China have used serum-free media containing fetal bovine serum for in vitro amplification culture, but the use of media containing human peripheral blood serum to directionally induce human umbilical cord mesenchymal stem cells to neural stem cells and human peripheral blood serum to promote neural stem cells to differentiate into other nerve cells, so far there are few relevant studies.  OBJECTIVE: To observe the feasibility of inducing human umbilical cord mesenchymal stem cells cultured with human peripheral blood serum into neural stem cells.  METHODS: (1) Human umbilical cord mesenchymal stem cells were cultured in DMEM/F-12 culture medium containing 10% human peripheral blood serum by volume fraction. Flow cytometry analysis was performed at the third passage to identify surface markers and alizarin red staining was used to detect osteogenic differentiation function. (2) The third-generation human umbilical cord mesenchymal stem cells were induced into neural stem cells using DMEM/F-12 medium containing 0.5% N2, 1.5% B27, 20 ng/mL basic fibroblast growth factor, and 20 ng/mL epidermal growth factor, and their surface markers were identified. (3) Well-growing human umbilical cord mesenchymal stem cell-derived neural stem cells were taken to prepare a single cell suspension. They were evenly inoculated into 96-well plates and incubated with DMEM/F-12 culture medium containing 10% human peripheral blood serum for 8 days. Then, hematoxylin-eosin staining, microtubule-associated protein 2, and glial fibrillary acidic protein immunofluorescence staining were performed to detect the differentiation of neural stem cells into other neural cells.   RESULTS AND CONCLUSION: (1) Human umbilical cord mesenchymal stem cells cultured with human peripheral blood serum grew in a spiral-like pattern and were distributed in multiple layers, with directional arrangement. The surface of human umbilical cord mesenchymal stem cells highly expressed CD44, CD105, CD29, and CD73. Cells stained with alizarin red showed a color reaction. (2) Human umbilical cord mesenchymal stem cells cultured with human peripheral blood serum could be induced to differentiate into neural stem cells, and the surface of neural stem cells was highly expressed with CD44, CD105, CD29, CD73, Nestin, NF-L, and GALC. (3) On day 8 of induced differentiation of neural stem cells, after staining with hematoxylin and eosin, it was found that the protruding protrusions were longer, with more branches and adjacent cells connected, presenting typical neural cell morphology. Immunofluorescence staining for microtubule-associated protein 2 and glial fibrillary acidic protein was positive. It is concluded that human umbilical cord mesenchymal stem cells cultured by human peripheral blood serum can be directly induced to differentiate into neural stem cells. Under the action of human peripheral blood serum, neural stem cells can differentiate into other neural cells as the culture time prolongs.  Figures and Tables | References | Related Articles | Metrics

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Action mechanism by which fibronectin type III domain-containing protein 5 inhibits macrophage pyroptosis Zhao Guangjian, Liu Danan, Zhou Bo, Wang Yao 2024, 28 (25):  4005-4012.  doi: 10.12307/2024.196 Abstract ( 120 )   PDF (1713KB) ( 65 )   Save BACKGROUND: Fibronectin type III domain-containing protein 5 (FNDC5) is a muscle factor that can regulate glucose and lipid metabolism, and exert anti-inflammatory, anti-oxidative effects and improvement in insulin resistance. Moreover, FNDC5 could control various cell pyroptosis. OBJECTIVE: To explore the effect and potential mechanism of FNDC5 on macrophage pyroptosis.  METHODS: (1) After completing the construction of the lentivirus virus overexpressing FNDC5 or silencing FNDC5, the THP-1 cells were transfected with the lentivirus vector. The result of transfection was detected by the expression of green fluorescence, qPCR, and western blot assay. (2) Phorbol ester induced THP-1 cells to differentiate into macrophages. Oxidized low-density lipoprotein (ox-LDL) was added to induce the cell pyroptosis model. There were six groups, i.e., NC group, ox-LDL group, ox-LDL+MOCK1 group, ox-LDL+Ov-FNDC5 group, ox-LDL+MOCK2 group, and ox-LDL+shFNDC5 group. (3) The level of cell pyroptosis was evaluated by Hoechst 33342/propidium iodide fluorescence double staining and lactate dehydrogenase release assay. The expression levels of related molecules in THP-1 cells were analyzed by qPCR and western blot assay. The interleukin-18 and interleukin-1β in cell supernatant were detected by ELISA.  RESULTS AND CONCLUSION: Compared with the ox-LDL+MOCK1 group, overexpression of FNDC5 significantly reduced the pyroptosis rate of macrophages and the release levels of lactate dehydrogenase, interleukin-1β and interleukin-18, significantly inhibited the mRNA expression of NF-κB p65, NF-κB p50, NLRP3, ASC, Caspase-1, and GSDMD, and significantly inhibited the protein expression of NF-κB p65, NF-κB p50, NLRP3, ASC, cleaved Caspase-1 and GSDMD-N. Compared with the ox-LDL+MOCK2 group, the silence of FNDC5 showed the opposite result. These findings suggest that FNDC5 attenuates pyroptosis in macrophages by inhibiting the NF-κB/NLRP3 pathway. Figures and Tables | References | Related Articles | Metrics

