清热活血方对胶原诱导关节炎大鼠miR-140、TLR4、NF-κB的影响

Effect of Qingre Huoxue Recipe on miR-140,TLR4 and NF-κB in CIA Rats

目的 探讨清热活血方对胶原诱导关节炎(collagen type Ⅱ-induced arthritis, CIA)大鼠miR-140/Toll样受体4(toll-like receptors 4,TLR4)/核因子κB(nuclear factor kappa-B,NF-κB)信号通路的影响。方法 6周龄SPF级大鼠24只,随机分为正常组、模型组、甲氨蝶呤组(0.2 mg/kg)、清热活血方组(34 g/kg),每组6只,通过对大鼠尾根部注射Ⅱ型胶原乳剂构建类风湿关节炎动物模型,连续给药6周。连续监测大鼠关节肿胀程度,Elisa法测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin 1β,IL-1β)、IL-6含量白细胞介素6(interleukin 6,IL-6),TRAP染色观察破骨细胞(osteoclast,OC)数量,Western blot法检测大鼠骨组织中TLR4、NF-κB的蛋白表达,实时荧光定量PCR检测大鼠骨组织中miR-140、TLR4、NF-κB的mRNA表达。结果 与正常组比较,模型组大鼠造模开始后足趾容积逐渐增加,第21天起逐渐下降。模型组大鼠血清IL-1β、IL-6、TNF-α浓度上升,OC数量明显增多,TLR4、NF-κB的蛋白与mRNA表达增加,miR-140 mRNA表达下降;与模型组比较,甲氨蝶呤组和清热活血方组大鼠血清IL-1β、IL-6、TNF-α浓度下降,OC数量减少,TLR4、NF-κB的蛋白与mRNA表达下降,miR-140 mRNA表达增加;与甲氨蝶呤组比较,清热活血方组大鼠上述指标改善更明显,以上差异均有统计学意义(P<0.05,P<0.01)。结论 清热活血方能够干预miR-140/TLR4/NF-κB信号通路,进而调控下游靶点分子,调节OC的成熟分化,从而达到延缓RA骨破坏的作用。

Objective To investigate the effect of QRHXF Recipe on miR-140/TLR/NF-κB signal pathway in collagen induced arthritis rats.Methods 24 rats were randomly divided into normal group,model group,methotrexate group(0.2 mg/kg),and QRHXF group(34 g/kg),with six rats in each group.The rheumatoid arthritis animal model was established by injecting typeⅡcollagen emulsion into the tail root of rats,and the drug was administered continuously for six weeks.Continuously monitored the degree of joint swelling in rats,and detected serum TNF-α、IL-1βand IL-6 with Elisa.TRAP staining was used to observe the number of osteoclasts,real-time fluorescence quantitative PCR was used to detect miR-140,TLR4 and NF-κB mRNA expression,and Western blot was used to detect TLR4 and NF-κB in rat bone tissue.Results Compared with the normal group,the toe volume of rats in model group increased gradually from the beginning of modeling,and then decreased gradually after 21 days.In the model group,the concentrations of serum IL-1β,IL-6 and TNF-αof rats increased,the number of osteoclasts increased significantly,the protein and mRNA expression of TLR4 and NF-κB increased,and the expression of miR-140 decreased.Compared with model group,the concentrations of IL-1β,IL-6 and TNF-αdecreased,the number of osteoclasts decreased,the protein and mRNA expression of TLR4 and NF-κB decreased,and the expression of miR-140 increased in methotrexate group and QRHXF group.Compared with methotrexate group,all the indexes above improved more obviously in QRHXF group,and all the differences had statistical significance(P<0.05,P<0.01).Conclusion Qingre Huoxue Recipe can interfere with miR-140/TLR4/NF-κB signal pathway,thereby regulating downstream target molecules,regulating the maturation and differentiation of osteoclasts,so as to delay bone destruction in RA.