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18 April 2023, Volume 27 Issue 11 Previous Issue    Next Issue

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Proliferation and differentiation of osteoblasts promoted by the two extracts of Sambucus adnata Wall. Zheng Meimei, Zheng Jianbin, Jiang Zixian, Jia Zhaoyu, Wang Tao, Wang Wenjing 2023, 27 (11):  1677-1682.  doi: 10.12307/2022.947 Abstract ( 393 )   PDF (1357KB) ( 152 )   Save BACKGROUND: Osteoblasts and osteoclasts play an important role in bone remodeling through the release of regulatory factors to remain the bone homeostasis. When the balance between functions and activities of the two types of cells is disturbed, bone diseases such as osteoporosis and osteoarthritis will occur. Sambucus adnata Wall. is commonly used by the ethnic Yi people to treat injuries caused by falls and fractures. Our previous studies have found that the water extract (SAW-A) and dichloromethane extract (SAW-B) of Sambucus adnata Wall. can promote fracture healing and improve osteoporosis in the active parts of fractures. However, their effects on osteoblast proliferation and differentiation in the active parts are still unclear. OBJECTIVE: To investigate the biological effects of SAW-A and SAW-B extracts from Sambucus adnata Wall. on the proliferation and differentiation of osteoblasts.  METHODS: Osteoblasts were extracted from the skull of newborn Sprague-Dawley rats by collagenase digestion, and cell counting kit-8 was used to detect the cytotoxicity of SAW-A and SAW-B. The proliferation activity of osteoblasts was detected by cell counting kit-8 at 24, 48, and 72 hours after extract intervention, to investigate the effects of 312.5 mg/L SAW-A and 156.15 mg/L SAW-B on osteoblast proliferation. Flow cytometry was used to detect the effect of SAW-A and SAW-B on cell activity at 6 days of intervention. The proliferation and differentiation of osteoblasts after SAW-A and SAW-B intervention were detected by alkaline phosphatase staining and alizarin red staining. Real-time fluorescence quantitative PCR assay was performed to detect the relative expression of osteogenesis-related genes RUNX2, bone sialoprotein, osteocalcin and type I collagen A1. RESULTS AND CONCLUSION: SAW-A and SAW-B could significantly improve the proliferation of osteoblasts at 48 and 72 hours after intervention, and SAW-B was stronger than SAW-A. Flow cytometry results showed that SAW-A and SAW-B inhibited apoptosis in osteoblasts after 6 days of intervention. The results of alkaline phosphatase and alizarin red staining showed that SAW-A and SAW-B could promote osteoblast mineralization. The relative expression levels of RUNX2, bone sialoprotein, osteocalcin and type I collagen A1 increased, and SAW-B showed a better effect on the expression of bone sialoprotein and type I collagen A1 than SAW-A. To conclude, SAW-A and SAW-B can both promote osteoblast differentiation. Figures and Tables | References | Related Articles | Metrics

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Mechanism by which naringin regulates osteogenic differentiation in osteoblasts Wu Jingjing, Lin Haixiong, Sun Weipeng, Li Zige, Jiang Ziwei 2023, 27 (11):  1722-1727.  doi: 10.12307/2022.1021 Abstract ( 479 )   PDF (1231KB) ( 108 )   Save BACKGROUND: Studies have found that naringin affects bone metabolism directly through Wnt, transforming growth factor β, MAPK signaling pathway, and osteoclast signaling pathway, as well as indirectly through P13K-Akt and vascular endothelial growth factor signaling pathway. Naringin can promote bone repair and retard osteoporosis.  OBJECTIVE: To investigate the effect of naringin on the proliferation and osteogenic differentiation of osteoblasts by targeting the activation of connexin 43/ERK1 signaling pathway through down-regulation of miR-206. METHODS: Rat osteoblasts (ROB cells) were treated and cultured with different concentrations of naringin (1, 10, 100 mg/L and 1, 10 g/L), and the cell proliferation was detected by cell counting kit-8 assay. Naringin at 100 mg/L and 1, 10 g/L were used for subsequent experiments. Osteoblasts with non-drug culture were used as controls. RT-qPCR was used to detect the relative expression levels of miR-206, connexin 43 and ERK1 mRNAs in ROB cells to verify their targeting relationship. Western blot was used to detect the protein expression levels of ERK1/2 and P-ERK in ROB cells. Alkaline phosphatase activity and calcification ability in ROB cells were observed by alkaline phosphatase staining and alizarin red staining, respectively. RESULTS AND CONCLUSION: Naringin significantly promoted the proliferation activity of ROB cells (P < 0.05). The absorbance value and relative proliferation rate of ROB cells increased as the administration concentration of naringin increased in a certain range. When the mass concentration was 1 g/L, the cell proliferation activity was the strongest. Naringin significantly inhibited the expression of miR-206 mRNA in ROB cells and promoted the expression of connexin 43 mRNA and ERK1/2 and P-ERK protein (P < 0.05). Naringin could significantly promote the activity and expression of alkaline phosphatase and the level of cellular calcification in ROB cells (P < 0.05). Moreover, naringin at 1 g/L showed the best effect. To conclude, naringin can target and activate the connexin 43/ERK1 signaling pathway by down-regulating the expression of miR-206, thereby promoting the proliferation and osteogenic differentiation of ROB cells. Figures and Tables | References | Related Articles | Metrics

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Calcitonin for postmenopausal osteoporotic hip fractures: a prospective randomized controlled trial Liu Zhi, Zhu Yong, Lin Zhangyuan, Zhao Ruibo, Long Haitao, Lu Bangbao, Sun Buhua, Cheng Liang, Zhao Shushan 2023, 27 (11):  1739-1743.  doi: 10.12307/2023.167 Abstract ( 411 )   PDF (797KB) ( 144 )   Save BACKGROUND: Calcitonin acts as a classic anti-osteoporosis drug and has been widely used in clinical anti-osteoporosis treatment. Increasing studies have found that it also has a marked effect in the treatment of osteoporotic fractures. OBJECTIVE: To evaluate the clinical efficacy and safety of calcitonin in the treatment of postmenopausal osteoporotic hip fractures.  METHODS: A prospective randomized control trial was conducted. Sixty elderly patients with osteoporotic hip fractures were enrolled and randomly assigned to an experimental group or a control group in a 1:1 ratio. The experimental group was given domestic calcitonin (once a week) combined with calcium carbonate+vitamin D (once a day) for 6 months; and the control group was given calcium carbonate+vitamin D (once a day) for 6 months. Clinical efficacy and safety of calcitonin were then evaluated by comparing bone turnover marker, fracture healing rate, bone mineral density, serum calcium, serum phosphorus and adverse effects between two groups.  RESULTS AND CONCLUSION: Both experimental group (n=26) and control group (n=28) completed the experiment and their baseline data were basically the same. Calcitonin could significantly inhibit the mass concentration of C-terminal crosslinked telopeptides of type 1 collagen  at 8, 12, and 24 weeks (P < 0.01) and improve visual analogue scale score at 4 and 8 weeks (P < 0.01) but have no effect on fracture healing, serum calcium and serum phosphorus levels. (3) There were no significant differences in bone mineral density and adverse effects between the two groups. To conclude, calcitonin can reduce postoperative high conversion state, reduce acute bone loss, relieve postoperative pain, and have good safety in postmenopausal osteoporotic fracture patients. Figures and Tables | References | Related Articles | Metrics

