TLR4抑制剂预处理对脓毒性心肌病小鼠心肌损伤的改善作用及其机制研究

Improvement effect and mechanism of TLR4 inhibitor pretreatment on myocardial injury in septic cardiomyopathy mice

目的探讨Toll样受体4(TLR-4)抑制剂对脂多糖(LPS)诱导脓毒性心肌病(SIC)小鼠心肌损伤的改善作用及其可能机制。方法2023年3—6月在武汉科技大学动物实验中心进行实验。采用随机数字表法将SPF级雄性C57BL/6小鼠9只随机分为对照组、SIC组和TLR4抑制剂干预组(干预组),每组3只。SIC组腹腔注射LPS(15 mg/kg)构建SIC小鼠模型。干预组于构建SIC小鼠模型前1 h经尾静脉以0.3 mg·kg^(-1)·h^(-1)速度泵入TLR4抑制剂预处理。对照组腹腔注射生理盐水(15 mg/kg)。于制模24 h后处死小鼠留取心脏,采用苏木精—伊红(HE)染色法观察小鼠心肌组织病理形态变化,酶联免疫吸附试验(ELISA)法测定各组小鼠血清肌钙蛋白(cTnI)、肌酸激酶同工酶(CK-MB)、血浆脑利钠肽(BNP)、肿瘤坏死因子α(TNF-α)、白介素6(IL-6)、IL-1β的含量,蛋白质免疫印迹试验(Western Blot)法检测TLR4、NF-κB蛋白表达情况。结果对照组心肌细胞结构正常;SIC组心肌纤维排列紊乱,部分心肌细胞水肿,间质内可见明显炎性细胞浸润;干预组组织形态结构相对完整,心肌纤维排列规则,间质内可见零散炎性细胞浸润。小鼠心肌损伤标志物cTnI、CK-MB、BNP:SIC组>干预组>对照组(F=437.637、631.009、378.443,P均<0.001);炎性因子TNF-α、IL-1β、IL-6表达:SIC组>干预组>对照组(F=546.621、329.266、665.091,P均<0.001);TLR4蛋白表达水平:SIC组>对照组>干预组(F=388.491,P<0.001),NF-κB蛋白表达水平:SIC组>干预组>对照组(F=77.421,P<0.001)。且通过进一步行相关性分析示炎性因子TNF-α、IL-1β、IL-6水平与TLR4、NF-κB蛋白表达呈正相关(r=0.990、0.988、0.994,0.976、0.981、0.971,P均<0.001)。结论TLR4抑制剂可通过下调TLR4/NF-κB信号通路降低脓毒性心肌病小鼠炎性反应,对SIC小鼠的心肌损伤起改善作用。

Objective To explore the improvement effect and possible mechanism of Toll like receptor 4(TLR-4)inhibitors on lipopolysaccharide(LPS)induced myocardial injury in septic cardiomyopathy(SIC)mice.Methods The experiment will be conducted at the Animal Experiment Center of Wuhan University of Science and Technology from March to June 2023.Nine SPF grade male C57BL/6 mice were randomly divided into a control group,SIC group,and TLR4 inhibitor intervention group(intervention group)using a random number table method,with three mice in each group.The SIC group was injected intraperitoneally with LPS(15 mg/kg)to construct a SIC mouse model.The intervention group received pre-treatment with TLR4 inhibitors at a rate of 0.3 mg·kg^(-1)·h^(-1) via the tail vein 1 hour before constructing the SIC mouse model.The control group was intraperitoneally injected with physiological saline(15 mg/kg).After 24 hours of modeling,the mice were euthanized and their hearts were collected.Hematoxylin eosin(HE)staining was used to observe the pathological changes in the myocardial tissue of the mice.Enzyme linked immunosorbent assay(ELISA)was used to determine serum troponin(cTnI),creatine kinase isoenzyme(CK-MB),plasma brain natriuretic peptide(BNP),and tumor necrosis factorα(TNF-α),Interleukin-6 and IL-1βin each group of mice.Detection protein expression status of TLR4 and NFκB by Western Blot.Results The myocardial cell structure in the control group was normal;The arrangement of myocardial fibers in the SIC group was disordered,with some myocardial cells showing edema and obvious inflammatory cell infiltration in the interstitium;The intervention group had relatively intact tissue morphology and structure,with regular arrangement of myocardial fibers and scattered inflammatory cell infiltration in the interstitium.Mouse myocardial injury markers cTnI,CK-MB,BNP:SIC group>intervention group>control group(F=437.637,631.009,378.443,P<0.001);Inflammatory factor TNF-α,IL-1β,IL-6 expression:SIC group>intervention group>control group(F=546.621,329.266,665.091,all P<0.001);TLR4 protein expression level:SIC group>control group>intervention group(F=388.491,P<0.001),NF-κB protein expression level:SIC group>intervention group>control group(F=77.421,P<0.001).Further correlation analysis revealed the inflammatory factor TNF-α,IL-1β,IL-6 levels and TLR4,NF-κB were positively correlated(r=0.990,0.988,0.994,0.976,0.981,0.971,all P<0.001).Conclusion TLR4 inhibitors can downregulate TLR4/NF-κThe B signaling pathway reduces inflammatory response in septic cardiomyopathy mice and improves myocardial injury in SIC mice.