苍膝通痹胶囊调控circRNA_0008365/miR-1271/p38 MAPK通路轴促进膝骨关节炎软骨细胞自噬的机制研究

Cangxi Tongbi Capsules promote chondrocyte autophagy by regulating circRNA_0008365/miR-1271/p38 MAPK pathway to inhibit development of knee osteoarthritis

该研究旨在探讨苍膝通痹胶囊通过调控circRNA_0008365/miR-1271/p38 MAPK通路轴促进软骨细胞自噬抑制膝骨关节炎(KOA)进展的机制。于体内、体外分别构建软骨细胞和大鼠的骨关节炎模型,分别给予苍膝通痹胶囊、circRNA_0008365干扰、空载对照、以及苍膝通痹胶囊合用circRNA_0008365干扰进行干预。应用流式细胞术观察细胞凋亡,透射电镜观察自噬小体,Western blot观察各组软骨细胞自噬指标微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)Ⅱ/Ⅰ、自噬效应蛋白-1(Beclin-1)、选择性自噬接头蛋白p62/sequestosome 1(selective autophagy junction protein p62/sequestosome 1,p62)、软骨细胞外基质代谢(collagenⅡ、ADAMTS-5)及p-p38 MAPK的蛋白表达,qRT-PCR观察各组软骨细胞circRNA_0008365、miR-1271、collagenⅡ、ADAMTS-5的表达水平,hematoxylin-eosin(HE)染色观察各组大鼠膝关节软骨组织形态学的改变,ELISA观察各组软骨细胞炎性因子(IL-1β、TNF-α)的表达变化。体外实验结果显示,IL-1β诱导的软骨细胞circRNA_0008365表达量下降,miR-1271、p38 MAPK表达量上升,自噬水平下降而凋亡率上升,软骨外基质的分解代谢增加;苍膝通痹胶囊的干预使KOA软骨细胞的circRNA_0008365表达量回升,miR-1271、p38 MAPK表达量下降,自噬水平升高而凋亡率降低,软骨外基质的分解代谢减弱;而干扰circRNA_0008365后,抑制了苍膝通痹胶囊的干预趋势。体内实验提示,苍膝通痹胶囊剂量依赖性的抑制了p38 MAPK通路的表达,增强了软骨细胞自噬,降低了KOA大鼠关节软骨的损害和炎症反应,从而抑制了KOA的进展。该研究表明,苍膝通痹胶囊能通过调控circRNA_0008365/miR-1271/p38 MAPK通路轴促进软骨细胞自噬,抑制KOA的发展。

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA)progression by regulating the circRNA_0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK)pathway.The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules,si-circRNA_0008365,si-NC,and Cangxi Tongbi Capsules combined with si-circRNA_0008365.Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes,respectively.Western blotting was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3Ⅱ/Ⅰ),beclin-1,selective autophagy junction protein p62/sequestosome 1,collagenⅡ,a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5),and p38 MAPK.The mRNA levels of circRNA_0008365,miR-1271,collagenⅡ,and ADAMTS-5 were determined by qRT-PCR.Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee,and enzyme-linked immunosorbent assay to measure the levels of interleukin-1β(IL-1β)and tumor necrosis factor-alpha(TNF-α).The chondrocytes treated with IL-1βshowed down-regulated expression of circRNA_0008365,up-regulated expression of miR-1271 and p38 MAPK,lowered autophagy level,increased apoptosis rate,and accelerated catabolism of extracellular matrix.The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA_0008365,down-regulated the expression of miR-1271 and p38 MAPK,increased the autophagy level,decreased the apoptosis rate,and weakened the catabolism of extracellular matrix.However,the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA_0008365.The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway,enhanced chondrocyte autophagy,and mitigated articular cartilage damage and inflammatory response,thereby inhibiting the progression of KOA in rats.This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA_0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.