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Significance of miR-27b/peroxisome proliferators-activated receptor gamma 2 axis for proliferation and osteoblast differentiation of mouse embryonic osteogenic precursor cells Wang Lei, Bai Xuesong, Du Yu, He Aimin, Zheng Jun, Zhang Zhipeng, Lyu Huicheng 2023, 27 (11):  1780-1786.  doi: 10.12307/2023.129 Abstract ( 292 )   PDF (1190KB) ( 84 )   Save BACKGROUND: Previous studies have confirmed that miR-27b at the lipid level can control multiple genes that have an important impact on dyslipidemia. It is speculated that miR-27b may function in osteoporosis by targeting peroxisome proliferators-activated receptors γ2 (PPARγ2). OBJECTIVE: To investigate the effect of miR-27b/PPARγ2 axis in the dexamethasone-induced osteoporosis model in mouse embryonic osteogenic precursor cells MC3T3-E1 and related mechanisms of action. METHODS: After MC3T3-E1 cells were dexamethasone-induced and cultured in vitro, miR-27b mimic, miR-27b inhibitor, NC-mimic, NC-inhibitor, siPPARγ2, and siNC were transfected with Lipofectamine®2000, and dimethyl sulfoxide was used as a control. After the cells were induced and cultured, the cell viability and alkaline phosphatase activity were detected to determine the osteogenesis and differentiation levels of the cells. The mRNA and protein expression levels of osteogenic differentiation genes miR-27B, PPARγ2, Runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 and osteocalcin were detected by real-time fluorescence quantitative PCR and western blot methods. Bioinformatics analysis predicted the downstream target genes of miR-27b followed by verification with dual luciferase gene reporter experiments. RESULTS AND CONCLUSION: Dexamethasone treatment significantly reduced cell viability and miR-27b expression levels in MC3T3-E1 pre-osteoblasts. Compared with dimethyl sulfoxide, dexamethasone significantly inhibited MC3T3-E1 cell viability and downregulated miR-27b expression levels at 24 and 48 hours. miR-27b could directly regulate PPARγ2. Compared with the corresponding NC-mimic, the miR-27b mimic significantly upregulated the expression level of miR-27b, while the miR-27b inhibitor significantly downregulated the expression level of miR-27b. Furthermore, PPARγ2 miRNA and protein expression levels were inhibited by the miR-27b mimic and significantly enhanced by the miR-27b inhibitor. miR-27b overexpression attenuated the inhibitory effect of dexamethasone on the proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts. Dexamethasone significantly inhibited osteoblast differentiation compared with the dimethyl sulfoxide + NC-mimic group. In contrast, the dexamethasone-mediated inhibition was reversed by the miR-27b mimic. Cellular alkaline phosphatase activity and bone morphogenetic protein 2, Runx2 and osteocalcin protein expression levels were partially restored. Inhibition of miR-27b suppressed proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts through the upregulation of PPARγ2. Knockout of miR-27b significantly increased PPARγ2 expression and decreased cell viability, osteoblast differentiation, alkaline phosphatase activity, and expression levels of bone morphogenetic protein 2, Runx2 and osteocalcin. PPARγ2 may be a direct target of miR-27b. Figures and Tables | References | Related Articles | Metrics

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Effects of epidermal growth factor receptor on proliferation, differentiation and apoptosis of mouse articular cartilage surface cells Zhu Zhoujun, Zhang Jiahui, Gao Jian, Xu Bin, Wang Chao, Xiang Wei, Jia Haoruo, Wang Guosheng 2023, 27 (11):  1728-1732.  doi: 10.12307/2023.190 Abstract ( 351 )   PDF (1059KB) ( 87 )   Save BACKGROUND: Epidermal growth factor receptor is a key factor regulating the cell cycle and its downstream signaling pathway is widely involved in the regulation of various biological processes of cells. OBJECTIVE: To isolate and culture articular cartilage surface cells from mice and to verify the effect of epidermal growth factor receptor signaling pathway on the proliferation, differentiation and apoptosis of articular cartilage surface cells. METHODS: Articular cartilage surface cells from mice were isolated and cultured in vitro by fibronectin adhesion and enzyme digestion and identified. Passage 3 cells were separately treated with transforming growth factor α (100 μg/L), Gefitinib (10 μmol/L), and conventional medium (control group). Cell proliferation was detected by cell counting kit-8 and Ki67 gene expression was detected by RT-PCR. Cell apoptosis was induced by transforming growth factor α (25 μg/L) and AOEB staining was used to identify the apoptosis. RT-PCR was used to detect the expression of Bcl2 and Bax genes. Chondrogenic, osteogenic, and adipogenic induction were conducted for 14-21 days in each group and the mRNA expressions of Sox9, Runx2, and lipoprotein lipase were detected by RT-PCR. RESULTS AND CONCLUSION: Articular cartilage surface cells isolated and cultured in vitro were long-shaped and positively expressed PRG4 identified by immunofluorescence. Cell counting kit-8 results showed that after treatment with transforming growth factor α, cell proliferation ability and the mRNA level of Ki67 were significantly increased (P < 0.05). AOEB staining results showed that after treatment with transforming growth factor α, the apoptotic level was significantly decreased, the mRNA level of Bcl2 was significantly increased (P < 0.05), and the mRNA level of Bax was significantly decreased (P < 0.05). RT-PCR results revealed that the mRNA level of Runx2 was significantly increased (P < 0.05) and the mRNA level of Sox9 was significantly decreased after treatment with transforming growth factor α. To conclude, the epidermal growth factor receptor signaling pathway is involved in the proliferation, differentiation, and apoptosis of articular surface chondrocytes. Epidermal growth factor receptor mediated by transforming growth factor α can promote the proliferation and osteogenic differentiation of articular cartilage surface cells and inhibit the chondrogenic differentiation and apoptosis of articular cartilage surface cells. Figures and Tables | References | Related Articles | Metrics

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Effect of maxillary protraction on temporomandibular joint changes in skeletal class III children: a correlation analysis Zhang Haolin, Wang Yalin, Liu Yafei, Zuo Yanping, Zhang Xiaohuan 2023, 27 (11):  1641-1646.  doi: 10.12307/2023.100 Abstract ( 354 )   PDF (1177KB) ( 36 )   Save BACKGROUND: Maxillary protraction treatment can effectively change the craniofacial structural relationship and the temporomandibular joint structure of children with skeletal Class III malocclusion. However, there are no definitive findings regarding the correlation between changes in the temporomandibular joint and craniomaxillofacial changes after maxillary protraction.  OBJECTIVE: To analyze the correlation between dentofacial changes and temporomandibular joint changes in children with skeletal class III malocclusion before and after maxillary protraction and to investigate the effect of maxillary protraction on the temporomandibular joint of children.  METHODS: A total of 29 children with skeletal class III malocclusion, including 16 boys and 13 girls, aged 8-11 years, were treated by maxillary protraction. The changes of dentofacial and temporomandibular joint were subjected to cephalometric analysis and coordinate system positioning measurement. Paired t-test, Mann-Whitney test and Pearson correlation analysis were conducted.  RESULTS AND CONCLUSION: The sagittal relationship of the jaw changed obviously before and after maxillary protraction. The glenoid fossa and condyle changed significantly in the X axis but not in the Y axis. The posterior space of the temporomandibular joint was significantly reduced. Correlation analysis revealed a moderate correlation between the temporomandibular joint and dentofacial structural changes. To conclude, after maxillary protraction, both dentofacial and temporomandibular joint structures have significant changes, and there is a moderate correlation between temporomandibular joint remodeling and dentofacial changes, but no close correlation exists between them. Figures and Tables | References | Related Articles | Metrics

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Effects of carvacrol on osteoblasts and common oral pathogens Zhang Zihan, Wang Wenli, Li Jinnuo, Li Yourui 2023, 27 (11):  1765-1771.  doi: 10.12307/2023.124 Abstract ( 429 )   PDF (1089KB) ( 218 )   Save BACKGROUND: The natural plant extract carvacrol has the characteristics of broad-spectrum antibacterial activity, low drug resistance and low cytotoxicity, which has become a research hotspot of antibiotic substitutes with a good prospect in recent years. OBJECTIVE: To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvacrol against Streptococcus mutans and Staphylococcus aureus, two common oral pathogens and to explore the antibacterial activity of carvacrol against these two bacteria and its effect on osteoblast proliferation, thereby providing a new choice for oral disease prevention and treatment.   METHODS: A microplate reader was used to determine the MIC and MBC values of carvacrol against Streptococcus mutans and Staphylococcus aureus. The antibacterial activity of carvacrol was tested by a zone of inhibition test. Cell counting kit-8 was used to detect the proliferation of MC3T3-E1 cells induced by different concentrations of carvacrol at 1, 4, and 7 days. Alkaline phosphatase activity and Runx2 and osteoprotegerin gene expression levels were detected by alkaline phosphatase and real-time fluorescence quantitative PCR kits at 1, 7, and 14 days, respectively.   RESULTS AND CONCLUSION: The MIC values of carvacrol against Streptococcus mutans and Staphylococcus aureus were 0.20% and 0.04%, and the MBC values were 0.60% and 0.12%, respectively. In the inhibition zone test, carvacrol showed an obvious antibacterial zone, and the difference was statistically significant (P < 0.05). Cell counting kit-8 assay showed that the proliferation activity of cells treated with carvacrol was significantly increased (P < 0.05). The activity of alkaline phosphatase increased in all carvacrol concentration groups. The increase of alkaline phosphatase activity was more obvious on day 7 and the effect of 1MIC group was the most obvious (P < 0.05). Results from the real-time fluorescence quantitative PCR showed that different genes were highly expressed at the mRNA level after treated with carvacrol and all of them occurred in the 1MIC group (P < 0.05). To conclude, carvacrol has strong antibacterial activity against two common oral pathogens, Streptococcus mutans and Staphylococcus aureus, can improve the osteogenic activity of MC3T3-E1 cells, and promote their proliferation and differentiation. Figures and Tables | References | Related Articles | Metrics

