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目的观察单味中药鹿茸对大鼠骨关节炎(osteoarthritis,OA)软骨靶器官Smad2、3表达的影响。方法选择3个月龄健康雌性SD大鼠100只,体质量(200±20)g。常规喂养1周后,随机数字表法分成5组:鹿茸低剂量组、鹿茸高剂量组、盐水组、模型组及正常组,每组20只。除正常组外采用经典Hulth方法造模,造模后6周,行病理学观察,确认造模成功,鹿茸低剂量组按0.021 g/100 g给药,鹿茸高剂量组按0.084g/100 g给药,盐水组每天灌服相应的生理盐水,正常组及模型组不予任何处理。于灌胃后2、4、6周,分别取大鼠双侧膝关节软骨,采用免疫组织化学、荧光定量-PCR和蛋白免疫印迹杂交方法分别检测Smad2、3 mRNA和蛋白表达。结果病理观察可见成功制备OA模型,免疫组织化学可见Smad2、3蛋白在软骨层广泛表达,且定位于软骨细胞膜内。与模型组比较,鹿茸低、高剂量组在灌胃后2、4、6周Smad2、3 mRNA表达量明显升高,差异有统计学意义(P<0.05);与同组灌胃后4周比较,灌胃后6周,鹿茸高、低剂量组的Smad2、3mRNA表达量有所下降,差异有统计学意义(P<0.05)。与模型组比较,鹿茸低、高剂量组Smad2、3蛋白在灌胃后2、4周时,其在软骨细胞中的表达量有明显的升高,差异有统计学意义(P<0.01);与同组灌胃后2周比较,鹿茸低、高剂量组灌胃后4周升高更显著,差异有统计学意义(P<0.01);与同组灌胃后4周比较,灌胃后6周时鹿茸低、高剂量组Smad2、3蛋白表达降低,差异有统计学意义(P<0.01)。结论 (1)鹿茸通过调控软骨细胞内Smad2、3基因和蛋白的表达而起到修复软骨的作用。(2)软骨细胞内的Smad2、3基因表达及蛋白水平的上调可能是OA发病的重要机制之一。
Objective To observe the effect of single Chinese herb antler on the expression of Smad2, 3 in the target organ of cartilage of osteoarthritis rats. Methods 100 healthy female SD rats of 3 months old were selected and their body weight was (200 ± 20) g. One week after routine feeding, the random number table was divided into five groups: low antler group, high antler group, saline group, model group and normal group, with 20 rats in each group. In addition to the normal group, the classic Hulth method was used to establish the model. After 6 weeks of model establishment, pathological observation was performed to confirm the successful model establishment. The low-dose pilose antler group was given at 0.021 g / 100 g and the high-dose pilose antler group at 0.084 g / 100 g The rats in the saline and saline groups were given the corresponding saline daily, without any treatment in the normal group and the model group. At 2, 4, and 6 weeks after gavage, rat bilateral articular cartilage were taken respectively and the expression of Smad2,3 mRNA and protein were detected by immunohistochemical staining, fluorescence quantitative PCR and Western blotting. Results The pathological observation showed that the OA model was successfully prepared. Immunohistochemistry showed that the Smad2,3 protein was widely expressed in the cartilage layer and localized in the chondrocyte membrane. Compared with the model group, the expression of Smad2 and 3 mRNA in the antler low and high dose groups were significantly increased at 2, 4, and 6 weeks after gavage (P <0.05). Compared with the model group, After 6 weeks of gavage, the expression of Smad2 and 3 mRNA in high and low antler groups decreased, with statistical significance (P <0.05). Compared with the model group, the expression of Smad2 and 3 in the pilose antlers low and high dose groups significantly increased in the chondrocytes at 2 and 4 weeks after gavage, the difference was statistically significant (P <0.01). Compared with the same group after 2 weeks of gavage, the antler low and high dose groups increased more significantly at 4 weeks after gavage, the difference was statistically significant (P <0.01) At 6 weeks, the expression of Smad2, 3 in pilose antlers low and high dose groups decreased, with statistical significance (P <0.01). Conclusion (1) Pilose antlers play a role in repair of cartilage by regulating the expression of Smad2, 3 gene and protein in chondrocytes. (2) The expression of Smad2 and 3 in chondrocytes may be one of the important mechanisms in the pathogenesis of OA.