巴西苏木素对骨关节炎模型中软骨保护作用及内质网应激通路变化的实验研究

Experimental study on the protective effect of brazilin on cartilage and the changes of endoplasmic reticulum stress pathway in osteoarthritis model

目的研究巴西苏木素(Brazilin)对IL-1β诱导的骨关节炎(OA)细胞模型中的软骨细胞保护作用及内质网应激通路(ERS)的变化。方法甲苯胺蓝对软骨细胞进行鉴定,CCK8检测Brazilin对正常软骨细胞增殖的影响;实验分组为正常软骨细胞组(Control组),其余组均用IL-1β(10 ng/mL)处理48 h建立OA体外模型,分为模型组(IL-1β组)、Brazilin低剂量组、Brazilin中剂量组、Brazilin高剂量组。显微镜拍照观察各组细胞形态变化,qRT-PCR和Western blot分别检测GRP78、CHOP、Collagen TypeⅡ、MMP-13的基因和蛋白的表达。结果本实验获得纯度较高的大鼠乳鼠原代软骨细胞;Brazilin在小于10μg/mL的浓度对细胞的增殖无影响;IL-1β诱导的OA模型中,IL-1β组较多细胞发生皱缩、染色质固缩、细胞质和细胞核裂解碎片现象,提示细胞凋亡的发生,Brazilin可以降低IL-1β诱导的凋亡发生,且成剂量依赖;MMP-13的表达上调,Collagen TypeⅡ的表达下调,导致了软骨细胞的损伤,Brazilin处理后有效地逆转了相关因子的表达和激活(P均<0.05);IL-1β组ERS通路标志分子GRP78表达水平下调、CHOP表达水平上调,提示IL-1β诱导的软骨损伤机制可能与ERS通路有关,而Brazilin处理后通过促进GRP78的表达,抑制CHOP的表达,在软骨细胞修复中发挥作用(P均<0.05)。结论Brazilin改善IL-1β诱导的骨关节炎细胞模型中软骨损伤,其机制可能与激活ERS通路有关。

Objective To investigate the protective effect of Brazilian hematoxylin(Brazilin)on chondrocytes and changes in the endoplasmic reticulum stress(ERS)pathway in a cellular model of IL-1β-induced osteoarthritis(OA).Methods Toluidine blue was used to identify chondrocytes,and CCK8 was used to detect the effect of Brazilin on the proliferation of normal chondrocytes.The experimental groups were divided into the normal chondrocytes group(Control group),and the other groups were treated with IL-1β(10 ng/ml)for 48 h to establish an in vitro model of OA.The latter can be divided into model group(IL-1βgroup),the low-dose Brazilin group,the medium-dose Brazilin group,and the high-dose Brazilin group.Both were treated with the following experiments:the morphological changes of cells in each group were observed by microscope,and the gene and protein expressions of GRP78,CHOP,Collagen TypeⅡand MMP-13 were detected by qRT-PCR and Western blot,respectively.Results In this experiment,primary chondrocytes of neonatal rats with high purity were obtained.Brazilin had no effect on cell proliferation at concentrations less than 10μg/ml.In the IL-1β-induced OA model,more cells in the IL-1βgroup showed shrinkage,chromatin fixation,cytoplasmic and nuclear fragmentation,suggesting the occurrence of apoptosis.Brazilin can reduce the occurrence of IL-1β-induced apoptosis in a dose-dependent manner.The expression of MMP-13 was upregulated and the expression of Collagen TypeⅡwas downregulated,leading to chondrocyte damage,and the expression and activation of related factors were effectively reversed by Brazilin treatment.The decreased expression levels of GRP78 and increased expression of CHOP in IL-1βgroup,suggesting that IL-1β-induced cartilage injury mechanism may be related to the ERS pathway.While Brazilin treatment played a role in chondrocyte repair by promoting the expression of GRP78 and inhibiting the expression of CHOP.Conclusion Brazilin ameliorates cartilage damage in an IL-1β-induced osteoarthritis cell model by a mechanism that may be related to activation of the ERS pathway.