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08 November 2022, Volume 26 Issue 31 Previous Issue    Next Issue

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Mitochondrial dysfunction affects osteogenic differentiation potential of bone marrow mesenchymal stem cells Gu Chao, Chen Weikai, Liu Tao, Yang Huilin, He Fan 2022, 26 (31):  4921-4927.  doi: 10.12307/2022.727 Abstract ( 464 )   PDF (2707KB) ( 33 )   Save BACKGROUND: The occurrence of osteoporosis is closely related to the damage of cellular antioxidant system, and mitochondria is the main part of cellular energy metabolism and oxidative stress. The effect of mitochondrial injury on the osteogenic differentiation of bone marrow mesenchymal stem cells and its specific mechanism remain to be explored.   OBJECTIVE: To investigate the effect of high level of oxidative stress on mitochondrial function and antioxidant capacity, and the effect of mitochondrial injury on the osteogenic differentiation potential of bone marrow mesenchymal stem cells. METHODS:  The rat model of osteoporosis was established by ovariectomy. Bone marrow mesenchymal stem cells were isolated from femurs of female rats in ovariectomized group and sham operation group. Reactive oxygen species detection kits and MitoSOX Red fluorescent probe were used to detect reactive oxygen species levels in bone marrow mesenchymal stem cells and mitochondria. Subsequently, JC-1 fluorescent probe was used to detect the mitochondrial membrane potential level of the two kinds of stem cells. ATP production was measured by the ATP detection kit. The changes of mitochondrial function were analyzed in both groups. qPCR and western blot assay were used to detect the mRNA and protein expression of key genes of respiratory chain and antioxidant enzyme superoxide dismutase 2 in two kinds of stem cells. Finally, the two groups of stem cells were subjected to osteogenic induction for 14 days. Alizarin red staining and qPCR were utilized to determine osteogenic related gene expression.   RESULTS AND CONCLUSION: (1) There was high oxidative stress level in rat bone marrow mesenchymal stem cells of the ovariectomized group, and the superoxide in the mitochondria also maintained a high level. (2) The mitochondrial membrane potential and ATP content of rat bone marrow mesenchymal stem cells were decreased significantly, and the mitochondrial function was impaired in the ovariectomized group. (3) In the ovariectomized group, the respiratory chain was damaged and the expression of antioxidant enzyme superoxide dismutase 2 was decreased in rat bone marrow mesenchymal stem cells. (4) The osteogenic differentiation potential of rat bone marrow mesenchymal stem cells was decreased in the ovariectomized group. (5) These findings confirm that the level of oxidative stress in bone marrow mesenchymal stem cells from osteoporosis is increased, and the mitochondrial function is impaired, which ultimately leads to the decrease of osteogenic differentiation potential of stem cells. Figures and Tables | References | Related Articles | Metrics

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Effects of concentrated growth factors on the proliferation, osteogenic differentiation and migration of rat bone marrow mesenchymal stem cells Tang Kai, Zhao Wenhua, Shang Qi, Shen Gengyang, Zhang Zhida, He Jiahui, Zhang Peng, Yu Fuyong, Chen Guifeng, Ren Hui, Jiang Xiaobing, Yu Xiang 2022, 26 (31):  4935-4939.  doi: 10.12307/2022.732 Abstract ( 479 )   PDF (1737KB) ( 76 )   Save BACKGROUND: On the basis of studying the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, it is one of the hot topics in regenerative medicine to reveal their migration mechanism.   OBJECTIVE: To investigate the effects of concentrated growth factor on proliferation, osteogenic differentiation, and migration of rat bone marrow mesenchymal stem cells. METHODS:  Concentrated growth factor was extracted from rat heart vein by variable speed centrifugation. Rat bone marrow mesenchymal stem cells were cultured in vitro and divided into experimental and control groups. Cells in the experimental groups were cultured in α-MEM complete medium supplemented with 5%, 10% and 20% concentrated growth factors by volume fraction, separately. Cells in the control group were cultured in α-MEM complete medium without concentrated growth factor. Cell proliferation was detected by CCK-8 assay. Cell migration was detected by scratch test and Transwell migration chamber assay. Osteogenic differentiation was detected by alizarin red staining.   RESULTS AND CONCLUSION: (1) Concentrated growth factor with different volume fractions could promote the proliferation of rat bone marrow mesenchymal stem cells with different volume fractions. (2) The scratch test showed that the migration of rat bone marrow mesenchymal stem cells could be promoted in the experimental groups, and the migration rate of 10% concentrated growth factor group was fastest at 12 hours. Transwell migration chamber assay showed that the number of migrated rat bone marrow mesenchymal stem cells in the experimental groups was higher than that in the control group. The number of migrated rat bone marrow mesenchymal stem cells was highest in the 10% concentrated growth factor group. (3) Alizarin red staining showed that the total number of calcium nodules in the experimental groups was higher than that in the control group on day 7. (4) The results showed that all concentrated growth factors with different volume fractions could promote the proliferation, differentiation, and migration of rat bone marrow mesenchymal stem cells in vitro. The 10% concentrated growth factor had a faster ability to promote the migration of rat bone marrow mesenchymal stem cells. Figures and Tables | References | Related Articles | Metrics

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Hydrogel combined with bone marrow mesenchymal stem cells in the treatment of damaged endometrium in rats Lyu Yan, Guan Yongge, Song Yang, Li Yue 2022, 26 (31):  4940-4945.  doi: 10.12307/2022.776 Abstract ( 433 )   PDF (2228KB) ( 108 )   Save BACKGROUND: As a scaffold material for tissue engineering, hydrogels provide a good physical support for the proliferation and differentiation of transplanted cells in vivo.  OBJECTIVE: To investigate the effect of hydrogel combined with bone marrow mesenchymal stem cells on endometrial reproduction in rats.  METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were isolated, cultured, and labeled with CM-Dil and were prepared for Pluronic F-127-bone marrow mesenchymal stem cells complex. A total of 30 Sprague-Dawley rats aged 8 weeks old were randomly divided into sham operation group, model group, hydrogel group, stem cell group, and complex group. Except for the sham operation group, endometrial mechanical injury models were established in the other four groups and each uterine horn was injected with 0.2 mL of PBS, Pluronic F-127 hydrogel, bone marrow mesenchymal stem cell suspension, and Pluronic F-127 hydrogel-bone marrow mesenchymal stem cell suspension, respectively. After 7 days of observation, the uterus tissues were removed for hematoxylin-eosin staining, immunfluorescence staining, and enzyme-linked immunosorbent assay.  RESULTS AND CONCLUSION: (1) Compared with the sham operation group, the endometrial thickness of the rats was thinner in the model group, hydrogel group, stem cell group, and complex group (P < 0.05). Compared with the model group, the endometrial thickness of hydrogel, stem cell, and complex groups was significantly improved (P < 0.05). The complex group had the most obvious effect, while there was no significant difference between hydrogel group and stem cell group (P > 0.05). (2) Compared with stem cell group, the number of stem cells labeled with CM-Dil migrated to the endometrium was more in the complex group (P < 0.01) and both groups had the potential to differentiate into endometrial stromal cells. (3) Compared with the model group, the levels of interleukin-1β, interleukin-6, and tumor necrosis factor α were significantly decreased in the endometrium of the hydrogel, stem cell, and complex groups (P < 0.01). The levels of interleukin-1β, interleukin-6, and tumor necrosis factor α were significantly lower in the stem cell and complex groups than those in the hydrogel group (P < 0.05). The levels of interleukin-6 and tumor necrosis factor α were lower in the stem cell group than those in the complex group (P < 0.01); however, there were no significant differences in interleukin-1β levels between the two groups (P > 0.05). (4) The results suggest that Pluronic F-127 combined with bone marrow mesenchymal stem cells is feasible for the treatment of endometrial injury and provides new ideas for endometrial repair.  Figures and Tables | References | Related Articles | Metrics

