骨炎消巴布剂对膝骨关节炎家兔软骨细胞凋亡及Bcl-2、Bax表达的影响

Effects of Guyanxiao cataplasm on chondrocyte apoptosis and Bcl-2 and Bax expressions in rabbits with knee osteoarthritis

目的:研究骨炎消巴布剂对膝骨关节炎(KOA)家兔的疗效并探究其相关分子机制。方法:50只家兔随机分为正常组、模型组、巴布剂基质组、奇正消痛贴组、骨炎消巴布剂组,每组10只。除正常组外,其余各组膝关节注射碘乙酸钠制备兔KOA模型,2周造模成功后,模型组不做任何处理,其余各组分别给予巴布剂基质、奇正消痛贴、骨炎消巴布剂外敷。治疗6周后,HE染色观察各组家兔膝关节软骨组织病理变化,TUNEL染色观察各组家兔膝骨关节中软骨细胞凋亡情况,ELISA检测各组家兔滑膜液中IL-1β、IL-6、IL-17、IL-18和TNF-α的分泌水平,实时荧光定量PCR及Western blot检测软骨组织中Bcl-2和Bax mRNA及蛋白表达水平。结果:与正常组相比,模型组软骨组织结构紊乱,大量炎症细胞渗出,巴布剂基质组各层分界不清,细胞排列稍紊乱,细胞出现增生,模型组和巴布剂基质组软骨细胞凋亡率显著升高(P<0.01),滑膜液中IL-1β、IL-6、IL-17、IL-18和TNF-α水平均显著升高(P<0.01),软骨组织Bcl-2 mRNA与蛋白表达显著降低(P<0.01),Bax mRNA及蛋白表达升高(P<0.05)。与模型组相比,骨炎消巴布剂组和奇正消痛贴组软骨细胞形态较规则,胞核呈椭圆形,染色分布均匀,骨炎消巴布剂组和奇正消骨痛贴组软骨细胞凋亡率显著降低(P<0.01),滑膜液中IL-1β、IL-6、IL-17、IL-18和TNF-α表达水平显著下降(P<0.01),软骨组织Bcl-2 mRNA及蛋白表达水平显著上调(P<0.01),Bax mRNA及蛋白表达水平显著降低(P<0.01)。结论:骨炎消巴布剂可抑制炎症因子分泌,促进Bcl-2表达,降低Bax水平,抑制软骨细胞凋亡,保护软骨细胞,进而延缓KOA的病情进展。

Objective:To investigate the effect of Guyanxiao cataplasm on rabbits with knee osteoarthritis(KOA)and explore its related molecular mechanisms.Methods:A total of 50 rabbits were randomly divided into normal group,model group,Cataplasm base group,Qizhengxiaotong Plaster group and Guyanxiao cataplasm group,with 10 rabbits in each group.Except for normal group,knee joints of other groups were injected with sodium iodoacetate to create a rabbit KOA model.After two weeks of successful model building,the model group did not do any treatment,other groups were given topical Cataplasm base,Qizhengxiaotong Plaster group and Guyanxiao for external application.After 6 weeks of treatment,HE staining was used to observe pathological changes of cartilage tissues of knee joints of rabbits in each group,TUNEL staining was used to observe apoptosis of chondrocytes in knee joints of rabbits in each group,and ELISA was used to detect secretion levels of IL-1β,IL-6,IL-17,IL-18 and TNF-αin synovial fluid of rabbits in each group,expression levels of Bcl-2 and Bax mRNA and protein in cartilage tissues were determined by quantitative real-time PCR and Western blot.Results:Compared with normal group,cartilage tissue structure of model group was disordered,a large number of inflammatory cells were exuded,boundaries of the various layers of Cataplasm matrix group were unclear,cell arrangement was slightly disordered,and the cells were proliferating,apoptosis rate of chondrocytes in model group and Cataplasm matrix group was significantly increased(P<0.01),levels of IL-1β,IL-6,IL-17,IL-18 and TNF-αin synovial fluid were increased significantly(P<0.01),and expression of Bcl-2 mRNA and protein in cartilage tissue were significantly reduced(P<0.01),while Bax mRNA and protein expression were increased(P<0.05).Compared with model group,morphology of chondrocytes in Guyanxiao cataplasm group and Qizhengxiaotong Plaster group were relatively regular,the nucleus is oval,and the staining was evenly distributed,apoptosis rate of chondrocytes in Guyanxiao cataplasm group and Qizhengxiaotong Plaster group was significantly decreased(P<0.01),expression levels of IL-1β,IL-6,IL-17,IL-18 and TNF-αin synovial fluid were decreased significantly(P<0.01),and expression of Bcl-2 mRNA and protein in cartilage tissue were significantly up-regulated(P<0.01),while expression of Bax mRNA and protein were significantly down-regulated(P<0.01).Conclusion:Guyanxiao cataplasm can inhibit secretion of inflammatory factors,promoting expression of Bcl-2,reducing activation of Bax,inhibiting the occurrence of chondrocyte apoptosis,protecting cartilage cells,and can delay the progression of KOA.