苗药验方“四大血”对类风湿性关节炎大鼠关节炎症的作用及机制

Effect of sidaxue, a Miao ethnomedicine recipe, on rat rheumatoidarthritis and its mechanism

目的探讨苗药验方“四大血”(SX)对类风湿性关节炎(RA)大鼠关节炎症的作用及机制。方法42只健康SD大鼠随机均分为空白组、阳性对照组、模型组及10 g/kg(低剂量)SX组、20 g/kg(中剂量)SX组、40 g/kg(高剂量)SX组,后4组采用Ⅱ型胶原诱导建立RA模型,空白组大鼠予等容积生理盐水皮下注射;五花血藤∶鸡血藤∶见血飞∶黑骨藤按15∶22∶15∶8制取受试药,按体质量40、20、10 g/kg予SX高、中、低剂量灌胃,阳性药物组予4000 g/L雷公藤多苷片(GTW)水溶液灌胃,空白组、模型组予等容积生理盐水灌胃,持续3周,给药期间检测关节足肿度(ER);干预结束后处死各组大鼠,取膝关节滑膜组织、心脏取血,HE染色法观察各组大鼠膝关节滑膜组织的形态学特征,酶联免疫吸附试验(ELISA)检测各组大鼠血清的IL-18表达情况,采用即时聚合酶链式反应(Real-time PCR)和蛋白质免疫印迹法(Western blot)检测各组大鼠中NLRP3、Caspase-1的mRNA和蛋白表达。结果给药21 d后,与模型组比较,各给药组大鼠ER均不同程度的减轻(P<0.05);HE染色结果显示,与模型组比较,SX组大鼠炎症浸润、滑膜增生程度均有改善,且高剂量SX组大鼠更明显;与空白组比较,模型组大鼠血清IL-18水平明显升高(P<0.01),高剂量、中剂量SX组和阳性对照组大鼠血清IL-18含量降低(P<0.05);Real-time PCR结果显示,与模型组比较,中剂量SX组大鼠膝关节滑膜组织中NLRP3、Caspase-1 mRNA明显下调(P<0.01);Western blot结果显示,与空白组比较,模型组大鼠膝关节滑膜组织中NLRP3、Caspase-1蛋白表达上调(P<0.05),但中、低剂量SX组下调(P<0.05),且中剂量SX组蛋白下调与阳性对照组作用相当(P>0.05)。结论SX可抑制CIA大鼠关节炎症的反应,其机制可能与NLRP3炎症体轴激活受到抑制有关。

Objective To investigate the effect of Sidaxue(SX),a Miao ethnomedicine recipe,on rat rheumatoid arthritis(RA)and its mechanism.Methods Forty-two healthy SD rats were randomly divided into blank group,positive control group,model group and low dose-SX group(10 g/kg),medium dose-SX group(20 g/kg),and high dose-SX group(40 g/kg).Model group and three SX groups were subcutaneously injected with type II collagen to induce RA model.SX was prepared by mixing Sargentgloryvine Stem∶Suberect Spatholobus Stem∶Root of Spinyleaf Pricklyash∶Forrest silkvine stem at a ratio of 15∶22∶15∶8.According to rat body weight,high dose(40 g/kg),medium dose(20 g/kg),and low dose(10 g/kg)of SX were given by gavage.Positive control group was given with 4000 g/L tripterygium glycosides(GTW)solution by gavage.Blank and model groups were treated with equal volume of saline by gavage for 3 weeks.Swelling degree(ER)of the joints was detected during the administration period.After the intervention,the rats in each group were sacrificed,and the synovial tissues of rat knee joints and blood in rat hearts were collected.HE staining was applied to observe morphological characteristics of the synovial tissues of rat knee joints in each group.Enzyme-linked immunosorbent assay(ELISA)was used to measure rat serum IL-18 expression.mRNA and protein expression levels of NLRP3,Caspase-1 were detected by real-time polymerase chain reaction(Real-time PCR)and Western blot,respectively.Results After 21 d of administration,when compared with model group,ER of SX groups showed different degrees of reduction(P<0.05).HE staining results showed that the inflammatory infiltration and synovial hyperplasia in SX groups were improved relative to model group,more obvious in high-dose SX group.When compared with blank group,serum IL-18 level was remarkably elevated(P<0.01).Rat serum IL-18 levels were decreased in high-and medium-dose SX groups as well as positive group(P<0.05).Real-time PCR results showed that mRNA levels of NLRP3 and Caspase-1 were significantly down regulated in the synovial tissues of rat knee joints in medium-dose SX group when compared with model group(P<0.01).Western blot results showed that the protein expression levels of NLRP3 and Caspase-1 were upregulated in the synovial tissues of rat knee joints in model group when compared with blank group(P<0.05),but downregulated in low-and medium-dose SX groups(P<0.05),and their downregulation in medium-dose SX group was equivalent to that in positive control group(P>0.05).Conclusion SX may inhibit rat joint inflammation response in collagen-induced arthritis(CIA),and its mechanism may be related to the inhibition of NLRP3 inflammasome axis activation.