基于TLR4/MyD88/TRIF通路探讨芪参合剂对耐药肺炎克雷伯菌脓毒症大鼠的固有免疫调节机制

Investigation on intrinsic immune regulation mechanism of Qishen mixture in drug-resistant Klebsiella pneumoniae septic rats based on TLR4/MyD88/TRIF pathway

目的:基于TLR4/MyD88/TRIF通路探讨芪参合剂对耐药肺炎克雷伯菌脓毒症大鼠的固有免疫调节机制。方法:将40只SD大鼠随机分成空白组、模型组、芪参合剂组及TAK242组,气管注射耐药肺炎克雷伯菌复制脓毒症大鼠模型,造模后6 h,空白组和模型组灌胃给予生理盐水,芪参合剂组灌胃给予芪参合剂,TAK242组腹腔注射给予TAK242,1次/d,干预3 d后收集标本,检测BALF炎症因子水平及血清生化指标。RT-PCR、Western blot及免疫组化方法观察芪参合剂对肺组织中TLR4、MyD88、TRIF、NF-κB p65 mRNA和蛋白表达的影响,ELISA法观察芪参合剂对肺组织中TNF-α、IL-6、MIP-2、CXCL1水平的影响。结果:与空白组相比,模型组大鼠BALF中TNF-α、IL-6水平及血清生化指标明显升高(P<0.05),肺组织中TLR4、MyD88、TRIF、NF-κB p65 mRNA及蛋白水平明显升高(P<0.05),肺组织中炎症因子(TNF-α、IL-6、MIP-2、CXCL1)水平明显升高(P<0.05)。芪参合剂和TAK242均可显著下调TLR4信号通路中TLR4、MyD88、TRIF、NF-κB p65 mRNA和蛋白水平(P<0.05),同时显著下调炎症因子(TNF-α、IL-6、MIP-2、CXCL1)水平(P<0.05)。结论:芪参合剂和TAK242作用效果相似,均可显著下调TLR4信号通路中TLR4、MyD88、TRIF、NF-κB p65的蛋白水平和mRNA水平,减轻炎症因子释放,从分子生物学水平阐明了芪参合剂的固有免疫调节机制可能与抑制TLR4信号通路的过度活化有关。

Objective:To investigate intrinsic immune regulation mechanism of Qishen mixture in drug-resistant Klebsiella pneumoniae septic rats based on TLR4/MyD88/TRIF pathway.Methods:40 SD rats were randomly divided into blank group,model group,Qishen Mixture group and TAK242 group.Drug-resistant Klebsiella pneumoniae tracheal inoculation to construct septic rat model,then the blank group and model group were administered normal saline 6 h later as well as the Qishen mixture intervention groups were administered Qishen mixture by means of intragastric administration,and TAK242 was given by intraperitoneal injection,once a day for 3 d with collection of specimen 3 d later.Inflammatory factors in BALF and serum biochemical indexes were tested.Effect of Qishen mixture on mRNA and protein expressions of TLR4,MyD88,TRIF,NF-κB p65 in lung tissue were observed by RT-PCR,Western blot and immunohistochemistry,as well as effect of Qishen mixture on levels of TNF-α,IL-6,MIP-2 and CXCL1 in lung tissue were observed by ELISA.Results:Compared with blank group,levels of TNF-α,IL-6 in BALF and serum biochemical indexes were significantly increased(P<0.05).TLR4,MyD88,TRIF,NF-κB p65 mRNA and protein levels were significantly increased(P<0.05),and levels of inflammatory factors(TNF-α,IL-6,MIP-2,CXCL1)were also significantly increased in model group(P<0.05).Otherwise Qishen mixture and TAK242 could significantly down-regulate TLR4,MyD88,TRIF,NF-κB p65 mRNA and protein levels in TLR4 signaling pathway(P<0.05),and significantly down-regulated levels of inflammatory factors(TNF-α,IL-6,MIP-2,CXCL1).Conclusion:Effects of Qishen mixture and TAK242 were similar,which could significantly down-regulate TLR4,MyD88,TRIF and NF-κB p65 protein and mRNA levels in TLR4 signaling pathway,as well as reduce the release of inflammatory factors,which elucidate mechanism of innate immune regulation of Qishen mixture may be related to inhibition of over-activation of TLR4 signaling pathway from molecular biology level.