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Intervention with platelet-rich fibrin in rabbit models after microvascular anastomosis surgery of the femoral artery: changes in vascular structure and soft tissue Lu Yangyang, Yan Ruihong, Qiu Zhongpeng, Dai Yi, Wang Zixin, Wang Weishan, Shi Chenhui, Du Xinhui, Li Gang 2023, 27 (11):  1659-1668.  doi: 10.12307/2023.182 Abstract ( 343 )   PDF (5885KB) ( 383 )   Save BACKGROUND: Recently, numerous basic and clinical data have shown the superiority of platelet-rich fibrin in the regeneration and repair of soft and hard tissues, while there are few relevant reports in the field of microvascular anastomosis.   OBJECTIVE: To evaluate the efficacy of platelet-rich fibrin applied to the femoral artery after microvascular anastomosis surgery in a rabbit model.   METHODS: Thirty-six adult New Zealand white rabbits of either sex were randomly divided into experimental group and control group (n=18 per group) after establishing femoral artery transection-reanastomosis models. In the experimental group, autologous ear venous blood were extracted and centrifuged to prepare platelet-rich fibrin gel, and the prepared gel was immediately applied to the anastomotic site of the femoral artery. The control group was given the same volume of normal saline. Incision infection, healing, and adhesion were evaluated at 3, 7, 14 days after surgery. Vascular structure of the femoral artery at the anastomotic site was observed using hematoxylin-eosin staining. Immunohistochemistry detection was performed to analyze the expression levels of vascular endothelial growth factor, platelet-derived growth factor, and transforming growth factor β1.  RESULTS AND CONCLUSION: The overall number of incision infections in the experimental group was lower than that in the control group animals (P < 0.05), and the number of incision infections showed no significant changes in each group (P > 0.05). The overall incision healing level in the experimental group was better than that in the control group (P < 0.05), and the incision healing showed no significant changes in the experimental group (P > 0.05) but changed significantly in the control group at different time points (P < 0.05). The degree of femoral artery adhesion in the operation area was generally lighter in the experimental group than the control group (P < 0.05). The degree of adhesion had insignificant changes in the experimental group at different time points (P > 0.05) but gradually increased in the control group after operation (P < 0.05). The results of hematoxylin-eosin staining showed better repair of vascular endothelial cells and inner elastic lamina in the experimental group than the control group. Compared with the control group, the reconstruction of the intima was more complete in the experimental group, with less necrosis, proliferation, and migration of vascular smooth muscle cells in the media and traumatic hyperplasia of the extima. The overall healing condition of the vascular wall was better in the experimental group than the control group. Immunohistochemical results revealed that the expression levels of vascular endothelial growth factor, platelet-derived growth factor, and transforming growth factor β1 in the experimental group were higher than those in the control group (P < 0.05). To conclude, platelet-rich fibrin can promote the growth of vascular endothelial cells, contribute to the in situ reconstruction of various layers of vascular tissue, reduce postoperative complications, such as infection and adhesion, in the surgical area, and shorten the repair and healing time after vascular injury. This novel treatment method is expected to improve the therapeutic effect of arterial anastomosis in microsurgery.  Figures and Tables | References | Related Articles | Metrics

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Radial extracorporeal shockwave combined with resistance exercise for treating senile primary sarcopenia Shen Lianwei, Wang Wei 2023, 27 (11):  1647-1652.  doi: 10.12307/2023.085 Abstract ( 353 )   PDF (912KB) ( 160 )   Save BACKGROUND: Various treatment methods for senile primary sarcopenia have been developed, but all of them are restrictedly used in clinical practice due to their limitations. Therefore, it is necessary to find a safe treatment with good patient compliance. OBJECTIVE: To explore the clinical effect of radial extracorporeal shockwave therapy combined with resistance exercise on senile primary sarcopenia.  METHODS: Forty elderly patients with primary sarcopenia admitted in the First Affiliated Hospital of Jinzhou Medical University from February to August 2021 were collected and randomly divided into control group and treatment group with 20 cases in each group. Both two groups were subjected to resistance exercise. The treatment group was treated with radial extracorporeal shockwave therapy based on resistance exercise. Skeletal muscle index, walking speed, knee extension muscle strength, thigh circumference, and serum insulin growth factor-1 level were recorded before treatment and after 4 weeks and 8 weeks of treatment. All data were statistically analyzed. RESULTS AND CONCLUSION: Significant differences were found in skeletal muscle index, walking speed, knee extensor strength, thigh circumference and serum insulin growth factor-1 level in both control and treatment groups at different time points (P < 0.05). After 4 and 8 weeks of treatment, knee extensor strength and serum insulin growth factor-1 level were significantly better in the treatment group than the control group (P < 0.05). After 8 weeks of treatment, the circumference of the thigh was significantly improved in the treatment group compared with the control group (P < 0.05). These findings indicate that compared with resistance training alone, radial extracorporeal shockwave therapy combined with resistance training can better promote the recovery of muscle quality and function and delay the occurrence and development of senile primary sarcopenia. Figures and Tables | References | Related Articles | Metrics

