羟基积雪草苷对佐剂关节炎大鼠滑膜成纤维细胞活化的影响

Effect of Madecassoside on Activation of Synovial Fibroblasts from Rats with Adjuvant Arthritis

目的：研究羟基积雪草苷对佐剂关节炎大鼠滑膜成纤维细胞活化的影响.方法：将完全弗氏佐剂于大鼠右后足趾皮内注射0.1 mL致炎,建立佐剂关节炎大鼠模型,造模30 d后脱臼处死,取出关节处的滑膜,培养原代大鼠滑膜成纤维细胞（FLS）,第3～6代细胞用于体外细胞实验.将对数生长期FLS以105个/mL接种于96孔板,利用白介素1β（IL-1β）诱导白介素-6（IL-6）的表达,羟基积雪草苷（终浓度1,5,10,20 μmol·L-1）和IL-1β（终浓度10 μg·L-1）作用24 h后收集上清.酶联免疫吸附试验（ELISA）检测上清中炎症因子IL-6的水平;实时定量PCR（Real time PCR）检测IL-6 mRNA的表达;蛋白印记法检细胞丝裂原激活的蛋白激酶（MAPK）、蛋白激酶C（PKC）、反应元件结合蛋白（CREB）的表达.结果：与正常组比较,模型组（IL-1β组）IL-6水平和IL-6 mRNA的表达明显升高.羟基积雪草苷预孵24 h后,浓度依赖性抑制IL-6的分泌并下调IL-6mRNA的表达.模型组中细胞外信号调节激酶（ERK）、p38及PKC的磷酸化水平显著提高,羟基积雪草苷逆转了IL-1β诱导的ERK、p38及PKC的磷酸化;同时抑制IL-1β引起的CREB磷酸化.结论：羟基积雪草苷抑制佐剂关节炎大鼠滑膜成纤维细胞活化,其机制可能与抑制MAPK和PKC通路活化,降低CREB的磷酸化有关.

Objective：To investigate the effect of madecassoside on interleukin-6 （IL-6） production from synovial fibroblasts （FLS）.Method：The rat adjuvant arthritis model was established by cultivating the original generation of FLS in rats.Interleukin-1 beta （IL-1β） was used to induce the expression of Interleukin-6 （IL-6） in FLS.Enzyme-linked immunosorbent assay （ELISA） method was used to detect FLS inflammatory factor levels of IL-6.Real time PCR method was used for detection of IL-6 and IL-1 mRNA expression.Western bloting method was used for mitogen-activated protein kinase （MAPK） pathways,protein kinase C （PKC） protein,and cAMP response element binding protein （CREB） protein expression.Result：Our results showed that IL-1β （10 μg ·L-1） significantly increased the production of IL-6,and mRNA of IL-6,the activation of MAPK,PKC,and CREB which was relative to the synthesis of IL-6.Madecassoside （1,5,10,20 μmol ·L-1） concentrationdependently decreased the production of IL-6 from FLS due to IL-1β.Conclusion：The mechanisms probably involve the inhibition of MAPK activation,PKC phosphorylation.