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Vascular endothelial growth factor-Notch signaling pathways in endothelial precursor cells in promoting the transformation of bone marrow mesenchymal stem cells into hepatocyte-like cells Yu Yunbao, Chen Lin, Wu Xiya, Yan Lerong, Miao Zhang, Man Yang, Jing Renyi, Lei Zhen, Chu Zhiqiang, Zhang Hongwei 2022, 26 (31):  4946-4953.  doi: 10.12307/2022.736 Abstract ( 413 )   PDF (3719KB) ( 46 )   Save BACKGROUND: Previous studies have shown that endothelial precursor cells produced factors that could promote bone marrow mesenchymal stem cells differentiation by paracrine in vitro, and vascular endothelial growth factor was one of them.   OBJECTIVE: To investigate whether vascular endothelial growth factor-Notch signaling pathway participates in the way that endothelial precursor cells promote the transformation of bone marrow mesenchymal stem cells to hepatocyte-like cells. METHODS:  (1) Bone marrow mesenchymal stem cells and endothelial precursor cells of Sprague-Dawley rats were isolated and cultured. The bone marrow mesenchymal stem cells at passage 3 were divided into three groups. In the experimental group, the serum-free and factor endothelial precursor cells-conditioned medium was mixed with hepatocyte transformation medium in a volume ratio of 1:1. In the model control group, serum-free alpha-minimal essential medium was mixed with the hepatocyte transformation medium in a volume ratio of 1:1. In the blank control group, normal alpha-minimal essential medium was used. The expression levels of alpha-fetoprotein and albumin were detected by immunofluorescence at 3, 5, 7, and 9 days of culture. (2) The third generation of bone marrow mesenchymal stem cells was taken to transform into hepatocyte-like cells and grouped. The serum-free and factor endothelial precursor cells-conditioned medium, vascular endothelial growth factor, and serum-free and factor vascular endothelial growth factor-siRNA-endothelial precursor cells-conditioned medium were added in hepatocyte transformation medium in a volume ratio of 1:1. In the control group, the samples were cultured with the hepatocyte transformation medium. At 48 hours after culture, the expression changes of Dll4, Hes1, and Hey1 (downstream gene of Notch) in bone marrow mesenchymal stem cells were detected by RT-PCR and western-blot assay.   RESULTS AND CONCLUSION: (1) Alpha-fetoprotein and albumin were not found in the blank control group. Alpha-fetoprotein and albumin levels were increased in the model control group and the experimental group (P < 0.05), and alpha-fetoprotein and albumin levels were significantly increased in the experimental group compared with the model control group (P < 0.05). (2) After adding serum-free and factor endothelial precursor cells-conditioned medium or vascular endothelial growth factor, the expression of Dll4 and Hey1 protein and mRNA increased (P < 0.05), but the expression of Hes1 protein and mRNA decreased (P < 0.05). After adding serum-free and factor vascular endothelial growth factor-siRNA-endothelial precursor cells-conditioned medium, the expression of Dll4 and Hey1 protein and mRNA decreased, and the expression of Hes1 protein and mRNA increased (P < 0.05). (3) The results show that the bone marrow endothelial precursor cells may play a certain role in the transformation of bone marrow mesenchymal stem cells into hepatocyte-like cells, and the vascular endothelial growth factor-Notch signaling pathway may be involved in this process. Figures and Tables | References | Related Articles | Metrics

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Preparation of wogonoside polycaprolactone-polyethylene glycol micelles delivered by adipose stem cells Wang Xiao, Liu Qing, Hu Yaorui, Gu Chengxu, Guo Qixuan, Zhu Yonglin, Zhang Luping 2022, 26 (31):  4996-5001.  doi: 10.12307/2022.780 Abstract ( 371 )   PDF (3219KB) ( 29 )   Save BACKGROUND: Wogonoside has been proven to have anti-inflammatory, neuroprotective and other pharmacological activities. Wogonoside is difficult to dissolve in water and has a short half-life. In an organic solvent, wogonoside may bring patients with serious adverse reactions. Polycaprolactone-polyethylene glycol can combine highly hydrophobic drugs and achieve sustained release, but cannot accurately target the lesion. Micelles, because of their small size, can penetrate cell membranes. OBJECTIVE: To prepare wogonoside polycaprolactone-polyethylene glycol micelles and adipose stem cells as a carrier to deliver wogonoside polycaprolactone-polyethylene glycol micelles so as to overcome the insolubility and short half-life of wogonoside and enable the drug to target the damage site. METHODS: Wogonoside polycaprolactone-polyethylene glycol micelles were prepared by dialysis method and characterized. Encapsulation rate, drug loading and in vitro drug release rate were detected. SD rat adipose stem cells were prepared by collagenase digestion. The expression of CD90, CD44, and CD45 was detected by flow cytometry, immunohistochemistry, and immunofluorescence. After 14 days of adipocytes induction, oil red O staining was performed. CCK8 assay was used to detect cytotoxicity of wogonoside polycaprolactone-polyethylene glycol micelles to adipose stem cells. The micelles of coumarin 6 and wogonoside were co-incubated with adipose stem cells for cell localization.  RESULTS AND CONCLUSION: (1) The wogonoside polycaprolactone-polyethylene glycol micelles had a mean particle size of (26.7±0.4) nm, a Zeta potential of (-33.2±0.3) mV, and a relatively uniform shape of round ball. (2) The encapsulation rate of wogonoside polycaprolactone-polyethylene glycol micelles was (90.2±2.04)% and the drug loading was (9.18±0.31)%. (3) The cumulative release rate of free wogonoside was 98% for 24 hours, and that of wogonoside polycaprolactone-polyethylene glycol micelles was 87% for 96 hours. (4) Flow cytometry, immunohistochemistry, and immunofluorescence showed that CD90 and CD44 were highly expressed, while CD45 was almost not. Oil red O staining showed red droplets after lipid induction. (5) CCK8 assay showed that the micellar mass concentration in the range of 10-200 mg/L had no effect on cells. (6) Confocal laser confocal results showed that coumarin 6 and wogonoside polycaprolactone-polyethylene glycol micelles could enter the mitochondria of adipose stem cells. (7) The results showed that the wogonoside polycaprolactone-polyethylene glycol micelles could enter adipose stem cells, and adipose stem cells could carry wogonoside polycaprolactone-polyethylene glycol micelles.  wogonoside; polycaprolactone; polyethylene glycol; micelles; characterization; in vitro release; adipose stem cells Figures and Tables | References | Related Articles | Metrics

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Bax/Bcl-2 protein and apoptosis during adipose-derived stromal cell differentiation into astrocytes Zhang Lili, Ou Ya, Yuan Xiaodong, Zhang Pingshu 2022, 26 (31):  4982-4987.  doi: 10.12307/2022.777 Abstract ( 443 )   PDF (2434KB) ( 74 )   Save BACKGROUND: Neurodegenerative diseases are a large class of major neurological diseases that can lead to the loss of patients’ ability to work and live. At present, there is no effective treatment for this disease. Adipose-derived stromal cells can induce differentiation into nerve cells in vitro, which is expected to provide a new method for the treatment of this kind of diseases. OBJECTIVE: To explore the expression changes of Bax/Bcl-2 protein during the differentiation of adipose-derived stromal cells into astrocytes, and to further investigate whether there is mitochondrial pathway-mediated apoptosis during the induction.  METHODS: Immunocytochemistry and western blot assay were used to analyze the expression of glial fibrillary acidic protein, Bax, Bcl-2, cytochrome C, and casepase-3 in the process of adipose-derived stromal cell differentiation into astrocytes when induction for 48 hours and 7, 14, and 21 days. Transmission electron microscopy was used to observe the ultrastructure of cells and mitochondria.  RESULTS AND CONCLUSION: (1) After being induced for 48 hours and 7, 14, and 21 days, the expression of glial fibrillary acidic protein reached the peak on day 7 (P < 0.05). There was no significant difference in glial fibrillary acidic protein expression between 14 days and 7 days after induction (P > 0.05). (2) The expression of Bax, cytochrome C, and casepase-3 increased gradually with the induction time extending and reached the peak on day 14 (P < 0.05). The expression of Bcl-2 was decreased gradually and reached the lowest level on day 14 after induction (P < 0.05). (3) Typical apoptotic bodies and mitochondrial damage were observed using transmission electron microscopy. (4) Adipose-derived stromal cells were in the best survival and differentiation state at the 7th to 14th day of differentiation. During adipose-derived stromal cell differentiation into astrocytes, the inhibitory effect of Bcl-2 on apoptosis was significantly weakened, and the pro apoptotic effect of Bax was gradually enhanced. Cytochrome C was released into the cytoplasm by damaged mitochondria, which led to apoptosis in mitochondrial pathway. Figures and Tables | References | Related Articles | Metrics