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Effects of short-term low-frequency pulsed electrical magnetic field-induced classical transient receptor potential channel 1 on maximum voluntary contraction and strength endurance of the biceps brachii Li Zhongshan, Wang Chunlu, Liu Jie, Yang Tieli, Kong Weiqian, Li Wei, Zhang Qinyang, Chen Song, Che Tongtong, Li Zhiyuan, Guan Rongxin, Bai Shi 2023, 27 (11):  1796-1804.  doi: 10.12307/2023.200 Abstract ( 424 )   PDF (1502KB) ( 140 )   Save BACKGROUND: Strength quality is an essential element for human physical activity. Short-term low-frequency pulsed electrical magnetic field (PEMF) stimulation can activate classical transient receptor potential channel 1 (TRPC1) and trigger skeletal muscle growth and remodeling in mice, thereby producing a series of physiological support effects on muscle tissue. However, there is no report on whether this mechanism will alter the physiological structure and working ability of human skeletal muscle and act as a new way to improve muscle strength. OBJECTIVE: To select a specific low-frequency PEMF that can activate TRPC1 as an exogenous stimulus to observe and verify the effect of short-term stimulation on the maximum voluntary contraction force and strength endurance of human biceps brachii. METHODS: A total of 27 normal adult healthy subjects were selected and randomly divided into exercise group, PEMF group, and exercise+PEMF group, with 9 cases in each group. The training group was subjected to resistance training. The exercise+PEMF group received 10-min low-frequency PEMF stimulation (intensity 1.5 mT, frequency 3300 Hz) immediately after resistance training. The irradiation group only received 10-minute low-frequency PEMF stimulation. Training or irradiation was performed every 48 hours for 9 days. Electromyography information on the maximum voluntary contraction force was collected before and after five training sessions in the exercise group and the exercise+PEMF group, to observe whether the combination of low-frequency PEMF and resistance training will produce a gain effect. In the PEMF group, the maximum voluntary contraction force was tested only at the 1st, 3rd, and 5th sessions to track the changes in muscle strength. The maximum voluntary contraction force, one-repetition maximum force, endurance duration and median frequency were observed in each group. RESULTS AND CONCLUSION: (1) During the trial, there were significant interaction effects between the changes in maximum voluntary contraction force and time in all the subjects (P < 0.01) as well as in each group as time goes by (P < 0.05). However, there was no interaction effect between groups. (2) There was a significant improvement in the maximum voluntary contraction force, one-repetition maximum force, endurance duration, and median frequency in each group after completion of the trial. These indicators were successively improved by 19%, 23%, 28%, and 18% in the exercise group, 11%, 10%, 53%, and 18% in the exercise+PEMF group, and 28%, 18%, 27%, and 6% in the PEMF group. (3) The median frequency of the exercise+PEMF group was significantly higher than that of the PEMF group (P < 0.05), but there was no significant difference between the exercise+PEMF group and exercise group. (4) Compared with the exercise+PEMF group, the exercise group showed a significant decrease in the root mean square of maximum voluntary contraction after the first two exercise sessions (P < 0.05). (5) All these findings indicate that the short-term PEMF stimulation with intensity of 1.5 mT and frequency of 3 300 Hz can significantly improve the maximum voluntary contraction force and strength endurance of human biceps brachii. That is, low-frequency PEMF can induces TRPC1 to promote the working ability of muscle tissue, which has been effectively verified in human body. Low-frequency PEMF stimulation shares similar effects with conventional resistance training in terms of enhancing the maximum strength. Resistance training combined with PEMF stimulation shows better anti-fatigue ability and stabilizes the increase of muscle strength. This combination can stimulate more motor units of local muscle groups under the same load in the early stage of training and improve the overall training efficiency. In terms of strength endurance improvement, low-frequency PEMF stimulation can prolong isometric muscle contraction time and improve anti-fatigue ability. Compared with simple low-frequency PEMF stimulation, low-frequency PEMF stimulation combined with resistance training is more effective to develop strength endurance.   Figures and Tables | References | Related Articles | Metrics

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miR-382-3p inhibits the proliferation of human hypertrophic scar fibroblasts He Xi, Ma Fang, Wan Yu, Tang Yuting, Ma Shengchao, Jiang Yideng, Shen Jiangyong 2023, 27 (11):  1758-1764.  doi: 10.12307/2023.143 Abstract ( 381 )   PDF (1766KB) ( 88 )   Save BACKGROUND: At present, numerous studies have shown that miRNA is involved in the occurrence and development of hypertrophic scars and STAT1 is involved in the proliferation of scar fibroblasts. It is speculated that miR-382-3p may also be related to the occurrence and development of hypertrophic scars. OBJECTIVE: To investigate the mechanism of miR-382-3p on the proliferation of human hypertrophic scar fibroblasts. METHODS: Hypertrophic scar and normal skin of the same individual were collected from the Department of Burn Plastic Surgery, General Hospital of Ningxia Medical University, and human hypertrophic scar fibroblasts and human normal skin fibroblasts were then extracted. Cell transfection was conducted as follows: (1) the cells were divided into control group (no treatment), miR-382-3p negative control group, and miR-382-3p overexpression group; (2) the cells were divided into control group (no treatment), STAT1 interference control group (si-NC) and STAT1 interference groups (si-STAT1-1, si-STAT1-2, si-STAT1-3). Hematoxylin-eosin staining was used to identify normal skin and hypertrophic scar, and immunofluorescence was used to identify fibroblasts. Quantitative real-time PCR was applied to detect the mRNA expression of miR-382-3p, STAT1, proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase inhibitor (p27). Western blot assay was performed to detect the protein expressions of STAT1, PCNA and p27. Cell counting kit-8 and EdU were used to detect cell proliferation activity and proliferation level. Targetscan was used to predict the downstream target genes of miR-382-3p and dual luciferase was used to verify the binding of miR-382-3p to STAT1. RESULTS AND CONCLUSION: Compared with normal skin and normal skin fibroblasts, miR-382-3p was lowly expressed in hypertrophic scar and hypertrophic scar fibroblasts (tissue: P < 0.01, cell: P < 0.01), and STAT1 was highly expressed in hypertrophic scar and hypertrophic scar fibroblasts (mRNA level in tissue: P < 0.01, protein level in tissue: P < 0.01; mRNA level in cells: P < 0.01, protein level in tissue: P < 0.01). After overexpression of miR-382-3p, the cell proliferation ability was weakened (P < 0.05), the number of EdU positive cells was decreased (P < 0.01), the expression of PCNA was decreased (mRNA: P < 0.01, protein: P < 0.05), and the expression of p27 was increased (mRNA: P < 0.05, protein: P < 0.05). miR-382-3p could target and regulate the expression of STAT1 (P < 0.01), and overexpression of miR-382-3p could reduce the expression of STAT1 (mRNA: P < 0.01, protein: P < 0.01). Interference with STAT1 reduced the expression of PCNA (P < 0.05), increased the expression of p27 (P < 0.05), and reduced the number of EdU positive cells (P < 0.01). To conclude, miR-382-3p could inhibit the proliferation of hypertrophic scar fibroblasts by inhibiting the expression of STAT1, which provides a certain theoretical basis for identifying effective targets for the treatment of hypertrophic scars. Figures and Tables | References | Related Articles | Metrics

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Correlation analysis between thyroid function-related Thrβ and Thrsp genes and mechanical properties of Achilles tendon development in rats Niu Jingwei, Zhang Qingtao, Wang Na, Guan Shuo, Wang Ying 2023, 27 (11):  1653-1658.  doi: 10.12307/2023.091 Abstract ( 302 )   PDF (1300KB) ( 147 )   Save BACKGROUND: Although thyroid diseases are rarely thought to be related to tendon diseases and tendon tears, increasing evidence has indicated that thyroid diseases are involved in idiopathic tendon diseases in recent years. However, there are few studies on the effects of thyroid function on the development and mechanical properties of Achilles tendon during growth and development. OBJECTIVE: To clarify the correlation between thyroid hormone receptor beta (Thrβ) and thyroid hormone responsive (Thrsp) related to thyroid function and the mechanical properties and histological morphology of the Achilles tendon and to confirm the potential influence of thyroid function on the development of the Achilles tendon. METHODS: The expression profiles of developmental genes in Achilles tendon were analyzed by bioinformatics, and Thrβ and Thrsp genes related to thyroid function were identified. Then the mechanical properties of the Achilles tendon of female Sprague-Dawley rats at different growth and development stages (28, 42, 56 and 70 days of age, 6 females at each stage) were measured in a tensile test. Hematoxylin-eosin staining combined with histomorphometry was used for histological evaluation and the expression levels of Thrβ and Thrsp genes were determined by fluorescence quantitative PCR. Finally, correlation analysis was conducted to determine the relationship between Thrβ and Thrsp gene expression levels and mechanical properties and histological morphology of the Achilles tendon. RESULTS AND CONCLUSION: With the growth and development of rats, the maximum load, maximum tensile length and stiffness of the Achilles tendon did not change significantly after peaking. These mechanical properties of Achilles tendon may be related to the content, type and arrangement of collagen fibers in the tissue. Thrβ and Thrsp gene expression in the Achilles tendon at different growth stages were significantly correlated with the mechanical properties and histological scores of the Achilles tendon. These findings suggest that thyroid hormone plays a potential role in the development of the Achilles tendon and it is expected to offer new therapeutic ideas for the repair of Achilles tendon injury. Figures and Tables | References | Related Articles | Metrics