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Extraction and differentiation of mature and immature dendritic cells from Lewis rat bone marrow Li Liqiang, Li Minghao, Li Liang, Li Wenbo, Zhang Jun, Kong Lingmei 2024, 28 (25):  4013-4017.  doi: 10.12307/2024.190 Abstract ( 132 )   PDF (872KB) ( 81 )   Save BACKGROUND: Dendritic cells exhibit extremely strong antigen phagocytic function in the immature stage, and they can demonstrate great advantages in immune tolerance, cancer immunotherapy, and other aspects. However, due to the extremely low content of immature dendritic cells in living organisms, its clinical and scientific applications are severely limited.  OBJECTIVE: To study the extraction and identification of mature and immature dendritic cells from Lewis rat bone marrow.  METHODS: Bone marrow precursor cells were isolated from the bone marrow of Lewis rats, and immature dendritic cells were induced by 20 ng/mL of granulocyte colony-stimulating factor and 10 ng/mL of interleukin-4 for 7 days, and then mature dendritic cells were induced by adding 1 μg/mL of lipopolysaccharide to immature dendritic cells for 2 days. The morphology of dendritic cells was observed using inverted fluorescence microscopy. The surface-specific molecules of mature and immature dendritic cells were identified by flow cytometry, and the secretion levels of supernatant interleukin-10, interleukin-12, and interleukin-17A in mature and immature dendritic cells were detected by ELISA. The response of mature and immature dendritic cells to T lymphocyte stimulation was measured by mixed lymphocyte reaction. RESULTS AND CONCLUSION: (1) The dendritic cells showed an obvious protrusion structure under an ordinary inverted fluorescence microscope. (2) Flow cytometry showed low expression of CD40, CD86, and other co-stimulatory molecules in immature dendritic cells. On the contrary, mature dendritic cells highly expressed the above co-stimulatory molecules. (3) The secretion of interleukin-10 and interleukin-17A in immature dendritic cells was much higher than that in mature dendritic cells (P < 0.01). Interleukin-12 secretion in immature dendritic cells was much lower than that in mature dendritic cells (P < 0.05). (4) Mature dendritic cells stimulated T cells significantly better than immature dendritic cells, and the stimulation ability was stronger when the ratio of mature dendritic cells to T lymphocytes reached 1:10. (5) The results indicate that Lewis rat bone marrow precursor cells can differentiate into dendritic cells and distinguish between mature and immature dendritic cells by flow cytometry identification, related factor detection, and mixed lymphocyte reaction. Figures and Tables | References | Related Articles | Metrics

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Dynamic expression of fibroblast growth factor receptors 1 and 2 in mouse kidney development Bo Shuangling, Ma Taifang, Bai Huijian, Yang Yutian, Sun Yajie, Zhao Xinchen 2024, 28 (25):  4018-4021.  doi: 10.12307/2024.172 Abstract ( 123 )   PDF (1977KB) ( 48 )   Save BACKGROUND: The temporal and spatial expression of fibroblast growth factor receptors 1 and 2 remains a controversial issue during kidney development, so the relationship between them and kidney development remains unclear.  OBJECTIVE: To observe the dynamic expression of fibroblast growth factor receptors 1 and 2 during kidney development of mice, and to investigate the relationship between them and kidney development.   METHODS: The kidneys of fetal mice [embryotic days (E) 12, 14, 16, and 18] and neonatal mice [neonatal days (N) 1, 3, 7, 14, 24, and 40] were selected to examine the temporal and spatial expression of fibroblast growth factor receptors 1 and 2 by immunohistochemistry method in kidney tissues, and quantitative analysis was performed using western blot assay. RESULTS AND CONCLUSION: (1) Immunohistochemistry showed that fibroblast growth factor receptor 1 was mainly localized in metanephric tissue surrounding the tip of the ureteral bud at E12. Subsequently, fibroblast growth factor receptor 1 was expressed in immature renal corpuscles at various stages, some distal convoluted tubules and capillary loops. The positive site was mainly concentrated in the generative region. Fibroblast growth factor receptor 2 was initially expressed in both ureteral buds and metanephric tissue. Fibroblast growth factor receptor 2 was localized in immature renal corpuscles, distal tubules, collecting ducts and thin segments of medullary loops with kidney development. However, the expression of renal corpuscles was weak. (2) Stereology and western blot assay showed that the expression of fibroblast growth factor receptor 1 was high before birth and gradually decreased after birth, while the expression was very low after N7 day. The expression level of fibroblast growth factor receptor 2 increased gradually with the kidney development and tended to be stable after N7 day. (3) The results exhibit that fibroblast growth factor receptors 1 and 2 are expressed spatially and temporally during kidney development. It is speculated that fibroblast growth factor receptors 1 and 2 may influence nephron development and maturation, and fibroblast growth factor receptor 2 is critical during the formation of ureteral buds and morphology.  Figures and Tables | References | Related Articles | Metrics

