温胆汤对肥胖痰湿证大鼠相关炎症因子及JAK2/STAT3通路关键分子STAT3表达的影响

Effect of Wendantang on Related Inflammatory Factors and Expression of STAT3 as a Key Molecule in JAK2/STAT3 Pathway of Rats with Obesity Phlegm-dampness Syndrome

目的:观察温胆汤对肥胖痰湿证大鼠外周血肿瘤坏死因子-α(TNF-α),白细胞介素(IL)-6,IL^(-1)7,IL-22等相关炎症因子以及下丘脑组织Janus激酶2/信号传导子及转录激活子3(JAK2/STAT3)信号通路关键分子STAT3 mRNA和蛋白表达的影响,探讨温胆汤干预肥胖痰湿证的内在作用机制。方法:将100只大鼠随机分成2组(空白组30只和造模组70只),空白组采用基础饲料喂养,造模组采用高脂饲料喂养,共6周,制作肥胖痰湿证动物模型。造模成功后,视体质量情况,按顺序淘汰体质量过轻者,遴选出16只肥胖大鼠,并随机分成模型组和温胆汤干预组,每组8只;另在空白组大鼠中随机选取8只设为正常组。温胆汤干预组按剂量15 g·kg^(-1)(以生药量计算)进行灌胃,模型组和正常组均给予等量蒸馏水进行灌胃,每天1次,共6周。麻醉处理前12 h禁食不禁水,麻醉后进行样本取材,检测并计算大鼠体质量,Lee's指数和肥胖率,根据试剂盒要求采用全自动生化分析仪检测大鼠总胆固醇(TC),甘油三酯(TG),低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)水平;采用酶联免疫吸附测定法(ELISA)检测大鼠外周血血清中相关细胞因子TNF-α,IL^(-1)7,IL-22,IL-6的表达;采用实时荧光定量聚合酶链式反应(Real-time PCR)技术检测大鼠下丘脑组织中STAT3 mRNA的表达;采用蛋白免疫印迹法(Western blot)测定大鼠下丘脑组织中STAT3蛋白的表达。结果:高脂饲料喂养成功复制了肥胖大鼠模型,造模组大鼠肥胖率> 20%,且与空白组比较,造模组体质量和Lee's指数显著升高(P <0. 05,P <0. 01)。与正常组比较,模型组大鼠体质量显著升高(P <0. 01),血脂水平显著改变(P <0. 01),相关炎性因子(TNF-α,IL-6,IL^(-1)7和IL-22)的水平显著升高(P <0. 01),下丘脑组织中STAT3 mRNA和蛋白的表达显著升高(P <0. 01)。与模型组比较,温胆汤干预组大鼠体质量和Lee's指数显著下降(P <0. 05,P <0. 01),血脂水平显著变化(P <0. 01),相关炎性因子(TNF-α,IL-6,IL^(-1)7和IL-22)的水平显著下降(P <0. 01),下丘脑组织中STAT3 mRNA和蛋白的表达显著下降(P <0. 01)。结论:温胆汤可改善肥胖大鼠痰湿病理状态,其作用机制可能与调节JAK2/STAT3信号通路进而改善机体的慢性炎症状态有关,为温胆汤干预肥胖痰湿证提供了实验依据。

Objective:To observe the effect of Wendantang on tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-17,IL-22 and other related inflammatory factors in peripheral blood and the expression of STAT3[the key molecule of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signal pathway in hypothalamus]mRNA and protein of obese rats with syndrome of phlegm-dampness,so as to explore the internal mechanism of Wendantang in interfering obesity with syndrome of phlegm-dampness.Method:A total of 100 rats were randomly divided into blank group(30 rats)and modeling group(70 rats),rats in the blank group was fed with basic feed and the modeling group was fed with high-fat feed for 6 weeks.Animal model of obesity with syndrome of phlegm-dampness was established by the method in literature.After successful modeling,16 obese rats were selected and randomly divided into the model group and Wendantang intervention group with 8 rats in each group,and another 8 rats in the blank group were randomly selected as the normal group.Rats in Wendantang intervention group were given 15 g·kg-1 of crude drug by gavage,while the model group and the normal group were given the same amount of distilled water for gavage once a day for 6 weeks.No eating but no prohibiting drinking before dealing with 12 h and then taking samples after anesthesia.The body weight,Lee’s index and obesity rate of rats were measured,the levels of blood lipids[total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)]of rats were detected with a full-automatic biochemical analyzer according to the requirements of the kit,the expression of TNF-α,IL-17,IL-22 and IL-6 in peripheral blood of rats was detected with enzyme-linked immunosorbent assay(ELISA),STAT3 mRNA expression in hypothalamus of rats was detected with real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and the expression of STAT3 protein in hypothalamus of rats was detected with Western blot.Result:The high-fat feed feeding could successfully replicate the obese rat model,and the obesity rate of rats in the modeling group was greater than 20%,and compared with the blank group,the body weight and Lee’s index of rats in the modeling group were significantly increased(P<0.05,P<0.01).Compared with the normal group,the body weight of rats in the model group was significantly increased(P<0.01),blood lipid level changed greatly(P<0.01),levels of related inflammatory factors were significantly increased(P<0.01),the STAT3 mRNA and protein expression in hypothalamus tissue of rats was significantly increased(P<0.01).Compared with the model group,the body weight and Lee’s index of rats in Wendantang intervention group significantly decreased(P<0.05,P<0.01),blood lipid level significantly changed(P<0.01),levels of related inflammatory factors were significantly decreased(P<0.01),the STAT3 mRNA and protein expression in hypothalamus tissue of rats in Wendantang intervention group was significantly decreased(P<0.01).Conclusion:Wendantang has a good effect on improving phlegm-dampness in obese rats,and the mechanism may be related to regulating JAK2/STAT3 signal pathway then to improve the chronic inflammatory state of the body,and all of which provides a scientific basis for Wendantang in intervening obesity with syndrome of phlegm-dampness.