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Comparison of cerebral microcirculation perfusion in rat models of middle cerebral artery occlusion prepared through common carotid artery insertion and external carotid artery insertion Li Yong, Yuan Jianmei, Lu Danni, Ren Mihong, Deng Bowen, Wang Jiajun, Ma Rong, Xie Qian, Li Jinxiu, Xu Zhuo, Wang Jian 2023, 27 (11):  1683-1691.  doi: 10.12307/2023.127 Abstract ( 469 )   PDF (2306KB) ( 146 )   Save BACKGROUND: Animal models of middle cerebral artery occlusion (MCAO) prepared by common carotid artery insertion or external carotid artery insertion are commonly used in preclinical studies of cerebral ischemia. However, there are few comparative studies on the difficulty of model preparation and microcirculatory perfusion during ischemic brain injury between the two methods of insertion. OBJECTIVE: To compare the MCAO models prepared by the common carotid artery insertion approach (CCAIA group) and external carotid artery insertion approach (ECAIA group) in the difficulty of model preparation and the differences in microcirculation perfusion. METHODS: Sprague-Dawley rats were randomly divided into sham-operated group, CCAIA group, and ECAIA group. Rats in the sham-operated group were anesthetized but without blood vessel ligation and suture insertion, and the other operations were the same as the above two methods. The modeling time, success rate of insertion, postoperative mortality and success rate of modeling were recorded. After the middle cerebral artery was blocked by suturing, laser speckle imaging technique was used to observe the changes of cerebral blood flow in the ischemic hemisphere and blood vessel diameter in the ischemic penumbra within 90 minutes after ischemia and ischemia-reperfusion. The neurological deficit score was evaluated by Longa’s method and modified neurological severity score at 2, 24, 48, and 72 hours postoperatively. TTC staining, dry-wet weight method, and Evans blue staining were used to calculate cerebral infarction rate, brain water content, and blood-brain barrier permeability at 72 hours postoperatively. Levels of endothelin-1, calcitonin gene-related peptide, cyclic adenosine monophosphate, prostaglandin E2, and nitric oxide in serum were determined by ELISA.   RESULTS AND CONCLUSION: Compared with the ECAIA group, the CCAIA group had shorter operation time (P < 0.01) and higher success rate of modeling (P < 0.05). The cerebral blood flow, blood vessel diameter, and cyclic adenosine monophosphate level after reperfusion were significantly lower in the CCAIA group than the ECAIA group (P < 0.05). However, there was no significant difference between ECAIA and CCAIA groups in cerebral infarction rate, brain water content and blood-brain barrier permeability as well as in the serum levels of endothelin-1, calcitonin gene-related peptide, prostaglandin E2 and nitric oxide content (P > 0.05). To conclude, compared with the external carotid artery insertion, the common carotid artery insertion has the advantages of simple operation and high success rate of modeling, but results in an obvious microcirculation perfusion disorder in the cerebral cortex after reperfusion. Insufficient microcirculatory perfusion may be related to the decreased cyclic adenosine monophosphate level in serum. Figures and Tables | References | Related Articles | Metrics

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Hyperoxia-induced differential expression of microRNAs mediates lung injury in neonatal rats Wang Lina, Zhang Rong, Kang Lan, Lei Xiaoping, He Hongxia, Dong Wenbin 2023, 27 (11):  1692-1700.  doi: 10.12307/2023.155 Abstract ( 291 )   PDF (3247KB) ( 100 )   Save BACKGROUND: The main pathological features of bronchopulmonary dysplasia are stagnation of lung development and minor tissue damage. Fetal lung development needs to go through five stages, and a preterm infant cannot survive after birth until the cystic stage. Therefore, research on the pathogenesis of premature birth- and hyperoxia-induced bronchopulmonary dysplasia has focused on cystic and alveolar phases. OBJECTIVE: To investigate the regulatory role of differentially expressed microRNAs in hyperoxia-associated bronchopulmonary dysplasia during two developmental stages – cystic and alveolar stages. METHODS: Newborn Sprague-Dawley rats were randomly into four groups: in 0 day group (n=10), rat lung tissue was taken immediately after birth; 4-, 7-, 14-day groups were further divided into two subgroups – hyperoxia group (exposed to persistent hyperoxia with an oxygen concentration of 85%-90%, n=10) and air group (exposed to the air with FiO2: 21%, n=10). Lung samples tissue were then taken at postnatal days 4, 7, 14. MicroRNAs in lung tissue were acquired using miRNA-Seq technology to build a library, and sequenced on the Illumina platform. Differently expressed microRNAs between different groups were screened by Deseq2 (Version 1.10.1) and Venn map. Gene ontology enrichment and protein-protein interaction analysis were performed to predict target proteins of differentially expressed microRNAs.  RESULTS AND CONCLUSION: There were 633 microRNAs in the 0-day group, 609, 614, and 584 microRNAs in the hyperoxia group at postnatal days 4, 7, 14, respectively, and 629, 608, and 581 in the corresponding air groups. The hyperoxia group obtained 130, 3, and 114 differentially expressed microRNAs during postnatal days 0-4 (cystic stage), 4-7, and 4-14 (alveolar stage), respectively. And the corresponding air group received 106, 0, 111 differentially expressed microRNAs respectively. There were 91, 0, and 79 differentially expressed microRNAs in the two groups at different stages. During the cystic and alveolar phases, miR-34a-5p and miR-21-5p were significantly expressed in the lung tissue of neonatal rats in the hyperoxia group. miR-34a-5p had 198 target proteins, which concentrated around the Pdgfrb/Pdgfra/Ngfr; miR-21-5p had 302 target proteins that mainly revolved around the Mapk1/Stat3. To conclude, miR-34a-5p and miR-21-5p are highly expressed in the cystic and alveolar stages under hyperoxia exposure, targeting the proteins about PDGFRB/PDGFRA/NGFR and MAPK1/Stat3 respectively, which mediate the process of lung development and participate in the occurrence and development of bronchopulmonary dysplasia.  Figures and Tables | References | Related Articles | Metrics

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Mechanism by which tetramethylpyrazine improves inflammatory microenvironment after spinal cord injury in rats Zhang Houjun, Jiang Shengyuan, Deng Bowen, Liu Gang, Bai Huizhong, Tao Jingwei, Fan Xiao, Zhao Yi, Ren Jingpei, Xu Lin, Mu Xiaohong 2023, 27 (11):  1701-1707.  doi: 10.12307/2023.122 Abstract ( 336 )   PDF (1685KB) ( 49 )   Save BACKGROUND: The imbalance of inflammatory microenvironment after spinal cord injury can aggravate secondary spinal cord injury and hinder the repair of nerve function. Tetramethylpyrazine can inhibit inflammatory responses and protect nerve cells; however, its research in the field of spinal cord injury is less. OBJECTIVE: To observe whether tetramethylpyrazine can improve inflammatory responses and recover nerve function after spinal cord injury in rats and to explore its potential mechanism. METHODS: Thirty-six Sprague-Dawley rats were randomly divided into sham operation group, model group, and tetramethylpyrazine group, with 12 rats in each group. The sham operation group received T10 laminectomy; the model and tetramethylpyrazine groups received T10 spinal cord complete transection by self-made double-edge microshear. After modeling, the tetramethylpyrazine group was given tetramethylpyrazine hydrochloride injection via intraperitoneal injection (200 mg/kg/d) for consecutive 5 days. Basso, Beattie and Bresnahan score and modified Rivlin oblique plate test were used to evaluate the recovery of motor function at 1, 3, 7, and 14 days after surgery. Tissue samples were stained with hematoxylin-eosin and Nissl staining to observe the morphological changes at 14 days after surgery. Changes in C-reactive protein were detected by enzyme-linked immunosorbent assay. The expression of inflammatory factors, including nuclear factor-кB, tumor necrosis factor-α, interleukin-10, was analyzed by immunohistochemical staining. RESULTS AND CONCLUSION: At each time point after spinal cord injury, Basso, Beattie and Bresnahan score and modified Rivlin slant angle in the sham operation group were significantly higher than those in the other groups (P < 0.05), while there were no significant differences between the model and tetramethylpyrazine groups (P > 0.05). Hematoxylin-eosin staining results showed that the number of inflammatory cells in the tetramethylpyrazine group was significantly lower than that in the model group. Nissl staining results revealed that the number of neurons at both broken ends of the spinal cord was significantly higher in the tetramethylpyrazine group than the model group. After spinal cord injury, C-reactive protein expression increased firstly and then decreased, and tetramethylpyrazine could significantly reduce the expression of C-reactive protein (P < 0.05). At 7 days after modeling, the expression of nuclear factor-кB and tumor necrosis factor-α in the model group was significantly higher than that in the sham operation group (P < 0.05), while the expression of interleukin-10 was lower than that in the sham operation group. Compared with the model group, tetramethylpyrazine could significantly decrease the expression of nuclear factor-кB and increase the expression of interleukin-10 (P < 0.05). At 14 days after modeling, the expression levels of nuclear factor-кB and tumor necrosis factor-α increased significantly in the model group compared with the sham operation group, while the expression level of interleukin-10 was significantly lower than the sham operation group (P < 0.05). Compared with the model group, tetramethylpyrazine could significantly increase the expression of interleukin-10 (P < 0.05). All these findings suggest that after spinal cord injury, the expression of pro-inflammatory factors is significantly increased and the inflammatory microenvironment becomes unbalanced. Tetramethylpyrazine can regulate the immune inflammatory response after spinal cord injury, improve the inflammatory microenvironment, and promote the repair of spinal cord injury.  Figures and Tables | References | Related Articles | Metrics