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Behavior of cartilage-derived microtissue and ability of cartilage formation in three-dimensional dynamic and static culture conditions Liu Wei, Jiang Hongyu, Chen Jiajie, Gao Yuyang, Guan Yanjun, Jia Zhibo, Jiao Ying, Hua Zhen, Jiang Gehan, He Ying, Wang Aiyuan, Peng Jiang, Qi Jianhong 2024, 28 (25):  4022-4026.  doi: 10.12307/2024.191 Abstract ( 111 )   PDF (1331KB) ( 42 )   Save BACKGROUND: Compared with traditional two-dimensional culture, three-dimensional microtissue culture can show greater advantages. However, more favorable cultivation methods in three-dimensional culture still need to be further explored. OBJECTIVE: To evaluate the cell behavior of microtissue and its ability to promote cartilage formation under two three-dimensional culture methods. METHODS: Cartilage-derived microcarriers were prepared by chemical decellularization and tissue crushing. DNA quantification and nuclear staining were used to verify the success of decellularization, and histological staining was used to observe the matrix retention before and after decellularization. The microcarriers were characterized by scanning electron microscopy and CCK-8 assay. Cartilage-derived microtissues were constructed by combining cartilage-derived microcarriers with human adipose mesenchymal stem cells through three-dimensional static culture and three-dimensional dynamic culture methods. The cell viability and chondrogenic ability of the two groups of microtissues were detected by scanning electron microscopy, live and dead staining, and RT-qPCR.  RESULTS AND CONCLUSION: (1) Cartilage-derived microcarriers were successfully prepared. Compared with before decellularization, the DNA content significantly decreased after decellularization (P < 0.001). Scanning electron microscope observation showed that the surface of the microcarrier was surrounded by collagen, maintaining the characteristics of the natural extracellular matrix of cartilage cells. CCK-8 assay indicated that microcarriers had no cytotoxicity and could promote cell proliferation. (2) Scanning electron microscopy and live and dead staining results showed that compared with the three-dimensional static group, the three-dimensional dynamic group had a more extended morphology of microtissue cells, and extensive connections between cells and cells, between cells and matrix, and between matrix. (3) The results of RT-qPCR showed that the expressions of SOX9, proteoglycan, and type II collagen in microtissues of both groups were increased at 7 or 14 days. The relative expression levels of each gene in the three-dimensional dynamic group were significantly higher than those in the three-dimensional static group at 14 days (P < 0.05). At 21 days, the three-dimensional static group had significantly higher gene expression compared with the three-diomensional dynamic group (P < 0.001). (4) The results showed that compared with three-dimensional static culture microtissue, three-dimensional dynamic culture microtissue could achieve higher expression of chondrogen-related genes in a shorter time, showing better cell viability and chondrogenic ability. Figures and Tables | References | Related Articles | Metrics

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Bone morphogenetic protein-2 mediated homocysteine promotes vascular calcification Pei Jiansheng, Yang Wenjuan, He Jing, Yan Ru, Huang Hui, Jia Shaobin 2024, 28 (25):  4027-4033.  doi: 10.12307/2024.183 Abstract ( 105 )   PDF (1957KB) ( 25 )   Save BACKGROUND: There is an internal relationship between hyperhomocysteinemia and vascular calcification. However, the pathogenesis of hyperhomocysteinemia promoting vascular calcification is still unclear.    OBJECTIVE: To investigate the role of bone morphogenetic protein-2 in hyperhomocysteinemia-induced vascular calcification. METHODS: Human carotid wax samples were divided into a calcified group (n=29) and a non-calcified group (n=13) according to the presence or absence of calcified plaque. Sixteen ApoE-/- mice were randomly divided into a control group and a hyperhomocysteinemia group, with 8 mice in each group. Bone morphogenetic protein-2 vector was used to transfect rat thoracic artery smooth muscle A7r5 cells, and gradient concentration of homocysteine (50, 100, 200, and 400 μmol/L) was utilized to treat A7r5 cells. Calcification was detected by alizarin red staining and hematoxylin-eosin staining. The interaction of bone morphogenetic protein 2 with Runt-related transcription factor 2 was detected by immunofluorescence, and the expressions of bone morphogenetic protein 2, Runt-related transcription factor 2, and α-smooth muscle actin were detected by immunohistochemistry and western blot assay. RESULTS AND CONCLUSION: (1) Human carotid artery tissue staining revealed that compared with the non-calcification group, inflammatory cells increased and calcification positive rate increased in the calcification group (P < 0.05). Compared with the non-calcification group, the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated, and the expression of α-smooth muscle actin was decreased in the calcification group (all P < 0.05). (2) The staining of mouse arterial specimens exhibited that, the positive rate of calcified area in the hyperhomocysteinemia group was significantly higher than that in the control group (P < 0.05); serum homocysteine level in the hyperhomocysteinemia group was significantly higher than that in the control group (P < 0.05). Compared with the control group, the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated, and the expression of α-smooth muscle actin was decreased in the hyperhomocysteinemia group (all P < 0.05). (3) A7r5 cell culture analysis demonstrated that with the increase of homocysteine concentration gradient, the degree of calcification, the content of bone morphogenetic protein-2 and Runt-related transcription factor 2 protein in A7r5 cells increased (P < 0.05), and the content of α-smooth muscle actin protein decreased (P < 0.05). (4) The A7r5 cell culture analysis of overexpressed bone morphogenetic protein 2 showed that the calcification degree of the overexpressed bone morphogenetic protein 2 group was increased compared with the corresponding control group, the β-sodium glycerophosphate group, and the homocysteine group. RUNt-related transcription factor 2 expression up-regulated (P < 0.05) and α-smooth muscle actin expression down-regulated (P < 0.05). (5) The expression of bone morphogenetic protein 2 increased in A7r5 cells cultured with homocysteine in calcified medium, and the expression of Runt-related transcription factor 2 increased with the increase of bone morphogenetic protein 2 expression. (6) The results confirm that bone morphogenetic protein-2 is a key target gene in the regulation of smooth muscle cell phenotypic transformation resulting in vascular calcification by hyperhomocysteinemia. Targeted regulation of bone morphogenetic protein-2 reduces hyperhomocysteinemia-induced vascular calcification. Figures and Tables | References | Related Articles | Metrics

