基于IKK/IKB/NF-κB信号通路背俞穴埋线治疗非酒精性脂肪性肝炎的机制研究

Mechanism of catgut embedding at back-shu points for nonalcoholic steatohepatitis based on IKK/IKB/NF-κB signaling pathway

目的:基于IKK/IKB/NF-κB信号通路及下游炎性因子,探讨背俞穴穴位埋线干预非酒精性脂肪性肝炎(nonalcoholicsteatohepatitis,NASH)大鼠的机制。方法:80只SPF级SD大鼠,随机取10只作为正常组,剩余70只高脂饲料喂养制备NASH大鼠模型,12周末随机取10只验证造模是否成功。余60只随机分为模型组、背俞穴埋线组、腹部穴埋线组、背俞穴针刺组、假埋线组和西药组,每组10只。背俞穴埋线组、腹部穴埋线组、背俞穴针刺组分别予相应取穴治疗,背俞穴取"肝俞""脾俞""胃俞""肾俞",腹部穴取"大横""腹结""滑肉门""天枢";假埋线组在背俞穴处刺激,但不进入皮下组织,不留线;西药组给予维生素E软胶囊灌胃,疗程均4周。正常组不予任何干预,正常饲料喂养至第16周末;模型组继续予高脂饲料喂养至第16周末。干预结束后,计算各组大鼠肝指数;HE染色观察各组大鼠肝脏组织形态;血清生化检测大鼠肝功能[丙氨酸氨基转移酶(ALT)、γ-谷氨酰转肽酶(γ-GGT)、碱性磷酸酶(ALP)]及血脂[血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)]水平;ELISA法检测血清肿瘤坏死因子α(TNF-α)、白介素6(IL-6)和白介素1β(IL-1β)含量;Western blot法检测肝组织IKK-α、NF-κBp65、IL-6、IL-1β、TNF-α蛋白表达;红外热像仪检测大鼠任督脉温度。结果:与正常组比较,模型组大鼠脂肪变性、炎性细胞浸润明显,大鼠体质量、肝脏湿重、肝指数均增加(P<0.01);与模型组比较,背俞穴埋线组、腹部穴埋线组、背俞穴针刺组、西药组肝组织形态均有不同程度改善,肝指数下降(P<0.05),其中以背俞穴埋线组最明显(P<0.05)。与正常组比较,模型组大鼠ALT、γ-GGT、ALP、TG、TC、LDL、TNF-α、IL-6、IL-1β升高(P<0.01);各干预组与模型组比较,ALT、γ-GGT、ALP、TG、TC、LDL、TNF-α、IL-6、IL-1β均不同程度下降(P<0.01,P<0.05),其中背俞穴埋线组改善显著优于其他干预组(P<0.01)。模型组IKK-α、NF-κBp65、TNF-α、IL-6、IL-1β蛋白表达较正常组升高(P<0.01);各干预组与模型组比较,均有不同程度下降(P<0.05),背俞穴埋线组改善效果优于其他干预组(P<0.01)。模型组任督二脉温度较正常组降低(P<0.01);背俞穴埋线组、腹部穴埋线组、背俞穴针刺组大鼠任督脉温度与模型组比较,均有提高(P<0.01);背俞穴埋线组任脉温度与腹部穴埋线组比较差异无统计学意义(P>0.05);背俞穴埋线组督脉温度改善优于腹部穴埋线组(P<0.05)。结论:背俞穴埋线可能通过抑制IKK/IKB/NF-κB信号通路的活化,中断炎性因子引起的"瀑布样"效应,减少炎性因子对肝脏的"二次打击",从而阻断NASH进展,发挥防治NASH的作用。

Objective To explore the mechanism of catgut embedding at back-shu points on nonalcoholic steatohepatitis(NASH) in rats based on IKK/IKB/NF-κB signaling pathway and downstream inflammatory factors. Methods Eighty SPF SD rats were selected, among them 10 rats were selected divided into a normal group(group A), and the remaining 70 rats were fed with high-fat diet to establish NASH model. At the end of 12 weeks, 10 rats were randomly selected to verify whether the model establishment was successful. Then the remaining 60 rats were randomly divided into a model group(group B), a catgut embedding at back-shu points group(group C), a catgut embedding at abdominal points group(group D), an acupuncture at back-shu points group(group E), a sham catgut embedding group(group F) and a western medication group(group G), 10 rats in each group. The rats in the group C were treated with catgut embedding at "Ganshu"(BL 18), "Pishu"(BL 20), "Weishu"(BL 21) and "Shenshu"(BL 23);the rats in the group D were treated with catgut embedding at "Daheng"(SP 15), "Fujie"(SP 14), "Huaroumen"(ST 24) and "Tianshu"(ST 25);the rats in the group E were treated with acupuncture at the same acupoints as the group C;the rats in the group F were treated with catgut embedding at back-shu points but the needle did not enter subcutaneous tissue gamma;the rats in the group G were treated with intragastric administration of vitamin E capsule. All the treatment was given for 4 weeks. The rats in the group A were fed with normal diet until the end of 16 weeks without any intervention. The rats in the group B continued to be fed with high-fat diet until the end of 16 weeks. After the intervention, the liver index was calculated;the liver histomorphology was observed by HE staining;the liver function [alanine aminotransferase(ALT), gamma glutamyl transferase(γ-GGT), alkaline phosphatase(ALP)] and blood lipid [serum total cholesterol(TC), triglyceride(TG), low density lipoprotein(LDL)] were measured by serum biochemistry. The serum levels of TNF-α, IL-6 and IL-1βwere detected by ELISA, and the expressions of IKK-α, NF-κBp65, IL-6, IL-1β and TNF-α proteins in liver tissue were detected by Western blot. The temperature of the conception vessel and the governor vessel was measured by infrared thermography. Results Compared with the group A, the obvious steatosis and inflammatory cell infiltration were observed in the group B, and the body weight, liver wet-weight and liver index were all increased(P<0.01). Compared with the group B, the liver tissue morphology in the group C, the group D, the group E and the group G was improved in varying degrees, and the liver index was decreased(P<0.05), which was the most significant in the group C(P<0.05). Compared with the group A, the ALT, γ-GGT, ALP, TG, TC, LDL, TNF-α, IL-6 and IL-1β were all increased in the group B(P<0.01);compared with the group B, the ALT, γ-GGT, ALP, TG, TC, LDL, TNF-α, IL-6 and IL-1β in all intervention groups were all decreased in varying degrees(P<0.01, P<0.05), which was the most significant in the group C(P<0.01). Compare with the group A, the expressions of IKK-α, NF-κBp65, TNF-α, IL-6 and IL-1βproteins in the group B were all increased(P<0.01);compared with the group B, the expressions of IKK-α, NF-κBp65, TNF-α, IL-6 and IL-1βproteins in all intervention groups were decreased in varying degrees(P<0.05), which was the most significant in the group C(P<0.01). Compared with the group A, the temperature of the conception vessel and governor vessel was decreased in the group B(P<0.01). Compared with the group B, the temperature of the conception vessel and governor vessel was all increased in the group C, the group D and the group E(P<0.01);the temperature of the conception vessel in the group C was similar to that in the group D(P>0.05), while the temperature of the governor vessel in the group C was superior to that in the group D(P<0.05). Conclusion The catgut embedding at back-shu points might inhibit the activation of IKK/IKB/NF-κB signaling pathway to interrupt the inflammatory cascade, and reduce the "second hit" of inflammatory factors on liver, which could slow down NASH progress and prevent and treat NASH.