基于Wnt/β-catenin通路探讨尪痹片促进去势后胶原诱导性关节炎大鼠骨形成的机制

Mechanism of Wang-bi Tablet Promoting Bone Formation in Ovariectomized Collageninduced Arthritis Rats Based on Wnt/β-catenin Pathway

目的基于果蝇无翅基因/β-连环蛋白(Wnt/β-catenin)信号通路,研究尪痹片(WBT)干预去势后胶原诱导性关节炎(CIA)大鼠的骨形成机制。方法48只雌性SD大鼠经随机数字表法分为空白对照组、去势组、CIA组、去势CIA组、去势+WBT组、CIA+WBT组、CIA+甲氨蝶呤(MTX)组、去势CIA+WBT组,每组6只。去势法制备肾虚证模型,CIA法制备类风湿关节炎模型,WBT采用每日0.63 g/kg灌胃,MTX采用每周1.05 mg/kg结合生理盐水灌胃,其余组均予生理盐水灌胃,56日后取材。酶联免疫吸附试验(ELISA)检测血清环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)水平,蛋白质免疫印迹(Western Blot)检测Wnt3a、Wnt10b、β-catenin、糖原合成酶激酶-3β(GSK-3β)、Dickkopf相关蛋白-1(DKK-1)、Runt相关转录核心因子2(Runx2)、碱性磷酸酶(ALP)、核酸内切酶Dicer蛋白表达量,逆转录-聚合酶链反应(RT-qPCR)检测DKK-1、微小RNA(miR)-335-5p mRNA表达量。结果去势后大鼠逐渐出现弓背蜷缩、小便色淡、体温下降、毛发枯槁、垂尾等症状,CIA造模后出现关节肿胀变形、活动受限。与空白对照组比较,去势组Runx2表达降低,CIA组DKK-1、cAMP、cGMP表达增加,去势CIA组GSK-3β、Runx2、ALP表达降低,DKK-1、cAMP、cGMP表达增加,去势CIA+WBT组miR-335-5p表达上调(P<0.05)。与去势组比较,去势+WBT组cAMP、cGMP、cAMP/cGMP、Wnt3a、Runx2、ALP、Dicer蛋白表达升高(P<0.05)。与CIA组比较,CIA+WBT组Runx2表达上调(P<0.05)。与去势CIA组比较,去势CIA+WBT组的Wnt3a、Wnt10b、β-catenin、GSK-3β、Runx2、ALP表达均上调,DKK-1降低(P<0.05)。结论尪痹片可以抑制DKK-1表达、激活Wnt/β-catenin通路、提高miR-335-5p水平,促进去势CIA大鼠的骨形成。

Objective To study the mechanism of bone formation in ovariectomized collageninduced arthritis(CIA)rats after Wang-bi Tablet(WBT)intervention based on Wingless/Integrated(Wnt)/β-catenin signaling pathway.Methods Totally 48 female SD rats were randomly divided into blank control group,ovariectomized group,CIA group,ovariectomized CIA group,ovariectomized+WBT group,CIA+WBT group,CIA+methotrexate(MTX)group,and ovariectomized CIA+WBT group.The renal deficiency model was prepared by the ovariectomy method,and the rheumatoid arthritis(RA)model was prepared by the CIA method.WBT 0.63 g/kg per day was administered by gastrogavage.MTX 1.05 mg/kg per week combined with saline was administered by gastrogavage.And normal saline was administered by gastrogavage for the rest groups.The material was collected 56 days later.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum cyclic adenosine monophosphate(cAMP)and cyclic guanosine monophosphate(cGMP)levels.The expressions of Wnt3a,Wnt10b,β-catenin,glycogen synthase kinase-3β(GSK-3β),Dickkopf-related protein 1(DKK-1),Runt-related transcription factor 2(Runx2),alkaline phosphatase(ALP),Dicer were detected by Western Blot.And the expressions of DKK-1 and microRNA(miR)-335-5p mRNA were detected by real-time polymerase chain reaction(RT-qPCR).Results The rats gradually showed symptoms such as bowed back and curled up,pale urine,decreased body temperature,withered hair and drooping tail after ovariectomy,and swollen and deformed joints as well as liminted activites after CIA modeling.Compared with the blank control group,the expression of Runx2 decreased in the ovariectomized group.DKK-1,cAMP,and cGMP increased in the CIA group.GSK-3β,Runx2,and ALP decreased;DKK-1,cAMP,and cGMP increased in the ovariectomized CIA group.miR-335-5p was up-regulated in the ovariectomized CIA+WBT group(P<0.05).Compared with the ovariectomized group,cAMP,cGMP,cAMP/cGMP,Wnt3a,Runx2,ALP,and Dicer were elevated in the ovariectomized+WBT group(P<0.05).Runx2 expression was upregulated in the CIA+WBT group,as compared with the CIA group(P<0.05).Wnt3a,Wnt10b,β-catenin,GSK-3β,Runx2,and ALP expressions were up-regulated and DKK-1 decreased in the ovariectomized CIA+WBT group,as compared with the ovariectomized CIA group(P<0.05).Conclusion WBT inhibited DKK-1 expression,activated the Wnt/β-catenin pathway,and elevated miR-335-5p level to promote bone formation in ovariectomized CIA rats.