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Suture-occluded method versus chemical destruction method to induce swallowing disorders in model rats Hua Xiaoqiong, Li Yanjie, Li Sijin, Zhang Shuqin, Liu Haoyuan, Ding Huimin, Zhao Nannan 2023, 27 (11):  1708-1714.  doi: 10.12307/2023.183 Abstract ( 227 )   PDF (1648KB) ( 106 )   Save BACKGROUND: The establishment of animal models is one of the important methods to study swallowing disorders after stroke. Suture-occluded method is the most commonly classic model of ischemic stroke. Recent studies have shown that the chemical destruction of the nucleus ambiguus can also cause swallowing disorders in rats. However, its modeling success rate, clinical similarity, and controllability remain uncertain. OBJECTIVE: To observe the changes in swallowing function in the rat model established by two different modeling methods, namely, suture-occluded method and chemical destruction method, and to investigate the clinical and pathological characteristics of swallowing disorders similar to those in humans.  METHODS: Forty Sprague-Dawley rats were randomly divided into three groups: normal group (n=10), suture-occluded group (n=15), and chemical destruction group (n=15). The left-sided ischemic stroke models were prepared using the suture-occluded method through reperfusion after 90 minutes of transient cerebral ischemia in the suture-occluded group, while rat models of swallowing disorders were established through the chemical destruction of the nucleus ambiguus by gooseberry muscarinic acid in the chemical destruction group. Survival numbers per week, body mass, and 24-hour food and water intake in each group were recorded. The rats were examined on days 2, 7, 14, and 30 after modeling using a biosignal collector and a tension transducer to detect the swallowing initiation response time and the number of swallows. Hematoxylin-eosin staining was used to observe the pathomorphological changes of the nucleus ambiguus of the medulla oblongata swallowing center. ELISA method was used to detect the levels of serum interleukin-6, interleukin-1β and tumor necrosis factor-α. RESULTS AND CONCLUSION: The mortality rate in the suture-occluded group was significantly lower than that in the chemical destruction group on days 2 and 7 (P < 0.05), but there was no significant difference between the two groups at other observation time points (P > 0.05). The final mortality rate in the suture-occluded group was significantly lower than that in the chemical destruction group (40% vs.73%, P < 0.05). Compared with the normal group, body mass, 24-hour food and water intake, swallowing initiation response time, and the number of swallows were significantly worse in the chemical destruction group on day 7 (P < 0.05) but showed no significant changes in the suture-occluded group (P > 0.05). Compared with the normal group, the other two model groups showed a significant reduction in body mass, 24-hour food and water intake, and the number of swallows and significantly prolonged swallowing initiation response time (P < 0.05). There were significant differences in body mass, 24-hour food and water intake, swallow initiation response time, and the number of swallows between the suture-occluded group and the chemical destruction group on day 7 (P < 0.05), but no significant difference at other observation time points (P > 0.05). Rats in the two model groups showed obvious pathological changes in the medulla oblongata with a significant increase in inflammatory cells. However, there was no significant difference between the two groups (P > 0.05). To conclude, both the suture-occluded method and the chemical destruction method could cause swallowing disorders in rats. The mortality rate in the suture-occluded group was significantly lower than that in the chemical destruction group at all times after modeling. Therefore, the suture-occluded method with reperfusion following 90 minutes of transient ischemia is more replicative in terms of the final success rate. Figures and Tables | References | Related Articles | Metrics

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Apelin promotes locomotor recovery and spinal cord morphology repair in rats with spinal cord injury Liu Qing, Wang Xiao, Gu Chengxu, Guo Qixuan, Li Xikai, Zhang Luping, Huang Fei 2023, 27 (11):  1744-1749.  doi: 10.12307/2022.1018 Abstract ( 386 )   PDF (1508KB) ( 205 )   Save BACKGROUND: Apelin/APJ exhibits functions in regulating cell proliferation, apoptosis, inhibiting inflammatory responses and vascular remodeling. Therefore, it is speculated that Apelin has similar functions in spinal cord injury. OBJECTIVE: To evaluate the effects of Apelin in rats with spinal cord injury. METHODS: A rat model of spinal cord transection was used in an in vivo experiment. Forty-five female Sprague-Dawley rats were randomly divided into sham group, spinal cord injury group and Apelin-13 group. Rats in the latter two groups were submitted to make animal models of spinal cord transection. Rats were given the intraperitoneal injection of 0.2 mg/kg Apelin-13 in the Apelin group or the same amount of normal saline in the other two groups for 14 consecutive days. The motor function of rat’s hind limbs was assessed by Basso, Beattie, Bresnahan locomotor rating scale at the 1st, 3rd, 7th, and 14th days after modeling. The spinal cord tissues were collected at the 14th day after spinal cord injury and used for immunohistochemical, immunofluorescence and RT-PCR analyses. In in vitro experiments, H2O2 was used to induce PC12 cell injury followed by treatment with different concentrations (1, 2, 4 μmol/L) of Apelin-13. We then used cell counting kit-8 to detect the effect of Apelin-13 on the viability of injured PC12 cells, thereby exploring the neuroprotective effect of Apelin. RESULTS AND CONCLUSION: (1) After spinal cord injury, the hindlimb function of the rats was completely lost and motor recovery was observed 3 days after injury. However, there was no significant difference in the spinal cord injury group at different time points. Apelin promoted motor function recovery post spinal cord injury. (2) Spinal cord injury disrupted the local continuity of the spinal cord, and Apelin restored the injured morphology induced by spinal cord injury. (3) There were only a few cells positive for ionized calcium binding adapter protein 1 in the sham group. Spinal cord injury led to a sharp increase in the number of local positive cells and astrocytes. Apelin could alleviate the local activation of astrocytes and microglia (P < 0.05). (4) Severe demyelination occurred after spinal cord injury and the positive area of Luxol Fast Blue staining was significantly reduced. Apelin could attenuate demyelination caused by spinal cord injury. (5) The expression of growth-related protein 43 was significantly increased after spinal cord injury (P < 0.001). Apelin could further promote the expression of growth-related protein 43 (P < 0.05). (6) Apelin increased the cell viability of H2O2-stimulated PC12 cells in vitro (P < 0.05). To conclude, Apelin promotes locomotor recovery and spinal cord morphology repair in rats after spinal cord injury. Figures and Tables | References | Related Articles | Metrics