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Modified primary culture and identification of human adipose-derived mesenchymal stem cells Wang Hui, Wang Cuiju, Zheng Junyuan, Wang Hua, Guo Baojuan, Chen Pei, Yang Xufang 2022, 26 (31):  5002-5007.  doi: 10.12307/2022.969 Abstract ( 465 )   PDF (1819KB) ( 46 )   Save BACKGROUND: Human adipose-derived mesenchymal stem cells were extracted by conventional enzymatic digestion. The long-term digestion resulted in poor cell viability. To meet the demand for the number of seed cells in tissue engineering, this experiment aimed to find a modified human adipose-derived mesenchymal stem cell culture method.   OBJECTIVE: Human adipose-derived mesenchymal stem cells were obtained by collagenase I digestion combined with tissue block culture method and the relevant biological characteristics were identified. METHODS: The adipose tissue was cut up and cultured with collagenase I digestion combined with tissue block culture and conventional collagenase digestion method. Cells were counted by trypan blue exclusion method. The yield, viability and proliferation ability of cells obtained by different methods were compared. The expression of cell surface antigens CD105, CD73 and CD44 was tested by flow cytometry in the modified group. After osteogenic or adipogenic induction, alkaline phosphatase staining and oil red O staining were used to identify cell differentiation ability. RESULTS AND CONCLUSION: (1) The number of cells obtained by collagenase I digestion combined with tissue block culture method was far more than that obtained by conventional enzyme digestion method. (2) The cells obtained by collagenase I digestion combined with tissue block culture had the strong proliferation ability and good cell viability. They entered the plateau phase 1 day earlier than the conventional collagenase digestion method, and each passage took about 5 days. (3) The expression of CD105, CD73 and CD44 was positive in the modified group of P3 generation, and the purity was over 93%. (4) Human adipose-derived mesenchymal stem cells obtained by the modified method had good osteogenic and adipogenic differentiation capabilities. (5) The results showed that the collagenase I digestion method combined with tissue block culture method could obtain a large number of human adipose-derived mesenchymal stem cells with better viability and proliferation ability than the conventional enzymatic digestion method, and they could be passaged stably with a good differentiation ability, which met the needs of tissue engineering.  Figures and Tables | References | Related Articles | Metrics

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Neuroprotective effect of umbilical cord mesenchymal stem cell-derived exosomes on hippocampal neurons in mice with intracerebral hemorrhage Wang Xue, Liu Yang, Xu Jianfeng, Long Qianfa, Wang Tong, Zhong Jun 2022, 26 (31):  4928-4934.  doi: 10.12307/2022.953 Abstract ( 549 )   PDF (4184KB) ( 222 )   Save BACKGROUND: Exosomes derived from mesenchymal stem cells can inhibit neuroinflammation and some damage after intracerebral hemorrhage. There are few studies on the mechanism of neuroprotective effect of exosomes derived from mesenchymal stem cells on hippocampal neurons after intracerebral hemorrhage through heme oxygenase-1.   OBJECTIVE: To explore the protection and mechanism of exosomes derived from umbilical cord mesenchymal stem cells on hippocampal neurons after intracerebral hemorrhage.  METHODS: (1) A mouse model of intracerebral hemorrhage was established using autologous blood infusion, and then exosomes derived from umbilical cord mesenchymal stem cells were injected into the tail vein at 8 hours after intracerebral hemorrhage. Western blot assay was used to verify the expression of heme oxygenase 1, high mobility group box B1 and Toll-like receptor 4 at 24 hours, 4 and 30 days after intracerebral hemorrhage. (2) In vitro, the mouse hippocampal neuron cell (HT22 cell) intracerebral hemorrhage model was established by hemin stimulation, and cells were pretreated with exosomes derived from umbilical cord mesenchymal stem cells for 12 hours before modeling, and then the HT22 cells were stimulated by hemin for 6 hours. Heme oxygenase 1 expression was detected by western blot assay. (3) HT22 cells were transfected with lentivirus to reduce the expression of heme oxygenase 1, and then pretreated with exosomes derived from umbilical cord mesenchymal stem cells for 12 hours, stimulated with hemin for 6 hours. The expression of heme oxygenase 1 was detected by western blot assay.  RESULTS AND CONCLUSION: (1) At 24 hours, 4 and 30 days after intracerebral hemorrhage, the expression of heme oxygenase 1 in the hippocampus on the injured side was significantly increased (all P < 0.05). Compared with the sham-operated group, the protein expression levels of heme oxygenase 1, high mobility group box B1 and Toll-like receptor 4 were significantly increased in the mice of the cerebral hemorrhage group on day 4 after cerebral hemorrhage (all P < 0.05). Compared with the intracerebral hemorrhage group, the protein expression levels of heme oxygenase 1, high mobility group box B1 and Toll-like receptor 4 in the intracerebral hemorrhage + exosome group were significantly decreased (all P < 0.05). (2) In the in vitro cerebral hemorrhage model, compared with the control group, the expression of heme oxygenase 1 protein in HT22 cells was significantly increased in the hemin+PBS group. Compared with the hemin+PBS group, the heme oxygenase 1 protein expression was significantly decreased in HT22 cells in the hemin+exosome group (all P < 0.05). (3) After lentiviral transfection of HT22 cells to knock down the expression of heme oxygenase 1, there was no significant difference in the protein expression of heme oxygenase 1 between the knockdown heme oxygenase 1+hemin+PBS group and the knockdown heme oxygenase 1+hemin+exosome group. (4) The results showed that exosomes derived from umbilical cord mesenchymal stem cells had a neuroprotective effect on injured hippocampal neurons after intracerebral hemorrhage. The mechanism may be related to the regulation of high mobility group box B1 and Toll-like receptor 4-related inflammatory pathways through heme oxygenase 1.  Figures and Tables | References | Related Articles | Metrics

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Omission of day 11 methotrexate in regimen for the prophylaxis of graft-versus-host disease after haploid hematopoietic stem cell transplantation Kong Dai, Chen Xiangli, Pei Xiaohang, Niu Xiaona, Chen Yuqing, Zhu Zunmin, Lei Pingchong, Sun Kai, Liu Zhongwen 2022, 26 (31):  5026-5031.  doi: 10.12307/2022.775 Abstract ( 1066 )   PDF (1178KB) ( 32 )   Save BACKGROUND: The regimen of cyclosporin combined with four times of short-range methotrexate is still recognized as the classic prevention regimen for acute graft-versus-host disease. Previous studies have shown that whether day 11 methotrexate is used in sibling transplantation has no effect on the incidence of acute graft-versus-host disease. However, the effect of reducing day 11 methotrexate on the incidence of acute graft-versus-host disease in haploid hematopoietic stem cell transplantation patients remains unclear.  OBJECTIVE: To investigate the efficacy of the omission of day 11 methotrexate in the regimen for the prophylaxis of graft-versus-host disease in haploid hematopoietic stem cell transplantation.   METHODS: The clinical data of 63 patients with malignant hematologic diseases who received haploid hematopoietic stem cell transplantation from January 2017 to December 2019 were retrospectively analyzed. The graft-versus-host disease prevention regimen was cyclosporine combined with methotrexate 15 mg/m2 on day 1, 10 mg/m2 on day 3, day 6 and day 11. In the observation group (n=19), oral mucositis was grade III-IV at day 11, and day 11 methotrexate was cancelled. In the control group (n=44), oral mucositis was grade 0-II at day 11, and day 11 methotrexate was applied. The implantation situation, incidence of acute graft-versus-host disease, overall survival rate, and recurrence rate of the two groups were analyzed.    RESULTS AND CONCLUSION: (1) The median follow-up time was 30(3-54) months and all neutrophils were successfully implanted in both groups. The median implantation time was 12(9-29) days and 12(8-25) days, respectively, showing no significant difference (P=0.682). There was one patient with poor platelet implantation in the observation group, and four patients with poor platelet implantation in the control group. The median time of platelet implantation was 12(9-18) days and 13(9-31) days in the two groups, respectively, (P=0.71), showing no statistical difference. (2) The overall incidence of acute graft-versus-host disease was 44.4%, and grade II-IV acute graft-versus-host disease was 28.6%. The incidence of II-IV acute graft-versus-host disease in the observation group and control group was 31.5% and 27.3%, respectively, (P=0.728), and there was no statistical difference between the two groups. (3) The results showed that for haploid hematopoietic stem cell transplantation, the omission of day 11 methotrexate did not increase the incidence of acute graft-versus-host disease compared with the standard methotrexate regimen.  Figures and Tables | References | Related Articles | Metrics

