橙皮苷调控mTOR/HIF-1α通路对高糖诱导的肾小球系膜细胞自噬的影响  

Effect of hesperidin on high glucose induced autophagy in glomerular mesangial cells by regulating the mTOR/HIF-1αpathway

作　　者：李淼 李瑞 杨晓芳 丰文君 LI Miao;LI Rui;YANG Xiaofang;FENG Wenjun(Department of Nephrology,Zhengzhou Yihe Hospital,Zhengzhou 450018,China)

机构地区：[1]郑州颐和医院肾内科,郑州450018

出　　处：《天津中医药》2025年第1期78-83,共6页Tianjin Journal of Traditional Chinese Medicine

基　　金：河南省医学科技攻关计划项目(LHGJ20200493)。

摘　　要：[目的]探讨橙皮苷调控哺乳动物雷帕霉素靶蛋白(mTOR)/缺氧诱导因子-1α(HIF-1α)通路对高糖诱导的肾小球系膜细胞自噬的影响。[方法]取生长良好的SV40 MES-13细胞,依次分为对照组(不进行处理)、甘露醇组(30 mmol/L甘露醇处理)、高糖组(30 mmol/L葡萄糖)、不同剂量橙皮苷组(30 mmol/L、60 mmol/L橙皮苷处理)、60 mmol/L橙皮苷+胰岛素样生长因子-1(IGF-1)组(60 mmol/L橙皮苷、100 ng/mL IGF-1处理),噻唑蓝(MTT)及5-乙炔基-2'-脱氧尿苷(EdU)检测细胞增殖;酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β水平;流式细胞仪检测细胞凋亡水平;实时荧光定量PCR(qRT-PCR)检测微管相关蛋白1轻链3(LC3)、p62、自噬相关基因5(ATG5)mRNA表达水平;蛋白免疫印迹法(Western blot)检测p-PI3K、p-Akt、p-mTOR、PI3K、Akt、mTOR、HIF-1α相关蛋白表达水平。[结果]与对照组相比,甘露醇组吸光度值、EdU阳性率、TNF-α、IL-1β水平、凋亡率、LC3、p62、ATG5 mRNA表达、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、HIF-1α表达差异无统计学意义(P>0.05);高糖诱导细胞后,吸光度值、EdU阳性率、TNF-α、IL-1β水平、p62 mRNA表达、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、HIF-1α表达较对照组、甘露醇组显著增加,凋亡率、LC3、ATG5 mRNA表达显著降低(P<0.05);经不同剂量的橙皮苷处理后,吸光度值、EdU阳性率、TNF-α、IL-1β水平、p62 mRNA表达、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、HIF-1α表达较高糖组显著降低,凋亡率、LC3、ATG5 mRNA表达显著增加,组间有差异(P<0.05),60 mmol/L橙皮苷联合IGF-1处理细胞,吸光度值、EdU阳性率、TNF-α、IL-1β水平、p62 mRNA表达、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、HIF-1α表达较60 mmol/L橙皮苷单独处理显著增加,凋亡率、LC3、ATG5 mRNA表达显著降低(P<0.05)。[结论]橙皮苷调控mTOR/HIF-1α通路促进高糖诱导的肾小球系膜细胞自噬,抑制其增殖。[Objective]To investigate the effect of hesperidin on autophagy in glomerular mesangial cells induced by high glucose by regulating the mammalian target of rapamycin(mTOR)/hypoxia-inducible factor-1α(HIF-1α)pathway.[Methods]SV40 MES-13 cells with good growth were randomly separated into control group(without treatment),mannitol group(30 mmol/L mannitol treatment),high glucose group(30 mmol/L glucose),different doses hesperidin groups(30 mmol/L,60 mmol/L hesperidin treatment),and 60 mmol/L hesperidin+insulin-like growth factor-1(IGF-1)group(60 mmol/L hesperidin,100 ng/mL IGF-1 treatment).Methylthiazolyldiphenyl-tetrazolium bromide(MTT)and 5-ethynyl-2’-deoxyuridine(EdU)were used to detect cell proliferation.Enzyme linked immunosorbent assay(ELISA)was applied to detect levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-1β.Flow cytometry was applied to detect the level of cell apoptosis.Real-time quantitative PCR(qRT-PCR)was applied to detect the mRNA expression levels of microtubule-associated protein 1 light chain 3(LC3),p62,and autophagy related gene 5(ATG5).Western blot was applied to detect the expression levels of p-PI3K,p-Akt,p-mTOR,PI3K,Akt,mTOR,and HIF-1αrelated proteins.[Results]Compared with the control group,there was no statistically obvious difference in absorbance value,EdU positivity rate,TNF-α,IL-1βlevels,apoptosis rate,LC3,p62,ATG5 mRNA expression,p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR,and HIF-1αexpression in the mannitol group(P>0.05).After high glucose induction,the absorbance value,EdU positivity rate,TNF-α,IL-1βlevels,p62 mRNA expression,p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR,HIF-1αexpression were obviously increased compared to the control group and mannitol group,while apoptosis rate,LC3,and ATG5 mRNA expression were obviously decreased(P<0.05).After treatment with different doses of hesperidin,the absorbance value,EdU positive rate,TNF-α,IL-1βlevels,p62 mRNA expression,p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR,and HIF-1αexpression were obviously reduced compared to the high glucose gr

关 键 词：橙皮苷 mTOR/HIF-1α通路 高糖 肾小球系膜细胞 自噬 

分 类 号：R587.2[医药卫生—内分泌]