摘要
目的:白藜芦醇对高糖作用下肾小管上皮细胞(HK-2)氧化应激介导的凋亡影响及作用机制。方法:体外培养肾小管上皮细胞,高糖作用于肾小管上皮细胞诱导其凋亡,设置空白对照组、模型组(30 mmol·L~(-1)高糖处理)、不同浓度(5、10μmol·L~(-1))白藜芦醇组,LY294002(PI3K抑制剂)组(10μmol·L~(-1))。使用MTT法检测各组细胞活力;流式细胞术检测各组细胞凋亡率;JC-1检测线粒体膜电位(MMP);DHE荧光探针检测ROS的变化;特定试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH-PX)含量;Western-blot法检测各组细胞色素C(CYT-C)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平以及Akt的磷酸化情况。结果:与空白对照组相比高糖损伤组能够显著降低肾小管上皮细胞的活力,增加细胞中ROS的含量和凋亡率,LDH(细胞外)、MDA含量显著增加,SOD、GSH-PX活性显著下降,同时降低线粒体膜电位,增加了CYT-C、Caspase-3的表达,而p-Akt/Akt表达降低(P<0.01)。不同浓度白藜芦醇(起效剂量5μmol·L~(-1),高剂量10μmol·L~(-1))和高糖同时作用肾小管上皮细胞时,细胞存活率逐渐上升,ROS含量及凋亡率逐渐下降,LDH、MDA含量逐渐下降,SOD、GSH-PX活性随之上升,线粒体膜电位提升,CYT-C、Caspase-3表达显著下降,p-Akt/Akt表达增加(P<0.01)。LY294002(PI3K抑制剂)组上述指标的检测结果与白藜芦醇组相反。结论:白藜芦醇可能部分通过激活PI3K/Akt通路抑制氧化应激及其诱导的凋亡,从而对肾小管上皮细胞发挥保护作用。
Objective:To investigate the effect of resveratrol on oxidative stress-mediated apoptosis in renal tubular epithelial cells(HK-2)under high glucose action and its mechanism of action.Methods:HK-2 was cultured in vitro.High glucose acted on HK-2 to induce apoptosis mediated by oxidative stress.The cell viability of each group was detected by MTT assay.The apoptosis rate of cells in each group was detected by flow cytometry.The MMP of each group was detected by JC-1.The ROS of each group was detected by DHE.The LDH,MDA,SOD and GSH-PX of each group was detected by specific kit.Western blot was used to detect the expression of protein(CYT-C,Caspase-3 and p-Akt/Akt)in each group.Results:Resveratrol significantly enhanced HK-2 viability and increased the activities of antioxidant enzymes(superoxide dismutase,catalase),scavenged reactive oxygen species(ROS),raised MMP.Resveratrol also reduced the rate of apoptosis of cells.Resveratrol downregulated oxidative stress(CYT-C)and its mediated apoptosis pathway related protein(Caspase-3)expression.Resveratrol also activated phosphorylation of Akt,whereas the addition of LY294002,a pharmacological inhibitor of PI3 K,blocked resveratrol-induced downregulation of protein(CYT-C,Caspase-3)expression and cytoprotection.Conclusion:Resveratrol can prevent apoptosis of HK-2 through inhibiting apoptosis mediated by oxidative stress,which may protect vascular endothelial cells and cardiovascular system.The main mechanism responsible for the inhibition of oxidative stress is the activation of the PI3 K-Akt pathway.
引文
[1] THAKUR S,VISWANADHAPALLI S,KOPP J B,et al.Activation of AMP-activated protein kinase prevents TGF-β1-induced epithelial-mesenchymal transition and myofibroblast activation[J].Am J Pathol,2015,185(8):2168-2180.
[2] 吴海江,邓新娜,史永红,等.白藜芦醇对高糖刺激下人肾小管上皮细胞发生EMT的作用[J].中国细胞生物学学报,2015,37(11):1522-1527.
[3] 宋碧辉,谢红萍.白藜芦醇对高糖诱导肾小管上皮细胞凋亡的保护作用及机制[J].中国老年学,2016,36(19):4686-4689.
[4] 游娜,王雅云,董成龙,等.白藜芦醇对高糖状态下大鼠肾小管上皮细胞增殖、凋亡的影响及机制[J].南京医科大学学报:自然科学版,2015,13(4):485-489.
[5] 张苏皖,李素梅.糖尿病肾病与线粒体氧化应激[J].临床与病理杂志,2011,31(6):535-538.
[6] 崔伟,王高频,王洪新,等.黄芪甲苷通过激活PI3K/Akt通路抑制内皮细胞内质网应激介导的细胞凋亡的研究[J].中药药理与临床,2018,34(5):39-44.
[7] KANG K A,WANG Z H,ZHANG R,et al.Myricetin Protects Cells against Oxidative Stress-Induced Apoptosis via Regulation of PI3K/Akt and MAPK Signaling Pathways[J].International Journal of Molecular Sciences,2010,11(11):4348-4360.
[8] LI Q,YUE Y F,CHEN L,et al.Resveratrol Sensitizes Carfilzomib-Induced Apoptosis via Promoting Oxidative Stress in Multiple Myeloma Cells[J].Front Pharmacol,2018,9(24):334.
[9] WANG R,MOYUNG K C,ZHAO Y J,et al.A Mechanism for the Temporal Potentiation of Genipin to the Cytotoxicity of Cisplatin in Colon Cancer Cells[J].Int J Med Sci,2016,13(7):507-516.
[10] THILO F,LEE M,XIA S,et al.High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in humanpodocytes[J].Biochem Biophys Res Commun,2014,450(1):312-317.
[11] JHA J C,GRAY S P,BARIT D,et al.Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy[J].J Am Soc Nephrol,2014,25(6):1237-1254.
[12] HUNJOO H A,HIBAHL L E.Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose[J].Kidney Int Suppl,2000,77:S19-S25.
[13] GU D M,CHEN Y,ZHANG X B,et al.Effect of ketamine on oxidative stress in Lipopolysaccharide-stimulated rat alveolar macrophage cell line[J].J Clin Med Pract,2008,12(13):1-7.
[14] SHAW R J,CANTLEY L C.Ras,PI(3)K and mTOR signalling controls tumour cell growth[J].Nature,2006,441(70):424-430.
[15] ALESSANDRA G,MINGCHUAN L.Phosphoinositide 3-kinase:friend and foe in cardiovascular disease[J].Front Pharmacol,2015,6(13):16-19.
[16] 刘亭,李月婷,李勇军.荭草花醇提物对H2O2诱导的H9c2细胞氧化损伤的保护作用机制研究[J].天然产物研究与开发,2018,14(2):23-26.