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Cell-of-origin for heterotopic ossification induced by bone morphogenetic protein 4 in skeletal muscle Yu Yangyi, Lian Qiang, Wu Jianqun, Zhang Xuan, Ren Jinke, Li Guangheng 2024, 28 (25):  4034-4040.  doi: 10.12307/2024.180 Abstract ( 142 )   PDF (1892KB) ( 63 )   Save BACKGROUND: Heterotopic ossification of skeletal muscle is a clinically serious complication. For heterotopic ossification of skeletal muscles, the cells involved in the process of heterotopic ossification remain unclear. OBJECTIVE: To investigate the involvement of myocytes, fascia cells, and endothelial cells in the process of heterotopic ossification in skeletal muscle and to observe the cell origin of heterotopic ossification in skeletal muscle induced by bone morphogenetic protein 4.  METHODS: Both C2C12 cells and the myotubes formed by the C2C12 cells in the induction medium were cultured, and 500 ng/mL bone morphogenetic protein 4 was added to the medium respectively, and whether the C2C12 cells and myotubes continued to proliferate within 10 days under the treatment were observed under a microscope. Myogenic cells (L6, derived from rats) and fibroblast-derived cells (derived from human) were co-cultured. After treatment with 500 ng/mL bone morphogenetic protein 4 and 10 ng/mL transforming growth factor-β, osteogenic and chondrogenic differentiation potential within 21 days were observed using Safranine O staining and Alcian blue staining. Using transgenic animal FVB/N-TgN (TIE2-LacZ) 182Sato mice, 15 μL of adeno-associated virus-bone morphogenetic protein 4 (5 × 1010 PFU/mL) were implanted in the thigh muscle space of genetic mice for 10 and 14 days. X-gal staining was used to observe the formation of new blood vessel endothelium in the differentiated bone.  RESULTS AND CONCLUSION: (1) Bone morphogenetic protein 4 caused myotube breakdown and increased C2C12 cell proliferation. Compared with other groups, the pure fibroblast-derived cell group had a higher area of positive alcian blue and safarin O staining (P < 0.05) and a lower area of alkaline phosphatase staining (P < 0.05), while the pure L6 group had a bigger area of alkaline phosphatase staining (P < 0.05) but a smaller area of positive alcian blue and safarin O staining (P < 0.05). (2) Transplantation of adeno-associated virus-bone morphogenetic protein 4-adsorbed gelatin sponge into FVB/N-TgN (TIE2-LacZ)182Sato mice resulted in heterotopic ossification. (3) X-gal staining results demonstrated that there was no obvious staining in chondrocytes and differentiated bones and Tie2+ endothelial cells did not participate in the formation of the alienated bone. (4) These findings verify that fibroblasts are the primary source of osteoblasts during the adeno-associated virus-bone morphogenetic protein 4-induced ectopic endochondral ossification in skeletal muscle, but myogenic cells are the main source of osteoblasts. Tie2+ endothelial cells might not be the cell source for cartilage and bone.  Figures and Tables | References | Related Articles | Metrics

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Regulatory effect of Ganoderma lucidum polysaccharides on H2O2-induced apoptosis and mitochondrial dysfunction in SH-SY5Y cells Li Yanbing, Wang Jiwei, Liu Xiaoqin, Guo Minfang, Niu Xiaojie, Meng Tao, Su Qin, Wang Hanbin, Yang Lizhi, Ma Cungen, Yu Jiezhong 2024, 28 (25):  4041-4047.  doi: 10.12307/2024.200 Abstract ( 124 )   PDF (1997KB) ( 52 )   Save BACKGROUND: Current studies have confirmed that Ganoderma lucidum polysaccharides can promote nerve regeneration in neurodegeneration-related diseases. The occurrence of neurodegenerative diseases is closely related to mitochondrial dysfunction, but the role of Ganoderma lucidum polysaccharides on the regulation of apoptosis and mitochondrial function in neurodegenerative diseases is not yet clarified.  OBJECTIVE: To explore the regulatory effects and mechanisms of Ganoderma lucidum polysaccharides on apoptosis and mitochondrial dysfunction in H2O2-induced SH-SY5Y cells. METHODS: SH-SY5Y cells were divided into three groups: control group, H2O2 group, and Ganoderma lucidum polysaccharides group. Cells in the control group were normally cultured. Cells in the H2O2 group were treated with 300 μmol/L H2O2 for 24 hours. In the Ganoderma lucidum polysaccharides group, the intervention with 300 μg/L Ganoderma lucidum polysaccharides was conducted first for 1-2 hours, followed by the addition of 300 μmol/L H2O2 for 24 hours. The mitochondrial membrane potential was detected by JC-1 kit. Apoptosis was detected by TUNEL staining kit. The activities of malondialdehyde and superoxide dismutase were detected by malondialdehyde test kit and superoxide dismutase test kit, respectively. The apoptosis and expression of mitochondrial dynamics-related proteins were detected by immunofluorescence staining and western blot assay. RESULTS AND CONCLUSION: (1) Compared with the control group, the mitochondrial membrane potential and superoxide dismutase activity were significantly reduced, as well as apoptotic rate and malondialdehyde levels were significantly increased in the H2O2 group (P < 0.05). After treatment with Ganoderma lucidum polysaccharides, the membrane potential and superoxide dismutase activities were significantly increased, and apoptotic rate and malondialdehyde levels were significantly reduced compared with the H2O2 group (P < 0.05). (2) The expression levels of pro-apoptotic proteins Bax and Caspase-3 were significantly increased, but the expression of anti-apoptotic protein Bcl-2 was significantly decreased in the H2O2 group compared with the control group (P < 0.05). Compared with the H2O2 group, the levels of Bax and Caspase-3 were significantly decreased, but the expression of anti-apoptotic protein Bcl-2 was significantly increased in the Ganoderma lucidum polysaccharides group (P < 0.05). (3) Compared with the control group, the expression of mitochondrial splitting proteins Fis1 and p-Drp1 was significantly increased, but the expression of mitochondrial fusion proteins OPA1, Mfn1, and Mfn2 was decreased in the H2O2 group (P < 0.05). Compared with the H2O2 group, Fis1 and p-Drp1 expression was significantly reduced, but the expression levels of OPA1, Mfn1, and Mfn2 were significantly increased in the Ganoderma lucidum polysaccharides group (P < 0.05). (4) The above results confirm that Ganoderma lucidum polysaccharides can attenuate H2O2-induced oxidative stress damage and apoptosis in SH-SY5Y cells by ameliorating mitochondrial dysfunction. Figures and Tables | References | Related Articles | Metrics

