骨灵膏及其拆方制剂对骨关节炎大鼠关节软骨Wnt/β-catenin信号通路的影响

Influence of Guling Gao and its disassembled prescriptions for Wnt/β-catenin signal pathway of osteoarthritis rat

目的:通过骨灵膏(GLG)及其拆方制剂对骨关节炎关节软骨组织Wnt/β-catenin信号通路作用的研究,探讨GLG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组、骨膏(GUG)组、灵膏(LG)组、塞来昔布组、模型组及正常组6组,每组10只,除正常组外其余各组采用冷固法(寒冷刺激结合石膏固定的方法)6周建立骨关节炎模型,造模成功后除正常组和模型组分别给予0.9%氯化钠溶液灌胃外,其余组分别给予GLG、GUG、LG及塞来昔布灌胃,给药10周后,取大鼠膝关节软骨组织,采用免疫组化检测软骨组织Wnt-2、β-catenin的含量,采用Western Blot法检测LRP-5的表达,通过RT-PCR检测软骨细胞BMP-2、MMP-13、Caspase-9、Caspase-3 mRNA表达。结果:GLG可以减轻OA软骨组织浅表至钙化层各层的病理损伤;GLG及其拆方制剂可以明显下调Wnt2、β-catenin的阳性表达,其中GLG与GUG、LG比较β-catenin阳性率明显下降(P<0.01)。Western Blot结果显示GLG组LRP-5蛋白表达较其他组显著降低;FQ-RT-PCR实验结果表明在骨灵膏组BMP-2 mRNA的表达较GUG组明显增加(P<0.01),而与LG组比较明显降低(P<0.01),与正常组比较无差异(P<0.05);GLG及其拆方制剂组BMP-2、MMP-13、Caspase-3、Caspase-9 mRNA的表达较模型组显著降低(P<0.01),GLG较GUG、LG及塞来昔布比较MMP-13、Caspase-3、Caspase-9 mRNA的表达明显降低(P<0.01)。结论:GLG可能通过平衡调节OA中BMP-2 mRNA的表达,下调Wnt/β-catenin信号通路依赖的LRP-5跨膜蛋白的含量,阻止Wnt/β-catenin信号通路的过度抑制和异常激活,进而下调MMP-13、Caspase-9、Caspase-3 mRNA的表达起到减轻其对软骨ECM的降解及软骨细胞的异常凋亡作用,阻止了OA关节软骨进行性退变,GLG的上述作用优于其拆方制剂GUG和LG及塞来昔布。

Objective： Through studying on the effects of Guling Gao（GLG） and its disassembled prescriptions on Wnt/β-catenin signal pathway in articular cartilage tissue of osteoarthritis rat, to discuss the mechanism underlying the GLG and its disassembled prescriptions in the treatment of osteoarthritis. Methods： A total of 60 famale SD rats were randomly divided into GLG group, Gu Gao（GUG） group, Ling Gao（LG） group, celecoxib group, model group and normal group, with 10 rats in each group. All groups except for the normal group were given a cold solid method（cold stimulation combined with method of gypsum fixed） for 6 weeks to establish an osteoarthritis model. After modeling, the model group and normal group were given normal saline by gavage, and GLG group, GUG group, LG group and celecoxib group given corresponding durgs by gavage, respectively. After 10 weeks of durg administration, all rats were sacrificed to remove their knee cartilage tissue. The content of Wnt-2 and β-catenin in cartilage tissue were detected by immunohistochemistry; the protein expression of LRP-5 were detected by Western blot; the mRNA expression of BMP-2, MMP-13, Caspase-9 and Caspase-3 in cartilage cells were detected by RT-PCR. Results： GLG could reduce the pathological damage in each layer of cartilage tissue, from superficial layer to the calcified one. GLG and its disassembled prescriptions could down-regulate the positive expression of Wnt-2, β-catenin, in which the positive rate of β-catenin in the GLG group was decreased significantly when compared with the LG group and GUG group（P〈0.01）. Western blot result showed that the protein expression of LRP-5 in the GLG group was higher than that in the other groups. RT-PCR results showed that mRNA expression of BMP-2 in the GLG group was higher than that in the GUG group, but lower than the LG group（P〈0.01） and equal to the normal group（P〉0.05）; when compared with the model group, the mRNA expression of BMP-2, MMP-13, Caspase-3 and Caspase-9 in the GLG group and its disassembled prescription groups was decreased significantly（P〈0.01）; when compared with the GLG group, the mRNA expression of MMP-13, Caspase-3 and Caspase-9 in the GUG group, LG group and celecoxib group were increased significantly（P〈0.01）. Conclusions： Through a regulation of mRNA expression of BMP-2, GLG could down-regulate the expression of LRP-5, a Wnt/β-catenin signal pathway dependence-transmembrane protein, inhibiting excessive suppression and abnormal activation of Wnt/β-catenin signal pathway. Meanwhile, through a down-regulation of mRNA expression of MMP-13, Caspase-9 and Caspase-3, GLG could reduce the abnormal apoptosis of chondrocytes and degradation of cartilage ECM, stoping the degeneration of Osteoarthritis articular cartilag. These actions of GLG were superior to its disassembled prescriptions and celecoxib.