左归降糖舒心方含药血浆对ox-LDL诱导小鼠巨噬细胞泡沫化和凋亡的影响  

Effect of Zuogui Jiangtang Shuxin Formula Medicated Plasma on Foaming and Apoptosis of Macrophage Induced by ox-LDL Through PERK/eIF2α/ATF4 Signaling Pathway

作　　者：杨金伟 赵灿 刘秀 吴勇军[4] 喻嵘 YANG Jin-wei;ZHAO Can;LIU Xiu;WU Yong-jun;YU Rong(The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, 410208, China;Graduate School, Hunan University of Chinese Medicine, Changsha, 410208, China;Key Laboratory of Colleges and Universities, Traditional Chinese Medicine Prescription and Transformation of Medical Laboratory, Changsha, 410208, China;School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, China)

机构地区：[1]湖南中医药大学第一中医临床学院,湖南长沙410208 [2]湖南中医药大学研究生院,湖南长沙410208 [3]中医方证研究转化医学湖南省重点实验室,湖南长沙410208 [4]湖南中医药大学药学院,湖南长沙410208

出　　处：《南京中医药大学学报》2020年第3期370-375,共6页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家自然科学基金(81573956);国家重点研发计划中医药现代化研究重点专项(2018YFC1704300);湖南省研究生科研创新项目(CX2018B471);湖南中医药大学中医学一级学科开放性基金(2018ZYX44)。

摘　　要：目的探讨左归降糖舒心方含药血浆对氧化低密度脂蛋白(ox-LDL)诱导巨噬细胞泡沫化和凋亡的影响及其作用机制。方法将巨噬细胞J774A.1随机分为正常组、模型组、牛磺熊去氧胆酸(TUDCA)组、左归降糖舒心方含药血浆组、西药联合含药血浆组,每组5个复孔。除正常组外其余各组以ox-LDL(100 mg/L)处理构建巨噬细胞泡沫化模型。正常组和模型组正常培养,其余各组分别用10%相应含药血浆培养24 h。CCK-8法比较各组J774A.1细胞增殖情况,计算细胞抑制率;油红O染色检测细胞内脂质蓄积水平;Western blot检测双链RNA样内质网激酶(PEPK)、活化转录因子4(ATF4)、真核翻译起始因子2α(eIF2α)、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)及半胱氨酸蛋白酶蛋白-12(Caspase-12)的表达水平。结果与正常组比较,模型组细胞抑制率增高,在ox-LDL浓度为100 mg/L时细胞抑制率为(50.06±0.09)%,有明显的脂质颗粒蓄积(P<0.01)。与模型组比较,各药物干预组细胞抑制率降低,可有效阻断ox-LDL的摄入及脂质堆积,其中左归降糖舒心方含药血浆组优于西药联合含药血浆组,与TUDCA组作用效应相当。ox-LDL可诱导细胞内质网应激相关蛋白p-PERK、p-eIF2α、ATF4、CHOP与Caspase-12表达上调。左归降糖舒心方含药血浆可显著抑制100 mg/L ox-LDL所致的p-PERK、p-eIF2α、ATF4的上调,降低内质网相关凋亡蛋白CHOP与Caspase-12的表达(P<0.01),和TUDCA组作用相当(P>0.05),其效果较西药联合含药血浆组明显(P<0.01)。结论左归降糖舒心方含药血浆能有效改善ox-LDL诱导的巨噬细胞泡沫化、细胞凋亡及内质网应激状态,其作用机制可能与调控内质网应激信号通路PERK/eIF2α/ATF4通路,降低细胞内CHOP与Caspase-12表达有关。OBJECTIVE To investigate the effect of Zuogui Jiangtang Shuxin Formula medicated plasma on ox-LDL-induced PERK/eIF2α/ATF4 integrated stress response and activation of apoptosis key signaling molecules CHOP and Caspase-12.METHEOD J774A.1 cells were randomly divided into 5 groups:normal group,model group,TUDCA group,Zuogui Jiangtang Shuxin Formula medicated plasma group and western medicine combination(metformin+atorvastatin calcium)medicated plasma group.All the drug intervention groups were pretreated with ox-LDL to establish an experimental model for macrophage foam cell formation.The cells of drug intervention groups were cultured in 10%different drug containing plasma,respectively.Cells of normal and model groups were cultured in normal culture medium.The growth inhibition of J774A.1 were tested by CCK-8 essay after 24 h culture.Oil Red O Dyeing experiment was used to show the cellular lipid droplets.The expression of p-PERK,p-eIF2α,ATF4,CHOP and Caspase-12 were detected by Western blot.RESULTS 100 mg/L ox-LDL suppressed the growth of J774A.1 cells with an inhibitory rate of(50.06±0.09)%.Oil Red O staining showed that the model group cells had more lipid droplets than normal group(P<0.01).Drug intervention groups effectively prevented the pervasion of ox-LDL to reduce lipid accumulation.Oil red O staining showed that lipid aggregated in Zuogui Jiangtang Shuxin Formula medicated plasma,compared with western medicine combination medicated plasma group(P<0.01),and the effect was equivalent to TUDCA group(P>0.05).ox-LDL up-regulated the expression of p-PERK,p-eIF2α,ATF4,CHOP and Caspase-12.Zuogui Jiangtang Shuxin Formula medicated plasma significantly restrained the expression of p-PERK,p-eIF2αand ATF4,and apparently decreased endoplasmic reticulum associated apoptosis protein CHOP and Caspase-12 expression(P<0.01).There was no significant difference in Zuogui Jiangtang Shuxin Formula medicated plasma group and TUDCA group.And Zuogui Jiangtang Shuxin Formula medicated plasma group was superior to the western

关 键 词：左归降糖舒心方含药血浆 J774A.1细胞 内质网应激 PERK/eIF2α/ATF4通路 

分 类 号：R285.5[医药卫生—中药学]