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miR-146a-3p regulates astrocyte proliferation, migration and apoptosis by inhibiting insulin-like growth factor 1 expression Ye Jiapeng, Wang Jianwei, Wu Mao, Li Shaoshuo, Wang Guopeng, Wang Haotian, Tang Zhi, Shao Yang 2024, 28 (25):  4048-4053.  doi: 10.12307/2024.174 Abstract ( 113 )   PDF (1303KB) ( 34 )   Save BACKGROUND: The alteration of miR-146a-3p level is a common event in the pathogenesis of most neurological diseases, and the specific mechanism of miR-146a-3p regulation of astrocytes has not been studied. OBJECTIVE: To verify that miR-146a-3p regulates astrocyte proliferation, migration and apoptosis through insulin-like growth factor 1. METHODS: 12 SD rats were divided into a sham operation group and a spinal cord injury group, with six rats in each group. RNA sequencing analysis was performed on the spinal cord tissues of all groups 2 weeks after surgery to screen out the differential genes (log2FC > 2), and to select spinal cord injury-related genes (Score > 20) in the Genecards database, and then to predict the target genes of miR-146a-3p by Targetscan. The intersection of three gene sets was obtained to screen out insulin-like growth factor 1 as one of the important target genes. qPCR, western blot assay and immunohistochemistry were performed to analyze the expression level of insulin-like growth factor 1 in spinal cord tissues. The primary astrocytes were divided into NC group, NC-mimics group and miR-146a-3p mimics group. Annexin-V/PI staining was used to detect cell apoptosis. CCK-8 assay was used to detect cell proliferation. Transwell assay was used to detect cell migration ability.  RESULTS AND CONCLUSION: The expression of miR-146a-3p in the spinal cord tissue of the spinal cord injury group was lower than that of the sham operation group (P < 0.05). The expression of insulin-like growth factor 1 in the spinal cord tissue of the spinal cord injury group was higher than that of the sham operation group (P < 0.05). Compared with the NC group and NC-mimics group, the apoptotic rate of astrocytes was increased (P < 0.01); the proliferation of astrocytes was decreased (P < 0.01) and the number of migration was decreased (P < 0.01) in the miR-146a-3p mimics group. To conclude, the expression of miR-146a-3p decreased and the expression of insulin-like growth factor 1 increased in spinal cord tissue after spinal cord injury. miR -146a-3p targeted regulation of insulin-like growth factor 1 in astrocytes, inhibited the proliferation and migration of astrocytes and promoted their apoptosis.  Figures and Tables | References | Related Articles | Metrics

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Role of synaptic input remodeling of corticospinal motor neurons after spinal cord injury Dai Jiafeng, Wang Lizhao, Han Qi, Shen Hongxing 2024, 28 (25):  4054-4059.  doi: 10.12307/2024.175 Abstract ( 135 )   PDF (2082KB) ( 56 )   Save BACKGROUND: The recovery of function after spinal cord injury depends on the functional remodeling of the motor cortex. However, the anatomical evidence underlying the functional remodeling of the motor cortex is still illusive. Analyzing the anatomical changes in the motor cortex after spinal cord injury can provide new ideas and research directions for regulating functional recovery and rehabilitation after spinal cord injury. OBJECTIVE: To analyze the neural circuit structural basis of functional remodeling of the primary motor cortex after spinal cord injury. METHODS: C57BL/6J mice were randomly divided into a sham operation group and a spinal cord injury group. The adeno-associated virus vectors expressing the fusion protein of Cre recombinase were injected into C4 of mice of both groups. The adeno-associated virus vectors with Cre recombinase-inducible expression of avian sarcoma/leukosis envelope glycoprotein receptor TVA and rabies glycoprotein were injected into the primary motor cortex. Fourteen days later, a C6 dorsal hemisection mice model was established in the spinal cord injury group. The pseudotyped rabies virus was injected into the primary motor cortex of mice of both groups. After 7 days, brain samples were collected and frozen sections were made. The distribution of input neurons innervating corticospinal motor neurons in the brain was observed and analyzed quantitatively.  RESULTS AND CONCLUSION: Fluorescence microscopy observation and quantitative analysis found that input neurons innervating corticospinal motor neurons of the primary motor cortex in mice of both groups were distributed in the cerebral cortex, thalamus and midbrain. Among them, in the sham operation group, the number of input neurons in the mouse cerebral cortex accounted for (84.0±3.6)% of total brain input neurons; that in the thalamus accounted for (10.6±2.3)%, and that in the midbrain accounted for (0.7±0.4)%. Direct synaptic input neurons in the spinal cord injury group accounted for (81.7±1.0)%, (13.1±0.5)%, and (1.6±0.8)% in the cerebral cortex, thalamus and midbrain, respectively. The proportion and number of primary motor cortex input neurons in the three regions of the spinal cord injury group did not differ significantly from that of the sham operation group. After spinal cord injury, the number of input neurons innervating corticospinal pyramidal motor neurons in various brain regions did not change significantly, suggesting that functional remodeling of the motor cortex after spinal cord injury may not only depend on changes in synaptic input related to injured corticospinal motor neurons, but also on transcriptional regulation changes within the injured neurons themselves. Figures and Tables | References | Related Articles | Metrics

