七厘散治疗兔膝骨关节炎及其相关分子机制研究

Experimental study on Qili San in treatment of knee osteoarthritis and its related molecular mechanism

目的:探讨七厘散对膝骨关节炎(KOA)兔的治疗作用及与Wnt/β-catenin信号通路的关系。方法:将清洁级新西兰兔20只随机分为对照组和七厘散组(n=10),雌雄各取5只。七厘散组采取2%木瓜蛋白酶溶液关节腔注射及复合强迫屈曲位法构建兔膝关节骨性关节炎(KOA)模型;造模结束后七厘散组兔进行灌胃七厘散(0.25 g/kg),对照组兔给予相同体积的生理盐水,连续14 d。治疗前后对所有兔行右后肢X线拍摄,采用免疫组化法检测家兔软骨组织中Wnt4a的表达情况;取5只幼龄SD大鼠骨膜通过密度梯度离心法分离纯化制备骨膜间质干细胞(BMSC),再取另外5只灌胃七厘散后6 h取血制备含药血清,制备所得的七厘散含药血清用于给药。实验分为正常组,七厘散血清组,DKK-1组(0.1μg/kg),DKK-1组(0.2μg/kg);在第1、2、3、7、14天时,通过MTT法检测细胞增殖情况,利用RT-PCR法检测Runx2,Osteocalcin,β-catenin,Wnt4a基因的表达。结果:七厘散治疗后,七厘散组兔膝关节表面恢复光滑,少数软骨下骨出行高密度影;七厘散含药血清组促进了BMSCs增殖,增加Runx2、Osteocalcin、β-catenin,Wnt4a基因的表达;免疫组织化学染色观察显示,与治疗前相比,七厘散治疗后Wnt4a表达量明显增加。结论:七厘散能够通过调控Wnt/β-catenin信号通路促进骨髓间质干细胞的增殖从而改善兔膝骨关节炎。

Objective:To investigated the effect of Qili San in the treatment of knee osteoarthritis in rabbits and its relationship with Wnt/β-catenin signal pathway.Methods:20 New Zealand rabbits were randomly divided into two groups,the control group and the Qili San group(n=10),half male and female.Combined the 2%papain was injected into the right knees with forced flexion method,was used to build the KOA model in rabbits.After modeling,the Qili San group was given Qili San by gavage(0.25 g/kg),and the control group was given the same volume of normal saline for 14 consecutive days.X-ray images of the right hind limb of all rabbits were taken before and after treatment.After last medication,blood was taken to prepare Qili San Containing serum.The expression of Wnt4a in the cartilage tissue of the rabbits was detected by immunohistochemistry.Periosteal mesenchymal stem cells(BMSCS)were isolated and purified from the periosteum of young SD rats by density gradient centrifugation.The experiment was divided into normal group,drug-containing serum group,DKK-1 group(0.1μg/kg),DKK-1 group(0.2μg/kg).At day 1,2,3,7,14,cell proliferation was detected by MTT assay,and the expressions of Runx2,Osteocalcin,-catenin,Wnt4a,genes were detected by RT-PCR assay.Results:The X-ray showed that the surface of the knee joint of the Qili San group was restored to be smooth,and a few subchondral bones had high density shadow after the treatment of Qili San.The Qili San containing serum promoted BMSC proliferation and the expression of Runx2,Osteocalcin,β-catenin,Wnt4a and Wnt7 genes.Immunohistochemical staining showed that Wnt4a expression was significantly increased after Qili San treatment,compared with the group before treatment.Conclusion:Qili San can promote the proliferation of bone marrow mesenchymal stem cells to improving the osteoarthritis of rabbit knee.by regulating the Wnt/β-catenin signal pathway.