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28 August 2023, Volume 27 Issue 24 Previous Issue    Next Issue

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Up-regulation of long non-coding RNA SNHG12 expression attenuates hydrogen peroxidation-induced decline in neuronal cell activity, apoptosis and excessive autophagy Zheng Yuchen, Zhang Jian, Zhang Rui, Chen Xiaosheng, Lan Tao, Zhou Wenyu 2023, 27 (24):  3773-3779.  doi: 10.12307/2023.681 Abstract ( 216 )   PDF (4078KB) ( 149 )   Save BACKGROUND: After spinal cord injury, secondary injury is considered to be a major factor in its high morbidity rate. Inhibiting the pathological cascade of oxidative stress and apoptosis and delaying the disease process are the primary therapeutic direction for alleviating spinal cord injury. The long non-coding RNA SNHG12 is thought to be related to cellular redox homeostasis, and its function on regulating the oxidative damage in neurons caused by H2O2 overload remains unknown.   OBJECTIVE: To investigate the function and mechanism of long non-coding RNA SNHG12 in H2O2-induced oxidative damage of HT22 cells. METHODS: (1) The HT22 cells were exposed to 100 μmol/L H2O2 for 6 hours to establish the cellular oxidative damage model. (2) The expression level of SNHG12 in HT22 cells was increased by transfection. The influences of SNHG12 expression on HT22 cell viability, apoptosis level, and autophagy were evaluated using CCK8 assay, Tryphenol blue staining, TUNEL staining, and western blot analysis. (3) Luciferase assay was used to verify the targeting relationship between SNHG12/miR-320a/SIRT5. (4) The upregulation of miR-320a and SIRT5 in HT22 cells was induced by transfection, respectively, and the regulation of miR-320a and SIRT5 in the protective role of SNHG12 was explored by reverse experiments.   RESULTS AND CONCLUSION: SHNG12 overexpression could alleviate the decline in cell viability, apoptosis, and excessive autophagy caused by H2O2. Luciferase reporter assay confirmed the binding relationship among SHNG12/miR-320a/SIRT5. miR-320a overexpression could reverse the effects of elevated SNHG12 on the above aspects, whereas SIRT5 overexpression can reverse the effect of up-regulation of miR-320a on the above indicators. It is concluded that SNHG12 suppressed autophagy and apoptosis of HT22 cells and alleviated oxidative damage via regulating miR-320a /SIRT5 axis. Figures and Tables | References | Related Articles | Metrics

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Biological characteristics of tumor necrosis factor-alpha-primed human umbilical cord mesenchymal stem cells Chen Zili, Cao Ning, Xu Meng, Jiang Yan, Ji Meichao, Zheng Yangyang, Yang Lili 2023, 27 (24):  3780-3787.  doi: 10.12307/2023.673 Abstract ( 264 )   PDF (3281KB) ( 35 )   Save BACKGROUND: In some preclinical and clinical studies, human umbilical cord mesenchymal stem cells have shown promising therapeutic results but limited efficacy. Therefore, enhancing the function of human umbilical cord mesenchymal stem cells by priming provides a new idea for the development of stem cell culture technology.   OBJECTIVE: To explore the changes of biological properties and mechanism of human umbilical cord mesenchymal stem cells primed with inflammatory factor tumor necrosis factor-α. METHODS: The human umbilical cord mesenchymal stem cells were cultured to the sixth generation and primed with inflammatory factor tumor necrosis factor-α. The changes of immunomodulatory ability, anti-inflammatory function, surface markers, doubling time, and adipogenesis and osteogenesis ability were evaluated in control group and tumor necrosis factor-α-primed group of human umbilical cord mesenchymal stem cells. In addition, the transcriptome analysis of the two groups was compared to illustrate the precise mechanism.   RESULTS AND CONCLUSION: (1) Compared with the control group, different mass concentrations of tumor necrosis factor-α-primed human umbilical cord mesenchymal stem cells inhibited T cell proliferation and promoted Treg cell proliferation significantly. The effect of 1 ng/mL tumor necrosis factor-α was the most significant. (2) Compared with the control group, different mass concentrations of tumor necrosis factor-α-primed human umbilical cord mesenchymal stem cells released lower levels of tumor necrosis factor-α and higher levels of interleukin-10 in inflammatory model in vitro, and their anti-inflammatory ability was significantly enhanced. (3) There was no significant difference in cell multiplication time and surface marker expression between the control group and tumor necrosis factor-α-primed group, and both groups had strong osteogenic and lipogenic differentiation abilities. (4) Transcriptome changes between control group and tumor necrosis factor-α-primed group were mainly reflected in the activation of nuclear factor-κB signaling pathway and tumor necrosis factor-α signaling pathway. (5) These results indicated that tumor necrosis factor-α-primed human umbilical cord mesenchymal stem cells significantly enhanced their immunomodulatory and anti-inflammatory functions. Figures and Tables | References | Related Articles | Metrics

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Effect of Hippo-YAP signaling pathway on osteogenic differentiation of human amniotic epithelial cells Zhang Wenjing, Zhi Xiaodong, Zhang Yuqiang, Wang Wei 2023, 27 (24):  3788-3794.  doi: 10.12307/2023.697 Abstract ( 274 )   PDF (2129KB) ( 50 )   Save BACKGROUND: Human amniotic epithelial cells, as a new type of seed cells, play an important role in bone injury repair.  OBJECTIVE: To investigate the potential regulatory mechanism of osteogenic differentiation of human amniotic epithelial cells in coculture with osteoblasts.   METHODS: Human amniotic epithelial cells were extracted by the enzyme digestion method. The proliferation of primary cells was described by the real-time cell analysis system xCELLigence RTCA S16. Human amniotic epithelial cells were co-cultured with osteoblasts by Transwell and corresponding samples were treated with siRNA-YAP1. The osteogenic differentiation of human amniotic epithelial cells was evaluated by detecting osteogenic associated proteins.  RESULTS AND CONCLUSION: (1) Primary human amniotic epithelial cells were adherent within 12 hours and entered an exponential growth period from 24 to 48 hours. (2) During coculture, human amniotic epithelial cells showed a trend of osteogenic differentiation, and the expression of YAP1 protein was increased in Hippo signaling pathway. (3) After siRNA-YAP1 was added during coculture, the expression of YAP1 protein in the Hippo signaling pathway was decreased and the osteogenic differentiation ability of human amniotic epithelial cells was weakened.   Figures and Tables | References | Related Articles | Metrics