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Effect of cyclic RNA hsa-circ-0001360 on homocysteine-induced apoptosis of human umbilical vein endothelial cells Kuang Yuanjun, Yu Sumei, Zhong Yingyi, Zhang Xuhong, Ma Shengchao, Yang Anning, Hao Yinju, Xiong Jiantuan, Jiao Yun, Jiang Yideng 2024, 28 (25):  4060-4064.  doi: 10.12307/2024.184 Abstract ( 114 )   PDF (999KB) ( 34 )   Save BACKGROUND: Increased homocysteine level induces apoptosis of human umbilical vein endothelial cells, but the mechanism remains unclear.  OBJECTIVE: To investigate the role of hsa-circ-0001360 in human umbilical vein endothelial cell apoptosis induced by homocysteine.  METHODS: In vitro cultured human umbilical vein endothelial cells were divided into control group, homocysteine group, interference control group, interference control + homocysteine group, hsa-circ-0001360 interference group, hsa-circ-0001360 + homocysteine interference group, overexpression control group, overexpression control + homocysteine group, hsa-circ-0001360 overexpression group and hsa-circ-0001360 + homocysteine overexpression group. All groups were treated with 100 μmol/L homocysteine. After 72 hours of intervention, the expressions of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 were detected by western blot assay. The apoptotic rate was detected by flow cytometry. Quantitative real-time PCR was used to detect the expression of hsa-circ-0001360.   RESULTS AND CONCLUSION: (1) Compared with the control group, the expression of Caspase-3 and Bax was significantly increased (P < 0.01), and the expression of Bcl-2 was significantly decreased (P < 0.01), and the apoptotic rate was significantly increased (P < 0.01) in the homocysteine group. (2) Compared with control group, the expression of hsa-circ-0001360 was significantly increased in the homocysteine group (P < 0.01). (3) The expression of hsa-circ-0001360 was significantly higher in the cytoplasm than that in the nucleus (P < 0.01). (4) Compared with the interference control C group and interference control + homocysteine group, the expressions of Caspase-3 and Bax were significantly decreased (P < 0.01), while the expression of Bcl-2 was significantly increased (P < 0.01); the apoptotic rate was significantly decreased (P < 0.01) in sh-hsa-circ-0001360 interference group and sh-hsa-circ-0001360 + homocysteine interference group. (5) Compared with overexpression control group and overexpression control + homocysteine group, the expressions of Caspase-3 and Bax were significantly increased (P < 0.01), while the expression of Bcl-2 was significantly decreased (P < 0.01); the apoptotic rate was significantly increased (P < 0.01) in the hsa-circ-0001360 overexpression group and the hsa-circ-0001360 + homocysteine overexpression group. (6) In conclusion, hsa-circ-0001360 can promote the apoptosis of human umbilical vein endothelial cells induced by homocysteine.  Figures and Tables | References | Related Articles | Metrics

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Action mechanism and advantages of mesenchymal stem cells for treating flap ischemia-reperfusion injury He Bo, He Zhijun, Liu Tao, Ma Suilu, Wei Xiaotao, Wang Weiwei 2024, 28 (25):  4065-4071.  doi: 10.12307/2023.789 Abstract ( 104 )   PDF (917KB) ( 48 )   Save BACKGROUND: Mesenchymal stem cells are used in flap ischemia-reperfusion injury due to their antioxidant and inflammatory inhibition, and angiogenesis induction. OBJECTIVE: To review the mechanism and latest treatment progress of mesenchymal stem cells in the treatment of flap ischemia-reperfusion injury, and to provide a basis for further theoretical research and clinical rational application. METHODS: We searched the relevant articles indexed in CNKI, WanFang and PubMed databases. Chinese and English search terms were “mesenchymal stem cells; flap ischemia reperfusion injury; conditioned medium; exosomes; oxidative stress; inflammatory reactions; angiogenesis”. Relevant literature since 2010 was searched, and 74 articles were finally included after excluding the literature that had little to do with the topic of the article, poor quality and outdated content.   RESULTS AND CONCLUSION: (1) Mesenchymal stem cells play significant roles in antioxidation, inhibition of inflammation and induction of angiogenesis and have great potential in the treatment of flap ischemia-reperfusion injury. (2) However, the defects of mesenchymal stem cells themselves and the decline of therapeutic effect in recent years have put the development and application of mesenchymal stem cells into a bottleneck period, and the research on the plasticity of mesenchymal stem cells conditioned medium and its exosomes and mesenchymal stem cells came into being, and the therapeutic effect was significantly better than the use of mesenchymal stem cells alone. (3) Therefore, a more comprehensive understanding of the mechanism of action and the latest treatment progress of mesenchymal stem cells in the treatment of flap ischemia-reperfusion injury is of great significance for the research of mesenchymal stem cells and the treatment of flap ischemia-reperfusion injury.  Figures and Tables | References | Related Articles | Metrics

