基于PERK-eIF2α-CHOP通路调控巨噬细胞胆固醇稳态及冠心康含药血清干预机制研究

Perk-eIF2α-CHOP Pathway is Used to Regulate Cholesterol Homeostasis in Macrophages and the Intervention Mechanism of Guanxinkang Containing Serum

目的观察具有益气化痰活血作用的冠心康对人单核/巨噬细胞株THP-1细胞PERK-eIF2α信号通路及相关活化转录因子(ATF)和C/EBP同源转录因子(CHOP)的影响,探究冠心康对胆固醇在巨噬细胞内质网中的代谢稳态调节及作为“脂毒”流出后异常沉积的相关作用。方法采用100 ng·mL^(-1)PMA及80μg·mL^(-1)氧化低密度脂蛋白诱导THP-1细胞株,构建巨噬细胞模型,并随机分为模型组、冠心康组、辛伐他汀组、正常组。分别采用10%小牛血清及含药血清干预模型细胞72 h后收集细胞。HE染色观察巨噬细胞病理形态变化,同时进行胆固醇及脂滴含量测定。分别采用Real-time PCR及Western blot法检测各组细胞PERK、eIF2α、ATF、CHOP mRNA及蛋白的表达。结果HE染色观察到模型组细胞内呈现较多红色脂滴,经胆固醇含量测定,明显高于正常组,脂滴含量测定与前者一致(P<0.01)。模型组细胞ATF、CHOP基因表达水平均显著升高(P<0.01)。与模型组比较,冠心康组和辛伐他汀组细胞ATF、CHOP基因表达水平均显著降低(P<0.01)。PERK磷酸化以及CHOP蛋白表达水平在冠心康和辛伐他汀干预组表现为显著下调,与模型组比较,差异具有统计学意义(P<0.05)。结论冠心康可能通过调控PERK-eIF2α-CHOP通路相关基因及蛋白,减少泡沫细胞形成,从而减轻内质网应激,维持细胞内胆固醇稳态,减少细胞内胆固醇沉积。

Objective To explore the curative effect of Guanxinkang with Qi,phlegm and blood circulation on the PERKe IF2αsignaling pathway and related activated transcription factor(ATF)and C/EBP homologous transcription factor(CHOP)in human monocyte/macrophage THP-1 cell line,and To explore the related effects of Guanxinkang on the metabolic homeostasis regulation of cholesterol in the endoplasmic reticulum of macrophages and the abnormal deposition of cholesterol after efflux as"lipid poison".Methods THP-1 cell line was induced by 100 ng·mL^(-1)PMA and 80μg·mL^(-1)oxidized low density lipoprotein,and the macrophage model was constructed,and randomly divided into model group,Guanxinkang group,simvastatin group and normal group.The model cells were treated with 10%calf serum and each drug serum,and then collected 72 hours later.HE staining was used to observe the pathological changes of macrophages,and the content of cholesterol and lipid droplets was determined.The mRNA and protein expressions of PERK,eIF2α,ATF and CHOP were detected by Real-time PCR and Western blot in each group.Results There were more red lipid droplets in the cells of the model group by HE staining,which were significantly higher than that of the normal group by cholesterol content determination,and the lipid droplets content determination was consistent with the former(P<0.01).Compared with the normal group and the model group,The latter significantly increased the expression levels of ATF and CHOP genes(P<0.01).In comparison to model group,the gene expression levels of ATF and CHOP in Guanxinkang group and simvastatin group were significantly improvement(P<0.01).Guanxinkang group and simvastatin group could significantly downregulate the phosphorylation of PERK and the level of CHOP protein,and the difference was statistically significant compared with model group(P<0.05).Conclusions Guanxinkang may reduce the formation of foam cells by regulating the genes and proteins related to the PERK-eIF2α-CHOP pathway,thereby alleviating endoplasmic reticulum stress,maintaining intracellular cholesterol homeostasis and reducing intracellular cholesterol deposition.