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

摘要:

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-6 mRNA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。

Abstract:

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) concentration-dependently decreased the production of IL-6 from FLS due to IL-1β. Conclusion: The mechanisms probably involve the inhibition of MAPK activation, PKC phosphorylation.