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Application and mechanism of induced pluripotent stem cells in inherited heart disease models Ma Yangguang, Zhang Yayong, Meng Mingyao, Jin Zhihao, Li Yingming, Huang Yaoxuan, Han Shen, Li Yaxiong 2024, 28 (25):  4072-4078.  doi: 10.12307/2024.176 Abstract ( 126 )   PDF (896KB) ( 142 )   Save BACKGROUND: Inherited heart disease has a high prevalence and mortality rate, but its pathogenesis has not yet been clarified. Although relevant animal models have been established to provide a foundation for the pathogenesis research of inherited heart disease, the value of these research results has been significantly reduced due to differences among species. Therefore, a new model is needed to explore its occurrence and development.  OBJECTIVE: To review the current role of induced pluripotent stem cells in disease modeling and potential application prospects in various inherited heart diseases. METHODS: The first author searched the relevant articles published nearly 13 years in PubMed from January to March 2023. The search terms were “induced pluripotent stem cell, inherited heart disease, congenital heart disease”. Finally, 76 articles were included for analysis. RESULTS AND CONCLUSION: Since 2007, when induced pluripotent stem cells were induced from human somatic cells, many studies have been reported on disease-specific induced pluripotent stem cells. Due to the ability of disease-specific induced pluripotent stem cells to reproduce disease phenotypes, they are expected to become a new research tool for in vitro disease modeling, used to analyze pathogenesis and develop auxiliary drugs. In the research of cardiovascular genetic diseases, cardiomyocytes derived from patient-specific induced pluripotent stem cells contain gene mutations that are involved in cardiac dysplasia. Therefore, it can be used as a new tool to study the potential mechanisms of inherited heart disease. Up to now, induced pluripotent stem cells-derived cardiomyocytes have been widely used to study the molecular mechanisms of various genetic heart diseases, such as cardiac electrophysiological diseases, cardiomyopathy, and some syndromic inherited heart diseases. Figures and Tables | References | Related Articles | Metrics

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Current status and future of treatment of pulmonary fibrosis by mesenchymal stem cells and extracellular vesicles Wang Yanyang, Liu Chan, Yu Limei, He Zhixu 2024, 28 (25):  4079-4086.  doi: 10.12307/2024.193 Abstract ( 135 )   PDF (1289KB) ( 74 )   Save BACKGROUND: Despite a series of clinical treatment measures, the treatment of pulmonary fibrosis still faces challenges. In recent years, mesenchymal stem cells and their extracellular vesicles have attracted extensive attention as an emerging therapeutic strategy and are considered to be a promising means of treating pulmonary fibrosis.  OBJECTIVE: To systematically review the application of mesenchymal stem cells and their extracellular vesicles in the treatment of pulmonary fibrosis, to comprehensively understand their therapeutic mechanism, efficacy evaluation and problems, and provide reference and guidance for further research and clinical application in the future. METHODS: Using Chinese and English search terms “mesenchymal stem cells”, “ mesenchymal stem cell extracellular vesicles”, “pulmonary fibrosis”, we searched the CNKI and PubMed electronic journal databases. By means of manual reading and eliminating duplicate articles, 112 articles were selected, but 58 Chinese and English articles were finally included for summary. RESULTS AND CONCLUSION: (1) Mesenchymal stem cells and their extracellular vesicles have shown great potential in the treatment of pulmonary fibrosis, such as regulating inflammatory responses, inhibiting fibroblast proliferation, and promoting damaged tissue repair. Preliminary results from clinical trials have also shown some effects of the treatment, including improved lung function and quality of life in patients. (2) However, mesenchymal stem cells and extracellular vesicles in the treatment of pulmonary fibrosis still face some challenges. During treatment, technical challenges such as cell migration and intrachistological localization need to be addressed for it to accurately reach the damaged lung tissue. Furthermore, its long-term safety also needs to be further studied and improved. For translational medicine development, standardized procedures such as cell collection, cell isolation, cell culture, cell harvesting, and cell identification need to be refined. (3) Despite these challenges, through the joint efforts of scientific researchers and medical personnel, these problems are expected to be gradually solved. In the future, we can further improve treatment outcomes by optimizing treatment regimens and exploring individualized treatments. At the same time, in-depth research on the therapeutic mechanism of stem cells and their extracellular vesicles is expected to develop more efficient and safe therapeutic strategies.  Figures and Tables | References | Related Articles | Metrics