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Correlation of the level of plasma biomarkers with acute graft-versus-host disease in children Wen Jianyun, Miao Lili, Guan Di, Liu Xuan, Chen Libai, Feng Xiaoqin, Xu Xiaoxiao, Liu Qiujun, Wu Xuedong, He Yuelin 2022, 26 (31):  5032-5039.  doi: 10.12307/2022.739 Abstract ( 513 )   PDF (1514KB) ( 43 )   Save BACKGROUND: Acute graft-versus-host disease is one of the major complications of allogeneic hematopoietic stem cell transplantation, and it is also the key to the success of transplantation, which is particularly important to find specific biomarkers of acute graft-versus-host disease for the early diagnosis and treatment. The immune function of children is imperfect, and the changes of plasma biomarkers after acute graft-versus-host disease have their own characteristics.   OBJECTIVE: To explore the correlation of plasma levels of soluble growth stimulation expressed gene 2 (sST2), regenerating islet-derived protein 3 alpha (REG3α), tumor necrosis factor receptor 1 (TNFR1), interleukin 6 (IL6), and interleukin 8 (IL8) with acute graft-versus-host disease in children after allogeneic hematopoietic stem cell transplantation. It is expected to provide reliable detection biomarkers for early diagnosis of acute graft-versus-host disease and prediction of therapy effect and prognosis. METHODS:  Samples were collected from 127 pediatric patients who underwent allogeneic hematopoietic stem cell transplantation from March 2019 to December 2020 in the Department of Pediatrics, Nanfang Hospital. The plasma was collected at multiple time points, including 10 days before transplantation, 0, 7, 14, 28, and 90 days after transplantation, at the onset of acute graft-versus-host disease symptoms, 1, 2, and 4 weeks after acute graft-versus-host disease therapy. The plasma concentrations of sST2, REG3α, TNFR1, IL6 and IL8 were detected by the Luminex technology.   RESULTS AND CONCLUSION: (1) Plasma samples were collected from 100 of 127 patients at the point of occurrence of acute graft-versus-host disease. Sixty cases never developed acute graft-versus-host disease symptoms; 40 cases presented acute graft-versus-host disease, among which 9 cases developed II-IV gastrointestinal acute graft-versus-host disease according to EBMT-NIH-CIBMTR classification standard. (2) The plasma concentrations of sST2 and REG3α at onset point in patients were significantly higher in the acute graft-versus-host disease group compared with the non-acute graft-versus-host disease group (P < 0.05). sST2 was significantly increased at 7 days after transplantation in the acute graft-versus-host disease group than that in the non-acute graft-versus-host disease group (P < 0.05). The sST2 and REG3α levels were significantly higher in the II-IV gastrointestinal acute graft-versus-host disease group than those in the non-gastrointestinal acute graft-versus-host disease group (P < 0.01; P < 0.05). There were no significant differences in TNFR1, IL6 and IL8 levels at onset point and 7 days after transplantation in patients between the acute graft-versus-host disease group and the non-acute graft-versus-host disease group (P > 0.05). (3) In all acute graft-versus-host disease patients, the plasma concentrations of sST2 and REG3α in the steroid-resistant acute graft-versus-host disease group showed increasing tendency compared with steroid-sensitive acute graft-versus-host disease group. (4) It is concluded that the increate of plasma sST2 and REG3α levels at onset point after transplantation suggests the incidence of acute graft-versus-host disease. sST2 and REG3α in plasma can be helpful for the early prediction of acute graft-versus-host disease. By analyzing the levels of biomarkers at 1, 2, and 4 weeks after treatment, no decrease in the sST2 and REG3α levels in patients with acute graft-versus-host disease after treatment may be related to poor prognosis. Figures and Tables | References | Related Articles | Metrics

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Mechanism underlying the effect of Fuhebeihua Recipe on the proliferation of CD133+HepG2 hepatoma stem cells and related autophagy proteins Li Zulong, Rong Zhen, Sun Hua, Jiang Ruiyuan, Zhong Xiaoting, Mo Chunmei 2022, 26 (31):  4988-4995.  doi: 10.12307/2022.992 Abstract ( 338 )   PDF (1831KB) ( 217 )   Save BACKGROUND: Hepatoma stem cells are key cells for the continuous development and high heterogeneity of liver cancer. At present, there is no research on Chinese medicine compound that targets hepatoma stem cell-related autophagy pathways. Therefore, it is very necessary to study Chinese medicine compound prescription to inhibit hepatoma stem cell autophagy.   OBJECTIVE: To investigate the effect and mechanism of Fuhebeihua Recipe on PI3K/AKT/mTOR pathway and related autophagy proteins in CD133+HepG2 hepatoma stem cells. METHODS:  CD133+HepG2 cells were selected by immunomagnetic bead method. CD133+HepG2 cells were treated with different volume fractions of Fuhebeihua Recipe-containing serum of 2%, 4%, 8%, and 16%. The cell proliferation activity was detected by CCK-8 assay. The best volume fraction of Fuhebeihua Recipe-containing serum was selected. CD133+HepG2 cells were divided into blank control group, serum control group, rapamycin group, and Fuhebeihua Recipe group. Cells were separately cultured with DMEM/F12 containing serum of 16%, serum-free DMEM/F12, DMEM/F12 containing 0.05% rapamycin, and DMEM/F12 with Fuhebeihua Recipe-containing serum of 16%. The percentage of CD133+HepG2 hepatoma stem cells was detected by flow cytometry in each group. The mRNA expression levels of PI3K, AKT, mTOR, LC3-II, and beclin-1 were detected by RT-PCR. The protein expression levels of PI3K, p-AKT, AKT, p-mTOR, mTOR, and LC3-II were detected by western blot assay. The expression levels of LC3 and LC3-II were detected by immunofluorescence in each group.   RESULTS AND CONCLUSION: (1) The Fuhebeihua Recipe-containing serum inhibited the proliferation of CD133+HepG2 cells, and the inhibitory effect was the most obvious when the volume fraction was 16% (P < 0.05). (2) Compared with serum control group and blank control group, the percentage of CD133+HepG2 cells reduced (P < 0.05); protein expression levels of PI3K, AKT, p-mTOR, mTOR, and LC3-II were significantly decreased (P < 0.05); mRNA expression levels of PI3K, AKT, mTOR, LC3-II, and Beclin-1 were significantly down-regulated (P < 0.05); the fluorescence signal intensities of LC3 and LC3-II were significantly down-regulated (P < 0.05) in the Fuhebeihua Recipe group and rapamycin group. (3) Compared with the rapamycin group, the protein expression levels of PI3K, p-Akt, AKT, p-mTOR, mTOR, and LC3-II in CD133+HepG2 cells were significantly down-regulated (P < 0.05); the mRNA expression levels of PI3K, AKT, mTOR, LC3-II, and Beclin-1 were significantly decreased (P < 0.05); the fluorescence signal intensities of LC3 and LC3-II were significantly decreased in the Fuhebeihua Recipe group (P < 0.05). (4) It is concluded that Fuhebeihua Recipe can inhibit autophagy and proliferation of hepatoma stem cells, and its mechanism is related to the inhibition of PI3K/AKT/mTOR signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Expression of autophagy in rotator cuff tendon stem cells induced by oxidative stress Wei Hewei, Zheng Weipeng, Liu Zhijun, Zhao Guoyuan, Fang Weihua, Chen Sheng, Liao Zhihao, Wan Lei 2022, 26 (31):  4954-4961.  doi: 10.12307/2022.954 Abstract ( 410 )   PDF (6487KB) ( 162 )   Save BACKGROUND: The mechanism of rotator cuff tendon-bone healing has not yet been fully elucidated. Studies have shown that oxidative stress can lead to tissue cell damage, which is not conducive to wound healing. It is clinically found that the tendon-bone insertion point of rotator cuff injury often has a poor healing effect, and it is speculated that this may be related to the abnormal cell apoptosis in the wound under ischemia and hypoxia stress.  OBJECTIVE: To induce autophagy of rotator cuff tendon stem cells to promote cell survival as a breakthrough point by oxidative stress stimulation, it is envisaged that autophagy can alleviate cellular oxidative stress damage. METHODS: The rotator cuff tendon stem cells were cultured in vitro and treated with 0, 50, 100, 200, 400 μmol/L H2O2 respectively for 24, 48 and 72 hours, followed by CCK-8 assay to detect the cell viability. The oxidative stress injury model of rotator cuff tendon stem cells in vitro was established with an appropriate concentration of H2O2. The cultured rotator cuff tendon stem cells at passage 3 were divided into three groups: blank group, H2O2 (50 μmol/L) intervention group, and 3-methyladenine pretreatment (5 mmol/L, 30 minutes)+H2O2 group. At 48 hours after intervention, autophagy was detected by MDC and Lyso-Tracker Red staining. Autophagy related genes were detected by gene expression microarray. Reactive oxygen species level was detected by DCFH-DA. Apoptosis was detected by Annexin V/PI. Protein expression levels of Beclin-1, p-mTOR, mTOR, LC3A/B and Caspase-3 were detected by western blot assay.  RESULTS AND CONCLUSION: (1) With the increase of H2O2 concentration, the proliferation activity of rotator cuff tendon stem cells significantly decreased (P < 0.05). When the concentration of H2O2 was 50 μmol/L, the proliferation activity of rotator cuff tendon stem cells was the strongest. (2) Compared with the blank group and 3-methyladenine+H2O2 group, the autophagy level of H2O2 group was significantly increased (P < 0.05). (3) Compared with the blank group and 3-methyladenine+H2O2 group, the level of reactive oxygen species was significantly increased (P < 0.05); the expression of Beclin-1 protein was up-regulated (P < 0.05); the expression of mTOR protein was down-regulated (P < 0.05); the expression of LC3A/B protein was up-regulated (P < 0.05); and the expression of Caspase-3 protein was up-regulated (P < 0.05) in the H2O2 group. (4) These results indicate that the low concentration of H2O2 can promote the proliferation of human tendon stem cells, and the degree of apoptosis and reactive oxygen species level also increase slightly in a certain range, which is a state of high cell transformation. Autophagy may play a cytoprotective role and promote this high transformation state, but the specific mechanism needs to be further studied.  Figures and Tables | References | Related Articles | Metrics

