来源：风湿病与关节炎,2019,8（12）:5-8,26.

独活寄生汤抑制炎症介导软骨细胞基质降解的机制研究

（基础研究）

李　慧¹，马玉环¹，王圣杰¹，许丽梅²，许云腾²，曾建伟²，王丽丽²，李西海^2,3，叶蕻芝^2,4

　　【摘　要】目的：探讨独活寄生汤抑制炎症介导软骨细胞基质降解的机制。方法：取4周龄SD大鼠10只，采用机械-Ⅱ型胶原酶消化法建立软骨细胞体外培养体系。在光学显微镜下观察软骨细胞形态，用Ⅱ型胶原免疫组化鉴定软骨细胞。将第2代软骨细胞随机分为空白对照组、模型对照组、中药组，3组均连续干预培养8 h。采用酶联免疫吸附法（ELISA）检测干预后各组软骨细胞培养液中白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）含量。采用Western blot检测干预后各组软骨细胞中蛋白多糖1（PGS1）、基质金属蛋白酶（MMP）-13、MMP-9、MMP-3、蛋白聚糖酶（ADAMTS）-4、ADAMTS-5蛋白表达量。结果：①软骨细胞免疫组化鉴定：第2代软骨细胞胞浆呈棕黄色，细胞核不着色，具备软骨细胞的典型特征。②ELISA检测结果：与空白对照组比较，模型对照组IL-1β、TNF-α含量明显升高（P < 0.05）；与模型对照组比较，中药组IL-1β、TNF-α含量均明显下降（P < 0.05）。③Western blot法检测结果：与空白对照组比较，模型对照组PGS1蛋白表达量明显降低（P < 0.01），MMP-13、MMP-9、MMP-3、ADAMTS-4、ADAMTS-5蛋白表达量均明显升高（P < 0.01或P < 0.05）；与模型对照组比较，中药组PGS1蛋白表达量明显升高（P < 0.05），MMP-13、MMP-9、MMP-3、ADAMTS-4、ADAMTS-5蛋白表达量均明显降低（P < 0.05）。结论：独活寄生汤可以通过调控IL-1β、TNF-α、PGS1、MMP-13、MMP-9、MMP-3、ADAMTS-4、ADAMTS-5表达，明显抑制脂多糖介导软骨细胞的炎症反应，改善软骨细胞基质稳态失衡，从而延缓软骨退变。

　　【关键词】　骨关节炎；独活寄生汤；软骨细胞；炎症因子；软骨基质；大鼠

Study on the Mechanism of Duhuo Jisheng Tang（独活寄生汤）Inhibiting Inflammation Mediated Matrix Degradation of Chondrocytes

LI Hui,MA Yu-huan,WANG Sheng-jie,XU Li-mei,XU Yun-teng,ZENG Jian-wei,WANG Li-li,LI Xi-hai,YE Hong-zhi

　　【ABSTRACT】Objective:To explore the mechanism of Duhuo Jisheng Tang（独活寄生汤）inhibiting inflammation mediated matrix degradation of chondrocytes.Methods:Ten four-week-old SD rats were chosen to establish the chond-rocyte culture system in vitro by mechanical-Ⅱcollagenase digestion method.The morpho-logy of chondrocytes was observed under the optical microscope,and chondrocytes were identified by type-Ⅱcollagen immuno-histochemistry.The generation-Ⅱ chond-rocytes were randomly divided into three groups:the blank control group,the model control group and the Chinese medicine group.ELISA was used to detect the contents of IL-1β and TNF-α in the chondrocyte culture medium of each group.Western blot was used to detect the protein expressions of PGS1,MMP-13,MMP-9,MMP-3,ADAMTS-4 and ADAMTS-5 in chondrocytes.Results:①Immunohistochemical identification of chondrocytes showed that the cytoplasm of the generation-II chondrocytes was brownish yellow and the nucleus was not stained,having typical characteristics of chondrocytes.②ELISA results showed that compared with the blank control group,the contents of IL-1β and TNF-α in the model control group increased significantly（P < 0.05）;compared with the model control group,the contents of IL-1β and TNF-α in the Chinese medicine group decreased significantly（P < 0.05）.③Western blot results showed that compared with the blank control group,the protein expression of PGS1 in the model control group decreased significantly（P < 0.01）,and the protein expressions of MMP-13,MMP-9,MMP-3,ADAMTS-4 and ADAMTS-5 increased significantly（P < 0.01 or P < 0.05）;compared with the model control group,the protein expression of PGS1 in the Chinese medicine group increased significantly（P < 0.05）,and the protein expressions of MMP-13,MMP-9,MMP-3,ADAMTS-4 and ADAMTS-5 decreased significantly（P < 0.05）.Conclusion:Duhuo Jisheng Tang can obviously inhibit the inflammatory response of chondrocytes mediated by lipopolysaccharide,improve the matrix homeostasis of chondrocytes and delay the degeneration of cartilage by regulating the expressions of IL-1β,TNF-α,PGS1,MMP-13,MMP-9,MMP-3,ADAMTS-4 and ADAMTS-5.

　　【Keywords】 osteoarthritis;Duhuo Jisheng Tang（独活寄生汤）;chondrocytes;inflammatory factors;cartilage matrix;rats