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Proteomic analysis on cartilage differentiation of rabbit bone marrow mesenchymal stem cells induced by Achyranthes bidentata alcohol extract Ma Dujun, Zhu Houjun, Liu Leshi, Peng Liping, Zhao Jing, Liao Zhouwei 2023, 27 (24):  3795-3802.  doi: 10.12307/2023.641 Abstract ( 242 )   PDF (2952KB) ( 48 )   Save BACKGROUND: Preliminary studies have found that Achyranthes bidentata alcohol extract can induce the directional chondrocyte differentiation of bone marrow mesenchymal stem cells, but the specific protein targets and network mechanisms are unknown.   OBJECTIVE: To observe the proteomic analysis of cartilage differentiation of rabbit bone marrow mesenchymal stem cells induced by Achyranthes bidentata alcohol extract and the construction of protein interaction network. METHODS: Bone marrow mesenchymal stem cells of New Zealand white rabbits were isolated and cultured by density gradient centrifugation combined with bone marrow adherent method. Passage 3 cells were randomly divided into five groups: blank group, control group, low-dose Achyranthes bidentata alcohol extract group, medium-dose Achyranthes bidentata alcohol extract group, and high-dose Achyranthes bidentata alcohol extract group. After continuous induction and culture for 21 days, qRT-PCR was used to detect the expression of type II collagen mRNA and toluidine blue staining was used to identify the formation of chondrocytes. The differentially expressed proteins of different groups were identified by absolute quantitative isotope labeling (iTRAQ) combined with two-way liquid chromatography tandem mass spectrometry, and the differentially expressed proteins were analyzed by GO analysis, KEGG analysis and protein interaction network analysis.   RESULTS AND CONCLUSION: (1) The qRT-PCR results showed that the expression of type II collagen mRNA in the high-dose Achyranthes bidentata alcohol extract group was significantly higher than that in other groups (P < 0.05), and toluidine blue staining displayed positive reaction in the high-dose Achyranthes bidentata alcohol extract group. A total of 1 354 differential protein spots were identified by proteomic analysis, including 633 up-regulated proteins and 721 down-regulated proteins. (2) According to the results of qRT-PCR, the differentially expressed proteins of blank group, high-dose Achyranthes bidentata alcohol extract group and control group were analyzed. Go analysis showed that these differentially expressed proteins were involved in metabolism, cell differentiation, cell cycle and apoptosis, inflammatory response, immune regulation, oxidative stress, phosphorylation, ubiquitination, cancer and so on. KEGG analysis obtained 10 typical signal pathways most related to osteoarthritis: interleukin-17 signaling pathway, Toll like receptor signaling pathway, Wnt signaling pathway, PI3K-Akt signaling pathway, mTOR signaling pathway, JAK-STAT signaling pathway, NF-kappa B signaling pathway, MAPK signaling pathway, AMPK signaling pathway, and HIF-1 signaling pathway. According to the protein difference between groups, Cytoscape 3.6.0 software was used to successfully construct protein interaction network diagram. (3) These results suggest that Achyranthes bidentata alcohol extract can induce chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells by regulating oxidation, cell cycle and apoptosis, changes in cell structure, cell differentiation, metabolism and inflammatory injury. It has multi-target and multi-center network regulation, and its specific mechanism needs to be further studied. Figures and Tables | References | Related Articles | Metrics

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Icariin regulates apoptosis of nucleus pulposus-derived mesenchymal stem cells to repair intervertebral disc degeneration Zhang Wenjie, Zhang Yong, Shi Ming, Tang Guangjun, Shi Pengzhi, Wang Junwu, Hu Man, Wang Pingchuan, Zhang Liang 2023, 27 (24):  3803-3809.  doi: 10.12307/2023.668 Abstract ( 279 )   PDF (2961KB) ( 41 )   Save BACKGROUND: Intervertebral disc degeneration is one of the most common causes of low back pain. The decrease in the number and dysfunction of endogenous nucleus pulposus-derived mesenchymal stem cells may be an important cause of intervertebral disc degeneration. Icariin within a certain range may reduce the apoptosis of mesenchymal stem cells through the PI3K/Akt signaling pathway.   OBJECTIVE: To investigate the mechanisms underlying the effects of icariin on apoptosis of nucleus pulposus-derived mesenchymal stem cells. METHODS: The nucleus pulposus-derived mesenchymal stem cells were isolated from degenerated disc of Sprague-Dawley rats. Nucleus pulposus-derived mesenchymal stem cells of passage 3 were treated with different concentrations of icariin. CCK-8 assay was used to detect cell viability and proliferation. Nucleus pulposus-derived mesenchymal stem cells of passage 3 were divided into control, icariin, and LY294002 groups. The apoptosis was detected by flow cytometry and TUNEL staining at 1 week after treatment. The mRNA and protein expression of Caspase-3, Bcl-2, Bax and PI3K/Akt signaling pathway-related proteins p-Akt and p53 was detected by real-time polymerase chain reaction and western blotting. Sixteen Sprague-Dawley rat intervertebral disc degeneration models were equally divided into icariin (icariin 50 mg/kg per day intragastrically) and control groups (equivalent normal saline intragastrically). X-ray, MRI and hematoxylin-eosin staining were performed to evaluate disc degeneration before, 2 and 4 weeks after treatment.   RESULTS AND CONCLUSION: (1) Icariin promoted the proliferation of nucleus pulposus-derived mesenchymal stem cells at a certain concentration range, and the best concentration was 0.1 μmol/L (P < 0.05). (2) The apoptosis rate, the number of TUNEL-positive cells, mRNA and protein expression of Caspase-3, Bax and p53 in the icariin group were significantly decreased compared with the control group, while the expression levels of p-Akt and Bcl-2 were significantly increased (P < 0.05). LY294002 (PI3K/Akt signaling pathway inhibitor) reversed the protective effect of icariin on nucleus pulposus-derived mesenchymal stem cells (P < 0.05). (3) The disc degeneration was delayed after icariin treatment in the animal models. There were significant differences in the Disk Height Index, MRI scores, and hematoxylin and eosin staining scores between the icariin and control groups at 4 weeks (P < 0.05). (4) The results show that icariin can promote the proliferation of nucleus pulposus-derived mesenchymal stem cells in a concentration-dependent manner within a certain concentration range. Furthermore, icariin can inhibit nucleus pulposus-derived mesenchymal stem cell apoptosis by activating PI3K/Akt signaling pathway to delay disc degeneration. Figures and Tables | References | Related Articles | Metrics

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Three-dimensional cultured periodontal ligament stem cells promote osteoclastic differentiation of RAW264.7 under static compression Han Xing, Li Wenwen, Ma Wensheng 2023, 27 (24):  3810-3817.  doi: 10.12307/2023.698 Abstract ( 279 )   PDF (3827KB) ( 93 )   Save BACKGROUND: The osteogenic potential of periodontal ligament stem cells (as seed cells) has become a research hotspot. The regulation of periodontal ligament stem cells on osteoclast activity is less studied and deserves further exploration.  OBJECTIVE: To investigate the effect of three-dimensional cultured periodontal ligament stem cells under continuous static compression on the osteoclastic differentiation of RAW264.7 cells. METHODS: Periodontal ligament stem cells at passages 3 and 4 were taken for three-dimensional culture and randomly divided into 13 groups. Continuous static compression of 5 kPa (2, 6, and 12 hours), 25 kPa (2, 6, and 12 hours), 45 kPa (2, 6, and 12 hours), and 65 kPa (2, 6, and 12 hours) was applied by FLEXCELL 5000C, and group 13 with no compression. The cell supernatant was used as a conditioned medium to act on RAW264.7 cells. The expression of osteoclast differentiation genes tartrate resistant acid phosphatase, cathepsin K, and matrix metalloproteinase 9 was detected by qPCR and tartrate resistant acid phosphatase staining after 5 days.    RESULTS AND CONCLUSION: (1) The scaffold morphology was stable and the cell status was good in the compression experimental group and the non-compression control group under three-dimensional culture. (2) The conditioned medium of periodontal ligament stem cells in the compression loading group promoted the osteoclastic differentiation of RAW264.7 cells. (3) Within a certain range, the pro-osteoclastic effect was positively correlated with the force conditions. At 45 kPa and 12 hours, RAW264.7 cells had the strongest osteoclastic differentiation effect. The osteoclast-promoting ability of 65 kPa and 12-hour group was weakened. (4) These findings indicated that the osteoclastic differentiation of RAW264.7 cells was promoted by three-dimensional cultured periodontal ligament stem cells under static compression through the paracrine pathway, and the force conditions were correlated with the osteoclast effect. Figures and Tables | References | Related Articles | Metrics

