血色素加氧酶-1与甾醇异构酶的相互作用拮抗高脂刺激诱发心肌细胞损伤的作用机制

The study of the mechanism under the cytoprotective function of HO-1 on cardiomyocytes

【目的】探究血色素加氧酶-1(heme oxygenase-1,HO-1)在胆固醇诱导的高脂环境中对大鼠H9c2心肌细胞的保护作用机制。【方法】MTT法检测10、100和500 mmol/L胆固醇作用H9c2细胞12、24、36、48 h后对细胞增殖活力的影响,油红O染色法检测细胞内脂肪沉积水平。通过免疫共沉淀结合质谱鉴定策略(Co-IP coupled to LC-MS/MS)筛选HO-1相互作用蛋白,经生物信息软件Cytoscape重塑HO-1相互作分子信号网络。通过细胞免疫荧光(immunocytofluorescence,ICC)和Western blotting技术验证HO-1和EBP以及相关蛋白激酶B(protein kinase B,AKT)、雷帕霉素靶蛋白(mechanistic target of rapamycin,mTOR)和糖代谢过程等信号通路相关分子的表达量。【结果】MTT结果显示10,100和500 mmol/L胆固醇显著抑制H9c2细胞增殖活力(P<0.05),且抑制效应呈现时间-浓度依赖性。质谱结果显示HO-1可以与137个蛋白分子发生相互作用,其中互作蛋白甾醇异构酶(emopamil-binding protein,EBP)与HO-1的结合可能参与调节脂类代谢进程。Western blotting以及ICC结果证明了这一相互作用。Western blotting结果还显示胆固醇刺激后AKT和mTOR信号通路被活化,糖代谢相关蛋白表达受到抑制。【结论】HO-1通过与EBP相互作用,通过激活AKT、mTOR和抑制糖代谢进程继而抑制H9c2心肌细胞的有氧氧化过程,缓解胆固醇对细胞的损伤刺激,执行保护心肌作用。

【Objective】To explore the mechanism of protective function of HO-1 to H9c2 cells under high fat condition induced by cholesterol.【Methods】Using MTT assay to detect the influences on cell viability of 10, 100 and 500 mmol/L stimulated H9c2 cells for12, 24, 36 and 48 h, and using Oil Red O staining to detect the accumulation of lipid in H9c2 cells. Using Co-IP coupled to LC-MS/MS to screen interaction proteins of HO-1, the network of HO-1 interaction proteins was rebuilt through bioinformatics analysis. The expression levels of HO-1, EBP and the related molecular of AKT, mTOR signal pathways and glycometabolism using ICC and Western blotting.【Results】Result of MTT assay showed that 10, 100, and 500 mmol/L cholesterol could significantly inhibits the proliferation of H9c2 cells（P〈0.05）, and presents a time-dose dependency. Result of MS analysis showed that HO-1 could interacted with 137 proteins, the interaction of HO-1 and EBP could participate in the metabolic process of lipid. And the results of Western blotting and ICC also confirmed this result. AKT and mTOR signal pathways was also activated after cholesterol stimulation, meanwhile the expression of proteins related to glycometabolism were inhibited.【Conclusion】The interaction of HO-1 and EBP could activate AKT and mTOR signal pathways and inhibit glycometabolism process to inhibit aerobic oxidation in H9c2 cells, further alleviate the stimulation of cholesterol on H9c2 cells, and preform cytoprotective function.