基于线粒体自噬和内质网应激,探究黑果枸杞提取物(Lycium ruthenicum Murray extraction,LRME)对HepG2细胞自噬的调控作用.采用流式细胞术检测HepG2细胞周期,通过qRT-PCR和Western blot测定内质网应激、线粒体自噬和自噬关键基因的表达水平.结果表明:HepG2细胞G0/G1期向S期的转化过程被LRME有效抑制;LRME极显著上调肿瘤蛋白p53(tumor protein p53, TP53)、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3, LC3)和AMP依赖的蛋白激酶(adenosine 5'-monophosphate-activated protein kinase, AMPK),并显著下调细胞周期蛋白D1(cyclin D1, CCND1)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、周期素依赖性激酶-4(cyclin-dependent kinase-4, CDK4)和哺乳动物雷帕霉素靶点(mammalian target of rapamycin, mTOR)的mRNA水平;上调内质网转录激活子4(activating transcription factor 4, ATF4)和FUN14域蛋白1(FUN14 domain containing 1, FUNDC1)的mRNA和蛋白表达.由此推测LRME可通过在体外促进内质网应激和线粒体自噬,进而诱导HepG2细胞自噬.
The effect of Lycium ruthenicum Murray extraction(LRME)on autophagy in HepG2 cells was explored based on endoplasmic reticulum stress and mitochondrial autophagy. Cell cycle of HepG2 cells were detected by flow cytometry. Besides, the expression of mitochondrial autophagy-related gene, endoplasmic reticulum stress-related gene and autophagy-related genes were tested by qRT-PCR and Western blot. It was found that HepG2 cells induced by LRME were significantly arrested from G0/G1 phase to S phase while the expression of related key factors(TP53, LC3 and AMPK)were significantly up-regulated and the expression of related key factors(CCND1, PCNA, CDK4 and mTOR)were significantly down-regulated at mRNA level. In addition, LRME could up-regulate endoplasmic reticulum stress factor(ATF4)and mitochondrial autophagy factor(FUNDC1)at mRNA and protein level. In conclusion,- DOI:10.3969/j.issn.1000-1565.2022.01.011黑果枸杞提取物基于内质网应激和线粒体自噬调控HepG2细胞自噬王梦杰,吴华,刘嘉华,张龙飞(青海大学 农牧学院,青海 西宁 810000)摘 要:基于线粒体自噬和内质网应激,探究黑果枸杞提取物(Lycium ruthenicum Murray extraction,LRME)对HepG2细胞自噬的调控作用.采用流式细胞术检测HepG2细胞周期,通过qRT-PCR和Western blot测定内质网应激、线粒体自噬和自噬关键基因的表达水平.结果表明:HepG2细胞G0/G1期向S期的转化过程被LRME有效抑制;LRME极显著上调肿瘤蛋白p53(tumor protein p53, TP53)、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3, LC3)和AMP依赖的蛋白激酶(adenosine 5'-monophosphate-activated protein kinase, AMPK),并显著下调细胞周期蛋白D1(cyclin D1, CCND1)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、周期素依赖性激酶-4(cyclin-dependent kinase-4, CDK4)和哺乳动物雷帕霉素靶点(mammalian target of rapamycin, mTOR)的mRNA水平;上调内质网转录激活子4(activating transcription factor 4, ATF4)和FUN14域蛋白1(FUN14 domain containing 1, FUNDC1)的mRNA和蛋白表达.由此推测LRME可通过在体外促进内质网应激和线粒体自噬,进而诱导HepG2细胞自噬. 关键词:线粒体自噬;内质网应激;HepG2细胞;黑果枸杞提取物中图分类号:R932 文献标志码:A 文章编号:1000-1565(2022)01-0078-07Lycium ruthenicum Murray extraction regulates autophagy based on endoplasmic reticulum stress and mitophagy in HepG2 cellsWANG Mengjie, WU Hua, LIU Jiahua, ZHANG Longfei(College of Agriculture and Animal Husbandry, Qinghai University, Xining 810000, China)Abstract: The effect of Lycium ruthenicum Murray extraction(LRME)on autophagy in HepG2 cells was explored based on endoplasmic reticulum stress and mitochondrial autophagy. Cell cycle of HepG2 cells were detected by flow cytometry. Besides, the expression of mitochondrial autophagy-related gene, endoplasmic reticulum stress-related gene and autophagy-related genes were tested by qRT-PCR and Western blot. It was found that HepG2 cells induced by LRME were significantly arrested from G0/G1 phase to S phase while the expression of related key factors(TP53, LC3 and AMPK)were significantly up-regulated and the expression of related key factors(CCND1, PCNA, CDK4 and mTOR)were significantly down-regulated at mRNA level. In addition, LRME could up-regulate endoplasmic reticulum stress factor(ATF4)and mitochondrial autophagy factor(FUNDC1)at mRNA and protein level. In conclusion,- 收稿日期:2021-06-09 基金项目:青海省科技厅应用基础研究计划项目(2018-ZJ-759) 第一作者:王梦杰(1995—),女,河南周口人,青海大学在读硕士研究生,主要从事营养与免疫方向研究. E-mial: 2583700850@qq.com 通信作者:吴华(1971—),女,四川眉山人,青海大学教授,主要从事营养与免疫方向研究. E-mial: qhwuhua@qhu.edu.cn第1期王梦杰等:黑果枸杞提取物基于内质网应激和线粒体自噬调控HepG2细胞自噬LRME can promote the mitochondrial autophagy, endoplasmic reticulum stress and autophagy of HepG2 cells in vitro.
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