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Effects of mycophenolate mofetil on the development of rat embryonic auricle Chen Qian, Zhang Yang, Li Gaofeng 2023, 27 (24):  3818-3823.  doi: 10.12307/2023.446 Abstract ( 247 )   PDF (11862KB) ( 32 )   Save BACKGROUND: The pathogenesis of congenital microtia is not clear. Current studies believe that microtia is related to the joint action of environmental and genetic factors, and drug teratogenesis is an important aspect of environmental factors. OBJECTIVE: To observe the embryonic auricle development indexes of pregnant rats exposed to different concentrations of mycophenolate mofetil, in order to establish a drug-induced microtia model and provide a basis for further exploring the pathogenesis of microtia. METHODS: Adult Sprague-Dawley rats were caged according to the ratio of male to female at 2:1, and the female rats were checked the next day. The presence of a vaginal plug was designated as gestational day 0. Thirty-two pregnant rats were randomized into four groups (n=8 per group): control group, 50, 100, and 200 mg/kg mycophenolate mofetil groups. Rats in the four groups were intragastrically given corn oil, and 50, 100, and 200 mg/kg mycophenolate mofetil corn oil respectively at 8 a.m. on gestational days 9 and 10. The pregnant rats in each group were decapitated under anesthesia on gestational day 20.5. The embryonic development and auricle development indexes were generally observed. The development of middle and inner ears was observed by micro-CT imaging. Auricle cartilage and soft tissue changes were histologically observed. RESULTS AND CONCLUSION: (1) Compared with the control group, the numbers of dead fetuses and absorbed fetuses were increased and the number and rate of live fetuses decreased in different mycophenolate mofetil groups. There were significant differences in ear transverse length, ear longitudinal length, ear longitudinal length/ear transverse length, nasal occipital distance/biparietal diameter, eye distance, body length and body mass between different mycophenolate mofetil groups and control group (P < 0.05). The above-mentioned indexes were significantly different in the 50 mg/kg mycophenolate mofetil group from 100 and 200 mg/kg mycophenolate mofetil groups (P < 0.05). There were also significant differences in ear length, body length, body mass, and nasal occipital distance between 100 and 200 mg/kg mycophenolate mofetil groups (P < 0.05). (2) Compared with the control group, the main micro-CT changes in each mycophenolate mofetil group were dysplasia of the skull and incomplete development of auditory vesicle. (3) The results of hematoxylin-eosin staining, toluidine blue staining and type II collagen immunohistochemistry staining showed that mycophenolate mofetil affected the proliferation and differentiation of chondrocytes and the expression of type II collagen. (4) To conclude, mycophenolate mofetil can cause the dysplasia of rat embryonic auricle in a dose-dependent manner, because mycophenolate mofetil may inhibit the proliferation and differentiation of chondrocytes and the expression of type II collagen. Mycophenolate mofetil can be used to establish an animal model of microtia. Figures and Tables | References | Related Articles | Metrics

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Deer antler stem cell-derived exosomes prevent alcoholic liver injury via modulating nuclear factor kappa B signaling pathway in mice Wang Dongxu, Ren Jing, Li Jiping, Wang Yusu, Hu Pengfei, Zhang Guokun, Li Chunyi 2023, 27 (24):  3824-3830.  doi: 10.12307/2023.674 Abstract ( 339 )   PDF (6048KB) ( 85 )   Save BACKGROUND: Excessive alcohol intake can lead to many liver diseases, but there is currently no effective drug to prevent it. Previous studies have found that antler stem cells can significantly improve liver fibrosis, and it is speculated that the therapeutic effect may be achieved through paracrine effects.   OBJECTIVE: To investigate the preventive effects of antler stem cell-derived exosomes on acute liver injury in mice and its underlying mechanism. METHODS: Forty C57BL6 mice were randomly divided into the control, model, bone marrow mesenchymal stem cell-derived exosome prevention, and antler stem cell-derived exosome prevention groups (n=10). The prevention groups were intervened with exosomes for 7 consecutive days, and the model group was intervened with PBS, followed by 50% alcohol (15 mL/kg) gavage for modeling of acute alcoholic liver injury. At 24 hours after gavage, liver indexes were calculated; liver injury and redox indexes were detected; the pathological lesions of liver tissues were evaluated and the expression levels of inflammatory factors and nuclear factor-κB signaling pathway-related genes were detected.   RESULTS AND CONCLUSION: (1) Antler stem cell-derived exosome significantly reduced liver injury, including increasing body weight, decreasing alanine aminotransferase and aspartate aminotransferase levels in serum, improving liver histopathological lesions, promoting proliferation and division of the hepatocytes and increasing antioxidant levels (increasing glutathione peroxidase and superoxide dismutase levels and decreasing malondialdehyde levels). The effects were stronger than those of bone marrow mesenchymal stem cell-derived exosomes. (2) Further studies revealed that antler stem cell-derived exosome significantly reduced the mRNA expression levels of relevant inflammatory factors (interleukin-1β, interleukin-6 and tumor necrosis factor α) and protein expression levels of nuclear factor-κB signaling pathway-related genes (p65, p-p65, IKB and p-IKB) in liver tissues. (3) Overall, our results suggest that antler stem cell-derived exosomes can play an important role in protecting the liver by reducing the damage caused by alcohol, and its effects may be achieved via targeting the nuclear factor-κB signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Effects of high-mobility group box 1 on different subtypes of rat spinal reactive astrocytes after oxygen-glucose deprivation/restoration in vitro Wang Liping, Li Jisheng, Deng Li, Wang Zhiqiang, Liu Jinming, Deng Chen, Sun Lin 2023, 27 (24):  3831-3837.  doi: 10.12307/2023.194 Abstract ( 215 )   PDF (3350KB) ( 39 )   Save BACKGROUND: Astrocytes can be activated into A1 and A2 subtypes of reactive astrocytes, which are completely different in gene, molecule and function, and have different specific roles in spinal cord injury repair. High-mobility group box 1 (HMGB1) has been proven to be an important inflammatory and immune factor after spinal cord injury, which can cause cell edema, inflammatory reaction and apoptosis; however, its effect on reactive astrocytes remains unclear.   OBJECTIVE: To investigate the activation of rat spinal cord astrocytes into reactive astrocytes after oxygen-glucose deprivation/restoration (OGD/R) and to explore the effect of HMGB1 on different subtypes of reactive astrocytes and the relevant mechanism.  METHODS: Rat spinal cord astrocytes were cultured to the third generation. Cell morphology was observed after OGD/R treatment, activation of reactive astrocytes was detected by western blot assay, and migration ability of the cells was detected by cell scratch assay. After inhibition of HMGB1, the cells were divided into five groups: normal group, OGD6 h/R6 h group, HMGB1 interference group, negative sequence group, and OGD6 h/R6 h+12 μmol/L ethyl pyruvate group (an HMGB1 inhibitor). Western blot assay and immunofluorescence method were used to detect the expression of C3 and S100A10 in different subtypes of reactive astrocytes, and cell scratch assay was used to detect the migration of reactive astrocytes. To explore the possible mechanism underlying the effect of HMGB1 on reactive astrocytes, the cells were divided into four groups: normal group, OGD6 h/R6 h group, OGD6 h/R6 h+5 µmol/L CLI-095 group (a toll-like receptor 4 inhibitor), and OGD6 h/R6 h+5 µmol/L BAY 11-7082 group (a nuclear factor-κB inhibitor). Western blot assay was used to detect the expression of toll-like receptor 4, nuclear factor-B, C3, and S100A10. RESULTS AND CONCLUSION: (1) Oxygen-glucose deprived cells were shrunk in size and had an increased migration capacity. After reoxygen-glucose treatment, the cell volume was increased and A1 and A2 reactive astrocytes were activated. S100A10 expression was the highest at OGD6 h/R6 h and C3 expression was peaked at OGD6 h/R12 h (P < 0.05). (2) Effects of silencing or inhibition of HMGB1 expression on reactive astrocytes: Compared with the OGD6 h/R6 h group, inhibition of HMGB1 significantly reduced the expression of C3 (P < 0.05), increased the expression of S100A10 (P < 0.05), and enhanced cell migration ability. (3) Compared with the OGD6 h/R6 h group, the expression of toll-like receptor 4 decreased in the OGD6 h/R6 h+CLI 095 group (P < 0.05), while the expression of nuclear factor-κB and C3 decreased and the expression of S100A10 increased in the OGD6 h/R6 h+CLI 095 group and OGD6 h/R6 h+BAY 11-7082 group (P < 0.05). (4) To conclude, astrocytes can be activated into two different subtypes of reactive astrocytes after OGD/R. Inhibition of HMGB1 can reduce A1 reactive astrocytes, increase A2 reactive astrocytes, and enhance cell migration ability through the toll-like receptor 4/nuclear factor-κB pathway.   Figures and Tables | References | Related Articles | Metrics

