温化蠲痹方对胶原诱导性关节炎大鼠成纤维样滑膜细胞凋亡的影响

Effects of Wenhua Juanbi Recipe on Inducing Apoptosis of Fibroblast-like Synoviocytes in Collagen-induced Arthritis Rats

目的 研究温化蠲痹方对微小核糖核酸-146a(miRNA-146a)/核因子κB(NF-κB)环路诱导胶原诱导性关节炎(CIA)大鼠成纤维样滑膜细胞(FLS)凋亡作用及其相关分子机制。方法 选用雌性Wistar大鼠25只,按随机数字表法分为正常组(10只)和造模组(15只),造模组采用尾部皮内多点注射牛Ⅱ型胶原乳剂的方法进行免疫,建立CIA模型。在造模成功后(12只,造模成功率为80%)随机选取10只作为模型组,分别取正常大鼠和CIA大鼠的关节滑膜,用组织块培养法培养出原代FLS,培养至第3代进行干预实验。分别过表达、沉默miRNA-146a,并加入通路阻断剂和含药血清共培养,应用荧光定量聚合酶链式反应(RT-PCR)检测miRNA-146a、肿瘤坏死因子受体相关因子6(TRAF6)、白细胞介素-1受体相关激酶1(IRAK1)基因表达;Western Blot法检测磷酸化核因子κB p65(NF-κB p65,p-p65)、核因子κB抑制因子α(IκBα)、增殖细胞核抗原(PCNA)、含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)蛋白表达;流式细胞术检测各组FLS凋亡;CCK-8法检测各组FLS增殖。结果 与正常组比较,模型组大鼠足掌厚度明显增加(P<0.01);正常转染miRNA-146a组miRNA-146a、TRAF6、IRAK1基因表达及PCNA、p-p65蛋白表达增加,Caspase-3、IκBα蛋白表达及FLS凋亡率降低(P<0.05);正常转染反义miRNA-146a组miRNA-146a、TRAF6、IRAK1基因表达及PCNA、p-p65蛋白表达降低,Caspase-3、IκBα蛋白表达及FLS凋亡率增加(P<0.05)。与CIA对照组比较,CIA转染miRNA-146a组TRAF6、IRAK1基因表达及PCNA、p-p65蛋白表达增加,Caspase-3、IκBα蛋白表达及FLS凋亡率降低(P<0.05);CIA转染反义miRNA-146a组TRAF6、IRAK1基因表达及PCNA、p-p65蛋白表达降低,Caspase-3、IκBα蛋白表达及FLS凋亡率增加(P<0.05)。加入NF-κB通路阻断剂后,随着阻断时间延长及PCNA、p-p65蛋白表达降低,Caspase-3、IκBα蛋白表达增加(P<0.05)。与对照血清组比较,中药含药血清组细胞增殖率降低(P<0.05)。结论 温化蠲痹方可以通过mi RNA-146a/NF-κB环路诱导FLS凋亡、抑制FLS增殖。

Objective To study the apoptotic effects of Wenhua Juanbi Recipe(WJR) on the fibroblastlike synoviocytes(FLS) of collagen-induced arthritis(CIA) rats through the microRNA-146a/nuclear factor-κ-gene blinding(miRNA-146a/NF-κB) circuit and its related molecular mechanisms.Methods Totally 25 female Wistar rats were divided into normal control group(n=10) and model making group(n=15) by random number table.Rats in the model making group were induced by intradermal injection of bovine collagen type Ⅱ emulsion at multiple points of the tails to establish CIA model.The successfully modeled CIA rats(n=12,success rate of modeling was 80%) were randomly selected as the model group(10 rats).The joint synovium of normal rats and CIA rats were taken,and primary FLS were cultured by explant culture method to the third generation for intervention experiments.miRNA-146a was overexpressed and silenced respectively,and pathway blockers and drug-containing serum were added to co-culture.The expressions of miRNA-146a,tumor necrosis factor(TNF) receptor associated factor 6(TRAF6),and IL-1 receptor associated kinase 1(IRAK-1) genes were detected by quantitative real-time PCR(αRT-PCR).p-NF-κBp65(p-p 65),NF-kappa-B inhibitor alpha(IκBα)、proliferating cell nuclear antigen(PCNA),and Caspase-3 protein expressions were detected by Western Blot.FLS apoptosis in each group was detected by flow cytometry(FCM).FLS proliferation in each group was detected by cell counting kit-8(CCK-8) method.Results Compared with the normal group,the pads thickness of the model group became thicker(P<0.01).The expressions of miRNA-146a,TRAF6,IRAK1,PCNA,p-p65increased(P<0.05),the expressions of Caspase-3 and IκBα and FLS apoptosis rate decreased(P<0.05) in normal transfection miRNA-146a group.The expressions of miRNA-146a,TRAF6,IRAK1,PCNA,p-p65decreased(P<0.05),the expressions of Caspase-3 and IκBα and FLS apoptosis rate increased(P<0.05) in normal antisense transfection miRNA-146a group.Compared with CIA control group,the expressions of TRAF6,IRAK1,PCNA,p-p65 increased(P<0.05),the expressions of Caspase-3 and IκBα and FLS apoptosis rate decreased(P<0.05) in CIA transfection miRNA-146a group.The expressions of TRAF6,IRAK1,PCNA,p-p65decreased(P<0.05),the expression of Caspase-3 and IκBα and FLS apoptosis rate increased(P<0.05) in CIA antisense transfection miRNA-146a group.As the extending of blocking time after adding pathway blocker went by,the expressions of PCNA,p-p65 decreased(P<0.05),the expressions of Caspase-3 and IκBα increased(P<0.05).Compared with control serum group,the expressions of FLS proliferation rate decreased in Chinese herbs-containing serum group(P<0.05).Conclusion WJR induced the apoptosis of FLS and inhibited its proliferation through miRNA-146a/NF-κB circuit.