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18 July 2022, Volume 26 Issue 20 Previous Issue    Next Issue

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Effect of smoking on bone mineral density in rats Cai Zhencun, Gao Zhenhuai, Ren Lixuan, Zhang Zelin 2022, 26 (20):  3117-3120.  doi: 10.12307/2022.607 Abstract ( 547 )   PDF (688KB) ( 190 )   Save BACKGROUND: Osteoporosis is a systemic disease that causes abnormal bone metabolism and decreases bone mineral density due to various reasons. Clinical studies have found that most patients with osteoporosis have a long-term history of smoking, but whether smoking is directly related to bone mineral density reduction has not been determined yet. OBJECTIVE: To explore the relationship between smoking and bone mineral density in rats, and to evaluate the association between smoking and osteoporosis. METHODS: Forty Wistar rats were randomly divided into two groups (n=20 per group): non-smoking group and smoking group. Rats in the smoking group were subjected to passive cigarette smoking through a passive smoking animal exposure system that could simulate the process of human smoking. Each rat was fumigated with 20 cigarettes for 40 minutes, twice a day, for 8 weeks. Rats in the non-smoking group were used as controls and they were only raised in the passive smoking animal exposure system for 8 weeks without smoking. The natural experimental environment, feeding, water supply and other aspects of the two groups were completely the same. The bone mineral density of bilateral femurs and L5 lumbar vertebrae of rats were measured with dual-energy X-ray bone densitometer before the experiment and 4 and 8 weeks after the experiment, and the average value was taken. Body mass changes of rats were measured at the same time.  RESULTS AND CONCLUSION: There was no significant difference in bone mineral density and body mass between the two groups before the beginning of the experiment (P > 0.05). In the 4th week of the experiment, the bone mineral density of the smoking group was lower than that of the non-smoking group [(225.50±10.11) mg/cm2 vs. (238.86±11.53) mg/cm2, P=0.002]. In the 8th week of the experiment, the bone mineral density of the smoking group was further reduced, which was lower than that of the non-smoking group [(201.98±15.58) mg/cm2 vs. (240.26±13.69) mg/cm2, P=0.013]. In the 4th week of the experiment, the body mass of the rats in the smoking group was lower than that of the non-smoking group [(236.4±15.3) g vs. (258.8±19.6) g, P=0.026]. In the 8th week of the experiment, the body mass of the rats in the experimental group was still lower than that of the non-smoking group [(278.9±18.1) g vs. (339.5±13.3) g, P=0.008].The body mass gain of the experimental group in the last 4 weeks (5-8 weeks) was significantly less than that in the first 4 weeks (1-4 weeks) (P=0.006), but there was no significant difference in the body mass gain of the non-smoking group (P=0.081). Therefore, smoking can reduce bone mineral density and slow body mass growth in rats, indicating that smoking is closely related to osteoporosis. Control of smoking should be clinically concerned in patients with osteoporosis in order to achieve effective treatments.  Figures and Tables | References | Related Articles | Metrics

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Lipopolysaccharide stimulates the expression of interleukin-6 and nuclear factor kappa B receptor activator ligand in mouse MLO-Y4 osteoblasts Liu Qiqi, Liu Min, Yang Jian, Yu Ke 2022, 26 (20):  3121-3126.  doi: 10.12307/2022.608 Abstract ( 510 )   PDF (1125KB) ( 249 )   Save BACKGROUND: Previous studies have confirmed that lipopolysaccharide could up-regulated the expression of interleukin-6, an inflammation-related cytokine, in mouse MLO-Y4 osteocytes, and interleukin-6 could further up-regulate the expression of receptor activator of nuclear factor κB ligand (RANKL), a factor related to osteoclastogenesis, in osteoblasts. PI3K/AKT signaling pathway is an important regulatory pathway in bone metabolism. Verifying whether this signaling pathway is involved in the production of inflammatory factors induced by lipopolysaccharides is greatly beneficial to studying the mechanism of bone resorption that is caused by bacterial inflammation. OBJECTIVE: To verify whether PI3K/AKT signaling pathway is involved in the expression of interleukin-6 and RANKL in mouse MLO-Y4 osteocytes stimulated by lipopolysaccharides. METHODS: MLO-Y4 osteoblasts were stimulated with 0 or 100 µg/L lipopolysaccharides for 1, 2, 4, 6, and 8 hours. Then the mRNA expressions of interleukin-6 and RANKL were detected by real-time quantitative polymerase chain reaction, and the protein expression of RANKL and p-AKT were detected by western blot. MLO-Y4 osteocytes in logarithmic growth was divided into seven groups: no treatment group (control group), 100 µg/L lipopolysaccharide group, 1 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 2.5 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 5 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 10 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 20 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of interleukin 6 and RANKL. MLO-Y4 osteocytes were divided into four groups: no treatment group (control group), 100 µg/L lipopolysaccharide group, dimethyl sulfoxide+100 µg/L lipopolysaccharide group, and 10 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of RANKL, and western blot was used to detect the protein expression of RANKL and p-AKT. RESULTS AND CONCLUSION: After lipopolysaccharide stimulation, the mRNA expression of interleukin-6 and RANKL in osteocytes was increased first and then decreased; however, the mRNA expression levels were higher than those in the control group at each time point (P < 0.05). After lipopolysaccharide stimulation, the protein expression of RANKL and p-AKT in osteocytes was significantly higher than that in the control group (P < 0.05) at each time point, although the protein expression in lipopolysaccharide groups was increased first and then decreased. PI3K inhibitor of 1-10 µmol/L could decrease the mRNA expression of interleukin 6 and RANKL in lipopolysaccharide-induced osteocytes in a concentration-dependent manner, while 20 and 10 µmol/L PI3K inhibitor showed similar effects. Both dimethyl sulfoxide and PI3K inhibitors could inhibit the increase in the protein and mRNA expression of RANKL and the protein expression of p-AKT in osteocytes induced by lipopolysaccharide. Furthermore, PI3K inhibitor showed the better inhibitory effect. These results indicate that lipopolysaccharide stimulates the expression of interleukin-6 and RANKL in mouse MLO-Y4 osteocytes through the PI3K/AKT signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Abnormally expressed beta-amyloid affects bone metabolism Li Fangyu, Xia Wenfang, Cui Shun 2022, 26 (20):  3152-3157.  doi: 10.12307/2022.613 Abstract ( 528 )   PDF (1687KB) ( 180 )   Save BACKGROUND: Amyloid β exists widely in human tissues, and its abnormal expression is closely related to various chronic inflammatory diseases such as Alzheimer’s disease and osteoporosis. Studies have shown that amyloid β deposits in human vertebrae and femur and then causes bone destruction. However, the effect of low expression of amyloid β on bone metabolism remains unclear.   OBJECTIVE: To investigate the effect of overexpression and low expression of amyloid β on bone metabolism in mice. METHODS: There were three groups: an APPswe+/+ group with APPswe+/+ transgenic mice aged 12 months with overexpression of amyloid β, a BACE-/- group with β-secretase cleaving enzyme-1 gene knockout mice with low expression of amyloid β, and a wild type group with wild type mice. Amyloid β deposition in bone tissue was observed by Congo red staining. Immunohistochemical staining was used to detect the distribution of amyloid precursor protein in bone tissue, and micro-computed tomography was used to study the difference of overall bone structure. Alkaline phosphatase staining and tartrate-resistant acid phosphatase staining were used to analyze the differences between bone formation and bone resorption.   RESULTS AND CONCLUSION: Compared with the wild type group, amyloid β deposition of cancellous bone and compact bone was increased in the APPswe+/+ group, and decreased in the BACE-/- group (P < 0.05). Compared with the wild type group, the expression of amyloid precursor protein in mouse cancellous bone was increased in the APPswe+/+ and BACE-/- groups. Compared with the wild type group, the bone mass and thickness of trabecular bone were decreased, and trabecular bone became thinner, sparse, disordered or fractured in the APPswe+/+ and BACE-/- groups. There was also abnormal bone formation and enhanced activity of osteoclasts in the APPswe+/+ and BACE-/- groups  (P < 0.05). To conclude, overexpression or low expression of amyloid β can cause bone metabolism disorder and lead to bone loss. Abnormal lower expression of amyloid β caused by β-secretase cleaving enzyme-1 gene knockout can affect the expression of amyloid precursor protein in bone, and lead to an abnormal bone metabolism.  Figures and Tables | References | Related Articles | Metrics

