miR-138对类风湿关节炎大鼠免疫和软骨损伤的影响

Effects of miR-138 on Immunity and Cartilage Damage in Rats with Rheumatoid Arthritis

目的探究miR-138基因干扰对胶原诱导的类风湿关节炎大鼠免疫紊乱和软骨损伤的影响.方法选取健康雄性6周龄SD大鼠60只,采用随机数字表法分为:健康对照(Control)、模型组(RA)、模型+miR-138 NC组(RA+anti-miR-138 NC)和模型+anti-miR-138组(RA+anti-miR-138),每组15只;除Control组外各组采用胶原诱导类风湿性关节炎大鼠模型,RA+anti-miR-138 NC组采用无序siRNA质粒溶于1 ml生理盐水大鼠尾静脉注射;RA+anti-miR-138组使用有效的siRNA质粒溶于1 ml生理盐水大鼠尾静脉注射.RT-PCR检测各组大鼠miR-138表达情况;双能X线骨密度仪检测大鼠骨体积/总量、骨小梁表面/骨体积、骨小梁厚度和骨小梁间隙;HE染色、骨MASSON染色和软骨阿利新蓝染色观察大鼠骨组织病理损伤;统计骨小梁和软骨占比;蛋白质印迹法检测各组大鼠中骨特异性转录因子:成骨细胞特异性转录因子(osterix,Osx),1型胶原蛋白α1(collagen type I alpha 1,COL1A1)和骨钙素(osteocalcin,OC)的表达;ELISA检测外周血中炎性因子白细胞介素-6(interleukin-6,IL-6),白细胞介素-17(interleukin-17,IL-17),一氧化氮合酶(inducible nitric oxide synthase,iNOS)和单核细胞趋化蛋白1(monocyte chemoat-tractant protein 1,MCP-1)的含量水平.结果RT-PCR结果显示:RA组miR-138显著高于Control组(P<0.05);RA+anti-miR-138组miR-138表达显著低于RA+anti-miR-138 NC组(P<0.05).染色结果发现,干扰miR-138基因后,大鼠骨组织细胞排列更为整齐,炎性浸润和纤维化得到了显著的改善.相比Control组,RA组BV/TV、Tb.Th、骨小梁占比、软骨占比、Osx、COL1A1和OC相对表达量均显著降低(P<0.05),BSA/BV、Tb.Sp、IL-6、IL-17、iNOS和MCP-1水平均显著升高(P<0.05);相比RA+anti-miR-138 NC组,RA+anti-miR-138组BV/TV、Tb.Th、骨小梁占比、软骨占比、Osx、COL1A1和OC相对表达量均显著升高(P<0.05),BSA/BV,Tb.Sp、IL-6、IL-17、iNOS和MCP-1水平均显著降低(P<0.05).结论干扰miR-138基因表达可以有效缓解胶原诱导的类风湿关节炎大鼠软骨损伤,其作用机制与调节免疫紊乱降低炎性反应相关.

Objective To explore the effects of miR-138 gene interference on immune disorders and cartilage damage in rats with collagen-induced rheumatoid arthritis(RA).Methods Sixty six-week-old healthy male SD rats were divided into a healthy control group(control),a model group(RA),a model+miR-138 NC group(RA+anti-miR-138 NC)and a model+anti-miR-138 group(RA+anti-miR-138)by using random number table method,with 15 animals in each group.Except for the control group,collagen was administered to induce RA rat models in the other 3 groups.RA+anti-miR-138 NC group was injected 1 ml normal saline plus disordered siRNA plasmid via tail vein,while RA+anti-miR-138 group was given 1 ml normal saline with effective siRNA,plasmid via tail vein.The expression of miR-138 in each group was detected by RT-PCR.The bone volume/amount,trabecular surface/bone volume,trabecular thickness and space were determined by using a dual-energy X-ray bone densitometer.The pathological damage of bone tissues was observed by HE staining,MASSON staining and alcian blue staining.The proportions of trabeculae and cartilage were statistically analyzed.The expression of bone-specific transcription factors[osteoblast specific transcription factors(Osterix,Osx),collagen type I alpha 1(COL1A1),osteocalcin(OC)]in each group was detected by Western blotting.The levels of inflammatory factors[interleukin-6(IL-6),interleukin-17(IL-17),inducible nitric oxide synthase(iNOS)]and monocyte chemoattractant protein 1(MCP-1)in peripheral blood were detected by ELISA.Results RT-PCR showed that miR-138 was significantly higher in RA group than in the control group(P<0.05).The expression of miR-138 was significantly lower in RA+anti-miR-138 group than in RA+anti-miR-138 NC group(P<0.05).After miR-138 gene interference,cells of bone tissues were arranged more neatly,and inflammatory infiltration and fibrosis were significantly ameliorated.Compared with the control group,BV/TV,Tb.Th,proportions of trabeculae and cartilages,relative expression levels of Osx,COL1A1 and OC proteins were significantly decreased(P<0.05),while BSA/BV,Tb.Sp,levels of IL-6,IL-17,iNOS and MCP-1 were significantly increased in RA group(P<0.05).Compared to RA+anti-miR-138 NC groups BV/TV,Tb.Th,proportions of trabeculae and cartilages,relative expression levels of Osx,COLlAl and OC proteins were significantly elevated(P<0.05),while BSA/BV,Tb.Sp,levels of IL-6,IL-17,iNOS and MCP-1 were significantly reduced in RA+anti-miR-138 group(P<0.05).Conclusion Interference with miR-138 gene expression can effectively alleviate cartilage damage in rats with collagen-induced RA.The mechanism might be that immune disorders are regulated and thereby inflammation response is reduced.