独活寄生汤含药血清对膝关节OA大鼠滑膜细胞TNF-α、IL-6、IL-8、IL-10表达的影响及其机制

Effect of Duhuo Jisheng decoction drug-containing serum on expression of synovial cell TNA-α,IL-6,IL-8,IL-10 in knee osteoarthritis rats

目的探讨独活寄生汤含药血清对膝关节骨关节炎(OA)大鼠滑膜细胞炎症因子表达的影响及其可能机制。方法采用Hulth法建立OA大鼠模型,分离膝关节滑膜细胞,采用酶原消化法进行培养,并将其分为模型组及低、中、高剂量组,以正常大鼠膝关节滑膜细胞为对照组。低、中、高剂量组分别加入经独活寄生汤中药0.85、1.7、3.4 m L/kg灌胃大鼠的含药血清,对照组与模型组加入等量20%FBS,培养48 h。采用ELISA法检测各组细胞TNF-α、IL-6、IL-8及IL-10表达,超高效液相色谱-高分辨质谱法检测各组细胞脂氧素A4表达,Western blotting法检测各组细胞抗c-Jun氨基末端激酶(JNK)、磷酸化JNK(p-JNK)、激活子蛋白1(AP-1)及磷酸化AP-1(p-AP-1)表达。结果对照组、高剂量组、中剂量组、低剂量组和模型组细胞TNF-α、IL-6、IL-8表达均依次升高,IL-10、脂氧素A4表达依次降低,组间两两比较P均<0.05。与对照组比较,模型组细胞AP-1、p-AP-1、JNK及p-JNK表达均升高(P均<0.05);与模型组比较,高、中、低剂量组细胞AP-1、p-AP-1、JNK及p-JNK表达均降低,且高剂量组降低更明显(P均<0.05)。结论独活寄生汤含药血清可降低膝关节OA大鼠滑膜细胞TNA-α、IL-6、IL-8、IL-10表达,并呈剂量依赖性;提高滑膜细胞脂氧素A4表达、抑制JNK、AP-1及其磷酸化通路可能是其作用机制。

Objective To observe the effect of Duhuo Jisheng decoction drug-containing serum on the expression of inflammatory factors in synovial cells of knee osteoarthritis （OA） rats and to explore its possible mechanism. Methods The rat model of OA was established by Hulth method. The synovial cells of knee joint were cultured by enzymatic digestion. The synovial cells were divided into the model group and low-dose, medium-dose and high-dose groups. The normal rat synovial cells were used as the control group. The rats in the low-dose, medium-dose and high-dose groups were added with the serum containing Duhuo Jisheng decoction 0.85, 1.7 and 3.4 mL/kg by gavage, respectively, while rats in the control group and the model group were given the same amount of 20% fetal bovine serum, and then both of them were cultured for 48 h. The levels of TNF-α, IL-6, IL-8, and IL-10 were detected by ELISA. The expression of lipoxin A4 was detected by high performance liquid chromatograpt^y-high resolution mass spectrometry. Western blotting was used to detect the expression of Anti-c-Jun amino terminal kinase （JNK） , p-JNK, activator protein 1 （ AP-1 ）, and p-AP- 1 expression. Results The expression levels of TNF-α, IL-6, and IL-8 in the control group, high-dose group, mediumdose group, low-dose group, and model group gradually increased, the expression levels of IL-10 and lipoxin A4 gradually decreased, and significant difference was found between every two groups （ all P 〈 0.05 ）. Compared with the control group, the expression levels of AP-1, p-AP-1, JNK, and p-JNK in the model group increased （ all P 〈 0.05 ）. Compared with the model group, the expression levels of AP-1, p-AP-1, JNK, and p-JNK gradually decreased in the high-dose, medium-dose and low-dose groups, and the decrease was more significant in the high-dose group （ all P 〈 0.05 ）. Conclusion Duhuo Jisheng decoction can reduce the expression of TNA-α, IL-6, IL-8, IL-10 in the synovial cells of knee OA rats in a dose-dependent manner by inhibiting JNK, AP-1 and its phosphorylation pathway and increasing the expression of lipoxin A4 in synovial cells.