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Mechanism of osteoarthritis induced chondrocyte apoptosis and extracellular matrix degradation Wang Wei, Tang Xiangyu, Yi Zhiqian, Liu Zhaoxu 2022, 26 (20):  3133-3140.  doi: 10.12307/2022.610 Abstract ( 491 )   PDF (2299KB) ( 324 )   Save BACKGROUND: It has been found that long non-coding RNA (LncRNA) IFNG-AS1 plays a regulatory role in rheumatoid arthritis, but its role in osteoarthritis remains largely unknown. OBJECTIVE: To investigate the effects of LncRNA IFNG-AS1 on proliferation, apoptosis and extracellular matrix degradation of chondrocytes in osteoarthritis via regulating miR-376b-3p/AKT serine/threonine kinase 3 (AKT3) axis. METHODS: Real-time fluorescence quantitative PCR was used to detect the expression of IFNG-AS1, miR-376b-3p, and AKT3 in cartilage tissue of osteoarthritis and non-osteoarthritis patients. Cell proliferation, apoptosis, and the expression of extracellular matrix-related factors matrix metalloproteinase-3, matrix metalloproteinase-9 and matrix metalloproteinase-13 were analyzed by cell counting kit-8, flow cytometry and enzyme-linked immunosorbent assay. The interaction between miR-376b-3p and IFNG-AS1 or AKT3 was analyzed by dual luciferase reporter assay and RNA pull-down assay. RESULTS AND CONCLUSION: Compared with normal cartilage tissue, the expression of IFNG-AS1 and AKT3 was increased in osteoarthritis cartilage tissue, while the expression of miR-376b-3p was decreased. Knockdown of IFNG-AS1 promoted cell proliferation but inhibited apoptosis and extracellular matrix degradation in chondrocytes. There was a targeted relationship between IFNG-AS1 and miR-376b-3p, and down-regulation of miR-376b-3p partially reversed the effect of IFNG-AS1 knockdown on chondrocytes (all P < 0.05). Further analysis showed that IFNG-AS1 that acted as ceRNA adsorbed miR-376b-3p and regulated the expression of AKT3, and up-regulation of AKT3 partially saved the effect of IFNG-AS1 knockdown on chondrocytes (all P < 0.05). To conclude, LncRNA IFNG-AS1 can inhibit chondrocyte proliferation but promote apoptosis and extracellular matrix degradation by regulating miR-376b-3p/AKT3 axis. IFNG-AS1 is expected to be a potential target for osteoarthritis therapy. Figures and Tables | References | Related Articles | Metrics

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A circular RNA, circ_0040646, regulates the proliferation, differentiation, and apoptosis of knee osteoarthritis chondrocytes by targeted inhibition of microRNA-188-3p Zhang Xuepu, Wu Yuexin, Zhao Haosen, Ban Zhaoliang, Ma Xiaohu, Tong Gang, Yang Limin 2022, 26 (20):  3141-3146.  doi: 10.12307/2022.611 Abstract ( 558 )   PDF (1332KB) ( 330 )   Save BACKGROUND: Circular RNAs play an important regulatory role in various diseases. Recent studies have shown that circular RNAs are abnormally expressed in knee osteoarthritis. Therefore, it is very essential to study the effect of circular RNAs on the pathogenesis of knee osteoarthritis. OBJECTIVE: To investigate the effect of circ_0040646 regulating microRNA-188-3p (miR-188-3p) on the proliferation, differentiation and apoptosis of knee osteoarthritis chondrocytes. METHODS: Quantitative real-time fluorescence PCR was used to detect the expression level of circ_0040646 in knee osteoarthritis patients and knee osteoarthritis chondrocytes, in comparison with normal chondrocytes from meniscal injury patients. Chondrocytes from knee osteoarthritis patients were transfected with small-interfering negative control (lowly expressed circ_0040646 control plasmid), small-interfering circ_0040646, overexpressed negative control (overexpressed circ_0040646 control plasmid), overexpressed circ_0040646, miRNA negative control (control group), or microRNA-188-3p inhibitor. Quantitative real-time fluorescence PCR and western blot were used to detect the expression levels of proliferating cell nuclear antigen, bone morphogenetic protein 2 and runt-related transcription factor 2. Flow cytometry was used to detect cell apoptosis. Dual luciferase reporter assay was used to detect the targeted binding of circ_0040646 and miR-188-3p.     RESULTS AND CONCLUSION: Compared with the control group, circ_0040646 was highly expressed in knee osteoarthritis patients and knee osteoarthritis chondrocytes. In knee osteoarthritis chondrocytes, the low expression of circular RNA-0040646 could promote cell proliferation and differentiation, and inhibit cell apoptosis, while overexpression of circ_0040646 could inhibit cell proliferation and differentiation, and promote cell apoptosis. circ_0040646 could target miR-188-3p in knee osteoarthritis chondrocytes. Co-transfection of small-interfering circ_0040646 and miR-188-3p inhibitors could reverse the effect of small-interfering circ_0040646 alone on the proliferation, differentiation and apoptosis of knee osteoarthritis chondrocytes. These findings reveal that circ_0040646 can regulate the proliferation, apoptosis and differentiation of knee osteoarthritis chondrocytes by targeted inhibition of miR-188-3p, and therefore, circ_0040646 may become a target for the treatment of knee osteoarthritis. Figures and Tables | References | Related Articles | Metrics

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Relationship between apoptosis of osteoarthritis chondrocytes and reduction of histone deacetylase 4 content Gu Xiaodong, Li Fei, Che Xianda, Li Pengcui 2022, 26 (20):  3147-3151.  doi: 10.12307/2022.612 Abstract ( 438 )   PDF (1607KB) ( 453 )   Save BACKGROUND: Endoplasmic reticulum stress-mediated chondrocyte apoptosis plays an important role in the pathogenesis of osteoarthritis, which is mainly regulated by the activated transcription factor 4 (ATF4)/CAAT/enhancer-binding protein homologous protein (CHOP) signaling pathway. However, the mechanism of this signaling pathway in osteoarthritis remains unclear. OBJECTIVE: To investigate the mechanism of histone deacetylase 4 and ATF4/CHOP signaling pathway in osteoarthritis chondrocytes apoptosis. METHODS: According to Outerbridge classification, the articular cartilage of the tibial plateau cartilage obtained during knee joint replacement was divided into relatively normal cartilage (Outerbridge I) and osteoarthritis cartilage (Outerbridge III). Apoptosis of chondrocytes was observed by TUNEL staining. Immunohistochemistry staining was used to detect the expression of histone deacetylase 4, ATF4, and CHOP. Real-time fluorescence quantitative PCR was used to detect the mRNA expression of CHOP, while western blot and real-time fluorescence quantitative PCR were used to detect the protein expression of histone deacetylase 4 and ATF4. RESULTS AND CONCLUSION: TUNEL staining results showed that the apoptosis rate of chondrocytes in osteoarthritic cartilage was higher than that in relatively normal cartilage [(83.5±10.1)% vs. (20.5±5.2)%, P < 0.05]. Immunohistochemical staining results showed that the expression of ATF4 in osteoarthritic cartilage was significantly higher than that in relatively normal cartilage, and the expression of histone deacetylase 4 in osteoarthritic cartilage was lower than that in relatively normal cartilage. The expression of CHOP in osteoarthritis cartilage was higher than that in relatively normal cartilage. The mRNA expression of ATF4 and CHOP in osteoarthritic cartilage were higher than that in relative normal cartilage (P < 0.05), and the mRNA expression of histone deacetylase 4 in osteoarthritic cartilage was lower than that in relative normal cartilage (P < 0.05). The protein expression of histone deacetylase 4 in osteoarthritic cartilage was lower than that in relatively normal cartilage (P < 0.05), and the protein expression of ATF4 in osteoarthritic cartilage was higher than that in relatively normal cartilage (P < 0.05). Therefore, in the pathological process of osteoarthritis, the downregulated expression of histone deacetylase 4 and upregulated expression of ATF4/CHOP signal pathway molecules may be related to chondrocyte apoptosis in osteoarthritis. Figures and Tables | References | Related Articles | Metrics

