基于GRP78/PERK/ATF4的泽泻汤改善脂质诱导肝细胞内质网应激作用机制研究  

Mechanism Study of Zexie Decoction(泽泻汤)in Improving Endoplasmic Reticulum Stress of Lipid-Induced Hepatocytes Based on GRP78/PERK/ATF4

作　　者：王梦瑶[1] 谢治深 徐江雁[1] 丁婧 赵洁[1] 孙意冉 张效威 王辉[1] 王潘 张振强[1] WANG Mengyao;XIE Zhishen;XU Jiangyan;DING Jing;ZHAO Jie;SUN Yiran;ZHANG Xiaowei;WANG Hui;WANG Pan;ZHANG Zhenqiang(Academy of Chinese Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450046)

机构地区：[1]河南中医药大学中医药科学院,郑州450046

出　　处：《中药药理与临床》2023年第10期8-14,共7页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金项目(编号:82004019、82174267);河南省高校科技创新团队支持计划项目(编号:21IRTSTHN026);河南省高校科技创新人才支持计划资助(编号:22HASTIT048);河南省高等学校青年骨干教师(2021GGJS081)。

摘　　要：目的:基于GRP78/PERK/ATF4通路探讨泽泻汤改善肝细胞内质网应激的潜在机制。方法:采用油酸(OA)及高脂饮食诱导高脂细胞及动物内质网应激(Endoplasmic reticulum stress,ERS)模型。ER与Ca^(2+)荧光共定位观察泽泻汤对ER-Ca^(2+)稳态的调节情况;流式细胞术及Tunel法检测泽泻汤对凋亡的影响;Q-PCR检测ER核心信号1(Ern1)、转录激活因子6(Atf6)、CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein,Chop)、Grp78、Perk、X-盒结合蛋白1(Xbp1)、热休克蛋白90β1(Hsp90β1)、肿瘤坏死因子受体超家族成员10B(Tnfrsf10b)的mRNA表达;Western bolt法检测葡萄糖调节蛋白78/B细胞免疫球蛋白结合蛋白(GRP78/BIP)GRP78、类PKR的内质网激酶(PERK)、p-PERK和转录激活因子4(ATF4)的蛋白表达。结果:与正常对照组相比,模型对照组ER-Ca^(2+)明显失衡(P<0.05),肝细胞凋亡增加(P<0.01),Perk、Xbp1等ERS相关mRNA的表达上调(P<0.05),ERS伴侣标志蛋白GRP78、ATF4蛋白表达明显上调(P<0.05);与模型对照组相比,泽泻汤逆转了OA诱导的ER-Ca^(2+)失衡(P<0.01);显著改善OA及高脂饮食诱导的肝细胞凋亡(P<0.01);体内外下调ERS相关基因Ern1、Atf6、Chop、Grp78、Perk、Xbp1、Hsp90b1、Tnfrsf10b的表达(P<0.05);体内外逆转OA及高脂饮食诱导的ERS伴侣标志蛋白GRP78表达的显著上调(P<0.01),同时下调p-PERK及ATF4蛋白表达(P<0.05)。结论:泽泻汤抑制脂质诱导的ERS,其机制可能与调控GRP78/PERK/ATF4通路有关。Objective:To study the potential mechanism of Zexie Decoction(泽泻汤,ZXD)in improving endoplasmic reticulum stress(ERS)of hepatocytes based on the CRP78/PERK/ATF4 pathway.Methods:Oleic acid(OA)and high-fat diet(HFD)were used to induce high-fat cells and ERS model of animals.ER and Ca^(2+) fluorescence co-localization was used to observe the regulation of ER-Ca^(2+) by ZXD.Flow cytometry and Tunel method were used to detect the effect of ZXD on apoptosis.Western bolt method was used to detect the protein levels of glucose-regulated protein 78/B-cell immunoglobulin binding protein(GRP78/BIP),PKR-like endoplasmic reticulum kinase(PERK),p-PERK,and activating transcription factor 4(ATF4).Q-PCR was used to detect the mRNA levels of ER core signal 1(Ernl),activating transcription factor 6(Atf6),CCAAT/enhancer-binding protein-homologous protein(Chop),Grp78,Perk,X-box binding protein 1(Xbp1),heat shock protein 90β1(Hsp90β1),and tumor necrosis factor receptor superfamily 10B(Tnfrsf10b).Results:Compared with that in the normal control group,ER-Ca^(2+) was in an imbalance in the model control group(P<0.05),hepatocyte apoptosis was increased(P<0.01),the expression levels of ERS chaperone marker protein GRP78 and ATF4 protein were significantly up-regulated(P<0.05),and the mRNA expressions of ERS-related genes such as Perk and Xbpl were up-regulated(P<0.05).Compared with the model control group,ZXD reversed the imbalance of ER-Ca2 induced by OA(P<0.01).It also effectively improved hepatocyte apoptosis induced by OA and HFD(P<0.01),reversed the significant up-regulation of ERS chaperone marker protein GRP78 induced by OA and HFD in vitro and in vivo(P<0.05),and down-regulated the protein expressions of p-PERK and ATF4(P<0.05).ZXD could also down-regulated the mRNA level of ERS-related genes,namely Ernl,Atf6,Chop,Grp78,Perk,Xbpl,Hsp90b1,and Tnfrsf10b in vivo and in vitro(P<0.05).Conclusion:The mechanism of ZXD in inhibiting lipid-induced ERS may be related to the regulation of the GRP78/PERK/ATF4 pathway.

关 键 词：泽泻汤 内质网应激 葡萄糖调节蛋白78/B细胞免疫球蛋白结合蛋白/蛋白激酶R(PKR)样内质网激酶/转录激活因子4 

分 类 号：R285[医药卫生—中药学]