基于PI3K/Akt信号通路探讨活血通络法对膝骨关节炎小鼠细胞凋亡和相关蛋白水平表达的影响

Exploring the Effect of Activating Blood Circulation Method on Cell Apoptosis and Related Protein in KOA Mice Based on PI3K/Akt Signaling Pathway

目的观察以活血通络为治法的骨痹合剂对膝骨关节炎(KOA)模型小鼠关节软骨细胞凋亡、Bcl-2相关凋亡启动子(Bad)、天冬氨酸特异性半胱氨酸蛋白水解酶3和9(Caspase-3和Caspase-9)、核转录因子-κB(NF-κB)的影响。方法KOA小鼠模型由Hulth法制备,造模成功后分为3组,分别予骨痹合剂、氨糖美辛溶于等体积的蒸馏水和等体积生理盐水灌胃,每日2次,持续灌胃8周。苏木素-伊红染色观察关节软骨组织病理形态变化;免疫组化检测关节软骨组织Bad、Caspase-3、Caspase-9、NF-κB蛋白表达;实时荧光定量RT-PCR技术检测关节软骨组织Bad、Caspase-3、Caspase-9、NF-κB mRNA的表达。结果生理盐水组中软骨表面可见缺损且软骨细胞数量明显减少,软骨表面粗糙;与生理盐水组比,骨痹合剂组和氨糖美辛组软骨组织有明显改善,软骨表面细微裂隙减少,软骨细胞数目增加。生理盐水组中软骨表面Bad、Caspase-3、Caspase-9、NF-κB蛋白mRNA均高表达;与生理盐水组比,骨痹合剂组Bad、Caspase-3、Caspase-9、NF-κB蛋白和mRNA表达下降(P<0.05或P<0.01)。结论骨痹合剂通过下调软骨组织表面Bad、Caspase-3、Caspase-9、NF-κB蛋白和基因表达,从而有效延缓KOA小鼠关节软骨退变病理进程。

Objective To observe the effects of GuBi mixture on chondrocytes apoptosis,Bcl-2 associated apoptosis promoter(Bad),aspartic acid-specific cysteine proteolytic enzymes 3 and 9(caspase-3 and caspase-9),and nuclear transcription factor-κB(NF-κB)in KOA model mice.Methods KOA mouse model was prepared by Hulth method.After successful preparation of the model,KOA mouse were divided into three group,respectively used GuBii mixture、Osaminethacine dissolved in same volum of distilled water to full the stomach,and normal saline(NS)group used equal volum of NS to full the stomach,twice daily,continue 8weeks.The pathological changes of articular cartilage were observed by hematoxylin-Eosin(HE)staining.Immunohistochemistry(IHC)was used to detect the expression of Bad,Caspase-3,Caspase-9 and NF-κB proteins in articular cartilage.The expressions of Bad,Caspase-3,Caspase-9 and NF-κB mRNA in articular cartilage were detected by real-time fluorescence quantitative RT-PCR.Results In the saline group,cartilage defects were observed and the number of chondrocytes decreased significantly.Compared with the normal saline group,the cartilage tissue of GuBi mixture and Osaminethacine group was significantly improved,and the number of chondrocytes on the surface was increased.The expressions of Bad,Caspase-3,Caspase-9,NF-κB protein and mRNA on the cartilage surface in the saline group were all high.Compared with normal saline group,the expression of Bad,Caspase-3,Caspase-9,NF-κB protein and mRNA in GuBi group were decreased(P<0.05 or P<0.01).Conclusion By down-regulating the expression of Bad,Caspase-3,Caspase-9,NF-κB protein and genes on the surface of cartilage tissue,GuBi mixture can effectively inhibit the articular cartilage of KOA mice and delay the pathological process of articular cartilage degeneration.