非抗凝肝素通过抑制心肌细胞铁死亡减轻脂多糖诱导的脓毒症心肌损伤

N-acetylheparin alleviates lipopolysaccharide-induced sepsis myocardial injury by inhibiting cardiomyocytes ferroptosis

摘要

目的 探讨非抗凝肝素(N-acetylheparin,NAH)对脓毒症心肌损伤的作用及潜在的分子机制。 方法 将C57BL/6小鼠随机分成4组,正常组(Control组)、模型组(LPS组)、NAH治疗组(LPS+NAH组)和非抗凝肝素对照组(NAH组)。腹腔注射脂多糖(lipopolysaccharide, LPS)10 mg/kg构建脓毒症心肌损伤小鼠模型,18 h后眼球取血,收集血清和心脏组织。采用小动物超声心动图评估小鼠心功能变化,苏木精-伊红(hematoxylin-eosin, HE)染色观察心肌损伤程度,末端脱氧核苷酸转移酶介导的缺口末端标记法(terminal-deoxynucleoitidyl transferase mediated nick end labeling, TUNEL)观察心肌细胞凋亡率;酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测血清中肌酸激酶同工酶MB(creatine kinase isoenzyme MB, CKMB)、心肌肌钙蛋白I(cardiac troponin I, cTnI)和高迁移率蛋白1(high mobility group box 1 protein, HMGB1)的含量;试剂盒检测心肌组织中总谷胱甘肽过氧化物酶(glutathione, GSH)、超氧化物歧化酶(superoxide dismutase, SOD)、丙二醛(malondialdehyde, MDA)和组织铁的含量;Western blotting法检测心肌组织中高迁移率蛋白-1(high mobility group box-1 protein, HMGB1)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4, GPX4)蛋白水平的表达量。 结果 与Control组相比,LPS注射18 h后,LPS组小鼠左室射血分数、缩短分数均明显降低,血清中CK-MB和cTnI的水平升高,HE染色显示心肌组织中炎性细胞增加、组织出现扭曲变形和碎片,TUNEL染色显示心肌细胞凋亡率升高;心肌组织中铁死亡相关标志物铁含量和MDA显著升高,GSH、SOD和GPX4的表达降低。血清和心肌组织中HMGB1的表达升高。NAH干预治疗后,与LPS组相比,LPS+NAH组小鼠的左室收缩功能、心肌酶含量和心肌组织病理损伤的程度均得到改善;心肌细胞凋亡率、心肌组织中铁含量和MDA降低,GSH、SOD和GPX4的表达升高;ELISA和Western blotting检测表明血清和心肌中HMGB1的表达也显著降低;NAH组与Control组相比差异无统计学意义(P>0.05)。 结论 NAH可通过抑制HMGB1的表达,上调GSH、GPX4表达和总SOD活力,降低组织铁和MDA含量,减轻心肌细胞铁死亡,从而缓解LPS诱导的心肌损伤。

Abstract

Objective To investigate the effect of N-acetylheparin(NAH)on sepsis-induced myocardial injury and its underlying molecular mechanisms. Methods C57BL/6 mice were randomly divided into to four groups: control group(Control group), LPS-induced model group(LPS group), LPS combined with N-acetylheparin treatment group(LPS+NAH group), and N-acetylheparin(NAH group)alone group. A mouse model of sepsis-induced myocardial injury was established via intraperitoneal injection of lipopolysaccharide(LPS)at a dose of 10 mg/kg. Blood samples were collected by orbital venous puncture 18 hours post-injection, followed by serum separation and heart tissue harvesting. Cardiac function was evaluated using small animal echocardiography, while the extent of myocardial injury was assessed through hematoxylin-eosin(HE)staining, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)was used to observe the apoptosis rate of cardiomyocytes. The serum levels of creatine kinase isoenzyme MB(CK-MB), cardiac troponin I(cTnI)and high-mobility group box 1(HMGB1)were quantified using enzyme-linked immunosorbent assay(ELISA). The concentrations of total glutathione peroxidase(GSH), superoxide dismutase(SOD), malondialdehyde(MDA), and tissue iron in myocardial tissue were determined using commercial assay kits. Western blotting analysis was employed to quantify the protein expression levels of high mobility group box-1(HMGB1)and glutathione peroxidase 4(GPX4)in myocardial tissue. Results Compared with the Control group, mice in the LPS group exhibited significantly reduced left ventricular ejection fraction and fractional shortening at 18 hours post-injection. Serum CK-MB and cTnI levels were markedly elevated. HE staining revealed increased inflammatory cell infiltration and structural disarray in myocardial tissue, while TUNEL staining indicated a significantly higher rate of cardiomyocyte apoptosis. Additionally, myocardial iron content and MDA levels were significantly increased, whereas GSH, SOD, and GPX4 levels were decreased. Both serum and myocardial HMGB1 expression levels were upregulated. In contrast, NAH treatment in the LPS+NAH group effectively improved left ventricular systolic function, reduced myocardial enzyme release, and ameliorated histopathological damage. Furthermore, NAH administration attenuated cardiomyocyte apoptosis, lowered myocardial iron accumulation and MDA production, and restored antioxidant enzyme activities(GSH and SOD)as well as GPX4 expression. Notably, both serum and cardiac HMGB1 levels were significantly downregulated following NAH intervention, as confirmed by ELISA and Western blotting analysis. There was no statistically significant difference between the NAH group and the Control group. Conclusion NAH alleviates lipopolysaccharide-induced myocardial injury by suppressing HMGB1 expression, upregulating GSH and GPX4 levels and total SOD activity, reducing iron and MDA content in myocardial tissues, and thereby attenuating cardiomyocyte ferroptosis.

关键词

脓毒症 / 心肌病 / 非抗凝肝素 / 铁死亡 / 脂多糖

Key words

Sepsis / Cardiomyopathy / N-acetylheparin / Ferroptosis / Lipopolysaccharide

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基金资助

山东省医药卫生科技发展计划(202203100768)

滨州医学院科技计划项目(BY2019KJ10)