补肾调肝方含药血清促进大鼠软骨细胞自噬治疗关节炎  

Bushen Tiaogan Prescription containing serum in the treatment of osteoarthritis by promoting chondrocyte autophagy in rats

作　　者：范帅 吴春飞 梁祖建 徐兆辉 谢平金 朱根福 Fan Shuai;Wu Chunfei;Liang Zujian;Xu Zhaohui;Xie Pingjin;Zhu Genfu(Third Affiliated Hospital,Guangzhou University of Chinese Medicine,Guangzhou 510240,Guangdong Province,China;Luohu District Hospital of Traditional Chinese Medicine,Shenzhen 518000,Guangdong Province,China)

机构地区：[1]广州中医药大学第三附属医院,广东省广州市510240 [2]深圳市罗湖区中医院,广东省深圳市518000

出　　处：《中国组织工程研究》2022年第20期3178-3183,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然科学基金项目(2021A1515011469),项目负责人:梁祖建;广东省中医药局科研项目(20201171),项目负责人:范帅;广东省中医药局科研项目(20211211),项目负责人:吴春飞;广州中医药大学第三附属医院科研创新基金项目(sy201701,sy2018007),项目负责人:范帅;2020年深圳市科技创新委员会基础研究面上项目(JCYJ20190812170815559),项目参与人:谢平金。

摘　　要：背景:前期研究发现,补肾调肝方治疗骨关节炎有明显临床疗效,但缺乏对其作用机制的研究。目的:观察补肾调肝方含药血清对大鼠软骨细胞自噬的影响,并探讨其内在机制。方法:传代培养的第3代大鼠软骨细胞随机分为4组:二甲基亚砜组、10μg/L白细胞介素1β组、10μg/L白细胞介素1β+10%补肾调肝方含药血清组和雷帕霉素5μmol/L组,分别处理软骨细胞24 h。单丹磺酰尸胺染色法检测软骨细胞自噬小体表达;实时荧光定量PCR检测自噬相关基因ATG5、ATG7 mRNA的表达;Western blot检测Beclin1、LC3A/B和磷酸化蛋白激酶B(p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的蛋白表达水平。结果与结论:①与二甲基亚砜组比较,10μg/L白细胞介素1β组中细胞的自噬小体数量明显减少;雷帕霉素处理后的软骨细胞与二甲基亚砜组相比,自噬小体的数量明显增多;与10μg/L白细胞介素1β组相比,10μg/L白细胞介素1β+10%补肾调肝方含药血清组的自噬小体明显增多;②实时荧光定量PCR及Western blot结果显示,与二甲基亚砜组比较,10μg/L白细胞介素1β组中软骨细胞自噬相关基因ATG5、ATG7 mRNA及Beclin1和LC3A/B-Ⅱ/LC3A/B-Ⅰ蛋白表达水平均明显下降(P<0.05,P<0.01,P<0.01,P<0.05),而p-AKT和p-mTOR蛋白表达水平明显上升(P<0.05,P<0.05);与二甲基亚砜组相比,雷帕霉素处理后的软骨细胞中ATG5、ATG7 mRNA及Beclin1和LC3A/B-Ⅱ/LC3A/B-Ⅰ蛋白表达水平均明显上升(P<0.01,P<0.05,P<0.05,P<0.05),而p-AKT和p-mTOR蛋白表达水平明显下降(P<0.01,P<0.01);与10μg/L白细胞介素1β组相比,10μg/L白细胞介素1β+10%补肾调肝方含药血清组中软骨细胞自噬相关基因ATG5、ATG7 mRNA及Beclin1和LC3A/B-Ⅱ/LC3A/B-Ⅰ的蛋白表达水平均明显上升(P<0.01,P<0.01,P<0.05,P<0.01),而p-AKT和p-mTOR蛋白表达水平明显下降(P<0.05,P<0.05);③提示补肾调肝方含药血清可能通过抑制软骨细胞内AKT/mTOR信号通路增加软骨�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),10μg/L interleukin-1β(IL-1β),10μg/L IL-1β+10%Bushen Tiaogan Prescription medicated serum(BSTG),and 5μmol/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-1βgroup.Compared with the DMSO group,the number of autophagosomes was significantly increased in the rapamycin group.Compared with the IL-1βgroup,the number autophagosomes was significantly increased in the 10μg/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-1βgroup(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 decr

关 键 词：自噬 骨关节炎 补肾调肝方 关节软骨 AKT/MTOR 

分 类 号：R459.9[医药卫生—治疗学] R684.3[医药卫生—临床医学]