717解毒合剂通过抑制NF-κB信号通路和STAT3活性减轻蝮蛇咬伤大鼠肝脏炎症反应

Reduction Influences of 717 Jiedu Decoction on Liver Inflammation in Agkistrodon halys Rats by Inhibiting NF-κB Signaling Pathway and STAT3 Activity

目的研究717解毒合剂对蝮蛇伤大鼠肝脏NF-κB信号通路与STAT3活性相关基因表达的影响,探讨717解毒合剂抗蝮蛇毒急性肝损伤作用。方法将SD大鼠随机分为正常对照组、模型组、717解毒合剂(25、60和150 mg生药/kg)组和抗蝮蛇毒血清组。除正常对照组外,其余各组制备蝮蛇伤大鼠模型,造模成功后2 h,正常对照组和模型组以等量生理盐水灌胃,抗蝮蛇毒血清组尾静脉注射抗蝮蛇毒血清,中药组灌胃717解毒合剂,每天1次,第6天后收集血清、肝脏。镜下观察各组肝脏病理学变化,ELISA法检测各组血清中C-反应蛋白(C-reactive protein,CRP)、白细胞介素-4(Interleukine-4,IL-4)、白细胞介素-6(Interleukine-6,IL6)、肿瘤坏死因子α(Tumor necrosis factor-α,TNF-α)及β-干扰素(Interferon-β,INF-β)等炎性因子浓度水平,Western blot法检测肝核因子κB(Nuclear factor-kappa B,NF-κB)抑制蛋白α(Inhibitor kappa B alpha,IκBα)、信号转导和转录激活因子3(Signal transducer and activator of transcription 3,STAT3)、磷酸化信号传导与转录活化因子3(p-STAT3)的蛋白表达水平,RT-RCR检测IκBαmRNA的表达水平,免疫组化检测肝核因子κBP50(NF-кBP50)、核因子κBP65(NF-кBP65)蛋白表达。结果与模型组比较,717解毒合剂能降低血清炎症因子水平(P<0.05或P<0.01),显著提高IκBαmRNA及其蛋白的表达水平(P<0.05或P<0.01),抑制NF-кBP50蛋白的高表达(P<0.01),降低STAT3、p-STAT3蛋白的表达水平(P<0.05或P<0.01)。结论717解毒合剂可能通过抑制NF-κB信号通路及STAT3磷酸化,减轻蝮蛇伤大鼠肝脏炎症反应,促进肝损伤修复,从而降低因蛇伤致多脏器综合征的发生率,对蛇伤治疗具有积极意义。

Objective To study the effect of 717 Jiedu decoction on the expression of NF-κB signaling pathway in liver of Agkistrodon halys bite rats,and investigate the effect of 717 Jiedu decoction on acute liver injury of Agkistrodon halys rats.Methods SD rats were randomly divided into normal control group,model group,717 Jiedu decoction(25,60 and150 mg crude drug/kg)group and anti-Agkistrodon halys serum group.In addition to the normal control group,all other groups prepared Agkistrodon halys bite rat models.2 hours after successful modeling,the normal control group and the model group were given the same amount of normal saline by intragastric administration;the serum of anti-Agkistrodon halys venom was injected into the tail vein of the anti-Agkistrodon halys serum group;and the Chinese medicine group was given intragastric administration of 717 Jiedu Decoction,once a day.The serum and liver were collected on the 6th day.Pathological changes of liver in each group were observed under microscope.ELISA method detected serum CRP,IL4,IL6,TNF-αand INF-βinflammatory factor levels,Western Blot method detected IκBα,STAT3,p-STAT3 protein expression,and RT-RCR method detected IκBαmRNA expression levels of each group.Immunohistochemical methods detected NF-кB P50 and P65 protein expression of each group.Results Compared with model group,717 Jiedu Decoction reduced the level of serum inflammatory factors(P<0.05 or P<0.01),significantly improved IκBαmRNA and protein expression levels(P<0.05 or P<0.01),inhibited NF-кBP50 protein expression(P<0.01),decreased the expression level of STAT3 and p-STAT3(P<0.05 or P<0.01).Conclusion 717 Jiedu Decoction may inhibit NF-кB signaling pathway and STAT3 phosphorylation to reduce liver inflammation in Agkistrodon halys rats,promote liver injury repair,and thus reduce the incidence of multiple organ syndrome caused by snake bites.Therefore,717 Jiedu Decoction has positive significance for the treatment of snake bites.