热休克因子1通过上调蛋白质C减轻脓毒症凝血功能障碍保护小鼠急性肺损伤

HSF1 Attenuates Coagulatory Dysfunction and Protects Mice From Acute Lung Injury by Upregulating Protein C in Sepsis

目的探讨热休克因子1(HSF1)减轻脓毒症凝血功能障碍,保护小鼠急性肺损伤的机制。方法本研究采用盲肠结扎穿孔术(cecal ligation and puncture,CLP)制备脓毒症小鼠模型,检测凝血相关指标和观察小鼠肺部病理变化,通过酶联免疫吸附实验(ELISA)、q RT-PCR和蛋白质印迹法(Western blot)等方法检测蛋白质C表达水平,通过质粒转染抑制或增强HSF1表达从而观察蛋白质C表达水平的变化,并利用生物信息学、凝胶电泳迁移实验(EMSA)和双荧光素酶报告基因实验探讨HSF1调节蛋白质C转录的机制。结果在CLP脓毒症小鼠模型中,HSF^(-/-)组小鼠的凝血活性与HSF1;组相比明显增强,肺损伤明显加重。ELISA、qRT-PCR和Western blot检测发现,HSF;脓毒症小鼠血浆和肺组织中的蛋白质C表达水平显著低于野生型小鼠。体外bEnd.3血管内皮细胞的实验结果显示,HSF1抑制脂多糖(LPS)诱导的蛋白质C表达,HSF1过表达则增强蛋白质C表达。生物信息学数据分析提示,蛋白质C启动子区含有HSF1结合元件(HSE),通过EMSA和双荧光素酶报告基因实验显示HSF1与蛋白质C启动子区域的HSE结合,从而上调蛋白质C转录。结论本研究揭示了HSF1参与小鼠脓毒症急性肺损伤的发生,并通过上调蛋白质C转录,减轻脓毒症凝血功能障碍,从而对小鼠肺组织起到保护作用。

Objective To explore the mechanism of heat shock factor(HSFl)alleviating coagulatory dysfunction in sepsis and protecting mice from acute lung injury.Methods In this study,a mouse model of sepsis was established by cecal ligation and puncture(CLP).We tested the coagulation indexes and pathological changes in the lungs of mice.Protein C expression was detected by ELISA,q RT-PCR and Western blotting.The expression level of protein C was observed by inhibiting or enhancing the expression of HSF1 by plasmid transfection,and the mechanism of HSF1 regulating protein C transcription was explored by bioinformatics,EMSA and dual luciferase reporter gene experiments.Results In the mouse model of sepsis,we discovered that the coagulatory activity of the HSF;mice was significantly enhanced and the lung injury was aggravated after CLP,compared with the HSF1;mice.ELISA,qRT-PCR and Western blot showed that the expression level of protein C in the plasma and lung tissue of the HSF;mice was lower than that in the wild-type mice in sepsis.In vitro studies also demonstrated that HSF1 interference inhibited lipopolysaccharide(LPS)-induced protein C expression,while HSF1 overexpression enhanced protein C expression in bEnd.3 vascular endothelial cells.Further bioinformatics analysis indicated that the protein C promoter region contains HSF1 binding element(HSE).EMSA and dual luciferase reporter gene experiments showed that HSF1 bound to the HSE in the promoter region of protein C,thereby directly upregulating protein C transcription.Conclusion This study revealed that HSF1 was involved in acute lung injury in sepsis mouse model.HSF1 alleviated the coagulatory dysfunction in sepsis by directly upregulating protein C transcription,thus playing a protective role in mouse lung tissue.