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-1α pathway

关键词:  橙皮苷  mTOR/HIF-1α通路  高糖  肾小球系膜细胞  自噬

Key words:  hesperidin  mTOR/HIF-1α pathway  high glucose  glomerular mesangial cell  autophagy

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。

Abstract:

[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-1α related proteins. [Results] Compared with the control group，there was no statistically obvious difference in absorbance value，EdU positivity rate，TNF-α，IL-1β levels，apoptosis rate，LC3，p62，ATG5 mRNA expression，p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR，and HIF-1α expression in the mannitol group（P>0.05）. After high glucose induction，the absorbance value，EdU positivity rate，TNF-α，IL-1β levels，p62 mRNA expression，p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR，HIF-1α expression 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-1β levels，p62 mRNA expression，p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR，and HIF-1α expression were obviously reduced compared to the high glucose group. The apoptosis rate，LC3，and ATG5 mRNA expression were obviously increased，and there was a difference between groups（P<0.05）. After treatment with 60 mmol/L hesperidin combined with IGF-1，the absorbance value，EdU positive rate，TNF-α，IL-1β levels，p62 mRNA expression，p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR，and HIF-1α expression were obviously increased compared to 60 mmol/L hesperidin alone treatment. The apoptosis rate，LC3，and ATG5 mRNA expression were obviously reduced（P<0.05）. [Conclusion] Hesperidin promotes high glucose induced autophagy in mesangial cells and inhibits their proliferation by regulating the mTOR/HIF-1α pathway.