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Bushen Tiaogan Prescription containing serum in the treatment of osteoarthritis by promoting chondrocyte autophagy in rats Fan Shuai, Wu Chunfei, Liang Zujian, Xu Zhaohui, Xie Pingjin, Zhu Genfu 2022, 26 (20):  3178-3183.  doi: 10.12307/2022.617 Abstract ( 550 )   PDF (1236KB) ( 130 )   Save BACKGROUND: Previous studies have found that Bushen Tiaogan Prescription has obvious clinical efficacy in the treatment of osteoarthritis, but there is a lack of research on its mechanism. OBJECTIVE: To study the effect of Bushen Tiaogan Prescription containing serum on the autophagy of rat chondrocytes, and to explore its internal mechanism.  METHODS: The third-generation rat chondrocytes subcultured were randomly divided into four groups: dimethyl sulfoxide (DMSO), 10 μg/L interleukin-1β (IL-1β), 10 μg/L IL-1β+10% Bushen Tiaogan Prescription medicated serum (BSTG), and 5 μmol/L rapamycin groups, and chondrocytes in each group were treated correspondingly for 24 hours. Dansylcadaverine staining was used to detect the expression of chondrocyte autophagosomes. Real-time fluorescence quantitative PCR was used to detect the expression of autophagy-related genes ATG5 and ATG7 mRNAs, and the expression levels of Beclin1, LC3A/B, phosphorylated protein kinase B (p-AKT), and phosphorylated mammalian target of rapamycin (p-mTOR) proteins were analyzed by western blot assay.  RESULTS AND CONCLUSION: Compared with the DMSO group, the number of autophagosomes was significantly reduced in the IL-1β group. Compared with the DMSO group, the number of autophagosomes was significantly increased in the rapamycin group. Compared with the IL-1β group, the number autophagosomes was significantly increased in the 10 μg/L IL-1β+10% BSTG group. Compared with the DMSO group, the mRNA expressions of chondrocyte autophagy-related genes ATG5, ATG7 and the protein expressions of Beclin1 and LC3A/B-II/LC3A/B-I protein expression were significantly decreased in the IL-1β  group (P < 0.05, P < 0.01, P < 0.01, P < 0.05), while the protein expressions of p-AKT and p-mTOR were significantly increased (P < 0.05, P < 0.05). Compared with the DMSO group, the mRNA expressions of ATG5 and ATG7 and the protein expressions of Beclin1 and LC3A/B-II/LC3A/B-I were significantly increased in the rapamycin group (P < 0.01, P < 0.05, P < 0.05, P < 0.05), while the protein expressions of p-AKT and p-mTOR were significantly decreased (P < 0.01, P < 0.01). Compared with the IL-1β group, the mRNA expressions of ATG5 and ATG7 the protein expressions of Beclin1 and LC3A/B-II/LC3A/B-I were significantly increased in the 10 μg/L IL-1β+10% BSTG group (P < 0.01, P < 0.01, P < 0.05, P < 0.01), while the protein expressions of p-AKT and p-mTOR were significantly decreased (P < 0.05, P < 0.05). To conclude, Bushen Tiaogan Prescription containing serum increases the autophagy ability of chondrocytes by inhibiting the AKT/mTOR signaling pathway in chondrocytes and alleviating the degeneration of articular cartilage, and thereby plays a role in the treatment of osteoarthritis. Figures and Tables | References | Related Articles | Metrics

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Astragalus polysaccharides repair articular cartilage damage in a mouse osteoarthritis model Shi Guirong, Ren Bowen, Zhang Zhongbo, Wang Lisha, Zhang Qiwei, Shi Dongliang 2022, 26 (20):  3236-3242.  doi: 10.12307/2022.627 Abstract ( 531 )   PDF (1490KB) ( 148 )   Save BACKGROUND: Astragalus polysaccharide has a definite effect on osteoarthritis, but its mechanism of action is still unclear.   OBJECTIVE: To investigate the effects of astragalus polysaccharide on osteoarthritis through regulating ubiquitination of Notch receptor 1 mediated by WW domain containing E3 ubiquitin protein ligase 2 (WWP2). METHODS:  In the in vivo experiment, 30 C57BL/6 mice were randomly divided into 3 groups (n=10): a sham operation group, a model group, and an astragalus polysaccharide group. A mouse osteoarthritis model was established in the model and astragalus polysaccharide groups. Mice in the astragalus polysaccharide group were treated with astragalus polysaccharide. After 4 weeks of intervention, the knee joint tissue was taken under anesthesia. Safranin O staining method and Osteoarthritis Research Society International score were used to evaluate the damage of articular cartilage in mice. In the in vitro experiment, C28/12 cells were cultured with 5 μg/L interleukin 1β for 24 hours to establish osteoarthritis cell models, which were divided into a control group, an interleukin 1β group, a 50 mg/L astragalus polysaccharide group, and an astragalus polysaccharide-Notch receptor 1 overexpression and a WWP2 interference group. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect cell proliferation. Flow cytometry was used to detect apoptosis. Co-immunoprecipitation assay was used to verify the binding of WWP2 and Notch receptor 1. Ubiquitination experiment was used to analyze the effect of astragalus polysaccharides on the ubiquitination of Notch receptor 1 mediated by WWP2. Enzyme-linked immunosorbent assay was used to detect the levels of proteoglycans, type II collagen, matrix metallopeptidase 3, and matrix metallopeptidase 13 in mouse knee joint tissue and C28/12 cells. Western blot assay was used to detect the protein expression of WWP2, Notch receptor 1, jagged canonical Notch ligand 1, and Notch intracellular domain 1 in mouse knee joint tissue and C28/12 cells.   RESULTS AND CONCLUSION: The results of in vivo experiments confirmed that intervention of astragalus polysaccharides alleviated cartilage damage in mice, inhibited the levels of matrix metallopeptidase 3 and matrix metallopeptidase 13, and promoted the expression of proteoglycans and type II collagen in mouse knee joint tissue. The results of in vitro experiments confirmed that the intervention of astragalus polysaccharides promoted cell proliferation and the expression of proteoglycan, type II collagen, and WWP2, and inhibited cell apoptosis and the expression of matrix metallopeptidase 3 and matrix metallopeptidase 13, Notch receptor 1, jagged canonical Notch ligand 1, and Notch intracellular domain 1, while Notch receptor 1 overexpression and WWP2 interference reversed the effect of astragalus polysaccharides. Astragalus polysaccharides promoted the ubiquitination of Notch receptor 1 mediated by WWP2. To conclude, astragalus polysaccharides can play a certain therapeutic effect on mouse osteoarthritis by increasing the WWP2-mediated ubiquitination level of Notch receptor 1 and inhibiting the Notch signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Lipopolysaccharides mediate autophagy of mouse insulinoma βtc6 cells in high glucose state Cai Zhiguo, Du Shasha, Yang Kun, Zhao Na, Liu Qi 2022, 26 (20):  3127-3132.  doi: 10.12307/2022.609 Abstract ( 507 )   PDF (952KB) ( 105 )   Save BACKGROUND: Our previous studies have confirmed that diabetes mellitus promotes the occurrence and development of periodontitis. However, it is unclear whether periodontitis can promote the development of diabetes. OBJECTIVE: To explore the molecular mechanism by which periodontitis promotes the development of diabetes mellitus. METHODS: Mouse βtc6 insulinoma cells cultured in vitro were divided into control group, glucose group, lipopolysaccharide group, and glucose+lipopolysaccharide group. The cells in the latter three groups were treated with different concentrations of glucose (0, 25, 50, 100 mmol/L) and lipopolysaccharide (0, 10, 20, 40 mg/L) alone or in combination for 12 hours. Insulin secretion was measured in each group. In addition, 3-methyladenine (an autophagy inhibitor, 5 μmol/L) and rapamycin (an autophagy activator, 10 mmol/L) were used to intervene with the phosphatidylinositol 3-kinase/rotein kinase B/the mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. The cells were then further divided into rapamycin+lipopolysaccharide group, rapamycin+glucose group, rapamycin+glucose+lipopolysaccharide group, 3-methyladenine+lipopolysaccharide group, 3-methyladenine+glucose group, and 3-methyladenine+glucose+lipopolysaccharide group. The cell counting kit-8 method was used to detect the proliferation of βtc6 cells. Fluorescent probes were used to detect the amount of reactive oxygen species in βtc6 cells. Production of autophagosomes in βtc6 cells was observed by transmission electron microscopy. Western blot method was used to detect the expression of autophagy proteins and PI3K/AKT/mTOR signaling pathway-related proteins. RESULTS AND CONCLUSION: The insulin secretion was out of control as the glucose concentration exceeded 50 mmol/L (P > 0.05), whereas the insulin secretion was inhibited as the lipopolysaccharide concentration exceeded 20 mg/L (P < 0.05). After addition of 3-methyladenine, the expression levels of Becline1, p-PI3K, p-AKT/AKT, p-AKT/AKT, p-AKT/AKT, and p-PI3K were significantly decreased in the 3-methyladenine+glucose group, 3-methyladenine+lipopolysaccharide group, and 3-methyladenine+lipopolysaccharide+glucose group compared with the control group. While the expression of p62 protein showed the opposite trend (P < 0.05). After addition of rapamycin, the expression of Becline1, p-PI3K, and p-mTOR/mTOR was significantly increased in comparison with the control group (P < 0.05). These findings indicate that lipopolysaccharides can induce excessive autophagy of pancreatic β cells and downregulate insulin secretion. This mechanism may be related to the silencing of PI3K/AKT/mTOR signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Effects of concentrated growth factor combined with epidermal growth factor on the proliferation and aging of oral mucosa equivalents Li Tengyan, Nie Minhai, Liu Xuqian 2022, 26 (20):  3164-3172.  doi: 10.12307/2022.615 Abstract ( 696 )   PDF (2214KB) ( 242 )   Save BACKGROUND: Concentrated growth factor (CGF) and epidermal growth factor (EGF) are biological materials that have been extensively studied in recent years. Studies have shown that they can affect the biological behavior of cells, thereby promoting tissue repair and injury healing. OBJECTIVE: To explore the effects of CGF combined with EGF on the proliferation and aging of human gingival fibroblasts, epithelial cells, and human normal melanocytes. METHODS: CGF/EGF was co-cultured with human gingival fibroblasts, epithelial cells and human normal melanocytes in vitro. The MTT assay was used to screen the optimal concentrations of CGF, EGF and their combination. Experiments were divided into: human gingival fibroblast control group, 50% CGF group, 10 μg/L EGF group, 50% CGF+10 μg/L EGF group; epithelial cell control group, 30% CGF group, 10 μg/L EGF, 30% CGF + 10 μg/L EGF group; human skin melanocyte control group, 30% CGF group, 20 μg/L EGF group, 30% CGF + 10 μg/L EGF group, 30% CGF + 20 μg/L EGF group. Cell counting kit-8 assay was used to detect the proliferation of cells, Transwell technology used to detect the migration of cells, and Annexin V/PI double staining method used to detect the apoptosis of cells. An ethical approval was obtained from the Biomedical Science Research Ethics Committee of the Affiliated Stomatological Hospital of Southwest Medical University (approval No. 20181008001) and written informed consents were obtained from patients and volunteers. RESULTS AND CONCLUSION: The results of MTT assay suggested that the optimal concentrations of CGF on the cell proliferation were 50% for human gingival fibroblasts, 30% for epithelial cells and 30% for human normal melanocytes, while the optimal concentrations of EGF on the cell proliferation were 10 μg/L for human gingival fibroblasts, 10 μg/L for epithelial cells, and 20 μg/L for human normal melanocytes. Cell counting kit-8 assay, Transwel assay and Annexin V/PI double staining method showed that CGF combined with EGF could promote the proliferation and migration of human gingival fibroblasts, epithelial cells and human normal melanocytes and inhibit cell apoptosis. Their effects were ranked as follows: EGF > CGF + EGF > CGF. To conclude, CGF combined with EGF can promote cell proliferation and migration and inhibit cell apoptosis, and its effect is stronger than that of CGF alone but weaker than that of EGF alone. Figures and Tables | References | Related Articles | Metrics

