蠲痹方含药血清对绝经后膝骨关节炎大鼠软骨细胞自噬的影响及其作用机制  

Effects and mechanism of Juanbi Fang(蠲痹方)medicated serum on autophagy of chondrocytes in postmenopausal rats with knee osteoarthritis:an experimental study

作　　者：马靖哲 康武林 姚彬 黄文博 李越 陈小林 李思聪 袁普卫 MA Jingzhe;KANG Wulin;YAO Bin;HUANG Wenbo;LI Yue;CHEN Xiaolin;LI Sicong;YUAN Puwei(The Affiliated Hospital of Shaanxi University of Chinese Medicine,Xianyang 712046,Shaanxi,China;College of Pharmacy,Shaanxi University of Chinese medicine,Xianyang 712046,Shaanxi,China)

机构地区：[1]陕西中医药大学附属医院,陕西咸阳712046 [2]陕西中医药大学药学院,陕西咸阳712046

出　　处：《中医正骨》2024年第2期7-16,共10页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：陕西省教育厅青年创新团队建设科研计划项目(21JP035);陕西省教育厅服务地方专项计划项目(21JC010);咸阳市重点研发计划项目(2019k01-53)。

摘　　要：目的:探讨蠲痹方含药血清对绝经后膝骨关节炎(knee osteoarthritis,KOA)大鼠软骨细胞自噬的影响及其作用机制。方法:①绝经后KOA动物模型的建造和蠲痹方含药血清的制备。采用摘取大鼠卵巢,并切断膝关节内侧副韧带、前交叉韧带,摘除膝关节内侧半月板的方法,建造绝经后KOA大鼠模型。造模成功后,对模型大鼠进行蠲痹方浓缩液灌胃,制备蠲痹方含药血清。②大鼠软骨细胞的提取。分别取正常大鼠和模型大鼠的膝关节软骨分离、提取软骨细胞。③蠲痹方含药血清最佳作用浓度筛选。将模型大鼠软骨细胞分为模型组及1.25%、2.5%、5%、10%、20%含药血清组6组,除模型组外,其余各组分别加入相应体积分数的蠲痹方含药血清干预24 h。检测各组软骨细胞的活力,筛选蠲痹方含药血清最佳作用浓度。④蠲痹方含药血清对大鼠软骨细胞G蛋白耦联受体30(G protein-coupled receptor 30,GPR30)表达影响的检测。将模型大鼠软骨细胞分为模型组及1.25%、2.5%、5%、10%含药血清组,并取正常大鼠软骨细胞设为空白组。除模型组和空白组外,其他各组软骨细胞用相应体积分数的蠲痹方含药血清干预24 h。检测各组大鼠软骨细胞GPR30的相对表达量。⑤蠲痹方含药血清对大鼠软骨细胞自噬相关蛋白表达和AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路影响的检测。将模型大鼠软骨细胞分为模型组、含药血清组、含药血清+Compound C组、Compound C组,并取正常大鼠软骨细胞设为空白组。除模型组和空白组外,含药血清组、含药血清+Compound C组、Compound C组分别用10%蠲痹方含药血清、10%蠲痹方含药血清+Compound C及Compound C干预24 h。检测各组软骨细胞中自噬相关蛋白微管相关蛋白轻链3β(microtubule-associated protein light chain 3 beta,MAPLC3β)、Beclin-1、p62,及AMPObjective:To observe the effects of Juanbi Fang(蠲痹方,JBF)medicated serum on autophagy of chondrocytes in postmenopausal rats with knee osteoarthritis(KOA),and to explore its mechanism.Methods:①Twenty rats were subjected to ovariectomy,transection of knee medial collateral ligament(MCL)and anterior cruciate ligament(ACL),followed by knee medial meniscectomy for inducing postmenopausal KOA.After successful modeling,15 model rats were intragastric administrated with JBF concentrate for making JBF medicated serum.②The normal rats and model rats were sacrificed and their knee cartilages were harvested for isolating and extracting chondrocytes.③The chondrocytes extracted from the model rats were divided into model group,1.25%medicated serum group,2.5%medicated serum group,5%medicated serum group,10%medicated serum group,and 20%medicated serum group.Except for the model group,the chondrocytes in the other groups were intervened with JBF medicated serum in their corresponding volume fraction for consecutive 24 hours.After the end of intervention,the viability of chondrocytes in each group was detected,and the optimal acting concentration of the JBF medicated serum was screened.④The chondrocytes extracted from the model rats were assigned into model group,1.25%medicated serum group,2.5%medicated serum group,5%medicated serum group,and 10%medicated serum group,and the chondrocytes from the normal rats were taken as blank group.Except for the model group and blank group,the chondrocytes in other 4 groups were intervened with JBF medicated serum in their corresponding volume fraction for consecutive 24 hours.After the end of intervention,the relative expression level of G protein-coupled receptor 30(GPR30)in chondrocytes was detected by using Western blotting assay.⑤The chondrocytes extracted from the model rats were divided into model group,medicated serum group,medicated serum+Compound C group,and Compound C group,and the chondrocytes from the normal rats were taken as blank group.Except for the model group

关 键 词：骨关节炎 膝 绝经后期 软骨细胞 自吞噬 蠲痹方 大鼠 

分 类 号：R285.5[医药卫生—中药学]