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Action mechanism of traditional Chinese medicine and mesenchymal stem cells regulating immune response in treatment of amyotrophic lateral sclerosis Wang Shaona, Gao Chen, Fan Feiyan, Liu Feixiang, Zhang Yunke 2024, 28 (25):  4087-4093.  doi: 10.12307/2024.146 Abstract ( 146 )   PDF (990KB) ( 112 )   Save BACKGROUND: Amyotrophic lateral sclerosis is a progressive neurodegenerative disease, which often leads to the death of neurons in the brain and spinal cord. The pathogenesis of amyotrophic lateral sclerosis is extremely complex, with high refractory rate and mortality rate. There are only two kinds of drugs for its treatment, so it is urgent to develop new treatment methods to improve the prognosis of patients. OBJECTIVE: To review the mechanism of Chinese medicine and mesenchymal stem cells regulating the immune response in the treatment of amyotrophic lateral sclerosis.  METHODS: “Traditional Chinese medicine, medical stem cells, ALS, immune response” were Chinese and English search terms. Articles were retrieved from WanFang, CNKI, PubMed, Web of Science and other databases from 2010 to 2023. Finally, 69 articles were included for review. RESULTS AND CONCLUSION: (1) The article summarizes in detail the five mechanisms of traditional Chinese medicine regulating the immune response in the treatment of amyotrophic lateral sclerosis: mainly including the promotion of expression of closed zone protein-1 and closed protein-5 by traditional Chinese medicine such as borneol and astragaloside IV to rebuild the integrity of the blood central nervous system barrier. Fufangteng Mixture can regulate the receptor molecules on the surface of the natural killer cells to inhibit their autotoxicity. The complement system factors such as Scutellaria barbata and patchouli can inhibit their abnormal activation. Tripterygium wilfordii and Uncaria rhynchophylla inhibit the activation of microglia by mediating the production of extracellular signal-regulated kinase 1/2 attenuated inducible nitric oxide synthase. Zuogui Pill and Trichosanthes kirilowii Root promote the expression of interleukin-10 and regulate T cells to improve the immune environment. (2) Through existing research, five mechanisms of mesenchymal stem cells regulating the immune response in the treatment of amyotrophic lateral sclerosis have been summarized, mainly including reducing the expression of aquaporin 4 and reducing endothelial nitric oxide synthase signal transduction to repair the integrity of the immune barrier; releasing indoleamine 2,3-dioxygenase, prostaglandin E2 and other factors to resist natural killer cell toxicity; secretion factor H interferes with the activity of invertase and inhibits abnormal activation of the complement system; regulating the CX3CL1/CX3CR1 system axis or secreting transforming growth factors β, which can change the phenotype of microglia and inhibit its activity by other ways; increasing the expression of interleukin-10 or activating the STATS phosphorylation pathway to restore T cell function. (3) At present, there are few studies on the combination of traditional Chinese medicine and mesenchymal stem cells in the treatment of amyotrophic lateral sclerosis. Relevant research reports have shown that Jiweiling Injection can promote stem cell proliferation and differentiation and that Buyang Huanwu decoction combined with bone marrow mesenchymal stem cells can significantly improve the integrity of the blood-brain barrier. In the future, further exploration is needed to explore the synergistic treatment effect of both on refractory amyotrophic lateral sclerosis. Figures and Tables | References | Related Articles | Metrics

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Biological mechanism of satellite cell aging in skeletal muscles and potential coping strategies Xie Yingao, Kong Jianda, Chen Yun, Li Zhilin, Xu Peng 2024, 28 (25):  4094-4100.  doi: 10.12307/2024.198 Abstract ( 144 )   PDF (1607KB) ( 63 )   Save BACKGROUND: Satellite cells are myogenic stem cells located between the muscle fiber membrane and the basement membrane. However, a comprehensive review of the aging mechanisms of satellite cells and their potential mitigation strategies is still lacking. This gap in knowledge hinders the effective guidance for current strategies aimed at attenuating skeletal muscle aging. OBJECTIVE: To review the mechanisms of satellite cell aging in skeletal muscle and the relevant strategies for mitigating this aging process. METHODS: Major databases were searched up to May 2023, including Web of Science, PubMed, China National Knowledge Infrastructure (CNKI), WanFang Data, and VIP. Chinese and English search terms included “skeletal muscle, satellite cells, aging, mechanism, and solution strategy”. After strict inclusion and exclusion criteria were applied, 78 articles were finally included. RESULTS AND CONCLUSION: (1) Satellite cells, situated between the muscle fiber membrane and basement membrane, possess proliferative and differentiative potential. They usually remain in a quiescent state but become activated in response to muscle tissue stimuli, participating in processes of repair and restoration of normal tissue structure. Aging leads to a reduction in satellite cell numbers, resulting in symptoms such as muscle weakness and decreased endurance. (2) Mechanisms of satellite cell aging primarily involve diminished regenerative capacity, perturbed niche interactions with changing ecology, age-dependent loss, and heterogeneity changes. Reduced satellite cell numbers and activity due to aging lead to slower muscle regeneration and increased injury recovery time. Errors during differentiation may occur, resulting in decreased muscle quality and function deterioration. (3) Strategies for mitigating satellite cell aging encompass modulation of the receptor environment of intra-body satellite cells, peripheral interventions to promote satellite cell regeneration, construction of human muscle models, and exercise and nutritional interventions to induce satellite cell proliferation. These strategies hold promise in offering novel insights and methods for satellite cell regeneration and treatment of skeletal muscle diseases. (4) Future research should delve into the mechanisms of satellite cell aging, explore the interaction between satellite cells and their niches, investigate the relationship of satellite cells with the immune system and mitochondrial function, and develop human muscle models to enhance research depth and accuracy. Figures and Tables | References | Related Articles | Metrics