电针联合雷公藤红素干预膝骨关节炎模型小鼠滑膜和滑膜液中转化生长因子β的表达变化

Effect of electroacupuncture combined with triptolide on transforming growth factor beta expression in synovium and synovial fluid in a mouse model of osteoarthritis

背景:中医治疗膝骨关节炎的方法包括针灸疗法和中药药剂;雷公藤红素具有抗炎症、抗癌等功效,治疗关节炎有显著疗效,但是其具体机制仍不清楚。目的:探讨电针联合雷公藤红素对膝骨关节炎转化生长因子β信号通路的影响。方法:选取40只SPF级8周龄小鼠,适应性喂养1周后,随机分为5组,即空白对照组、模型组、电针组、雷公藤红素组、电针联合雷公藤红素组。除空白对照组外,其他组小鼠膝关节处注射单碘乙酸盐构建膝骨关节炎模型。模型构建成功后,空白对照组和模型组小鼠正常饲养,电针组小鼠电针刺激膝关节处干预治疗,雷公藤红素组小鼠雷公藤红素灌胃治疗,电针联合雷公藤红素组小鼠电针刺激膝关节处,且灌胃服用雷公藤红素治疗。每天电针治疗1次,雷公藤红素灌服1次,整个治疗周期为4周。观察治疗前后各组小鼠膝关节压力疼痛阈值;并通过宏观关节炎评分系统评价小鼠关节炎症状况;取小鼠外周血,通过ELISA试剂盒测定小鼠外周血中炎症因子肿瘤转化因子α、白细胞介素1β和白细胞介素6的含量;Western blot技术检测小鼠滑膜及滑膜液中基质金属蛋白酶2、基质金属蛋白酶9及转化生长因子β1蛋白表达水平。结果与结论:①干预治疗后,与模型组相比,电针组和雷公藤红素组小鼠膝骨关节炎得到极大改善(P<0.05),且二者联合改善更为显著(P<0.01);②疼痛阈值测定:干预治疗后,与模型组相比,电针组和雷公藤红素组小鼠痛觉阈值显著增加(P<0.05),且二者联合疼痛阈值增加更为显著(P<0.05);③关节炎评分:治疗过程中,模型组小鼠关节炎评分逐渐升高;与模型组相比,电针组和雷公藤红素组小鼠关节炎评分均有所下降,而电针和雷公藤红素联合组评分下降幅度更大;④外周血中炎症因子含量:干预治疗后,与模型组相比,电针组和雷公藤红素组小鼠外周血中肿瘤转化因子α、白细胞介素1β和白细胞介素6含量均显著下降(P<0.05),且联合组小鼠外周血中炎症因子含量极显著降低(P<0.01);⑤Western blot结果:干预治疗后,与模型组相比,电针组和雷公藤红素组小鼠滑膜和滑膜液中基质金属蛋白酶2、基质金属蛋白酶9和转化生长因子β1蛋白表达水平均显著增加(P<0.05),而二者联合组中相关蛋白表达水平极显著增加(P<0.01);⑥提示电针联合雷公藤红素可能通过转化生长因子β信号通路治疗小鼠膝骨关节炎。

BACKGROUND:Traditional Chinese medicine methods for treating knee osteoarthritis include acupuncture and traditional Chinese medicine.Triptolide has anti-inflammatory and anti-cancer effects,and it also has significant effects in treating arthritis.However,its specific mechanism is still unclear.OBJECTIVE:To investigate the effect of electroacupuncture combined with triptolide on the transforming growth factor-βsignal pathway related to osteoarthritis.METHODS:Forty 8-week-old SPF mice were selected and fed for 1 week.Mice were then randomized into five groups:blank control group,model group,electroacupuncture group,triptolide group,and electroacupuncture combined with triptolide group(combined use group).A mouse model of knee osteoarthritis was established using intraarticular injection of monoiodoacetate in the latter four groups.After the model was successfully constructed,the mice in the blank control group and model group were raised normally;the mice in the electroacupuncture group were treated with electroacupuncture to stimulate the knee joints;the mice in the triptolide group were treated with intragastric administration of triptolide;and the mice in the combined use group were given electroacupuncture at the knee joints and intragastric administration of triptolide.Electroacupuncture was given once a day,and triptolide was administered once a day.Treatments in each group lasted for 4 weeks.Pressure-pain thresholds were observed in each group before and after treatment.Inflammation of the knee joint was evaluated by the macro scoring system.The peripheral blood samples of mice were collected to detect the levels of inflammatory factors,tumor transforming factor-α,interleukin-1βand interleukin-6,by ELISA kit.Western blot assay was used to detect the protein expression levels of matrix metalloproteinase 2,matrix metalloproteinase 9,and transforming growth factorβ1 in mouse synovium and synovial fluid.RESULTS AND CONCLUSION:After intervention,compared with the model group,a great improvement in knee osteoarthritis was achieved in the electroacupuncture and triptolide groups(P<0.05),and their combined use showed a better effect(P<0.01).After intervention,the pain threshold of mice increased significantly in the electroacupuncture and triptolide groups compared with the model group(P<0.05),and the pain threshold in the combined use group increased more significantly(P<0.05).During the treatment,the arthritis score of mice in the model group gradually increased;compared with the model group,the arthritis scores decreased in the electroacupuncture and triptolide groups,and the score of the combined use group had a significant decline.After intervention,both electroacupuncture and triptolide significantly decreased the levels of tumor transforming factor-α,interleukin-1βand interleukin-6 in the peripheral blood of mice compared with the model group(P<0.05),and their combined use could extremely significantly decrease the levels of these inflammatory factors in the peripheral blood of mice(P<0.01).Western blot results revealed that after intervention,the expression levels of matrix metalloproteinase 2,matrix metalloproteinase 9,and transforming growth factorβ1 were significantly increased in the synovium and synovial fluid of mice in the electroacupuncture group and the triptolide group compared with the model group(P<0.05),while the expression levels of related proteins were extremely significantly increased(P<0.01).It is suggested that electroacupuncture combined with triptolide may treat knee osteoarthritis in mice through transforming growth factorβsignaling pathway.