骨灵膏及其拆方制剂对骨关节炎软骨细胞凋亡及胞外基质降解的影响

目的：通过骨灵膏及其拆方制剂对骨关节炎（OA）软骨细胞凋亡及胞外基质降解作用的研究，探讨骨灵膏及其拆方制剂对骨关节炎的作用机制。方法：选用60只雌性SD大鼠随机分为骨灵膏、骨膏、灵膏、塞来昔布、模型及正常组6组，每组10只，采用冷固法建立骨关节炎模型，造模成功后，骨灵膏、骨膏、灵膏均按10.41 mL·kg^-1·d^-1（相当于生药量33.31 g·kg^-1）灌胃，塞来昔布20.83 mg·kg^-1·d^-1灌胃，正常、模型组给予等量生理盐水灌胃，给药10周后，取大鼠膝关节软骨组织，分别以苏木精-伊红（HE）、番红-固绿（SA/FG）、番红-阿尔辛蓝（SA/AB）进行组织病理观察，根据改良的Mankin关节软骨病理评分标准进行分级评分，采用Western blot法检测基因153（CHOP/GADD153）蛋白的表达，通过荧光定量PCR检测软骨细胞B细胞淋巴瘤/白血病2（Bcl-2），Bcl-2相关X蛋白（Bax），基质金属蛋白酶-3（MMP-3），基质金属蛋白酶组织抑制剂-1（TIMP-1）mRNA表达。结果：与正常组比较模型组能显著减低Bcl-2，TIMP-1 mRNA表达（P<0.01）及增加Bax，MMP-3 mRNA表达（P<0.01）。与模型组相比骨灵膏能明显下调ERS相关CHOP/GADD153蛋白的生成（P<0.01）、促进抑凋亡基因Bcl-2mRNA表达及降低促凋亡基因BaxmRNA表达（P<0.01），进而上调Bcl-2/bax，抑制软骨细胞的异常凋亡；同时，骨灵膏较模型组能显著降低MMP-3 mRNA的表达（P<0.01）、升高TIMP-1 mRNA的表达（P<0.01），降低MMP-3 mRNA/TIMP-1 mRNA。结论：骨灵膏可能通过降低ERS途径相关蛋白GADD153的异常生成，进一步调控相关凋亡靶基因的表达而抑制软骨细胞的异常凋亡和阻止细胞外基质异常降解而阻止OA关节软骨退行性变，保护了关节软骨，其作用明显优于其拆方制剂骨膏、灵膏。

Influence of Guling Grease and Dismantlement Preparations on Extracellular Matrix Degradation and Chondrocyte Apoptosis on Osteoarthritis

Objective: To study the effects of Guling grease (GLG) and dismantlement preparations on extracellular matrix degradation and chondrocyte apoptosis on osteoarthritis(OA),and to discuss the mechanism. Method: A total of 60 famale SD rats randomly were divided into normal group(NG) and GLG group, Gugao group(GUG), Linggao group(LG),celecoxib(CLXB) group, model group(M),10 rats in each group osteoarthritis model was established using cold-solid method,after the modeling,LG,GUG,GLG were given according to the dosage of 10.41 mL·kg^-1·d^-1(equivalent to dosage 33.31 g·kg^-1·d^-1),CLXB was given celecoxib 20.83 mg·kg^-1·d^-1. After 10 weeks,the rat knee cartilage tissue was collected, the staining of Hematoxylin-eosin(HE),safranin-fast green(SA/FG), safranin-alcian blue(SA/AB) were used for histopathological observation, according to the improvement Mankin articular cartilage pathology score standard conducted classification score,C/EBP homologous protein/growth arrest and DNA damage inducible gene 153,(CHOP/GADD153) protein expression were detected by western blot method,the fluorescence quantitative real time PCR was used to detect the expression of B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax),matrix metallo-proteinase-3(MMP-3),tissue inhibitor of metalloproteinase-1(TIMP-1)mRNA on cartilage cells. Result: Compared with the M group GLG group down-regulated endoplasmic reticulum stress(ERS) associated CHOP/GADD153 protein generation obviously(P<0.01),promoted the expression of Bcl-2mRNA,reduced the expression of apoptosis gene BaxmRNA(P<0.01),then raised the ratio of Bcl-2/Bax;At the same time,compared with M group GLG group reduced the expression of MMP-3 mRNA significantly(P<0.01),increased the expression of TIMP-1 mRNA(P<0.01),reduced the ratio of MMP-3 mRNA/TIMP-1 mRNA.Compared with NG M group reduced the expression of Bcl-2,TIMP-1 mRNA (P<0.01)and increased the expression of Bax, MMP-3 mRNA significantly (P<0.01). Conclusion: GLG could regulate GADD153 abnormal formation, further regulate the expression of apoptosis-related target genes and inhibit cartilage cells abnormal apoptosis and block extracellular matrix abnormal degradation,then inhibit OA articular cartilage degeneration, protect articular cartilage,its function is superior than the dismantlement preparatio.