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Effect of MKRN1 silencing on proliferation and apoptosis of human embryonic kidney cells HEK-293T Zhang Yi, Wu Daoqiu, Tang Hongting, Li Mengxing, Liu Honglin, Chen Zhongliang, Li Qinshan 2023, 27 (24):  3838-3844.  doi: 10.12307/2023.165 Abstract ( 265 )   PDF (4612KB) ( 83 )   Save BACKGROUND: It has been reported that makorin ring finger protein 1 (MKRN1) knockdown can inhibit the growth of tumor cells, but whether MKRN1 plays a role in the body as a tumor-related protein needs further study. The siRNA-mediated gene silencing is transient, and therefore, the duration is short and the conventional lentivirus technique also has an off-target effect. A cell line stably silencing MKRN1 gene constructed using Tet-on lentivirus system has reversible advantages and can avoid the above shortcomings, which is of great significance for the further study on the biological function of MKRN1. OBJECTIVE: To construct a MKRN1 gene silencing vector mediated by the Tet-on lentivirus system in order to study the effect of MKRN1 gene silencing on the proliferation and apoptosis of human embryonic kidney cells HEK-293T. METHODS: Using RNAi technique, three short hairpin RNAs (shRNAs) were designed for MKRN1 gene, namely sh1, sh2, and sh3, and three shRNA sequences were ligated into tetracycline-induced expression vector pTripz. The correct sequence was verified by sequencing. HEK-293T cells were transfected with the three-plasmid system and cultured in the complete medium containing 1 mg/L doxycycline for lentivirus packaging. The virus solution was collected at 48 and 72 hours and concentrated to infect HEK-293T cells. Forty-eight hours after infection, the virus infection efficiency was observed by fluorescence microscope. The knockout efficiency of MKRN1 was detected by real-time fluorescence quantitative PCR and western blot assay. The successful construction of tetracycline-induced expression system was detected by western blot assay. Colony formation test and western blot assay were used to detect the effect of silencing MKRN1 expression on the proliferation and apoptosis of HEK-293T cells. RESULTS AND CONCLUSION: (1) Three MKRN1-shRNAs, named sh1, sh2, and sh3, were successfully inserted into the Tet-on vector, and the sequence of each vector was correct. HEK-293T cells were successfully transfected with lentivirus. (2) RT-qPCR and western blot results indicated that the MKRN1 knockdown level of the first sequence of the three shRNA sequences was significantly decreased (P < 0.05), and the expression of the target gene was rescued in the silent group without doxycycline induction. (3) The proliferation of HEK-293T cells decreased after MKRN1 silencing detected by colony formation experiments (P < 0.05). Western blot assay results revealed that after MKRN1 knockdown, the expression of proliferating cell nuclear antigen was down-regulated, the expression of BAX was increased, the expression of Bcl2 was down-regulated, and the Bcl2/BAX ratio was down-regulated (P < 0.001). (4) To conclude, the HEK-293T cell line stably silencing MKRN1 can be constructed by using Tet-on lentiviral vector system, and the silencing of MKRN1 can inhibit the proliferation of HEK-293T cells and promote their apoptosis. The construction of stably silencing MKRN1 HEK-293T cells can lay a foundation for the further study on the biological function of MKRN1 and also provide a methodological reference for the study of the biological function of other genes. Figures and Tables | References | Related Articles | Metrics

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Astrocyte-derived extracellular vesicles promote the differentiation of rat neural stem cells into neurons Cai Chuang, Huang Yonghui, Cao Xingbing, Sun Haitao, Guan Shihao, Huang Wenkang, Han Bo 2023, 27 (24):  3845-3851.  doi: 10.12307/2023.672 Abstract ( 298 )   PDF (2501KB) ( 135 )   Save BACKGROUND: Neural stem cells have the potential to differentiate into neurons. It has been reported that astrocytes are closely related to neurons, but whether astrocyte-derived extracellular vesicles can promote the efficient differentiation of neural stem cells into neurons has not been reported.   OBJECTIVE: To explore the possibility of inducing and promoting neuronal differentiation of neural stem cells from extracellular vesicles derived from astrocytes. METHODS: (1) Rat neural stem cells and astrocytes were cultured in vitro and identified by cell morphology and immunofluorescence. Astrocyte-derived extracellular vesicles were further extracted and identified by transmission electron microscopy, particle size analysis, western blot assay and other methods. (2) Passage 3 neural stem cells were divided into two groups: Neural stem cells were treated with the same volume of PBS and astrocyte-derived extracellular vesicles (1 mg/mL) for 6 days. (3) The expression levels of neuron markers after astrocyte-derived extracellular vesicle intervention were detected by immunofluorescence, qRT-PCR and western blot assay.   RESULTS AND CONCLUSION: (1) Under the microscope, neural stem cells were spherical in shape and distributed in cell clusters. Immunofluorescence showed that the characteristic markers CD133 and Nestin were highly expressed.  (2) Astrocyte morphology was irregular stellate under the microscope, and immunofluorescence showed that the characteristic marker glial fibrillary acidic protein was highly expressed.  (3) The shape of astrocyte-derived extracellular vesicles was round or oval cup and 10-200 nm in diameter by dynamic light particle scatterer. Western blot assay showed positive expression of CD63 and TSG101, related markers of extracellular vesicles.  (4) Compared with PBS group, astrocyte-derived extracellular vesicle group showed a large number of adherent differentiation of neural stem cells, and the differentiated cells were plump and obvious protrusions, which were mainly neuron-like, and the expression of tubulin β3 and neurofilament 200 was high (P < 0.05).  (5) These results suggest that astrocyte-derived extracellular vesicles can promote the differentiation of neural stem cells into neurons. Figures and Tables | References | Related Articles | Metrics

