摘要
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1h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24h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.
Abstract
Objective To explore the improvement effect and possible mechanismof Toll like receptor4(TLR-4)in-hibitors on lipopolysaccharide(LPS)induced myocardial injury in septic cardiomyopathy(SIC)mice.Methods The experi-ment 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 in-tervention 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 factor α(TNF-α),Interleukin-6 and IL-1 βin each group of mice.Detection protein expression status of TLR4 and NF κ B by West-ern 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 myo-cardial 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-1 β,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-κ B protein expression level:SIC group>intervention group>control group(F=77.421,P<0.001).Further correlation analysis revealed the inflam-matory 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-κ The B signaling pathway reduces inflamma-tory response in septic cardiomyopathy mice and improves myocardial injury in SIC mice.
基金项目
湖北省自然科学基金面上项目(2019CFB621)
国家科技期刊平台
NSTL
万方数据