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Effect and mechanism of conditioned medium from hypoxia preconditioned human Wharton’s Jelly derived mesenchymal stem cells on myocardial ischemia/reperfusion injury Li Yan, Zhang Yan, Zhang Ningkun, Chen Yu 2022, 26 (31):  5008-5013.  doi: 10.12307/2022.955 Abstract ( 440 )   PDF (2175KB) ( 41 )   Save BACKGROUND: Mesenchymal stem cells improve tissue injury mainly through their paracrine action, which is enhanced by hypoxic preconditioning. However, the effect of conditioned medium from hypoxia preconditioned human Wharton’s Jelly derived mesenchymal stem cells on myocardial ischemia/reperfusion injury and its mechanism still need to be further studied.  OBJECTIVE: To investigate the effect and mechanism of conditioned medium from hypoxia preconditioned human Wharton’s Jelly derived mesenchymal stem cells on the apoptosis of H9C2 cells after ischemia/reperfusion injury.  METHODS: Human Wharton’s Jelly derived mesenchymal stem cells were treated with normoxia or hypoxia to obtain conditioned medium. The H9C2 cells were subjected to hypoxia/reoxygenation and serum-free culture for 6 hours to mimic the myocardial ischemia/reperfusion injury model in vitro. Lactate dehydrogenase activity in H9C2 conditioned medium was detected using lactate dehydrogenase assay kit. Rat myocardial H9C2 cells were divided into four groups: normoxic culture group, model group, normoxic conditioned medium group and hypoxic conditioned medium group. After treatment with normoxic or hypoxic conditioned medium for 24 hours after hypoxia, the apoptosis rate of H9C2 cells was detected by flow cytometry. Western blot assay was applied to detect the expression of apoptotic markers (Bax and Bcl-2) and autophagic markers (Beclin-1, P62 and LC3) in each group. RESULTS AND CONCLUSION: (1) The morphology of H9C2 cells did not change significantly after treatment with ischemia/reperfusion or human Wharton’s Jelly derived mesenchymal stem cells-conditioned medium. (2) Compared with the normoxic culture group, lactate dehydrogenase activity in supernatant of H9C2 cells in the model group was significantly increased, but decreased in both conditioned medium groups. (3) Compared with the model group, the apoptosis rate of H9C2 and the expression of Bax, Beclin-1, and LC3 protein were downregulated and the expression of p62 protein was upregulated in H9C2 cells in the normoxic conditioned medium group and hypoxic conditioned medium group. The change was more significant in the hypoxic conditioned medium group than that in the normoxic conditioned medium group. (4) The results demonstrated that the conditioned medium from hypoxia preconditioned human Wharton’s Jelly derived mesenchymal stem cells could reduce the ischemia/reperfusion injury induced apoptosis of H9C2, and the protective effect may be related to the inhibition of autophagy. Figures and Tables | References | Related Articles | Metrics

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Human amniotic epithelial cells regulate the proliferation, apoptosis and extracellular matrix synthesis of articular chondrocytes by activating the EGFR/ERK1 signaling axis Wang Kang, Zhi Xiaodong, Zhang Yuqiang, Gong Chao, Wang Chenliang, Wang Wei 2022, 26 (31):  4967-4974.  doi: 10.12307/2022.993 Abstract ( 480 )   PDF (11302KB) ( 31 )   Save BACKGROUND: Previous studies have found that human amniotic epithelial cells have the effect of promoting the repair of osteoarthritis, but the mechanism of action of human amniotic epithelial cells on cartilage regeneration is not yet clear.   OBJECTIVE: To explore the effect of human amniotic epithelial cells on chondrocyte proliferation, apoptosis and extracellular matrix synthesis and its mechanism.  METHODS: The human amniotic epithelial cells were separated by enzymatic digestion. Human amniotic epithelial cells, articular chondrocytes, and epidermal growth factor receptor signaling pathway inhibitor (AG1478) were co-cultured in a Transwell chamber. The chondrocyte proliferation, apoptosis, and extracellular matrix synthesis were detected using CCK-8, flow cytometry, and RT-qPCR, respectively. The expression of related proteins in the epidermal growth factor receptor signaling pathway was determined by western blot assay.   RESULTS AND CONCLUSION: (1) After co-culture with human amniotic epithelial cells, the proliferation of chondrocytes was significantly enhanced; the apoptotic rate was significantly reduced; the expression levels of COL2, COL1, Aggrecan, and SOX-9 mRNA were significantly increased; and the protein expression level of p-ERK1 was significantly increased. (2) After the addition of epidermal growth factor receptor signaling pathway inhibitor AG1478, the proliferation of chondrocytes was significantly weakened; the apoptotic rate was significantly increased; COL2, COL1, Aggrecan, and SOX-9 mRNA expression decreased; protein expression level of p-ERK1 was significantly reduced. (3) It is concluded that human amniotic epithelial cells can promote the proliferation of chondrocytes and the synthesis of extracellular matrix, and inhibit their apoptosis, and its effect may be regulated by the EGFR/ERK1 signaling axis.  Figures and Tables | References | Related Articles | Metrics