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Role of thioredoxin-interacting protein-mediated inflammatory response and apoptosis in myocardial infarction Wang Xuejiao, Shi Wenjuan, Zhang Yan, Xing Dehai, Li Dongxue, Jiao Xiangying 2023, 27 (11):  1715-1721.  doi: 10.12307/2023.116 Abstract ( 352 )   PDF (2021KB) ( 197 )   Save BACKGROUND: In recent years, studies have found that the expression of thioredoxin-interacting protein is increased in the myocardial tissue of mice with myocardial infarction. Thioredoxin-interacting protein is the endogenous activator of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome. It is unclear whether it participates in the pathological process of myocardial infarction by activating the inflammasome. OBJECTIVE: To investigate the effects of thioredoxin-interacting protein on cardiomyocyte apoptosis and NLRP3 inflammasomes, thereby providing new ideas for the prevention and treatment of myocardial infarction.  METHODS: A total of 30 thioredoxin-interacting protein knockout mice and 30 wild-type littermate C57BL/6 mice were selected and randomly divided into myocardial infarction group and sham operation group with 15 mice in each group. Myocardial infarction models were established. Four days after the operation, some mice (10 mice in each group) were sacrificed and the heart tissue was collected. TdT-mediated dUTP nick end labeling was used to detect cardiomyocyte apoptosis in mice (5 mice in each group). Western blot (5 mice in each group) was used to detect the expression of thioredoxin-interacting protein, NLRP3, cleaved cysteinyl aspartate specific proteinase-1, apoptosis-associated speck-like protein containing CARD, cleaved interleukin-1β, and cleaved cysteinyl aspartate speacific proteinase-3. The remaining mice (5 mice in each group) were fed till 1 month after the operation. The cardiac function was detected by echocardiography before the mice were sacrificed and heart tissue samples were collected for later use.   RESULTS AND CONCLUSION: The expression level of thioredoxin-interacting protein was significantly upregulated in the wild-type myocardial infarction group than the wild-type sham operation group (P < 0.05). Compared with the sham operation groups, the expressions of NLRP3, cleaved cysteinyl aspartate specific proteinase-1, apoptosis-associated speck-like protein containing CARD, cleaved interleukin-1β, and cleaved cysteinyl aspartate specific proteinase-3 proteins in the myocardium were significantly upregulated in the two myocardial infarction groups (P < 0.05). Compared with the wild-type myocardial infarction group, the expression levels of the above-mentioned five proteins were significantly downregulated, the cardiomyocyte apoptosis was reduced, and the cardiac function was significantly improved in the thioredoxin-interacting protein knockout mice with myocardial infarction (P < 0.05). To conclude, thioredoxin-interacting protein knockout can inhibit the expression of NLRP3, cleaved cysteinyl aspartate specific proteinase-1, apoptosis-associated speck-like protein containing CARD, cleaved interleukin-1β, and cleaved cysteinyl aspartate specific proteinase-3 proteins, reduce cardiomyocyte apoptosis, and thereby improve cardiac function after myocardial infarction. It is expected to provide a target for the clinical treatment of myocardial infarction. Figures and Tables | References | Related Articles | Metrics

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Establishment of an in vitro model of H2O2-induced neuronal senescence Fu Xinya, Ma Jinjin, Li Meimei, Wang Xingran, Xie Jile, Saijilafu 2023, 27 (11):  1733-1738.  doi: 10.12307/2022.1012 Abstract ( 569 )   PDF (1343KB) ( 170 )   Save BACKGROUND: Aging leads to loss of neuronal morphology and function, which becomes the most important risk factor for neurodegenerative diseases. Current research on the mechanism of neuronal aging is still incompleted, and establishing an in vitro aging model will promote relevant research. As a small number of mature mammalian neurons, dorsal root ganglion is easy to isolate and culture in vitro, and therefore, it is a suitable target for establishing the in vitro aging model. OBJECTIVE: To explore suitable conditions for inducing neuronal senescence and establish an in vitro model of H2O2-induced neuronal senescence. METHODS: The dorsal root ganglia were obtained from 1-month-old ICR mice and cultured in vitro with 10, 20, and 50 μmol/L H2O2 for 1 and 2 hours. The appropriate conditions for inducing cell senescence were screened through detecting the activity of β-galactosidase and cell survival rate of neurons. These induction conditions were further identified by detecting the expression level of autophagy-related protein SQSTM1/p62, the content of reactive oxygen species, and the axon growth ability of neurons. Consequently, the in vitro aging model was successfully established in neurons.  RESULTS AND CONCLUSION: By detecting the activity of β-galactosidase and the survival rate of neurons, culture with 10 μmol/L H2O2 for 2 hours was selected as suitable induction conditions. Under these suitable conditions, the expression of SQSTM1/p62 was significantly down-regulated (P < 0.01), consistent with the characteristics of cell senescence; the production of reactive oxygen species increased significantly (P < 0.01), indicating impaired function of mitochondria and emergence of senescent cells; the growth ability of neuronal axons was significantly weakened (P < 0.001), conforming to the characteristics of neuron aging. In conclusion, culture with 10 μmol/L H2O2 for 2 hours can be successfully used for the establishment of neuronal aging models in vitro. Figures and Tables | References | Related Articles | Metrics

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piRNA-5938 can regulate cardiomyocyte apoptosis and mitochondrial fission Wu Xiaolei, Han Yu, Li Jialei, Wang Shuang, Cao Jimin, Sun Teng 2023, 27 (11):  1750-1757.  doi: 10.12307/2023.125 Abstract ( 271 )   PDF (1507KB) ( 114 )   Save BACKGROUND: Cardiomyocyte apoptosis plays a critical role in ischemia/reperfusion injury. Recent studies have shown that PIWI-Interacting RNA (piRNA), a reproductive system regulator, participates in the pathogenesis of cardiac disorder. However, the role of piRNA in cardiomyocyte apoptosis has not been clarified; especially the mitochondria-dependent pathway involved is poorly understood. OBJECTIVE: To study the role and molecular mechanism of piRNA-5938 in cardiomyocyte apoptosis and mitochondrial fission. METHODS: The abundance of piRNA in normal and ischemic/reperfusion hearts of mice and rats was detected by deep sequencing, and the results were verified by real-time fluorescence quantitative PCR. Cardiac apoptosis was induced by hydrogen peroxide or anoxia/reoxygenation, in which the expression level of piRNA-5938 was detected by real-time fluorescence quantitative PCR. piRNA-5938 antagomir was synthesized and transfected into cardiomyocytes and then the cells were treated with hydrogen peroxide. Apoptosis was detected by TUNEL and mitochondrial division was evaluated by MitoTracker. RNAhybrid software was used to predict microRNAs that interact with piRNA-5938. RESULTS AND CONCLUSION: Compared with the sham group, the expression levels of 422 piRNAs were changed in the mouse ischemic/reperfusion heart, of which 289 were up-regulated and 133 were down-regulated, and piRNA-5938 expression increased significantly (P < 0.01). The expression level of piRNA-5938 was significantly up-regulated after cardiomyocyte apoptosis induced by H2O2 in a concentration-dependent manner (P < 0.05). piRNA-5938 was significantly increased after anoxia/reoxygenation-induced cardiomyocyte apoptosis (P < 0.01). Compared with the negative control group, knockdown of piRNA-5938 significantly reduced the apoptosis rate of cardiomyocytes induced by hydrogen peroxide (P < 0.05). The mitochondrial fission induced by hydrogen peroxide was significantly inhibited by the knockdown of piRNA-5938 (P < 0.05). piRNA-5938 was highly conserved in complementation with mitochondrial fission regulators mir-324 and Mir-668. To conclude, piRNA-5938 regulates cardiomyocyte apoptosis and mitochondrial fission, which probably functions through targeting mir-324 or mir-668. Figures and Tables | References | Related Articles | Metrics