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Mechanism of dexamethasone against high glucose-induced oxidative damage to glomerular podocytes Jiang Yifan, Geng Yu, Zhang Xin, Zuo Zhongfu 2023, 27 (24):  3852-3852.  doi: 10.12307/2023.517 Abstract ( 271 )   PDF (2047KB) ( 57 )   Save BACKGROUND: Dexamethasone is a glucocorticoid drug commonly used for treating diabetic nephropathy in recent years. It can directly act on glomerular podocytes and increase cell survival by inhibiting inflammatory response and stabilizing cell cycle. However, its specific mechanism of action remains unclear.   OBJECTIVE: To investigate the mechanism of dexamethasone on high glucose-induced oxidative damage to glomerular podocytes. METHODS: Glomerular podocytes were divided into six groups: group A, routine culture with no treatment; group B, treatment with glucose; group C, treatment with glucose for 48 hours and valsartan for another 24 hours; groups D-F, treatment with glucose for 48 hours and 1×10-7, 1×10-6, 1×10-5 mol/L dexamethasone for another 24 hours, respectively. Specific touchdown nested PCR was used to detect the methylation level of Nogo receptor 1 (NgR1) in cells. MTT method was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Chemiluminescence method was used to detect oxidative damage-related indicators. Immunofluorescence method was used to detect peroxisome proliferator-activated receptor γ expression.   RESULTS AND CONCLUSION: (1) Compared with group A, group B had significantly increased methylation level of NgR1, cell apoptosis, and malondialdehyde level (P < 0.05), but significantly decreased cell proliferation, superoxide dismutase and glutathione levels, and peroxisome proliferator-activated receptor γ expression (P < 0.05). (2) Compared with group B, group C had significantly decreased methylation level of NgR1, cell apoptosis, and malondialdehyde level (P < 0.05), but significantly increased cell proliferation, superoxide dismutase and glutathione levels, and peroxisome proliferator-activated receptor γ expression (P < 0.05). (3) Compared with group C, groups E and F had significantly decreased methylation level of NgR1, cell apoptosis, and malondialdehyde level (P < 0.05), but significantly increased cell proliferation, superoxide dismutase and glutathione levels, and peroxisome proliferator-activated receptor γ expression (P < 0.05). Moreover, these changes were more obvious in group F than group E (P < 0.05); however, there was no significant difference between groups D and C (P > 0.05). (4) To conclude, dexamethasone can significantly improve oxidative damage to glomerular podoccytes induced by high glucose and promote the expression of peroxisome proliferator-activated receptor γ, which may be related to inhibition of Ngr1 gene methylation. Figures and Tables | References | Related Articles | Metrics

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Effect of electroacupuncture modulation of glycogen synthase kinase 3 beta/beta-catenin signaling pathway on CD133 protein expression in rat ventricular zone cells after spinal cord injury Duan Zhaoyuan, Wu Mingli, Luo Meng, Liu Chengmei, Gao Jing, Li Ruiqing, Feng Xiaodong 2023, 27 (24):  3858-3864.  doi: 10.12307/2023.422 Abstract ( 211 )   PDF (4769KB) ( 31 )   Save BACKGROUND: Preliminary studies have shown that electroacupuncture can improve functional impairment after spinal cord injury and promote the proliferation of endogenous neural stem cells in rats after spinal cord injury. However, its specific mechanism of action is unclear. OBJECTIVE: To investigate the effect of electroacupuncture on the expression of glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling pathway-related factors and CD133 protein in ventricular canal area cells in rats with spinal cord injury, and to explore the mechanism by which electroacupuncture promotes endogenous stem cell proliferation. METHODS: A total of 45 female Sprague-Dawley rats were randomly divided into sham-operated group, model group, and electroacupuncture group, with 15 rats in each group. Animal models of spinal cord injury were constructed in the latter two groups using a precision percussion device. The electroacupuncture group received electroacupuncture treatment at Baihui and Jizhong acupoints of the Governor’s Vessel and at Jiaji acupoints of the upper and lower segments of the injured spinal cord, with sparse and dense waves at a frequency of 1 Hz/20 Hz and an intensity of 1.0-1.2 mA, once for 30 minuntes, once a day, for 14 continuous days. The motor function of the rats was assessed using the Basso, Beattie and Bresnahan motor function score and Nissl staining was used to observe the histomorphological changes and the number of neurons in the spinal cord. Immunofluorescence was used to detect the fluorescence intensity of CD133 in ependymal cells in spinal cord tissue and Nestin in spinal cord gray matter and white matter. Real-time fluorescence quantitative PCR was used to detect Nestin, GSK-3β, and β-catenin mRNA in spinal cord tissue. Western blot assay was used to detect Nestin, GSK-3β, p-GSK-3β, β-catenin, and p-β-catenin protein expression in spinal cord tissue.  RESULTS AND CONCLUSION: (1) Compared with the model group, the Basso, Beattie and Bresnahan scores showed no significant changes in the electroacupuncture group at 1, 3, and 5 days after surgery (P > 0.05) but were significantly increased at 7 and 14 days after surgery (P < 0.05, P < 0.01). (2) The morphology of some nerve cells in the spinal cord tissue was relatively intact in the electroacupuncture group and Nissl bodies were slightly dissolved. Compared with the model group, the number of neurons in the spinal cord tissue was significantly increased in the electroacupuncture group (P < 0.05). (3) Compared with the model group, in the electroacupuncture group, the fluorescence intensity of CD133 in the ventricular zone of spinal cord tissue was increased (P < 0.001), and the fluorescence intensity of Nestin was significantly increased in the gray matter (P < 0.01) and white matter (P < 0.001). (4) Compared with the model group, in the electroacupuncture group, the expression of Nestin mRNA was significantly higher (P < 0.001), GSK-3β mRNA expression was significantly lower (P < 0.01), and β-catenin mRNA expression was also significantly lower (P < 0.001). (5) Compared with the model group, Nestin and p-GSK-3β protein expression was significantly higher (P < 0.001) and p-β-catenin protein expression was significantly lower (P < 0.001) in the electroacupuncture group. (6) All these findings indicate that electroacupuncture can promote CD133 protein expression in the ventricular canal area of rats after spinal cord injury, and the mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Osteogenic and angiogenic differentiation of dental pulp stem cells modified by hypoxia-inducible factor-1 alpha gene in vitro Tang Juan, Yu Donglin, Liu Guoqi, Song Jiaojiao, Zuo Jinhua, Fu Honghai 2023, 27 (24):  3865-3870.  doi: 10.12307/2023.699 Abstract ( 245 )   PDF (2137KB) ( 36 )   Save BACKGROUND: How to effectively repair and reconstruct critical bone defects has become a difficult problem in clinical medicine. Dental pulp stem cells have certain advantages as transgenic target cells to repair damaged tissues or organs. OBJECTIVE: To investigate the effect of hypoxia-inducible factor-1α on osteogenesis and angiogenesis of dental pulp stem cells in vitro.  METHODS: The pulp tissues of healthy and intact bicuspid teeth extracted by orthodontic treatment in patients aged 12 to 18 years were isolated to culture dental pulp stem cells. The hypoxia-inducible factor-1α gene was transfected into dental pulp stem cells in the experimental group. The control group did not do any treatment. The expression of the hypoxia-inducible factor 1α gene was detected by qPCR and the expression of osteogenic and angiogenic factors was detected by western blot assay.  RESULTS AND CONCLUSION: (1) Dental pulp stem cells were short spindle-shaped fibroblast-like cells, and the cells grew in colonies. (2) Hypoxia-inducible factor-1α gene was stably transfected into dental pulp stem cells. The expression of the hypoxia-inducible factor-1α gene increased gradually with the extension of time, reached a peak at 14 days, and significantly decreased at 21 days. (3) Compared with the control group, the expression level of angiogenesis-related factors in the experimental group was higher. The protein expression levels of vascular endothelial growth factor, stromal cell-derived factor 1, angiopoietin 2, and placental growth factor were significantly increased at 1 day after gene transfection and reached a peak at 4 days. After that, the expression of angiogenesis-associated protein in the experimental group decreased slightly, but was significantly higher than that in the control group. (4) Compared with the control group, the expression level of osteogenic-related factors in the experimental group was higher. The protein expression levels of human bone morphogenetic protein 2, osteocalcin, and Runt-related transcription factor 2 were significantly increased at 1 day after gene transfection, with the highest protein expression at 4 days. (5) It is concluded that hypoxia-inducible factor-1α can promote the osteogenesis and the expression of angiogenic factors in dental pulp stem cells.   Figures and Tables | References | Related Articles | Metrics