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Mechanism by which echinacoside delays the senescence of human umbilical vein endothelial cells Tian Tian, Ouyang·Juyan, Li Yu, Miyesai·Ainiwaer, He Juanli, Li Zhenhua, Wang Hong 2022, 26 (31):  5014-5019.  doi: 10.12307/2022.970 Abstract ( 416 )   PDF (1828KB) ( 51 )   Save BACKGROUND: Preliminary work has found that homocysteine accelerates the aging of umbilical vein endothelial cells through reactive oxygen species. This experiment preliminarily studies the relevant mechanisms affecting endothelial cell aging. OBJECTIVE: To investigate the effects of echinacoside on the expression of cytoplasmic polyadenylate binding protein 1 (CPEB1), sirtuin1 (SIRT1), P53 and P21 genes in human umbilical vein endothelial cells. METHODS: Human umbilical vein endothelial cells were cultured in vitro. D-galactose 10 g/L was used to construct cell aging model. The proliferation viability of cells treated with echinacoside (0, 10, 20, 50, 100, 200, 300, 400, 500 mg/L) for 12 and 24 hours was analyzed by CCK-8 assay, and the optimal mass concentration and intervention time of echinacoside were screened. Human umbilical vein endothelial cells were divided into normal control group, D-galactose group and echinacoside groups (100, 200, and 300 mg/L). β-Galactosidase staining was used to detect the positive staining rate of cells after echinacoside pre-intervention for 12 hours. Real time RT-PCR and western-blot assay were used to detect the mRNA expression levels of CPEB1, Sirt1, P53 and P21 and the protein expression levels of CPEB1, Sirt1 and P21 in human umbilical vein endothelial cells after echinacoside pretreatment for 12 hours.  RESULTS AND CONCLUSION: (1) The results of CCK-8 assay showed that the survival rate of echinacoside group at 12 hours was significantly higher than that at 24 hours. Compared with the D-galactose group, cell viability increased in the echinacoside 100, 200, and 300 mg/L groups to varying degrees (P < 0.000 1), and there was a significant difference among the three groups (P < 0.01). (2) Compared with the normal control group, the D-galactose group had the highest positive rate of β-galactosidase staining (P < 0.0001). 100, 200, and 300 mg/L echinacoside pretreatment for 12 hours could reduce the positive rate of β-galactosidase staining to a certain extent (P < 0.000 1). (3) Compared with the normal control group, the mRNA expression levels of CPEB1, P53 and P21 were increased (P < 0.000 1), and the expression level of SIRT1 mRNA was decreased (P < 0.000 1) in the D-galactose group. Compared with the D-galactose group, 100, 200, and 300 mg/L echinacoside pretreatment for 12 hours decreased the mRNA expression levels of CPEB1, P53, and P21 (P < 0.000 1), but increased Sirt1 mRNA expression (P < 0.000 1). Western blot assay results were consistent with real-time fluorescence quantitative polymerase chain reaction results. (4) These findings indicate that echinacoside may delay D-galactose-induced human umbilical vein endothelial cell senescence by down-regulating CPEB1 and P53/P21 expression levels and up-regulating Sirt1 expression levels. Figures and Tables | References | Related Articles | Metrics

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Effect of phosphocreatine modified chitosan hydrogel on polarization and inflammatory factor expression in rat bone marrow-derived macrophages Sheng Weibei, Xiong Ao, Liu Su, Deng Jiapeng, Weng Jian, Yu Fei, Chen Yingqi, Zeng Hui 2022, 26 (31):  5040-5046.  doi: 10.12307/2022.959 Abstract ( 500 )   PDF (1920KB) ( 130 )   Save BACKGROUND: Macrophage polarization-mediated inflammation plays an important role in chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. Phosphocreatine-modified chitosan hydrogels show good potential application prospects in tissue repair, but the effect of this new hydrogel on polarization and inflammatory factor expression in macrophages is still unclear.  OBJECTIVE: To explore the effect of phosphocreatine modified methacrylic anhydride chitosan hydrogel on macrophage polarization and inflammatory factor expression.  METHODS: One-step freeze-drying method was used to prepare phosphocreatine modified methacrylic anhydride chitosan with good water solubility. Hydrogels were further prepared under low toxicity initiators and ultraviolet irradiation. Bone marrow-derived macrophages were extracted from SD rats and cultured for 7 days, and then divided into three groups. Complete cell culture medium was added in the control group. Complete cell culture medium containing lipopolysaccharide was added in the lipopolysaccharide group. Freeze-dried hydrogel samples and cell complete medium containing lipopolysaccharide were added in the chitosan hydrogel group. Cell viability was detected using CCK-8 assay. RT-PCR, ELISA, western blot assay, and immunofluorescence staining were used to detect macrophage polarization and the expression of inflammatory factors.  RESULTS AND CONCLUSION: (1) CCK-8 assay showed that after 1, 3, and 5 days of culture, there was no significant difference in the cell viability among the three groups (P > 0.05). (2) Western blot assay and immunofluorescence staining showed that compared with the control group, the M2 macrophage surface marker CD206 protein expression was down-regulated (P < 0.05) and the ARG1 protein expression was up-regulated (P < 0.05) in the lipopolysaccharide group after 1 and 3 days of culture. Compared with the lipopolysaccharide group, the protein expression levels of CD206 and ARG1 in the chitosan hydrogel group were up-regulated after 3 days of culture (P < 0.05). (3) ELISA and RT-PCR results showed that compared with the control group, the protein and mRNA expression levels of pro-inflammatory factors interleukin-1β and interleukin-6 in the lipopolysaccharide group increased after 1 and 3 days of culture (P < 0.05). Compared with the lipopolysaccharide group, the protein and mRNA expression levels of interleukin-1β and interleukin-6 in the chitosan hydrogel group were decreased after 1 and 3 days of culture (P < 0.05). (4) These results have shown that phosphocreatine modified methacrylic anhydride chitosan hydrogel promotes M2-type polarization of macrophages and inhibits the expression of pro-inflammatory factors, which may provide a potential treatment for immune-related diseases in the future. Figures and Tables | References | Related Articles | Metrics

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Apoptosis of mouse bone marrow-derived macrophages induced by lipopolysaccharide Zheng Meijie, Yang Weizhe, Liu Jialin, Han Xiangzhen, Zhou Qiqi, He Huiyu 2022, 26 (31):  4962-4966.  doi: 10.12307/2022.738 Abstract ( 415 )   PDF (1846KB) ( 42 )   Save BACKGROUND: Macrophage is an immune defense line in response to external stimuli. The related reaction of any biological material implanted in vivo is related to the immune response mediated by macrophage.   OBJECTIVE: To investigate the effect of lipopolysaccharide on the apoptosis of bone marrow-derived macrophages in mice. METHODS:  The bone marrow-derived macrophages of C57BL/6 mice were isolated in vitro and cultured with DMEM containing 0 (control), 10, 100, and 1 000 µg/L lipopolysaccharide for 24 hours. Apoptotic rate was identified by flow cytometry. Hoechst staining was used to observe the cell morphology. RT-PCR was used to detect the mRNA expression levels of interleukin 1, interleukin 6, tumor necrosis factor alpha, and apoptosis-related factors Bax and P53.   RESULTS AND CONCLUSION: (1) The apoptotic rate of the lipopolysaccharide group at each mass concentration was higher than that of the control group (P < 0.05), and the apoptotic rates of the 100, 1 000 µg/L lipopolysaccharide groups were higher than that of the 10 µg/L lipopolysaccharide group (P < 0.05). (2) Hoechst staining showed that the nucleus of the control group was normal, and the chromatin was concentrated when the cells underwent apoptosis. The cells in the lipopolysaccharide group of various mass concentrations experienced different degrees of apoptosis, and the nuclei of the apoptotic cells were densely stained or fragmented densely stained; the color was slightly whitish, especially at 1 000 µg/L. (3) RT-PCR detection showed that the expression levels of interleukin 1, interleukin 6, tumor necrosis factor α, Bax, and P53 mRNA in the cells of the lipopolysaccharide group of various mass concentrations were higher than those of the control group (P < 0.01 or P < 0.001). With the increased concentration of lipopolysaccharide, the expression levels of interleukin 1, interleukin 6, and tumor necrosis factor α mRNA showed a trend of first increasing and then decreasing, and the expression of Bax and P53 mRNA showed an increasing trend. (4) Results have confirmed that lipopolysaccharide can induce the apoptosis of bone marrow-derived macrophages. Figures and Tables | References | Related Articles | Metrics