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Therapeutic efficacy of amniotic membrane transplantation in different stages and degrees of ocular alkali chemical burns Li Ying, Chen Yingxin, Gao Minghong 2023, 27 (11):  1772-1779.  doi: 10.12307/2023.128 Abstract ( 421 )   PDF (938KB) ( 64 )   Save BACKGROUND: Amniotic membrane transplantation has been used to treat chemical burns of the eyes at various stages. It promotes corneal epithelialization, reduces inflammation, and restores the integrity of the ocular surface, preventing thawing of the ocular tissue. OBJECTIVE: To analyze the efficacy of amniotic membrane transplantation in ocular alkali chemical burns in the acute phase (7 days after injury) and in the repair phase (8-21 days) and to compare the effects of amniotic membrane transplantation in ocular alkali chemical burns with different degrees, in order to explore the optimal timing and mechanism of amniotic membrane transplantation for ocular alkali chemical burns. METHODS: A retrospective analysis was performed in 47 cases (59 eyes) undergoing amniotic membrane transplantation for ocular alkali chemical burns in the General Hospital of Northern Theater Command, China from November 2015 to December 2021. There were 26 patients (35 eyes), 0.5 hours-7 days (including 7 days) after injury, in acute phase group and 21 patients (24 eyes), 8-21 days after injury, in repair period group. Of the 59 eyes, 39 eyes were grade II-III (moderate burns) and 20 eyes were grade IV-VI (severe burns). Patients from both groups underwent amniotic membrane transplantation the next day after admission. The corneal epithelial defect healing rate, vision, corneal clarity, degree of ocular surface inflammation, degree of corneal vascularization, and symblepharon were observed and recorded at preoperative and postoperative 3 weeks, 1 month, 2 months, and 3 months in the two groups. Statistical analyses were then performed. RESULTS AND CONCLUSION: The acute phase group was superior to the repair period group in terms of average epithelial defect area, corneal epithelialization rate, average complete epithelial healing time, best corrected visual acuity at 3 months after operation, postoperative corneal transparency grading, incidence of symblepharon, degree of corneal vascularization, and success rate after operation (P < 0.05). By the end of the follow-up, the success rate of amniotic membrane transplantation was significantly higher in the moderate burn group than the severe burn group (P < 0.05). Most of the patients with failed amniotic membrane transplantation (65.2%) suffered from severe ocular alkali chemical burns, accompanied by a large area of limbal ischemia. The ocular surface could not be stabilized after repeated amniotic membrane transplantation, corneal conjunctivization appeared with central corneal neovascular infiltration and severe symblepharon, and visual acuity was reduced to manual. To conclude, the acute phase is the optimal timing to apply amniotic membrane transplantation for ocular alkali chemical burns. For patients with severe corneal epithelium defects who suffer from severe ocular alkali chemical burns, amniotic membrane transplantation needs to be performed multiple times in the early stage, thereby supporting wound repair. For patients with severe ocular alkali chemical burns who still have persistent epithelial defect after a series of amniotic membrane transplantation and have a tendency to dissolve the cornea and conjunctiva, amniotic membrane transplantation should be terminated, and permanent blepharoplasty or conjunctival flap masking should be used. After stabilization, second-phase ocular surface reconstruction should be conducted. Figures and Tables | References | Related Articles | Metrics

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Bioinformatics analysis of cuproptosis genes in immune infiltration of osteoarthritis Wang Weiwei, Ou Zhixue, Zhou Yi, Li Tong 2023, 27 (11):  1669-1676.  doi: 10.12307/2023.181 Abstract ( 671 )   PDF (2072KB) ( 336 )   Save BACKGROUND: Immune infiltration plays an important role in the progression of osteoarthritis, while cuproptosis is a novel modality of programmed cell death recently discovered. There are no studies on the mechanisms by which cuproptosis genes regulate immune infiltration in osteoarthritis. OBJECTIVE: To integrate cuproptosis genes and GEO database-related chips, analyze the correlation between cuproptosis genes and immune infiltration, build a risk model, and perform gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis as well as miRNA and Chinese medicine prediction so as to provide some theoretical support for future exploration on the mechanism underlying cuproptosis in osteoarthritis immune infiltration. METHODS: Eligible osteoarthritis-related microarrays were retrieved through the GEO database and normalized. Immune infiltration was extracted and quantified based on the processed gene expression matrix, and the correlation between immune infiltrating cells and their functions was analyzed, as well as their differences between osteoarthritis group and control group. Integrated cuproptosis gene expression matrix and standardized chip gene expression matrix were used to screen out the cuproptosis genes related to osteoarthritis. A risk model was then constructed to analyze the risk probability of cuproptosis genes related to osteoarthritis. In addition, upstream miRNAs were predicted by FunRich software. Finally, gene ontology, KEGG enrichment analysis and traditional Chinese medicine prediction of osteoarthritis-related cuproptosis genes were carried out through the Enrichr website and Coremine Medical database. RESULTS AND CONCLUSION: Immune infiltration correlation results showed that there were the strongest positive correlation between dendritic cells and mast cells (r=0.87) and the strongest negative correlation between mast cells and tumor-infiltrating lymphocytes (r=-0.81). Differential immune infiltration results showed that in the osteoarthritis group, dendritic cells, immature dendritic cells, macrophages, mast cells, neutrophils, antigen presentation co-suppression, and chemokine C-C-Motif receptors were significantly increased (P < 0.05), while co-stimulation of B cells, Th2 cells, and T cells significantly decreased (P < 0.05). A total of 10 osteoarthritis-related cuproptosis genes were screened, namely SLC31A1, PDHB, PDHA1, LIPT1 , LIAS, DLD, FDX1, DLST, DLAT, DBT. The risk model results showed that PDHB may be a risk factor for osteoarthritis. Enrichment analysis results showed that osteoarthritis-related cuproptosis genes were mainly enriched in citric acid cycle, pyruvate metabolism, glycolysis/gluconeogenesis signaling pathways. A total of 27 herbal medicines and 1 herbal active ingredient, including rhizoma curcumae (0.004 75), scutellaria baicalensis (0.049), and rhodioloside (0.000 237), were predicted through the Coremine Medical database, of which rhodioloside was a potential herbal active ingredient for PDHB, the independent risk factor of osteoarthritis. A total of 29 upstream miRNAs of osteoarthritis-related cuproptosis genes, including has-miR-7a-5p, has-miR-7e-5p, and has-miR-96-5p, were predicted by FunRich software. To conclude, osteoarthritis-related cuproptosis genes are mainly involved in citric acid cycle, pyruvate metabolism, glycolysis/gluconeogenesis signaling pathways to regulate immune cells and functions, including dendritic cells, macrophages, B cells, antigen presentation co-suppression in patients with osteoarthritis. In this process, 29 miRNAs, including has-miR-7a-5p, has-miR-7e-5p, and has-miR-96-5p, may also play an important role. In addition, Chinese herbs and herbal active ingredients, such as sheep liver, rhizoma curcumae, and rhodioloside, may be potential sources of molecular drugs for osteoarthritis. Figures and Tables | References | Related Articles | Metrics

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Rheumatoid arthritis Hub genes and their significance with gene set enrichment analysis and weighted gene co-expression network analysis methods Sun Yumin, Song Jiexin, Yang Yanmei, Yang Dongliang 2023, 27 (11):  1787-1795.  doi: 10.12307/2023.147 Abstract ( 320 )   PDF (3808KB) ( 152 )   Save BACKGROUND: Rheumatoid arthritis patients are still negative for 30% of traditional serum markers detected by the existing immunological methods. In recent years, serum biomarkers have been found to have low sensitivity and specificity for the diagnosis of rheumatoid arthritis, which are not suitable for clinical promotion. OBJECTIVE: To analyze rheumatoid arthritis Hub genes and the significance with gene set enrichment analysis and weighted gene co-expression network analysis methods.  METHODS: Data sets GSE1919 and GSE55235 were downloaded from GEO database, differential expression genes were initially screened by R software "limma" package and "clusterProfiler" package for the weighted gene co-expression network analysis, and intersected genes were selected as candidate Hub genes. The results were imported into String database to construct a protein-protein interaction network. A Cytoscape plugin, cytoHubba, was used to screen hub genes and perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. At the same time, the two data sets were analyzed by the GSEA method. RESULTS AND CONCLUSION: A total of 10 Hub genes were identified, and their GO functions were concentrated in lymphocyte differentiation, T cell differentiation, plasma membrane signal receptor complex, major histocompatibility complex protein binding, and other biological functions. KEGG pathway was enriched in T cell receptor signaling pathway, Th1/Th2 cell differentiation, Th17 cell differentiation, tumor cell programmed death-ligand 1 expression and programmed death receptor 1 checkpoint pathway, and primary immune deficiency pathway. Gene set enrichment analysis showed that three pathways such as bronchial asthma, autoimmune thyroid disease, and cell adhesion molecules were significantly up-regulated in rheumatoid arthritis samples from two datasets. The receiver operating characteristic curve showed that five genes (CD27, IL2RG, CD8A, LCK, and NKG7) had good specificity and sensitivity (AUC > 0.85) for the diagnosis of rheumatoid arthritis. Altogether, CD4 may be a multipotent coordinator of the gene network, performing important functions in the immune response. Figures and Tables | References | Related Articles | Metrics