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Interpretation of the embryo laboratory: number of oocytes retrieved affects the pregnancy outcomes of in vitro fertilization Gao Wenyi, Zhang Dong, Li Caixia, Du Juan, Zhang Yanru, Deng Yun 2023, 27 (24):  3871-3876.  doi: 10.12307/2023.285 Abstract ( 243 )   PDF (1638KB) ( 182 )   Save BACKGROUND: Controlled ovarian hyperstimulation has gradually become a routine method to increase the number of oocytes retrieved, but there has been controversy over whether the more oocytes retrieved the better. There is still no unified conclusion about the ideal range of egg retrieval number.   OBJECTIVE: To explore the effect of number of oocytes retrieved on embryo fertilization outcome and clinical pregnancy outcome in fertilization in vitro treatment cycle. METHODS: 489 patients undergoing in vitro fertilization treatment (including conventional in vitro fertilization and intracytoplasmic sperm injection) were divided into the following five groups according to the number of oocytes retrieved: 1-4 group, 5-8 group, 9-12 group, 13-16 group, and ≥17 group. The fertilization rate, cleavage rate, transferable embryo rate, high-quality embryo rate, and clinical pregnancy outcome were compared in each group, and the effect of the number of oocytes retrieved on the outcome of in vitro fertilization was analyzed.   RESULTS AND CONCLUSION: The number of oocytes retrieved was significantly correlated with the normal fertilization rate, transferable embryo rate, and high-quality embryo rate. Too many or too few retrieved oocytes could lead to a decrease in the normal fertilization rate and an increase in the abnormal fertilization rate, a decrease in the rates of transferable embryos and high-quality embryos, a decrease in the clinical pregnancy rates, embryo implantation rates, and live birth rates. In conclusion, too many or too few oocytes retrieved can lead to the decline of embryo quality and affect the clinical outcome of in vitro fertilization. The number between 5 and 16 oocytes may be appropriate, while the number between 9 and 12 oocytes may be optimal. During ovulation induction, clinicians should not only aim to increase the number of oocytes retrieved, but should pursue enough high-quality oocytes to reasonably formulate personalized ovulation induction programs for different patients. Figures and Tables | References | Related Articles | Metrics

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Effects of enalapril folic acid on vascular endothelial cell activity and angiotensin II and angiotensin-converting enzyme levels in a rat model of lower extremity venous embolism Yue Ying, Hao Wenli, Gan Yongkang 2023, 27 (24):  3877-3882.  doi: 10.12307/2023.473 Abstract ( 216 )   PDF (1931KB) ( 39 )   Save BACKGROUND: Lower limb venous embolism is prone to cause complications such as venous thrombosis and pulmonary embolism. If not treated in time, patients may die suddenly. Therefore, it is particularly important to explore an effective treatment method. OBJECTIVE: To investigate the effects of enalapril folate on the activity of vascular endothelial cells as well as angiotensin II and angiotensin-converting enzyme levels in rats with lower extremity venous embolism.  METHODS: Forty SPF male Sprague-Dawley rats were randomly divided into normal group, model group, rivaroxaban group, and enalapril folic acid group (n=10 per group). In the latter three groups, animal models of lower extremity venous embolism were established using the modified Reyers method. After modeling, the rats were gavaged 30 mg/kg rivaroxaban in the rivaroxaban group, 20 mg/kg enalapril maleate folic acid tablet in the enalapril folic acid group, and the same volume of normal saline in the model and normal groups for 30 days. During administration, Tarlov score was used to evaluate the lower limb motor function of rats. After administration, hematoxylin-eosin staining was used to detect the pathological morphology of vascular tissue. Hladovec and ELISA were used to detect the activity of endothelial cells. The level of angiotensin II was detected by homogeneous competitive radioimmunoassay and the level of angiotensin-converting enzyme was detected by hippuric acid method. RESULTS AND CONCLUSION: (1) Compared with the normal group, Tarlov score and serum nitric oxide level were significantly decreased in the model group (P < 0.05), while the number of serum circulating endothelial cells, endothelin 1, hepatocyte growth factor, angiotensin-converting enzyme, and angiotensin II in plasma were significantly increased (P < 0.05). (2) Tarlov scores and serum nitric oxide levels were significantly increased in the rivaroxaban and enalapril folic acid groups compared with the model group (P < 0.05), while the number of serum circulating endothelial cells and levels of endothelin 1, hepatocyte growth factor, angiotensin-converting enzyme, and plasma angiotensin II were significantly increased (P < 0.05). Moreover, the above indicators were significantly improved in the enalapril folate group compared with the rivaroxaban group (P < 0.05). (3) In the normal group, the vascular structure was clear and complete and the intima was intact. In the model group, the rats showed loosed vascular wall tissue, thickened edema, and numerous thromboses, indicating serious organization. After treatment with rivaroxaban and enalapril folic acid, the vascular morphology was improved, with basically intact structure of the vascular wall, and vascular endothelial cells were partially regenerated. The enalapril folic acid group showed better outcomes than the rivaroxaban group. (4) To conclude, enalapril folic acid has dramatically therapeutic effects in the rats with lower extremity venous embolism, which can effectively improve the activity of vascular endothelial cells and reduce the levels of angiotensin II and angiotensin-converting enzyme. Figures and Tables | References | Related Articles | Metrics