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Huoxue Huayu Decoction and Dieda Wanhua Oil hot compress combined with occlusal splint improve soft tissue tension in patients with temporomandibular joint disorders Feng Bing, Pan Jingjing, Yang Bin, Xu Jianheng, Liu Deyu, Shi Jingqin, Song Zhenhua 2022, 26 (20):  3225-3229.  doi: 10.12307/2022.625 Abstract ( 734 )   PDF (848KB) ( 398 )   Save BACKGROUND: Occlusal therapy, with a long history, is a conservative treatment for temporomandibular joint disorders. However, its efficacy is still controversial. Patients undergoing occlusal therapy are prone to relapse. Traditional Chinese Medicine has achieved good results in the treatment of temporomandibular joint disorders, which has been gradually concerned. OBJECTIVE: To investigate the therapeutic effect of Huoxue Huayu Decoction and Dieda Wanhua Oil hot compress combined with occlusal splint in patients with temporomandibular joint disorders and the effect on the soft tissue tension of the affected side.  METHODS: Sixty patients with temporomandibular joint disorders who were treated at the Affiliated Haikou Hospital of Central South University Xiangya School of Medicine from January 2017 to October 2019 were recruited and randomly divided into two groups (n=30 per group): an observation group and a control group. Patients in the control group were treated with an occlusal splint on their upper jaw, while patients in the observation group were treated with Huoxue Huayu Decoction and Dieda Wanhua Oil hot compress based on the use of occlusal splints. The clinical efficacy, degree of mouth opening, temporomandibular joint disorder index (Fricton index), joint tenderness and soft tissue tension were compared between the two groups.  RESULTS AND CONCLUSION: After the treatment of Huoxue Huayu Decoction, Dieda Wanhua Oil, and occlusal splint therapy, the total effective rate of the observation group was 97%, which was higher than that of the control group (73%; P < 0.05). Compared with the control group, there was a significant increase in the number of patients with the mouth opening of 3.0-3.9 cm (P < 0.05). The Fricton index values were decreased in both groups (P < 0.05), and the Fricton index values in the observation group were lower than those in the control group (P < 0.05). The Visual Analog Scale scores were reduced in both group after treatment (P < 0.05), and the score of tenderness in the observation group was lower than that in the control group (P < 0.05). The soft tissue tension of patients was increased in the two groups, and the soft tissue tension in the observation group was higher than that in the control group (P < 0.05). All these findings indicate that the application of Huoxue Huayu Decoction and Dieda Wanhua Oil hot compress combined with occlusal splint has remarkable effects on temporomandibular joint disorders, which can increase the decrease of mouth opening and reduce the Fricton index, thereby improving the patient’s soft tissue tension on the affected side. Figures and Tables | Related Articles | Metrics

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Mechanism by which imiquimod inhibits the migration of fibroblasts in pathological scars and its targets Wang Rui, Lyu Tao, Huo Jing, Liu Zhendong, Song Jianbo 2022, 26 (20):  3173-3177.  doi: 10.12307/2022.616 Abstract ( 566 )   PDF (1636KB) ( 367 )   Save BACKGROUND: Studies have shown that imiquimod has a certain prevention and therapeutic effect on pathological scar, but its target and exact mechanism are not clear yet. OBJECTIVE: To explore and verify the target genes of imiquimod acting on pathological scar, and to observe the effect of imiquimod on the migration ability of scar fibroblasts. METHODS: Data sets were downloaded from GEO online database and analyzed to screen out the target genes of imiquimod acting on scar tissue. Scar-derived fibroblasts were extracted. The effect of imiquimod on cell migration was detected by cell scratch test and Transwell test. The expression of type 1 collagen and PCOLCE was detected by western blot assay.  RESULTS AND CONCLUSION: By screening the results of data set integration, two target genes, PCOLCE and ARHGEF25, were obtained. The results of cell experiment in vitro showed that compared with the control group, imiquimod treatment group significantly decreased the scratch healing rate and the number of migrated cells (P < 0.05), and the protein expression levels of type I collagen and PCOLCE in scar fibroblasts were also significantly decreased (P < 0.05). The results confirmed that the inhibitory effect of imiquimod on scar may be related to its down-regulation of PCOLCE expression, reduction of extracellular matrix, and reduction of cell migration rate. Figures and Tables | References | Related Articles | Metrics

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Shixiang Paste interferes with the expression of advanced glycation endproducts and its receptors and endothelial nitric oxide synthase in wound tissue of diabetic ulcer rats Fei Ji, Zhang Kaiwei, Zhou Yifu, Ling Yiming 2022, 26 (20):  3196-3201.  doi: 10.12307/2022.620 Abstract ( 598 )   PDF (1736KB) ( 187 )   Save BACKGROUND: Diabetic ulcer, as one of the common complications of diabetes, has caused serious harm to human health due to its low healing rate and high amputation rate. Although traditional Chinese medicine has been proved to improve wound healing, the molecular mechanism of this effect has not been clarified. OBJECTIVE: To investigate the possible mechanism of Shixiang Paste in treating diabetic ulcers. METHODS: Forty clean male Sprague-Dawley rats were randomly divided into four groups (n=10 per group): a normal control group, a diabetic ulcer group, a cell growth factor group and a Shixiang Paste group. Except for the normal control group, a diabetic rat model was established in the other three groups. After 1 week, an ulcer wound model was established in four groups by using the full-thickness skin defect method. One week after successful modeling, rats in the Shixiang Paste and cell growth factor groups were treated with external Shixiang Paste and cell growth factor, respectively. Wounds in the diabetic ulcer and normal control groups were only covered and fixed with double sterile dry gauze. The granulation tissue samples were taken from the wound on the back of rats on the 7th and 14th days after intervention. Western blot was used to detect the protein expression of receptors of advanced glycation endproducts and endothelial nitric oxide synthase in the granulation tissue from the back wound of rats. Hematoxylin-eosin staining was used to detect pathological changes in rat back wound samples. Immunohistochemical staining was used to detect the expression of advanced glycation endproducts in the granulation tissue from the wound on the back of rats. RESULTS AND CONCLUSION: The results of hematoxylin-eosin staining showed that large areas of unrepaired tissue were seen under the microscope in the normal control group and the diabetic ulcer group on the 7th and 14th days after intervention, while in the Shixiang Paste and cell growth factor groups, the wounds healed well on the 7th and 14th days after intervention. Western blot results showed that, compared with the normal control group, the expression of receptors of advanced glycation endproducts was decreased in the diabetic ulcer group was increased, and the expression of endothelial nitric oxide synthase (P < 0.05). In addition, the expression of receptors of advanced glycation endproducts was relatively decreased, and the expression of endothelial nitric oxide synthase was relatively increased in the Shixiang Paste group and cell growth factor group (P < 0.05). Compared with the diabetic ulcer group, the expression of receptors of advanced glycation endproducts was relatively decreased, and the expression of endothelial nitric oxide synthase was relatively increased in the Shixiang Paste group and the cell growth factor group (P < 0.05). Compared with the cell growth factor group, the expression of receptors of advanced glycation endproducts was relatively decreased, and the expression of endothelial nitric oxide synthase was relatively increased in the Shixiang Paste group (P < 0.05). The results of immunohistochemical staining showed that, compared with the normal control group, the expression of advanced glycation endproducts was increased in the diabetic ulcer group and the cell growth factor group, and the expression of advanced glycation endproducts was decreased in the Shixiang Paste group (P < 0.05). Compared with the diabetic ulcer group, the expression of advanced glycation endproducts was increased in the other groups (P < 0.05). Compared with the Shixiang Paste group, the expression level of advanced glycation endproducts was decreased in the cell growth factor group (P < 0.05). To conclude, Shixiang Paste can promote the repair of diabetic ulcer wound. Inhibiting the expression of advanced glycation endproducts and receptors and enhancing the expression of endothelial nitric oxide synthase in wound tissue may be one of the mechanisms. Figures and Tables | References | Related Articles | Metrics

