鹿茸调控骨关节炎大鼠软骨Smads基因/蛋白表达水平的作用研究

Effect of Pilose Antler on Expressions of Smads Gene/Protein in Osteoarthritis Rats Cartilage

目的：研究鹿茸对骨性关节炎软骨组织中Smad2、3、6、7基因和蛋白表达的影响。方法：选择3月龄健康雌性SD大鼠50只,体质量（200±20）g,随机分为5组（空白对照组、模型对照组、鹿茸灌喂2周组、鹿茸灌喂4周组、鹿茸灌喂6周组）,每组10只。常规喂养1周后,除空白对照组外均采用经典Hulth方法制备骨关节炎疾病模型,造模4周后按分组不同给予鹿茸灌喂2、4、6周,空白对照组、模型对照组予等量生理盐水灌喂6周,末次灌喂2 h后取各组大鼠双侧膝关节软骨,采用组织病理HE染色检测关节软骨组织病理改变情况,采用荧光定量-PCR和蛋白免疫印迹杂交方法分别检测Smad2、3、6、7 mRNA和蛋白表达。结果：病理观察可见模型对照组大鼠关节软骨结构紊乱、软骨表面溃烂缺损,细胞数量弥漫性增多,呈团簇状杂乱分布,而鹿茸灌喂组关节软骨的病理改变介于空白对照组与模型对照组之间,随着灌喂时间的推移,软骨结构破坏情况逐渐改善。Smad2、3、6、7mRNA和蛋白检测可见：模型对照组Smad2、3的表达量明显降低,Smad6、7的表达量明显升高;鹿茸灌喂组Smad2、3的表达量明显升高,Smad6、7的表达量明显降低。结论：①软骨细胞内的smad2、3、6、7基因及蛋白的异常表达可能是骨性关节炎发病的重要机制之一;②鹿茸通过调控软骨细胞内Smad2、3、6、7基因及蛋白的表达促进了关节软骨的修复,从而对OA大鼠起到治疗作用;③在鹿茸调控软骨细胞内Smads家族基因和蛋白表达的过程中,除TGF-β/Smad信号通路外可能还存在其他非Smad信号通路调控机制发挥作用。

Objective：To explore the effects of Pilose Antler on the expressions of Smad2/3/6/7 mRNA and protein in osteoarthritis cartilage tissue.Methods： Fifty 3-month old female healthy SD rats,weighted（200±20）g,were recruited and routinely fed for 1 week.They were randomly divided into 5 groups,i.e.the blank control group,the model control group,the PA 2 weeks group,the PA 4 weeks group,the PA 6 weeks group,10 in each group.After 1 week of routine feeding, the classic Hulth method was used to prepare the disease model of osteoarthritis except the blank control group. After 4 weeks modeling,the PA feeding group＇s rats were fed with Pilose Antler for 2,4,6 weeks according to different time pointsand the blank control group and the model control group were given the same amount of saline for 6 weeks.The bilateral knee joint cartilage was taken out from rats in each group after the last irrigation of 2 h. The pathological changes of articular cartilage were detected by histopathological HE stainingand the expressions of Smad2, 3, 6, 7 mRNA and protein were detected by fluorescence quantitative-PCR and Western blot hybridization.Results：Pathological result showed that the articular cartilage of the rats in the model control group was disordered and the cartilage surface was ulcerated. The number of cells increased in a diffuse, cluster and disorder distribution.The pathological conditions of articular cartilage in pilose antler feeding groups were between those in the blank control group and the model control groupand the damage of cartilage structure improved gradually according to the feeding time.The expressions of Smad2/3/6/7 mRNA and protein were the expressions of Smad2/3 in the model control group decreased significantlyand the expressions of Smad6/7 increased significantlywhile the expressions of Smad2/3 in the pilose antler feeding groups increased significantlyand the expression of Smad6/7 decreased significantly. Conclusions：①The abnormal expressions of Smad2/3/6/7 mRNA and protein in chondrocytes may be one of the important mechanisms of osteoarthritis.②Pilose antler promotes the repair of articular cartilage and treats the OA rats by regulating the expressions of Smad2, 3, 6, 7 genes and proteins in chondrocytes.③When pilose antler regulates the expressions of Smads gene and proteins in chondrocytes and there may be other non Smad signaling pathways mechanism besides the TGF-β/Smad signaling pathway.