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Exosome-mediated cellular communication: a potential biomarker for Parkinson’s disease Wang Yiying, Li Ruiqing, Li Jingwen, Mei Jinjin, Zhang Jianyun, Zhang Lihong, Fan Yongfu, Guo Jian 2023, 27 (24):  3883-3891.  doi: 10.12307/2023.680 Abstract ( 167 )   PDF (1813KB) ( 235 )   Save BACKGROUND: Parkinson’s disease is the second largest neurodegenerative disease in the world. Both motor symptoms and non-motor symptoms seriously affect the daily life of patients. Recent studies have found that exosomes and the substances they carry are involved in the pathogenesis of Parkinson’s disease. It also mediates cell communication and can be used as a potential biomarker for Parkinson’s disease.   OBJECTIVE: To explore the type and mechanism of exosome-mediated cell communication as a potential biomarker of Parkinson’s disease, to find a new target for early diagnosis and treatment, to slow down the pathogenesis of Parkinson’s disease and provide new ideas for its diagnosis and treatment.  METHODS: The related articles included in PubMed, CNKI, WanFang, VIP, Web of Science, EMBASE, Cochrane, EBSCO, and SinoMed databases from January 1987 to August 2022 were searched by computer, and 76 English and 2 Chinese articles were finally included for inductive analysis.  RESULTS AND CONCLUSION: (1) The exosomes transport substances to recipient cells or membrane surface through endocytosis and exocytosis membrane fusion to carry signaling factors and release them in the intercellular space and other means for the exchange of substances between cells. (2) Exosomes can participate in the pathological progress of Parkinson’s disease by mediating intercellular communication and information exchange and material transport, so they can be used as early biomarkers of Parkinson’s disease, help provide new diagnosis and treatment targets, and can be used as a diagnostic carrier for Parkinson’s disease in the future. (3) The exosomes participate in mechanism of the pathological development of Parkinson’s disease through intercellular transmission and substance transport and diffusion. The relationship between its main biomarkers and pathologic pathways is that in neurotoxicity, neuroinflammation, oxidative stress, autophagy and other processes mediated by exosomes α synuclein, exosomal microRNA can target Parkinson’s disease-related genes and participate in oxidative stress, and exosomal DJ-1 protein regulates the level of cellular oxidative stress and affects cytotoxicity. Leucine-rich repetitive kinase 2 of exosomes mediates the formation and accumulation of Lewy bodies and causes neurotoxicity. The above exosomes and their carrying substances can be used as the early stage of Parkinson’s disease biomarkers. (4) At present, there are few related researches on the biomarkers of Parkinson’s disease in exosomes, but the most representative and most widely used is exosome α-synuclein. Due to the large number and variety of exosomal microRNAs, the expression and role in Parkinson’s disease are not the same, and the research has been hot in the past two years. (5) At present, related research lacks corresponding clinical verification, so it can be used as a future research direction, and the research prospect is broad. Therefore, more potential biomarkers and their mechanisms of action need to be further verified and studied, so as to provide new targets for the early diagnosis and treatment of Parkinson’s disease. (6) The application of exosomes in the clinical diagnosis of Parkinson’s disease still has a lot of research space and development prospects. The application of exosome-mediated cell communication in the diagnosis and treatment of Parkinson’s disease based on clinical trials is the focus and urgent breaking point of future research.  Figures and Tables | References | Related Articles | Metrics

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Transamniotic stem cell therapy: a novel strategy for prenatal treatment of multiple congenital diseases Cheng Xi, Sun Baolan, Xie Yuanyuan, Zhang Yuquan 2023, 27 (24):  3892-3900.  doi: 10.12307/2023.686 Abstract ( 290 )   PDF (1793KB) ( 96 )   Save BACKGROUND: Advances in prenatal diagnosis have made it possible to prenatal management and treatment of various congenital diseases. Transamniotic stem cell therapy has emerged as a new strategy for prenatal treatment of congenital anomalies.   OBJECTIVE: To review the results of studies using transamniotic stem cell therapy in the prenatal treatment of various congenital diseases. METHODS: Databases of CNKI, VIP, WanFang and PubMed were searched from the inception to August 1, 2022. The keywords were “transamniotic stem cell therapy”, “transamniotic”, “intra-amniotic”, “stem cells” or “TRASCET” in English and Chinese. Finally, a total of 61 articles on transamniotic stem cell therapy were included for review.   RESULTS AND CONCLUSION: (1) Transamniotic stem cell therapy can be used to treat fetal abnormalities by fully magnifying the biological function of stem cells in amniotic fluid under physiological or pathological conditions. (2) Specific stem cells infused into amniotic cavity can not only be directly disseminated to defects exposed to amniotic fluid, but also be transported to placenta, amnion, chorion, umbilical cord, fetal bone marrow, spleen, hip bone and brain. (3) Intra-amniotic infusion of mesenchymal stem cells remarkably promoted tissue repair and/or significantly ameliorated adverse effects associated with major congenital abnormalities. For example, transamniotic stem cell therapy can induce partial or complete coverage of experimental spina bifida by forming new skin. Transamniotic stem cell therapy can also alleviate the bowel damage associated with gastroschisis; at the same time, transamniotic stem cell therapy can also affect lung development in experimental congenital diaphragmatic hernia and significantly diminish the thickness of pulmonary arteriole wall. Transamniotic stem cell therapy can reverse the adverse effects of intrauterine growth restriction in rats, including significantly improving placental efficiency and increasing fetal weight. (4) Transamniotic stem cell therapy has shown good therapeutic potential in congenital disease models such as spina bifida, gastroschisis, congenital diaphragmatic hernia and intrauterine growth restriction. However, there is no clinical report on transamniotic stem cell therapy so far, and it is necessary to further verify the effectiveness and safety of transamniotic stem cell therapy in large animals before clinical transformation. (5) Although transamniotic stem cell therapy is still in its infancy, its potential in prenatal treatment of fetal congenital diseases deserves further study. Figures and Tables | References | Related Articles | Metrics

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Mechanism of Kupffer cells in immune tolerance after liver transplantation Wang Yaoquan, Xuan Juanjuan, Zhang Jixiang, Chen Kaige, Chen Guoyong, Wei Sidong 2023, 27 (24):  3901-3909.  doi: 10.12307/2023.476 Abstract ( 282 )   PDF (1662KB) ( 84 )   Save BACKGROUND: Immune rejection after liver transplantation seriously affects the quality of life of patients. The establishment of long-term immune tolerance still faces many problems. Kupffer cells play an important role in inhibiting liver transplantation rejection and promoting immune tolerance. OBJECTIVE: To review the role of Kupffer cells in immune tolerance after liver transplantation and its mechanism, in order to provide reference ideas for establishing immune tolerance in liver transplantation. METHODS: In December 2021, the first author used computers to search the relevant literature from January 1970 to December 2021 in PubMed and CNKI databases using “Kupffer cells, liver transplantation” as the English and Chinese search terms, respectively. Finally, 73 articles were included for further analysis. RESULTS AND CONCLUSION: (1) Kupffer cells are macrophages resident in the liver. There are many specific receptors and binding sites on the surface of Kupffer cells, including complement receptors, Toll-like receptors, and other pathogen recognition receptors, which can quickly respond to the changes of tissue environment and present different cell phenotypes, that is, M1 and M2 Kupffer cells. Liver transplantation immune tolerance is closely related to M2 Kupffer cells. (2) Kupffer cells exert vital roles in the establishment of immune tolerance after transplantation through the mechanisms of cell phagocytosis, cell polarization, antigen presentation, cell membrane proteins, and cytokines. However, there is still a lack of high-quality basic and clinical research. (3) Existing research on Kupffer cells indicates that a variety of cytokines and non-specific factors may be associated with immune tolerance after transplantation, mainly including T cell immunoglobulin domain and mucin domain 4, F1 type scavenger receptor, antigenic calreticulin, protein disulfide isomerase, inhibitory histone deacetylase 11, heat shock protein, X-box binding protein 1, bone marrow mesenchymal stem cells, interleukin 2, interleukin 4, interleukin 6, interleukin 10, interleukin 17, interleukin 23, interleukin 34, complement, Fas-FasL, Toll-like receptors, prostaglandins, transforming growth factor β and interferon γ. (4) In terms of basic research, constructing an orthotopic liver transplantation animal model helps further explore the effect of different functions of Kupffer cells on the establishment of immune tolerance after liver transplantation. (5) In terms of clinical application, we can predict and promote the establishment of immune tolerance through the pathological analysis of the graft, Kupffer cytokine expression in transplants, gene detection, and donor infusion of bone marrow mesenchymal stem cells. However, multi-center, large-sample clinical studies are warranted in the future. Figures and Tables | References | Related Articles | Metrics

