基于Gln/GLS1/α-KG代谢轴和线粒体凋亡信号通路探讨木瓜总三萜抗D-半乳糖诱导GES-1细胞衰老的作用机制  

Exploration of mechanism of total triterpenoids from fruits of Chaenomeles speciosa against senescent GES-1 cells induced by D-galactose based on Gln/GLS1/α-KG metabolic axis and mitochondrial apoptosis signaling pathway

作　　者：贺君宇 杨家鑫 覃诗颖 郑婧琪 国怡然 马天行 HE Jun-yu;YANG Jia-xin;QIN Shi-ying;ZHENG Jing-qi;GUO Yi-ran;MA Tian-xing(Basic Medical College,China Three Gorges University,Yichang 443002,China;College of Biological and Pharmaceutical Sciences,China Three Gorges University,Yichang 443002,China;Medical College,Nantong University,Nantong 226001,China)

机构地区：[1]三峡大学基础医学院,湖北宜昌443002 [2]三峡大学生物与制药学院,湖北宜昌443002 [3]南通大学医学院,江苏南通226001

出　　处：《中国中药杂志》2024年第8期2230-2246,共17页China Journal of Chinese Materia Medica

基　　金：湖北省科技厅重点研发大健康计划项目(2022BCE017);湖北省科技厅自然科学基金项目(2022CFB357,2022CFB427);湖北省卫生健康委员会中医药重点项目(ZY2023Z015);湖北卫生健康委员会卫生健康科研项目(WJ2023M153);湖北省宜昌市科学技术局医疗卫生研究项目(A23-1-061)。

摘　　要：木瓜总三萜(total triterpenoids from the fruits of Chaenomeles speciosa,TCS)是防治胃黏膜损伤的活性组分,具有潜在的抗衰老作用。然而,TCS是否改善胃衰老,尤其是对于其抗胃衰老的分子机制目前仍不清楚。基于此,该研究旨在探讨TCS对D-半乳糖(D-galactose,D-gal)诱导人胃黏膜上皮细胞GES-1衰老的影响及作用机制,为TCS在临床上用于预防胃衰老提供科学数据。将体外培养的GES-1细胞和转染谷氨酰胺酶1(glutaminase 1,GLS1)过表达(overexpression GLS1,GLS1-OE)质粒的GES-1细胞用D-gal诱导衰老后,给予TCS和谷氨酰胺酶1(glutaminase 1,GLS1)抑制剂bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide(BPTES),考察各组细胞存活率、衰老标志物β-半乳糖苷酶(β-galactosidase,SA-β-gal)染色阳性细胞比例、线粒体膜电位(mitochondrial membrane potential,MMP)和细胞凋亡的变化;采用酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA)法和比色法检测各组细胞上清液和细胞内GLS1活性、谷氨酰胺(glutamine,Gln)、谷氨酸(glutamate,Glu)、α-酮戊二酸(α-ketoglutarate,α-KG)、尿素和氨水平;采用实时荧光定量PCR和Western blot法检测各组细胞中GLS1、线粒体凋亡信号通路相关基因mRNA和蛋白表达情况。结果显示,与D-gal模型组和GLS1-OE D-gal模型组相比,TCS可显著降低D-gal诱导衰老GES-1细胞和GLS1-OE衰老GES-1细胞SA-β-gal染色阳性细胞率和MMP,抑制衰老细胞存活,促进其细胞凋亡(P<0.01),降低上清液和细胞内GLS1活性和Gln、Glu、α-KG、尿素、氨含量(P<0.01),降低线粒体中细胞色素C(cytochrome C,Cyto C)浓度以及细胞中GLS1、增殖细胞核抗原mRNA和蛋白表达(P<0.01),降低细胞中Bcl-2、Bcl-xl mRNA表达和前体半胱氨酸蛋白酶(pro-caspase)-9、pro-caspase-3蛋白表达以及Bcl-2/Bax和Bcl-xl/Bad比值(P<0.01),升高胞质中Cyto C浓度和细胞中Bax、Bad、凋亡蛋白酶激活因子1(apoptosis protease activating factor 1,Apaf-1)mRTotal triterpenoids from the fruits of Chaenomeles speciosa(TCS)are active components in the prevention and treatment of gastric mucosal damage,which have potential anti-aging effects.However,it is still unclear whether TCS can improve gastric aging,especially its molecular mechanism against gastric aging.On this basis,this study explored the effect and mechanism of TCS on senescent GES-1 cells induced by D-galactose(D-gal)to provide scientific data for the clinical use of TCS to prevent gastric aging.GES-1 cells cultured in vitro and those transfected with overexpression GLS1(GLS1-OE)plasmid of glutaminase 1(GLS1)were induced to aging by D-gal,and then TCS and or GLS1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide(BPTES)were given.Cell survival rate,positive rate ofβ-galactosidase(SA-β-gal)staining,mitochondrial membrane potential(MMP),and apoptosis were investigated.GLS1 activity,levels of glutamine(Gln),glutamate(Glu),α-ketoglutarate(α-KG),urea,and ammonia in supernatant and cells were detected by enzyme-linked immunosorbent assay(ELISA)and colorimetric methods.The mRNA and protein expressions of GLS1 and the related genes of the mitochondrial apoptosis signaling pathway were measured by real-time fluorescence quantitative PCR and Western blot.The results manifested that compared with the D-gal model group and GLS1-OE D-gal model group,TCS significantly decreased the SA-β-gal staining positive cell rate and MMP of D-gal-induced senescent GES-1 cells and GLS1-OE senescent GES-1 cells,inhibited the survival of senescent cells,and promoted their apoptosis(P<0.01).It decreased the activity of GLS1 and the content of Gln,Glu,α-KG,urea,and ammonia in supernatant and cell(P<0.01),reduced the concentration of cytochrome C(Cyto C)in mitochondria and the mRNA and protein expressions of GLS1 and proliferating nuclear antigen in cells(P<0.01).The mRNA expression of Bcl-2 and Bcl-xl,the protein expression of pro-caspase-9 and pro-caspase-3,and the ratio of Bcl-2/Bax and Bcl-xl/Bad in cells wer

关 键 词：木瓜总三萜 GES-1细胞 D-半乳糖 衰老 谷氨酰胺/谷氨酰胺酶1/α-酮戊二酸代谢轴 线粒体凋亡通路 

分 类 号：R285[医药卫生—中药学]