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Laminaria japonica polysaccharide protects radiation-induced brain injury by regulating the blood-brain barrier in mice Ao Pian, Zhao Xin, Yu Hongrong, Zhang Siqin, Zhang Xinyue, Gu Weili, Wei Li 2022, 26 (20):  3158-3163.  doi: 10.12307/2022.614 Abstract ( 561 )   PDF (2003KB) ( 220 )   Save BACKGROUND: Radiotherapy is one of the main methods for the treatment of brain tumors, which significantly improves the clinical efficacy. However, its side effects on normal brain tissue seriously affect the quality of life of patients. Therefore, it is particularly important to find a way to effectively prevent the damage of normal brain tissue and to study its mechanism. OBJECTIVE: To explore the effect and mechanism of Laminaria japonica polysaccharide intervention on radiation-induced brain injury in mice. METHODS: Ninety-six male Kunming mice, SPF grade, were randomly divided into four groups (n=24 per group): a control group, an irradiation group, a Laminaria japonica polysaccharide (LJP) group, and an irradiation+LJP group. All mice were given 7-day continuous administration before radiation, and then 60Go γ-rays (30 Gy) were used to establish a radiation brain injury model in mice. Immunofluorescence method was used to detect the expression of fibrinogen and activation and deposition of astrocytes in the brain tissue. The ultrastructural changes of the cerebrovascular system were investigated under transmission electron microscope. The motion trails of the mice in the Morris water maze were observed before and after treatment.  RESULTS AND CONCLUSION: After radiotherapy, the expression of fibrinogen in brain tissue was significantly increased, and astrocytes were obviously activated, while the radiation plus LJP intervention significantly relieved the deposition of fibrinogen in mice. Under the electron microscope, the brain vascular endothelial cells swelled, the basement membrane was broken and shed, and the blood-brain barrier structure changed after radiation. However, the ultrastructural morphology of the mouse blood-brain barrier in the radiation+LJP group was similar to that of the control group. After radiation, the number of times and activity time of the mice passing through the platform area was significantly reduced in the Morris water maze test (P < 0.05), while the activities of the radiation mice in the platform area showed an increasing trend after the LJP intervention. These results suggest that the protective effect of LJP on radiation-induced brain injury may be mainly achieved by maintaining the stability of the blood-brain barrier structure and regulating its permeability.  Figures and Tables | References | Related Articles | Metrics

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Expression of PirB and NogoA and neurological deficits in rats with intracranial hemorrhage Yang Yang, Liu Menglan, Meng Renliang, Tao Tao 2022, 26 (20):  3202-3206.  doi: 10.12307/2022.621 Abstract ( 474 )   PDF (1168KB) ( 86 )   Save BACKGROUND: Neurite outgrowth inhibitor A (NogoA) and paired immunoglobulin-like receptor B (PirB) are myelin-related inhibitors and receptors. They are significantly increased in a variety of central nervous system injury models, and play a role in inhibiting axon regeneration and impeding the recovery of nerve function. Persistent and severe neurological deficits due to intracerebral hemorrhage may be related to both NogoA and PirB. OBJECTIVE: To investigate the protein expression of PirB and NogoA and their important effect on neurological deficits by constructing a rat model of intracranial hemorrhage. METHODS: Seventy-five male Sprague-Dawley rats were randomly assigned into a sham surgery group (n=25) and an intracranial hemorrhage group (n=50). According to the time point of sample collection, each group was randomly divided into five subgroups as a 12-hour group, a 1-day group, a 3-day group, a 7-day group, and a 14-day group. Rats in the sham surgery group did not receive any treatment, while a rat intracranial hemorrhage model was established by modified secondary injection with autologous rat tail blood in the intracranial hemorrhage group. After the neurological deficit score was performed at each time point, rats were decapitated under deep anesthesia and brain samples were taken. Western blot was used to detect the quantitative protein expression of PirB and NogoA, and the location expression of PirB was observed by immunofluorescence. RESULTS AND CONCLUSION: PirB could be expressed in neurons and astrocytes of health adult rats. The expression of PirB and NogoA in the intracranial hemorrhage group was significantly higher than that in the sham surgery group at each time point (P < 0.05). The expression peaked on the 3rd day after intracranial hemorrhage, and remained relatively high until the 14th day. The expression of PirB and NogoA in the intracranial hemorrhage group was negatively correlated with the neurological deficit scores. These results indicate that PirB and NogoA can be both expressed in the brain tissue of health adult rats, which is significantly up-regulated and inhibits axon regeneration after intracranial hemorrhage, thereby hindering the recovery of nerve function. Figures and Tables | References | Related Articles | Metrics

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Effects of silent information regulator 1 on chronic heart failure in a postmenopausal mouse model and its mechanism Zhang Haiyang, Bi Shengli, Feng Jingru, Li Fan, Wang Jing, Zhao Na, Li Xinjun 2022, 26 (20):  3184-3189.  doi: 10.12307/2022.618 Abstract ( 566 )   PDF (1095KB) ( 68 )   Save BACKGROUND: Decreased serum estradiol in postmenopausal women with coronary heart disease is related to decreased heart function and ventricular remodeling aggravation. The effect of menopause on chronic heart failure and its mechanism are still unclear. Silent information regulator 1 plays a protective role in the cardiovascular system. OBJECTIVE: To investigate the effect of silent information regulator 1 on aggravating chronic heart failure in ovariectomized mice and its mechanism.  METHODS: Experiment 1: Wild-type female C57BL/6J mice and female C57BL/6J mice with knockout of silent information regulator 1 were selected. The wild-type mice were randomly divided into four groups: a sham surgery group, an aortic stenosis group, an ovariectomy+aortic stenosis group, and an ovariectomy+aortic stenosis+estradiol group. From the first day after surgery, mice in the ovariectomy+aortic stenosis+estradiol group were given subcutaneous injection of estradiol benzoate at a dose of 0.2 mg/kg per day, and mice in the other groups were injected subcutaneously with the same dose of normal saline for 8 weeks. Experimental 2: Mice with knockout of silent information regulator 1 were randomly divided into a silent information regulator 1 knockout+sham surgery group and a silent information regulator 1 knockout+aortic stenosis group. Experimental 3: There were five experimental groups: a negative control adenovirus group, a negative control adenovirus+aortic stenosis group, an overexpression of silent information regulator 1 adenovirus+aortic stenosis group, a negative control+adenovirus ovariectomy+aortic stenosis group, and an adenovirus overexpressing silent information regulator 1+ovariectomy+aortic stenosis group. Overexpression of silent information regulator 1 was achieved by multi-point injection of negative control adenovirus or adenovirus overexpressing silent information regulator 1. Eight weeks after modeling, we detected the left ventricular end-systolic diameter, left ventricular end-diastolic diameter, left ventricular ejection fraction, serum estradiol level, brain natriuretic peptide level, uterine mass, myocardial pathological changes, and the expression level of silent information regulator 1.  RESULTS AND CONCLUSION: Compared with the sham surgery group, the left ventricular end-systolic diameter, left ventricular end-diastolic diameter, and serum brain natriuretic peptide level were significantly increased, and the left ventricular ejection fraction and the expression level of myocardial silent information regulator 1 were significantly decreased in the aortic stenosis group (P < 0.05). Compared with the aortic coarctation group, the left ventricular end-systolic diameter, left ventricular end-diastolic diameter, and serum brain natriuretic peptide level were significantly increased, and the left ventricular ejection fraction, serum estradiol level, and the expression level of myocardial silent information regulator 1 were significantly reduced in the ovariectomized+aortic stenosis group (P < 0.05). Compared with the aortic coarctation group, silent information regulator 1 was not expressed in the silent information regulator 1 knockout+aortic stenosis group, in which the left ventricular end-systolic diameter, left ventricular end-diastolic diameter, and serum brain natriuretic peptide level were significantly increased, and the left ventricular ejection fraction was significantly decreased (P < 0.05). Compared with the negative control adenovirus+ovariectomy+aortic stenosis group, the left ventricular end-systolic diameter, left ventricular end-diastolic diameter, and serum brain natriuretic peptide level were significantly reduced, and the left ventricular ejection fraction and the expression level of myocardial silent information regulator 1 were significantly increased in the adenovirus overexpressing silent information regulator 1+ovariectomy+aortic stenosis group (P < 0.05). These results indicate that ovariectomy can aggravate chronic heart failure in mice, and the molecular mechanism is to inhibit the expression of silent information regulator 1 in myocardium. Figures and Tables | References | Related Articles | Metrics