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Effect of eriodictyol on the imbalance of mitochondrial dynamics and apoptosis in SH-SY5Y cells induced by hydrogen peroxide Li Suyao, Guo Minfang, Yu Jingwen, Meng Tao, Mu Bingtao, Li Mengdi, Li Na, Song Lijuan, Ma Cungen, Yu Jiezhong 2022, 26 (31):  4975-4981.  doi: 10.12307/2022.709 Abstract ( 489 )   PDF (3077KB) ( 124 )   Save BACKGROUND: Abnormal mitochondrial dynamics is closely related to oxidative stress after affecting neurodegenerative diseases. Our previous studies have shown that eriodictyol can reduce nerve injury in Alzheimer’s disease, but whether it has a regulatory effect on mitochondrial dynamics is not clear.   OBJECTIVE: To explore the mechanism of eriodictyol on SH-SY5Y cell apoptosis induced by hydrogen peroxide (H2O2). METHODS:  SH-SY5Y cells were divided into three groups: PBS control group, H2O2 (250 μmol/L) group, and H2O2 (250 μmol/L) plus eriodictyol (10 μmol/L) treatment group (eriodictyol group). The intervention was performed for 24 hours. The kits were utilized to observe the level of malondialdehyde and the activity of superoxide dismutase. Cell morphology was observed under the microscope. TUNEL staining was used to observe the apoptosis. JC-1 staining was used to observe mitochondrial membrane potential. The expression levels of apoptotic proteins and mitochondrial fusion and fission protein-related proteins were detected by immunofluorescence staining and western blot assay.   RESULTS AND CONCLUSION: (1) A significant decrease of malondialdehyde levels and a significant increase of superoxide dismutase activity were found in eriodictyol treated group, when compared with the H2O2 group. (2) Compared with the PBS control group, the H2O2 group showed decreased cell density, cytosolic hypertrophy, increased apoptosis, decreased mitochondrial membrane potential, decreased Bcl-2 expression, and increased Bax expression. Compared with the H2O2 group, above indexes were significantly improved in the eriodictyol group. (3) Compared with PBS control group, the expression levels of mitochondrial fission-related proteins dynamin-related protein 1 (p-DRP1) and mitochondrial fission protein 1 (FIS1) were elevated and the expression levels of mitochondrial fusion-related proteins optic atrophic protein 1 (OPA1) and mitofusin 2 (Mfn2) were decreased in the H2O2 group. Compared with the H2O2 group, eriodictyol treatment decreased the expression levels of p-DRP1 and FIS1 and increased the expression of OPA1 and Mfn2. (4) The results indicate that eriodictyol can inhibit H2O2-induced apoptosis in SH-SY5Y cells by regulating mitochondrial dynamics. Figures and Tables | References | Related Articles | Metrics

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Effects of myeloid-derived growth factor on ventricular remodeling in aging mice Sun Ying, Xiang Guangda, Xu Xiaoli 2022, 26 (31):  5020-5025.  doi: 10.12307/2022.721 Abstract ( 396 )   PDF (2363KB) ( 105 )   Save BACKGROUND: Aging is a predominant risk factor for many developing diseases, such as cardiovascular disease, neurodegenerative disease, and osteoporosis. Cardiovascular disease has become one of the main causes of death in the elderly.   OBJECTIVE: To investigate the effect of myeloid-derived growth factor (MYDGF) on ventricular remodeling in aging mice. METHODS:  15-month-old C57BL/6 mice (WT mice) and MYDGF-/- mice (KO mice) were randomly divided into four groups as follows: WT group, KO group, adeno-associated virus-green fluorescent protein group, and adeno-associated virus-MYDGF group according to the presence and absence of AAV MYDGF intervention. After the intervention for 12 weeks, the mice were subjected to cardiac ultrasound to evaluate their cardiac structure and function, and to detect various blood biochemical indicators related to cardiac function. The heart weight was weighed and the length of the tibia was measured. Hematoxylin-eosin staining and Masson staining were used to observe the degree of cardiomyocyte hypertrophy and interstitial fibrosis.   RESULTS AND CONCLUSION: (1) Compared with the WT group, the heart weight, body weight, and heart weight to tibia length of KO group were increased (P < 0.05). (2) Left ventricular end-diastolic diameter and left ventricular end-systolic dimension in the KO group were enlarged compared with the WT group. Importantly, the ejection fractions and fractional shortening were decreased (P < 0.05). (3) Pathological results showed that compared with the WT group, the myocardial cells of the KO group were significantly enlarged, and the degree of myocardial fibrosis was deepened. (4) The sensitivity to insulin of the mice in the KO group decreased, and the rate of blood glucose decline was lower than that in the WT group (P < 0.05). There was a significant increase in brain natriuretic peptide, plasma renin activity, angiotensin II, aldosterone, and norepinephrine levels in the circulation of KO mice compared to WT mice (P < 0.05). (5) After treatment of MYDGF, the above indicators had been improved. (6) There was no significant difference in food intake, fasting blood glucose, total cholesterol, triacylglycerol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and glycosylated hemoglobin levels among the groups (P > 0.05). (7) It is indicated that the lack of MYDGF can lead to pathological structural changes and decreased cardiac function in mouse cardiomyocytes, and finally cause ventricular remodeling; aging ventricular remodeling can be improved after MYDGF replenishment. MYDGF may become a new drug for the treatment of ventricular remodeling in elderly mice. Figures and Tables | References | Related Articles | Metrics

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Effects of re-vitrification at blastocyst stage on histone epigenetic modification and pluripotency gene expression in mouse embryos Wang Jianwu, Zhao Yanhua, Li Jingyu, Wang Jiaqiang 2022, 26 (31):  5047-5052.  doi: 10.12307/2022.733 Abstract ( 352 )   PDF (1573KB) ( 117 )   Save BACKGROUND: In assisted reproduction work, re-vitrification can effectively improve embryo utilization. Our previous research results show that the secondary vitrification at different developmental stages significantly affects embryo development, but the mechanism is still unclear.   OBJECTIVE: To discuss the potential mechanism of secondary vitrification in different periods affecting embryo development potential. METHODS:  These 2-cell embryos fertilized in vivo were collected and cultured in vitro, and randomly divided into four groups: control, vitrified at the 8-cell stage (8C), vitrified at the 8-cell stage, and re-vitrified at the 8-cell (8C-8C) or early blastocyst stage (8C-BL). Immunofluorescence was utilized to analyze changes in the expression levels of H3K4me3, H3K9me2, and H3K9AC at the blastocyst stage. Real-time fluorescent quantitative PCR was applied to analyze changes in the expression levels of pluripotency genes Cdx2, Oct4, and Sox2.   RESULTS AND CONCLUSION: (1) Immunofluorescence results showed that first and second freezing led to a significant increase in the levels of H3K9me2 (P < 0.01) and H3K9AC (P < 0.000 1) at the blastocyst stage. (2)The first freezing did not affect the level of H3K4me3, but the level of H3K4me3 in the second freezing decreased significantly, and the decrease was more serious in the 8C-BL group (P < 0.000 1). (3) The results of real-time fluorescent quantitative PCR showed that the first freezing and the second freezing at the 8-cell stage did not affect the expression levels of embryo pluripotency genes Cdx2, Oct4 and Sox2, but the expression levels of embryo pluripotency genes in the 8C-BL group changed significantly. The level of Oct4 decreased significantly (P < 0.01), while the expression level of Cdx2 (P < 0.001) and Sox2 (P < 0.01) increased significantly. (4) The results show that the secondary vitrification has a certain negative impact on the epigenetics of mouse embryos, and the secondary freezing at the blastocyst stage significantly affects the H3K4me3 modification and pluripotency-related gene expression levels. Figures and Tables | References | Related Articles | Metrics

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Biological role of exosomes in denervated muscle atrophy Ye Hua, Yang Jiaming, Zhang Jiahong, Niu Yanlong, Wang Maoyuan 2022, 26 (31):  5062-5068.  doi: 10.12307/2022.782 Abstract ( 452 )   PDF (1296KB) ( 126 )   Save BACKGROUND: Peripheral nerve injury is very common in clinic, but its regeneration rate is very slow. During nerve recovery, muscle atrophy has already occurred irreversibly, which seriously affects the quality of life of patients. Therefore, it is particularly important to find an effective method for the treatment of denervated muscle atrophy.  OBJECTIVE: To summarize the biological characteristics of exosomes and their mechanisms of involvement in skeletal muscle formation and atrophy, focus on the process of denervated muscle atrophy and the role of exosomes in the treatment of denervated muscle atrophy, providing a new method for the treatment of denervated muscle atrophy. METHODS: Relevant articles were searched on PubMed database with English search words “exosome, skeletal muscle, muscle atrophy, muscle regenerate, peripheral nerve injury, denervation muscle atrophy”. Related articles were searched on Wanfang and CNKI databases, and the Chinese search words were “exosome, skeletal muscle, peripheral nerve injury, muscle atrophy, muscle regeneration, nerve repair, denervated muscle atrophy”, and some articles were searched with literature retrospective method.  RESULTS AND CONCLUSION: (1) Exosomes are widely distributed in all kinds of body fluids. They act on recipient cells through paracrine or autocrine and affect the phenotype of recipient cells. (2) The main function of exosomes in muscle may be related to the delivery of RNA. Exosomal miRNAs secreted or ingested by muscle cells are involved in muscle development, growth, adaptation and regeneration. (3) After peripheral nerve injury, muscle loses its motor control and nutritional support, and then apoptosis and proteolysis increase, leading to muscle atrophy. (4) Exosomes from different cell sources repair injured nerves directly or indirectly, thereby alleviating muscle atrophy. It has been preliminarily confirmed that exosomes may also mediate the expression regulation of related regenerative genes in a miRNA-dependent manner to promote neural regeneration. Therefore, exosomes can act on nerve cells and delay denervated muscle atrophy.   Figures and Tables | References | Related Articles | Metrics

