摘要
目的:探讨川芎嗪能否通过cGAS-STING通路抑制Ca^(2+)浓度,进而保护KOA大鼠软骨的机制研究。方法:采用前交叉韧带离断法复制KOA模型,将雄性SD大鼠随机分为对照组、模型组、川芎嗪组(20、40、80 mg·kg^(-1)·d^(-1)),每组8只。提取雄性SD大鼠膝关节的软骨细胞,分为对照组,IL-1β组,川芎嗪组(100、200μmol/L)和H^(-1)51组(STING通路抑制剂)。用IL^(-1)β诱导软骨细胞24 h建立KOA模型,川芎嗪和H^(-1)51干预24 h。CCK-8法检测川芎嗪对细胞活力的影响;采用番红-固绿染色法观察软骨组织以及软骨细胞的病理变化;免疫组化法检测组织cGAS、STING蛋白表达;RT-qPCR检测组织cGAS、STING、Calmodulin、Calpain、NF-κB mRNA水平;采用流式细胞术检测细胞Ca^(2+)浓度;Western blot检测细胞和组织cGAS、STING、Calmodulin、Calpain蛋白表达以及细胞p-IRF3蛋白相对表达。结果:CCK-8检测结果显示川芎嗪在浓度为0~400μmol/L范围内对细胞活力无显著影响。与模型组相比,川芎嗪干预后,番红-固绿染色的结果显示各组的软骨组织损伤得到了改善;免疫组化法检测软骨组织的cGAS、STING蛋白阳性区域比例明显降低;cGAS、STING、Calmodulin、Calpain、NF-κB mRNA的相对表达水平也呈下降趋势。此外,体外实验也再次验证,相较于IL^(-1)β组,川芎嗪组的软骨细胞Ca^(2+)浓度降低;cGAS、STING、Calmodulin、Calpain、p-IRF3蛋白的相对表达减少。结论:川芎嗪可以通过cGAS-STING通路从而抑制Ca^(2+)浓度保护KOA大鼠的软骨。
Objective:To investigate whether Tetramethylpyrazine(TMP)can inhibit Ca^(2+)concentration through the cGAS-STING pathway and thus protect cartilage in KOA rats.Methods:The KOA model was replicated using the anterior cruciate ligament disarticulation method,and male SD rats were randomly divided into the control group,the model group,and the TMP group(20,40,and 80 mg·kg^(-1)·d^(-1)),with 8 rats in each group.Chondrocytes from the knee joints of male SD rats were extracted and divided into the control group,the IL-1βgroup,the TMP group(100 and 200μmol/L)and the H-151 group(STING pathway inhibitor).The KOA model was established by inducing chondrocytes with IL-1βfor 24 h,and TMP and H-151 intervened for 24h.The effect of TMP on cell viability was detected by CCK-8 assay.Pathological changes in cartilage tissues and chondrocytes were observed using Senna-Solid Green staining.Detection of cGAS,STING protein expression in tissues was measured by immunohistochemical methods.RT-qPCR was used to detect cGAS,STING,Calmodulin,Calpain、NF-κB mRNA levels in tissues.Cellular Ca^(2+)concentration was measured by flow cytometry.Western blot was used to detect intracellular and tissue cGAS,STING,Calmodulin,Calpain protein expression and relative cellular p-IRF3 protein expression.Results:CCK-8 assay showed that TMP had no significant effect on cell viability in the concentration range of 0-400μmol/L.The results of Senna-Solid green staining after TMP intervention showed improved cartilage tissue damage in all groups compared to the model group.The proportion of cGAS and STING protein-positive areas of cartilage tissue detected by immunohistochemistry was significantly reduced.The relative expression levels of cGAS,STING,Calmodulin,Calpain and NF-κB mRNA also showed a decreasing trend.In addition,in vitro experiments also re-verified that the chondrocyte Ca^(2+)concentration was reduced in the TMP group compared to the IL^(-1)βgroup.The relative expression of cGAS,STING,Calmodulin,Calpain and p-IRF3 proteins was reduced.Conclusion:TMP protects cartilage in KOA rats via the cGAS-STING pathway and thus inhibits Ca^(2+)concentration.
作者
施蕾
廖太阳
刘德仁
刘江宇
王培民
吴鹏
茆军
SHI Lei;LIAO Taiyang;LIU Deren;LIU Jiangyu;WANG Peimin;WU Peng;MAO Jun(The Affiliated Hospital of Nanjing University of Chinese Medicine,Jiangsu Province Hospital of Chinese Medicine,Nanjing 210029,China;Key Laboratory for Metabolic Diseases in Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China)
出处
《海南医科大学学报》
北大核心
2025年第16期1201-1210,共10页
Journal of Hainan Medical University
基金
国家自然科学基金青年项目(82205143)
江苏省中医院优秀青年博士培养专项(2023QB0122)
江苏省中医院第三批高峰学术人才(y2021rc20)
江苏省中医院中医膝骨关节炎临床医学创新中心(Y2023zx05)
江苏省医学重点学科/实验室建设单位(JSDW202252)
江苏省教育厅研究生创新项目(KYCX24_2227)。