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Construction, phenotype and functional identification of vascular endothelial cell-specific PDHA1 knockout mice Hao Wei, Sun Yue, Yang Anning, Liu Taiyang, Bao Rui, Liu Yaoyang, Wang Qiushi, Wang Meng, Chang Sirong, Li Yuanyuan, Liu Zhihong 2022, 26 (20):  3207-3211.  doi: 10.12307/2022.622 Abstract ( 654 )   PDF (1252KB) ( 3322 )   Save BACKGROUND: PDHA1 gene is an indispensable gene in the aerobic respiratory chain. The energy metabolism of vascular endothelial cells is related to many diseases. Construction of vascular endothelial cell-specific PDHA1 knockout mice helps to study the role of energy metabolism in diseases related to vascular endothelial cells. OBJECTIVE: To breed vascular endothelial cell-specific PDHA1 knockout mice and identify their phenotypes. METHODS: PDHA1(iΔEC/ iΔEC) mice were co-bred with PDHA1(iΔEC/-) mice, and more PDHA1(iΔEC/ iΔEC) mice were obtained for subsequent experiments. PCR and agarose gel electrophoresis were used for gene identification. RT-PCR and western blot were used to detect the mRNA and protein expression of genes related to energy metabolism after PDHA1 knockout, respectively. The protein expression of PDHA1 after PDHA1 conditional knockout was determined by double immunofluorescence staining of VE-cadherin and PDHA1. RESULTS AND CONCLUSION: Genotypes of offspring mice were successfully detected by agarose gel electrophoresis, including 15 PDHA1(iΔEC/ iΔEC) and 2 PDHA1(iΔEC/-) mice. Results of reverse transcription PCR and western blot assay showed that the transcription and translation levels of PDHA1 were decreased, the protein and mRNA expressions of HK2 in the glycolysis pathway were increased, and the protein and mRNA expression of IDH in the aerobic phosphorylation pathway were decreased. Double immunofluorescence staining of VE-cadherin and PDHA1 showed a decrease in the expression of PDHA1. Figures and Tables | References | Related Articles | Metrics

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Effects of Dendrobium officinale flavonoid on oxidative stress and autophagy in the liver of an exhaustive exercise rat model Wang Sihan, Chen Junji, Mo Weibin 2022, 26 (20):  3212-3219.  doi: 10.12307/2022.623 Abstract ( 551 )   PDF (1045KB) ( 1200 )   Save BACKGROUND: Proper exercise can improve antioxidant capacity, inhibit and delay oxidative stress, and activate autophagy in the body. However, the mechanism of oxidative stress and autophagy in the liver tissue of rats after exhaustive exercise due to a long-term endurance training is still very complicated. Dendrobium officinale flavonoids have antioxidant capacity, immune regulation, anti-inflammatory and analgesic effects. It is still unclear whether Dendrobium officinale flavonoids can protect liver tissue before exercise.  OBJECTIVE: To investigate the effect of Dendrobium officinale flavonoids on oxidative stress and autophagy in the liver tissue of exhaustive exercise rats. METHODS: Sixty male Sprague-Dawley rats aged 10 weeks old were randomly divided into a quiet control group, an exercise control group, and low-, medium-, and high-dose Dendrobium officinale flavonoid groups. Except for the quiet control group, the rest rats underwent 5-day adaptive treadmill training, followed by 6-week formal exercise training. Exhaustion was achieved in the last training at the end of 6 weeks. Rats in the low-, medium- and high-dose Dendrobium officinale flavonoids groups were intragastrically administrated with 1.5, 3.0, and 4.5 g/(kg•d) Dendrobium officinale flavonoids half an hour before exercise, respectively. The intragastric volume was 5 mL/kg, once a day, until the end of the experiment. Hematoxylin-eosin staining was used to observe the pathological changes of rat liver tissue, and to detect rat serum alanine aminotransferase and aspartate aminotransferase, malondialdehyde, catalase, superoxide dismutase, and glutathione peroxidase levels, and total antioxidant capacity. Changes in autophagy-related indexes in rat liver tissue were detected by RT-qPCR and western blot assay.  RESULTS AND CONCLUSION: Hematoxylin-eosin staining results revealed that in the exercise control group, hepatocytes were arranged in a disorderly manner, the hepatic sinusoids were swollen, the portal area was infiltrated with inflammatory cells, and there were a large number of lipid droplets in the cytoplasm. The lipid droplets and inflammatory cell infiltration in the liver tissue of rats were significantly reduced in the low-dose Dendrobium officinale flavonoid group, while there were no significant changes in the liver tissue of the rats in the medium- and high-dose Dendrobium officinale flavonoid groups compared with the quiet group. Compared with the quiet control group, the expression levels of serum alanine aminotransferase, aspartate aminotransferase, malondialdehyde and total antioxidant capacity were significantly increased (P < 0.01), while the serum activities of catalase, superoxide dismutase and glutathione peroxidase were significantly decreased in the exercise control group (P < 0.01). Compared with the exercise control group, the activities of alanine aminotransferase and aspartate aminotransferase were significantly decreased (P < 0.01), and the activities of serum catalase, superoxide dismutase and glutathione peroxidase were significantly increased in the low-, medium- and high-dose Dendrobium officinale flavonoid groups (P < 0.01). Moreover, the serum level of malonaldehyde and total antioxidant capacity were decreased in the medium- and high-dose Dendrobium officinale flavonoid groups (P < 0.05). Compared with the quiet control group, the mRNA and protein expression levels of Beclin1, Atg7, LC3-II, p62 and Rab7 in the liver tissue of rats were significantly increased in the exercise control group (P < 0.01). Compared with the exercise control group, the mRNA and protein expression levels of Beclin1, Atg7, LC3 - II, p62 and Rab7 in the liver tissues of rats showed a downward trend in the medium- and high-dose Dendrobium officinale flavonoid groups (P < 0.05 or P < 0.01). To conclude, long-term endurance exercise until exhaustive exercise can induce oxidative stress and damage to liver tissue in rats. Dendrobium officinale flavonoids can maintain the stability of liver function by regulating liver oxidative metabolism and autophagy in rat liver tissue. Thereby, liver protection is achieved. Figures and Tables | References | Related Articles | Metrics

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Vimentin silenced by small interfering RNA inhibits glial scar formation in a rat model of acute spinal cord injury Li Jianfeng, Zhang Shulian, Yan Jinyu, Li jiayi, Jin Zhao, Deng Qi 2022, 26 (20):  3190-3195.  doi: 10.12307/2022.619 Abstract ( 463 )   PDF (2378KB) ( 189 )   Save BACKGROUND: A barrier with formation of glial scar after spinal cord injury is considered to be harmful for nerve repair.  OBJECTIVE: To explore the influence of small interfering RNA (siRNA)-mediated inhibiting Vimentin on reactive astrocytes and on the formation of glial scar after central nervous system injury.   METHODS: Third-generation astrocytes that grew well were selected and randomized into untransfected group, siRNA-NC group, and siRNA-Vimentin group. We used siRNA interference technology to silence vimentin expression in astrocytes, and Vimentin immunofluorescence staining was conducted at 24 hours after transfection. Cell migration and proliferation were observed through a cell scratch test. Sprague-Dawley rats were randomized into a control group (n=10), in which only the lamina was opened, without damage to the spinal cord; a model group (n=20), in which a spinal cord injury model was prepared, followed by direct injection of saline and empty plasmids into the spinal cord; and a treatment group (n=20), in which the spinal cord injury model was prepared followed by direct injection of Vimentin plasmids intervened with siRNA for 24 hours. Spinal cord continuity and thickness as well as scar adhesion with surrounding tissues were observed at 1 day, 4, and 8 weeks after surgery. RESULTS AND CONCLUSION: Integral asorbance value of Vimentin by fluorescence immunoassay was lower in the siRNA-Vimentin group than the untransfected and siRNA-NC groups (P < 0.05). The scratched width was larger in the siRNA-Vimentin group than the untransfected and siRNA-NC groups. The spinal cord tissue structure at the injured site was improved in the treatment group, and the range of glial scar formation was smaller in the treatment group than the model group at 4 and 8 weeks after surgery. To conclude, Vimentin has a significant inhibitory effect on the proliferation and migration of reactive astrocytes and inhibits the formation of glial scars after spinal cord injury, providing a favorable microenvironment for axonal regeneration and nerve function recovery after spinal cord injury. Figures and Tables | References | Related Articles | Metrics