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Effects of different signaling pathways on osteogenic differentiation of periodontal ligament stem cells Fu Qiuyue, Lan Xingming, Xu Rongwei, Wang Hao, Yang Kun 2023, 27 (24):  3910-3919.  doi: 10.12307/2023.677 Abstract ( 437 )   PDF (1905KB) ( 105 )   Save BACKGROUND: Periodontal membrane stem cells, as a kind of odontogenic mesenchymal stem cells, have osteogenic differentiation potential and can be applied to periodontal tissue regeneration and repair, while the osteogenic differentiation of periodontal membrane stem cells involves the regulation of multiple signaling pathways.   OBJECTIVE: To review the factor-mediated signaling pathways associated with osteogenic differentiation of periodontal membrane stem cells and their effects on osteogenic differentiation. METHODS: In PubMed and CNKI, the search time was set from 2010 to 2022. The research progress of eight major signaling pathways associated with osteogenic differentiation of periodontal ligament stem cells since 2010 was summarized. Finally, 60 articles were included for analysis and discussion.   RESULTS AND CONCLUSION: (1) Periodontal membrane stem cells, as odontogenic mesenchymal stem cells, have the characteristics of good proliferation, self-renewal and multidirectional differentiation potential. As seed cells for periodontal tissue regeneration and restoration, periodontal membrane stem cells have broad prospects for the treatment of periodontitis. (2) At present, a large number of studies have confirmed that the potential relationship between various factors and periodontal membrane stem cell osteogenic differentiation is generated by mediating each signaling pathway. Although each signaling pathway has a certain role in promoting the osteogenic differentiation of periodontal membrane stem cells, the most studied is the MAPK signaling pathway and the Wnt signaling pathway. (3) The MAPK signaling pathway includes four pathways, which are widely present in cells and play an important role in regulating cell proliferation and differentiation. The classic Wnt/β-catenin pathway is one of the most important signaling pathways for controlling bone homeostasis, and numerous studies have confirmed its association with osteogenic differentiation of periodontal membrane stem cells. (4) At present, most of the current research has only confirmed through in vitro mineralization experiments that certain factors can activate the MAPK signaling pathway and the classic Wnt/β-catenin pathway, improve the expression of mineralization genes, and thus promote the osteogenic differentiation of periodontal membrane stem cells, but further research is needed on how these pathways are activated and the specific mechanisms of this pathway affecting the osteogenic differentiation of periodontal membrane stem cells. (5) The activation or inhibition of various signaling pathways mediated by different research factors significantly affected the osteogenic differentiation of periodontal membrane stem cells. Understanding the signaling pathways associated with osteogenic differentiation of periodontal membrane stem cells will facilitate the application of periodontal membrane stem cells to periodontal tissue regeneration, thereby effectively reducing tooth loss due to periodontitis. Figures and Tables | References | Related Articles | Metrics

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Clinical application of vascular cryoprotectants Wang Shan, Wang Cheng, Shi Yehong 2023, 27 (24):  3920-3925.  doi: 10.12307/2023.533 Abstract ( 331 )   PDF (1501KB) ( 90 )   Save BACKGROUND: Cryopreserved allogeneic blood vessels have broad clinical application prospects in vascular transplantation and repair. Cryopreservation is a common method to achieve long-term preservation of allogeneic blood vessels. During cryopreservation, effective cryoprotectants are essential to prevent vascular tissue hypothermia damage and maintain cell viability. However, there is no consensus on the choice of the best cryoprotectant. OBJECTIVE: To summarize the cryoprotectants used in major vascular tissue banks in recent years and compare their clinical effects, thereby providing a reference for the application of cryopreservation of allogeneic blood vessels in the future. METHODS: The relevant literature included in CNKI, WanFang, PubMed, and Google Scholar databases from 2015 to 2022 was retrieved by the first author. The Chinese search terms were “allogeneic blood vessels, artery, vein, cryopreservation, cryoprotective agent, cryoinjury, vascular transplantation.” The English search terms were “cryopreservation, blood vessels, vascular allografts, arteries, veins, cryoinjury, cryoprotectants, cryoprotective agents.” The relevant literature at home and abroad on vascular cryoprotectants used in clinic and their short-term and long-term results were searched. Finally, 54 articles were selected for review.  RESULTS AND CONCLUSION: (1) In the study of cryopreservation of blood vessels, a combination of permeable and non-permeable cryoprotectant is often used to reduce damage to the structure of the vessel wall through programmed cooling and rapid thawing. (2) Despite the toxic effects of dimethyl sulfoxide, tissue bank product lists in many countries do not include any cardiovascular tissue cryopreserved with cryoprotectant other than dimethyl sulfoxide alone. At present, there are still many clinical complications after cryopreservation of allogeneic blood vessels for vascular transplantation, and some are even fatal. (3) As an essential component of protective agent, dimethyl sulfoxide is very important to balance its toxic effect and protective effect. Finding a new type of cryoprotectant that is safe, non-toxic and free of xenogeneic antigens is the main direction of current research, which is very important for the clinical development of allogeneic vascular grafts. Figures and Tables | References | Related Articles | Metrics

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Extracellular vesicles for acute kidney injury in preclinical research: a meta-analysis Li Qingru, Wang Yifan, Chen Guanting, Shi Ruoyu, Zhang Linqi 2023, 27 (24):  3926-3936.  doi: 10.12307/2023.701 Abstract ( 301 )   PDF (3632KB) ( 64 )   Save OBJECTIVE: Acute renal injury is one of the common clinical critical diseases, and the long-term prognosis of surviving patients is not optimistic. This study systemically assessed the effectivity of extracellular vesicles on animal models of acute kidney injury, hoping to inspire for clinical trials. METHODS: Articles related to the treatment of extracellular vesicles for acute kidney injury in preclinical animal models were retrieved from PubMed, Embase, Web of Science, Cochrane Library, WanFang, VIP, CNKI, and Chinese Biomedical Literature Database. The retrieval time was from the establishment of the database to August 1, 2022. Data were independently extracted by two researchers. SYRCLE Animal Experiment Bias Risk Assessment table was used to evaluate the literature quality. RevMan 5.4 software and Stata 14.0 software were used to perform meta-analysis on serum creatinine, urea nitrogen, tumor necrosis factor-α, tubular necrosis and tubular type outcome indicators.   RESULTS:  (1) A total of 19 randomized controlled animal experimental studies were included, with 399 rodents. There were 169 rodents in the extracellular vesicle treatment group, 168 rodents in the control group and 62 rodents in the stem cell treatment group. (2) The meta-analysis results demonstrated that serum creatinine level (SMD=-5.41, 95%CI:-6.95 to -3.88, P < 0.001) and urea nitrogen level (SMD=-4.57, 95%CI:-5.94 to -3.20, P < 0.001), and tumor necrosis factor-α level (SMD=-2.15, 95%CI:-2.63 to -1.68, P < 0.001) were significantly lower in the extracellular vesicle groups compared with the animal model control groups. Tubular necrosis (SMD=-6.15, 95%CI:-9.66 to -2.64, P=0.001) and casts (SMD=-3.33, 95%CI:-4.17 to -2.49, P=0.001) were significantly reduced. There was no significant difference in the improvement of serum creatinine between the extracellular vesicle group and the mesenchymal stem cell group (SMD=-1.23, 95%CI:-2.80-0.35, P=0.127) and in the improvement of urea nitrogen (SMD=-0.34, 95%CI:-2.19-1.50, P=0.715). CONCLUSION: Extracellular vesicles could improve the levels of serum creatinine and urea nitrogen in animal models with acute kidney injury, which could significantly reduce the level of tumor necrosis factor-α, tubular necrosis and casts. Extracellular vesicles for acute kidney injury provide an important reference for further clinical trials. Figures and Tables | References | Related Articles | Metrics