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Mesenchymal stem cells-derived extracellular vesicles in the treatment and repair of acute and chronic renal injuries Li Qingru, Zhang Linqi, Chen Xu, Shi Ruoyu, Wang Xixi 2022, 26 (31):  5069-5075.  doi: 10.12307/2022.729 Abstract ( 465 )   PDF (1251KB) ( 78 )   Save BACKGROUND: Stem cells, especially extracellular vesicles derived from mesenchymal stem cells, have the potential to promote regeneration in different renal injury models.   OBJECTIVE: To review the application progress of extracellular vesicles derived from mesenchymal stem cells in preclinical models of acute and chronic renal injuries and the latest data in renal transplantation. In addition, new engineering strategies to improve the therapeutic effect of extracellular vesicles were discussed. METHODS:  CNKI, Wanfang, and PubMed databases were searched. The Chinese search words included “acute renal injury, chronic kidney disease, extracellular vesicles, exosomes”. English search words included “acute renal injury, chronic kidney disease, extracellular vesicles, exosomes, regeneration”. The search literature was the original research works and reviews. The retrieval time limit was from the establishment of the database to 2021, and 82 articles were selected for analysis and summary.   RESULTS AND CONCLUSION: (1) The research on mesenchymal stem cell derived extracellular vesicles in the treatment of renal injury has been widely carried out. Animal experiments have confirmed that mesenchymal stem cell derived extracellular vesicles can play a renal protective role and delay the progress of disease in the treatment of acute and chronic renal injuries. The specific mechanism involves the reprogramming of injured cells, cell proliferation, angiogenesis and inhibition of apoptosis and inflammation. Immediate administration of extracellular vesicles after renal transplantation can improve acute and chronic ischemia-reperfusion injuries. (2) There are few clinical trials on the treatment of nephropathy with mesenchymal stem cell-derived extracellular vesicles. One clinical trial observed that one year after taking umbilical cord blood mesenchymal stem cell-derived extracellular vesicles, the renal function of patients with chronic kidney disease was improved. (3) Based on the above research, mesenchymal stem cells derived extracellular vesicles will help to provide a new experimental model and basis for the effective treatment and repair of acute and chronic renal injuries, and open up a new direction for the clinical treatment of renal diseases. Figures and Tables | References | Related Articles | Metrics

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Mechanism and application prospects of mesenchymal stem cell exosomes gene-modified microRNA in the treatment of diabetic foot Deng Xiaohui, Zhang Zengzeng, Zhang Zhihua, Zhu Lingyan 2022, 26 (31):  5076-5084.  doi: 10.12307/2022.726 Abstract ( 546 )   PDF (1428KB) ( 93 )   Save BACKGROUND: Diabetic foot is one of the most serious complications of diabetes. The treatment of foot complications is a major problem that needs to be solved urgently in the medical care of diabetic people. Recent studies have shown that mesenchymal stem cell exosomes genetically modified miRNA has a great potential for promoting diabetic foot’s angiogenesis and tissue repair.   OBJECTIVE: To summarize the research progress of mesenchymal stem cell exosomes gene-modified miRNA in the treatment of diabetic foot, focus on the role of exosomal gene-modified miRNA in the treatment of diabetic foot, and systematically explain the molecular mechanism of how mesenchymal stem cell exosomes gene-modified miRNA promotes angiogenesis and tissue repair in the inflammatory microenvironment so as to lay a solid theoretical foundation for the advancement of later clinical trials. METHODS:  PubMed, Web of Science, and Wiley electronic journal databases were searched to retrieve related articles with “mesenchymal stem cells; exosomes; gene modification; diabetic foot; miRNA; inflammation; insulin resistance; angiogenesis; wound healing; tissue engineering; tissue reparation” as the English search terms. Finally, 89 English documents were included for summary.   RESULTS AND CONCLUSION: (1) Mesenchymal stem cell exosomes gene-modified miRNA has a significant effect on diabetic foot tissue regeneration and wound healing. Its main mechanisms include promoting angiogenesis by participating in transcriptional regulation of gene expression, accelerating wound re-epithelialization, inhibiting apoptosis and inflammation, regulating cell regeneration, and protecting β cells and regulating insulin resistance, thereby playing an important role in healing diabetic foot wounds. (2) Mesenchymal stem cell exosomes gene-modified miRNA can repair pancreatic tissue, improve obesity and insulin resistance, and delay the progression of diabetes and its complications by inhibiting β-cell apoptosis and increasing the sensitivity of insulin target tissues. (3) Targeted editing of the genome through regular clusters of interval short palindrome repeats and Cas9 (CRISPR-Cas9) technology can achieve precise gene modification, change the biological characteristics of miRNAs, and regulate the gene network to change its transcription and translation in target cells and target tissues to achieve the therapeutic effect. (4) This article comprehensively summarizes how exosomes miRNA is regulated by different gene loci to achieve the healing effect on refractory wounds of diabetic foot. (5) Exosomes miRNA also shows a great potential as a biomarker for diabetic foot treatment targets, early diagnosis, efficacy evaluation and prognostic diagnosis. (6) Seeking the best mesenchymal stem cell exosomes gene-modified miRNA therapy is expected to become a new technology for diabetic foot treatment. Figures and Tables | References | Related Articles | Metrics

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Gene-modified stem cells therapy for osteoporosis: a meta-analysis of preclinical studies Xiang Qianru, Deng Xuejian, Chen Huafeng, Liang Jiamin, An Min, Yang Li 2022, 26 (31):  5053-5061.  doi: 10.12307/2022.734 Abstract ( 440 )   PDF (4315KB) ( 137 )   Save OBJECTIVE: Gene-modified stem cells therapy in osteoporosis is a promising method with controversy. This article analyzed the effects of gene-modified stem cells on bone histometrology and calcium phosphorus metabolism in osteoporosis animals by meta-analysis, and evaluated its efficacy compared with simple stem cells. METHODS: PubMed, MEDLINE, Web of Science, CNKI, and Wanfang databases were used to collect articles about gene-modified stem cells therapy in osteoporosis animals before June, 2021. R4.1.0 software was used for meta-analysis. The primary outcome measures were bone mineral density and trabecular bone volume (BV/TV). The secondary outcome measures were trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), blood phosphorus, blood calcium, and blood bone alkaline phosphatase.   RESULTS: Totally 22 articles of animal experiments were included. The meta-analysis results showed: (1) gene-modified stem cells of the experimental group increased bone mineral density (SMD=2.23, 95%CI:1.34-3.11, P < 0.01), BV/TV (SMD=1.95, 95%CI:1.07-2.83, P < 0.01), Tb.N (SMD=2.33, 95%CI:1.34-3.32, P < 0.01), Tb.Th (SMD=1.47, 95%CI:0.70-2.24, P < 0.01) and blood phosphorus (MD=0.10, 95%CI:0.08-0.12, P=0.08) in simple stem cells of the control group. (2) Tb.Sp (SMD=-2.77, 95%CI:-4.17 to -1.36, P < 0.01), blood calcium (MD=-0.04, 95%CI:-0.06 to -0.03, P=0.82), and blood bone alkaline phosphatase (MD=-4.46, 95%CI:-5.12 to -3.80, P=0.96) of the experimental group were lower than those in the control group. CONCLUSION: Compared with simple stem cells, positive gene-modified stem cells improve bone mineral density, bone quality, and calcium phosphorus metabolism in osteoporosis. Due to the included literature’s insufficient quality, higher quality and more random control trials are required to supplement the argument. Figures and Tables | References | Related Articles | Metrics