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Interaction of Nogo-A/NgR signaling pathway and NGF/TrkA signaling pathway during the regeneration of injured spinal cord nerve axons Yang Lin, Wu Yao, Zhou Binbin 2022, 26 (20):  3220-3224.  doi: 10.12307/2022.624 Abstract ( 581 )   PDF (1248KB) ( 634 )   Save BACKGROUND: Growth-associated protein 43, as a membrane-like phosphoprotein, is a marker protein of neuronal regeneration plasticity and participates in the key process of axonal regeneration and the connection of various stages. It is of great significance for central nerve regeneration. OBJECTIVE: To investigate the mechanism of nerve regeneration and recovery after spinal cord injury by observing the interaction of neurite outgrowth inhibitor A (Nogo-A)/neurite growth inhibitor receptor (NgR) signaling pathway and nerve growth factor (NGF)/tyrosine kinase (TrkA) signaling pathway on the expression of growth-associated protein 43.  METHODS: A total of 120 Sprague-Dawley rats were randomly divided into 5 groups: NgR blocking group, TrkA blocking group, double blocking agent group, injury control group, and sham surgery group. Rats in the sham surgery group underwent laminectomy (without injury to the spinal cord); and spinal cord injury rat models were established in the other groups. Immediately after successful modeling, rats in the NgR blocking group were injected with 25 μL of NEP1-40, a NgR blocker, and 25 μL of normal saline in the injured segment. Rats in the TrkA blocking group were injected with 25 μL of K252a, a TrkA blocker, and 25 μL of normal saline. Rats in the double blocking agent group were injected with 25 μL of NEP1-40 and 25 μL of K252a. Rats in the injury control group and the sham surgery group were injected with 50 μL of normal saline. On the 3rd, 7th, 14th, and 21st days after the intervention, the mRNA and protein expression of growth-associated protein 43 in rat spinal cord tissue were detected by immunohistochemistry, PCR, and western blot.  RESULTS AND CONCLUSION: The results of immunohistochemistry, PCR, and western blot showed that the mRNA and protein expression of growth-associated protein 43 in the NgR blocking group, the TrkA blocking group, the double blocking agent group and the injury control group was higher than that in the sham surgery group. The expression of growth-associated protein 43 in the NgR blocking group and the double blocking agent group was higher than that in the injury control group, and there was a significant difference on the 7th and 14th days (P < 0.05 or P < 0.01). The mRNA and protein expression of growth-associated protein 43 in the TrkA blocking group was lower than that in the injury control group, and the difference was significant on the 7th and 14th days (P < 0.05 or P < 0.01). To conclude, the results from the NgR blocking group proves that the Nogo-A/NgR signaling pathway can significantly increase the expression of growth-associated protein 43 and promote the regeneration of nerve axons. The results from the TrkA blocking group proves that the NGF/TrkA signaling pathway can reduce the expression of growth-associated protein 43 and inhibit the regeneration of nerve axons. The results from the double blocking agent group shows that the interaction of the Nogo-A/NgR signaling pathway and the NGF/TrkA signaling pathway can promote the expression of growth-associated protein 43. Figures and Tables | References | Related Articles | Metrics

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Effects of time-specific AMP-activated protein kinase alpha1/2 gene knockout on hippocampal energy metabolism and synaptic plasticity in mice Liu Yulu, Jia Weiwei, Dai Yaling, Xu Wenshan, Ding Yanyi, Liang Shengxiang, Liu Weilin, Chen Lidian 2022, 26 (20):  3230-3235.  doi: 10.12307/2022.626 Abstract ( 715 )   PDF (1186KB) ( 2278 )   Save BACKGROUND: AMP-Activated protein kinase (AMPK) is a molecular-level energy sensor that can be activated when the body is in a low energy state to provide energy for neuronal activity. However, the relationship between time-specific AMPK knockout and the development of cognitive dysfunction remains unclear.  OBJECTIVE: To investigate the effects of time-specific AMPKα1/2 knockout on hippocampal energy metabolism and synaptic plasticity in mice, and to search for key molecular targets between energy metabolism and cognitive function.  METHODS: Sixteen 6-month-old mice with AMPKα1/2FLOX/FLOX and AMPKα1/2FLOX/FLOX+Camk2a-cre/ERT2 were randomly divided into control group (n=8) and AMPKα1/2 knockout group (n=8). The AMPKα1/2 knockout group was intraperitoneally injected with 0.1 mL tamoxifen daily, while the control group was intraperitoneally injected with an equal dose of corn oil solvent daily. Injections in each group were conducted for 5 continuous days. After 7 days, Barnes maze test was used to test spatial learning and memory ability of mice. Chemical exchange saturation transfer imaging was used to observe the glucose metabolism in the hippocampus. Patch clamp electrophysiological techniques were used to detect the field potential of hippocampal CA3-CA1 neural circuit, including input-output curve, paired-pulse facilitation ratio and long-term synaptic plasticity induced by high frequency stimulation.  RESULTS AND CONCLUSION: Compared with the control group, the escape latency was prolonged (P < 0.001), the frequency of contact with the target hole decreased significantly (P < 0.05), and the time spent in the target quadrant decreased significantly (P < 0.05) in the AMPKα1/2 knockout group. Compared with the control group, the level of glucose metabolism in the hippocampus of mice was reduced in the AMPKα1/2 knockout group (P < 0.05). Compared with the control group, the voltage in the hippocampus was significantly decreased with different amounts of stimulation in the AMPKα1/2 knockout group (P < 0.05). At the base field potential, the paired-pulse facilitation ratios at different time intervals decreased significantly (P < 0.05). Compared with the control group, high frequency stimulation significant reduced the amplitude of excitatory postsynaptic potential in the hippocampus of mice in the AMPKα1/2 knockout group after (P < 0.05). These results suggest that time-specific AMPKα1/2 knockdown decreases glucose metabolism in the hippocampus, impairs presynaptic transmitter release, synaptic transmission efficiency, and synaptic plasticity, thereby leading to spatial learning and memory disorders. Figures and Tables | References | Related Articles | Metrics

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Role and regulatory mechanism of glycophagy in glycogen metabolism Lyu Yuhu, Cheng Lin, Lin Wentao, Peng Fenglin 2022, 26 (20):  3243-3249.  doi: 10.12307/2022.628 Abstract ( 1275 )   PDF (958KB) ( 213 )   Save BACKGROUND: Glycophagy is a selective autophagy, which plays a unique role in glycogen degradation, but the mechanism of its regulation is still limited. OBJECTIVE: To review the function and regulatory signaling pathway of glycophagy based on the morphology, function and degradation pathway of glycophagy substrate, and then to clarify the role and regulatory mechanism of glycophagy in glycogen metabolism. METHODS: Web of Science, PubMed, and EBSCO databases were searched for the relevant English literatures using the search terms of glycophagy [Topic], Glycogen [Title] AND autophagy [Title], Glycogen-storage-disease [Title], Lafora-disease [Title], pompe-disease[Title], and Von-Gierke-disease [Title]. CNKI database was also searched to retrieve the relevant Chinese literatures with the search terms of glycogenated [Topic], glycogen autophagy [Title], glycogen accumulation [Title], and glycogen storage [Title]. Relevant literature was also manually retrieved. Based on the exclusion criteria, 87 papers were included for final review by reading the title and abstract. RESULTS AND CONCLUSION: The glycogen degradation system in vivo consists of glycophagy and glycogen oxidative phosphorylation, which can provide substrates for the generation of glycolipids and fats, and prevent more serious events through storing a certain amount of glycogen in glycogenosomes during energy stress. However, glycophagy disorders can cause some diseases that are even life-threatening, such as Lafora disease, von Gierke disease and Pompe disease. The activation of PI3K/mTOR, Akt/FoxO and SGK1/mTOR signaling pathways could inhibit glycophagy, while glycophagy can be elicited through the activation of cAMP/PKA, G6PC/SIRT1/FoxO, Ca2+, cGMP/PKG/GSK-3β signaling pathways. However, There are many problems that need to be solved, such as: (1) whether there is sex difference during glycophagy; (2) what signals or stimuli lead to an increase in glycogen storage during glycophagy; (3) what substances trigger enzymes in lysosomes to degrade glycogen; (4) whether there is a balance point between glycogen degradation and storage during glycophagy; and (5) how the various regulatory pathways of glycophagy are coordinated, and what are the mechanisms of action and pharmacology. Figures and Tables | References | Related Articles | Metrics

