葡萄籽原花青素通过Nrf2信号通路拮抗高糖高脂诱导的MIN6细胞铁死亡

Grape Seed Procyanidin Extract Inhibited High Glucose and High Fat-Induced Ferroptosis through the Nrf2 Signaling Pathway in MIN6 Cells

目的:铁死亡可能是高糖高脂诱导胰岛β细胞死亡的一种主要形式,本研究探讨葡萄籽原花青素提取物(grape seed procyanidin extract,GSPE)对高糖高脂诱导胰岛β细胞铁死亡的拮抗作用机制。方法:用25 mmol/L葡萄糖和200μmol/L棕榈酸钠干预MIN6细胞建立胰岛β细胞损伤模型,进一步用不同质量浓度10、20、30、40、50、75、100 mg/L的GSPE以及核转录因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)小干扰RNA(small interfering RNA,siRNA)进行干预,用细胞计数试剂盒(cell counting kit-8 assay,CCK8)检测细胞活性,用试剂盒检测细胞内谷胱甘肽(glutathione,GSH)、丙二醛(malonaldehyde,MDA)含量以及活性氧(reactive oxygen species,ROS)水平,用Western blot检测Nrf2、血红素氧化酶1(heme oxygenas e-1,HO-1)、醌氧化还原酶1(NADPH:quinone oxido-reductase-1,NQO1)、铁代谢指标转铁蛋白受体1(transferrin receptor 1,TfR1)、二价金属离子蛋白转运体(divalent metal transporter 1,DMT1)、铁蛋白(Ferritin)、铁死亡的指标蛋白溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、酰基合成酶长链家族成员4(acyl-CoA synthetase long-chain family member 4,ACSL4)蛋白表达水平。结果:与对照组相比,高糖高脂组导致细胞内铁沉积,上调TfR1、DMT1、Ferritin蛋白表达水平,增加氧化产物MDA含量、ROS、ACSL4水平,降低GSH水平,下调GPX4、SLC7A11蛋白表达水平,而GSPE干预后能够激活Nrf2/HO-1信号通路进而上调GPX4、SLC7A11蛋白表达水平,增加GSH水平从而降低MDA、ROS、ACSL4水平和铁代谢指标水平(TfR1、DMT1、Ferritin蛋白表达水平),抑制高糖高脂诱导的MIN6细胞铁死亡。联合Nrf2 siRNA干预后发现,抑制Nrf2信号通路后会抑制GSPE的保护作用,造成抗氧化酶HO-1、GPX4、SLC7A11表达水平降低。结论:GSPE可能是通过激活MIN6细胞内的Nrf2信号通路,上调抗氧化酶表达,抑制高糖高脂诱导的细胞铁死亡,从而提高MIN6细胞存活率。

Objective:Ferroptosis may be an important mechanism of high glucose and high fat-induced pancreaticβcell death.This study investigated the effect and mechanism of grape seed procyanidin extract(GSPE)on ferroptosis in pancreaticβcells induced by high glucose and high fat.Methods:An isletβcell injury model was established by treating MIN6 cells with 25 mmol/L glucose and 200μmol/L sodium palmitate.Then,the cells were transfected with nuclear factor erythroid 2-related factor 2 small-interfering RNA(Nrf2 siRNA)and treated with 10,20,30,40,50,75 and 100 mg/L GSPE.The cell activity was detected by the cell counting kit-8(CCK8)assay,and the levels of intracellular glutathione(GSH),malonaldehyde(MDA)and reactive oxygen species(ROS)were detected by commercial kits.The protein expression of Nrf2,heme oxygenase-1(HO-1),NADPH:quinone oxidoreductase-1(NQO1),iron metabolism indicator transferrin receptor 1(TfR1),divalent metal transporter 1(DMT1),ferritin,solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long-chain family member 4(ACSL4)were measured by Western blot.Results:Compared with the control group,high glucose and high fat caused intracellular iron deposition,up-regulated the protein expression levels of TfR1,DMT1 and ferritin,increased the levels of MDA,ROS and ACSL4,decreased the level of GSH and down-regulated the protein expression levels of GPX4 and SLC7A11.Intervention with GSPE could activate the Nrf2 signaling pathway to up-regulate GPX4 and SLC7A11 protein expression and increase the level of GSH.GSPE could also decrease the levels of MDA,ROS and ACSL4 and iron metabolism indices(TfR1,DMT1 and ferritin protein expression),thereby preventing ferroptosis in MIN6 cells induced by high glucose and high fat.After inhibition of the Nrf2 signaling pathway by Nrf2-siRNA transfection,the protective effect of GSPE was inhibited and the expression of the antioxidant enzymes HO-1,GPX4 and SLC7A11 was decreased.Conclusion:GSPE reduces ferroptosis induced by high glucose and high fat possibly by activating the Nrf2 signaling pathway and up-regulating antioxidant enzymes,consequently improving the cell viability of MIN6 cells.