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N6-methyladenosine methylation and its regulation in metabolic diseases Chen Ziyang, Pu Rui, Deng Shuang, Liu Min 2022, 26 (20):  3250-3255.  doi: 10.12307/2022.629 Abstract ( 897 )   PDF (807KB) ( 196 )   Save BACKGROUND: N6-methyladenosine (m6A) methylation has gradually become a research hotspot and new direction in life science and sports medicine. Many studies have confirmed that m6A methylation is closely related to various diseases and plays a key role in the diagnosis and treatment of metabolic diseases, but its mechanism has not been clarified.   OBJECTIVE: To investigate the relationship between m6A methylation and various metabolic diseases, to review the latest research progress of m6A and related enzymes in the regulation of metabolic diseases, and to provide a detailed overview of the specific molecular mechanism, in order to provide a new direction for the diagnosis and treatment of metabolic diseases.   METHODS: The latest domestic and foreign research results related to m6A methylation and the occurrence and development of various metabolic diseases were searched in PubMed and CNKI with the search terms of “m6A, obesity, type 2 diabetes, nonalcoholic fatty liver disease, osteoporosis” in English and Chinese, respectively. Finally, according to the inclusion and exclusion criteria, 51 literatures were finally included and summarized.   RESULTS AND CONCLUSION: M6A methylation is widely involved in the occurrence and development of various metabolic diseases through a variety of signaling pathways. The dynamic regulation of m6A on RNA regulates the processes of adipogenesis, glucose and lipid metabolism, islet β cell function, insulin resistance, blood glucose homeostasis and bone formation through related enzymes.  In turn, it plays a key role in obesity, type 2 diabetes, nonalcoholic fatty liver disease and osteoporosis. M6A methylase fat mass and obesity-associated protein and related enzymes are closely related to the occurrence and development of various metabolic diseases, and can be used as biomarkers for their diagnosis.  In addition, m6A methylation may be an effective indicator of lipid metabolism, lipid differentiation and other processes, and provide more options for relevant diagnosis.  Figures and Tables | References | Related Articles | Metrics

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Motor coordination: evolution of theoretical model and quantitative method Wang Jiawei, Liu Ye 2022, 26 (20):  3256-3264.  doi: 10.12307/2022.630 Abstract ( 844 )   PDF (1162KB) ( 226 )   Save BACKGROUND: Motor coordination is an important part of human motor ability, and it is of great significance to explore its rules to understand the coordination and control of “nerves-muscles-joints” in human locomotor system. OBJECTIVE: To review the evolution of theoretical models and quantitative methods of motor coordination through systematic analysis of domestic and foreign research literature on motor coordination. METHODS: We retrieved PubMed, Web of Science, EBSCO and CNKI Full-text databases using the keywords of “motor coordination, segment coordination, muscle synergy, equilibrium point, uncontrolled manifold” in English and “motor coordination, joint coupling, joint coordination, muscle synergy, vector coding, motor control” in Chinese. There was no limitation to the time of literature retrieval. Finally, 111 eligible literatures were included and reviewed. RESULTS AND CONCLUSION: In the current theoretical model of motor coordination, the dynamic system theory, uncontrolled manifold hypothesis, and the muscle synergy hypothesis have been most useful and widely applied. The corresponding quantitative methods are also evolving. Continuity analysis has great potential to explore the coordination characteristics of the joint under periodic and aperiodic relative motion. Muscle synergy analysis can be used to identify the muscle synergy strategies in different groups. It is suggested that research on motor coordination in China should combine the analysis of joint coordination mode and neuromuscular cooperative control strategy based on improving the exploration of basic theories and the use of various quantitative methods, so as to better understand the coordination law of nerves-muscles-joints. Figures and Tables | References | Related Articles | Metrics

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Application of extracorporeal shock wave therapy in burn wound repair and post-burn scar treatment An Dong, Liu Yang, Yang Tongjiang 2022, 26 (20):  3265-3272.  doi: 10.12307/2022.631 Abstract ( 792 )   PDF (1133KB) ( 330 )   Save BACKGROUND: Extracorporeal shock wave therapy as a new technology for burn treatment has been carried out for 16 years. It has achieved significant clinical effects in promoting burn wound healing and improving scar contracture, pruritus, pain, and skin erythema after burn. Up to date, there is still no standardized treatment guideline. Its molecular mechanism, action pathway and treatment parameters are worthy of more extensive and in-depth research. OBJECTIVE: To review the application and research progress of extracorporeal shock wave therapy in burn wound repair and scar treatment after burn. METHODS: Related literatures were searched in PubMed, Web of Science, CNKI, VIP and WanFang databases from their inception to February 2021, with the keywords of “extracorporeal shock wave therapy, shock wave, burns, burn scar, scar” in English and Chinese, respectively. A total of 132 papers were initially retrieved, and 59 papers that followed the inclusion criteria were used for analysis, summarization and review. RESULTS AND CONCLUSION: Extracorporeal shock wave therapy can promote burn wound healing through cell mechanotransduction, regulation of inflammatory response, promotion of angiogenesis, improvement of circulation and acceleration of cell proliferation and epithelialization. It can also soften and regulate the scar tissue, improve the symptoms either of itching or pain, and improve the appearance and function of the skin along with the scar. The therapeutic effect of extracorporeal shock wave on burn is embodied in both physical and biological effects. Cell mechanotransduction plays an important role in shock wave regulation of burn wound healing and hypertrophic scar development. The conduction of shock waves can cause the change of protein and gene expression and regulate the biochemical reaction in cells. Figures and Tables | References | Related Articles | Metrics

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Effect of anodal transcranial direct current stimulation on muscle strength and endurance: a Meta-analysis Hao Zhixin, Wu Yixin, Wang Xin, Xia Zhongliang 2022, 26 (20):  3273-3280.  doi: 10.12307/2022.632 Abstract ( 549 )   PDF (1225KB) ( 192 )   Save OBJECTIVE: Transcranial direct current stimulation has shown positive effects in clinical medicine, but its application in sports biomechanics is still at its infant stage. Some scholars still question the effect of this technique on enhancing muscle strength and improving sports performance. This study systematically reviewed the effect of anodal transcranial direct current stimulation on muscle strength and its mechanism. METHODS: PubMed, Web of Science, Google Scholar, and Scopus databases were searched for randomized controlled trials regarding the effect of anodal transcranial direct current stimulation on muscle strength according to inclusion and exclusion criteria. Cochrane Handbook for Systematic Reviews of Interventions was used to evaluate the quality of the included literatures. RevMan 5.3 software was used for statistical analysis, and effect size was uniformly represented by ES. RESULTS: The total effect size of anodal transcranial direct current stimulation on muscle strength level was ES=0.38, 95% confidence interval (Cl) [0.20, 0.57] (P < 0.05). The effect sizes of anodal transcranial direct current stimulation on different brain regions were that ESdorsolateral prefrontal cortex (0.88) > ESmotor cortex (0.32) > EStemporal cortex (0.29). The effect sizes of different current intensities on anodal transcranial direct current stimulation were ES1.5 mA (0.57) > ES2 mA (0.35). The effect sizes of anodal transcranial direct current stimulation on different subjects were ESathlete (0.64) > EShealthy person (0.29). The effect sizes of anodal transcranial direct current stimulation on different task types were ESendurance task (0.45) > ESpower task (0.35).  CONCLUSION: Transcranial direct current stimulation could enhance the muscle strength of athletes and the muscle endurance performance of healthy individuals and athletes by changing the excitability of cerebral cortex and regulating the perception of effort. The study did not find the positive effect of anodal transcranial direct current stimulation on the maximum strength of healthy individuals. Application strategy of anodal transcranial direct current stimulation parameters: To enhance muscle strength, the motor cortex was selected and equipped with unilateral or bilateral electrodes, followed by anodal transcranial direct current stimulation, 2 mA, 10-20 minutes, a polar plate of 25-35 cm2. To enhance muscle endurance, the dorsolateral prefrontal cortex and motor cortex were selected and equipped with unilateral electrode alone or combined with other cortex electrodes, followed by anodal transcranial direct current stimulation, 1.5 mA, 10-20 minutes, a polar plate of 25-35 cm2. Transcranial direct current stimulation can be used as a neuroregulatory technology in sports biomechanics; however, further explorations on the neurophysiological mechanism of transcranial direct current stimulation are warranted.  Figures and Tables | References | Related Articles | Metrics