小檗碱对高糖诱导血管内皮细胞损伤中蛋白O-GlcNAc修饰和氧化应激的影响
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摘要
目的观察小檗碱(BBR)对高糖诱导的人脐静脉内皮细胞(HUVECs)损伤中蛋白O-GlcNAc修饰和氧化应激的影响及机制。方法将4～8代的HUVECs分为正常组、高糖组、高糖+腺苷酸激活蛋白激酶(AMPK)抑制剂Compound C(CC)组、高糖+BBR组、高糖+CC+BBR组。通过Western blot法检测AMPK总蛋白、磷酸化蛋白和OGlcNAc蛋白变化,采用DHE和JC-1染色分别检测细胞内活性氧(ROS)水平和线粒体膜电位变化。结果高糖刺激显著降低细胞AMPK蛋白磷酸化水平和线粒体膜电位,增加O-GlcNAc蛋白和ROS水平,差异有统计学意义(P<0.05)。BBR能够增加细胞AMPK蛋白磷酸化水平和线粒体膜电位,降低O-GlcNAc蛋白和ROS水平,差异有统计学意义(P<0.05)。CC能够显著抑制AMPK蛋白磷酸化水平,减弱BBR对高糖刺激下HUVECs降低O-GlcNAc蛋白和ROS水平和升高线粒体膜电位的保护作用。结论 BBR可能通过AMPK信号通路实现降低高糖刺激下HUVECs的蛋白O-GlcNAc修饰水平及氧化应激而发挥血管保护作用。
Objective To investigate the effects of berberine on protein O-GlcNAcylation and oxidative stress in vascular endothelial cells injury induced by high-glucose,and explore the mechanisms involved in the progression. Methods Human umbilical vein endothelial cells(HUVECs)were divided into five groups:control group,high glucose-induced model group,Compound C group(AMPK inhibitor Compound C treatment plus model),berberine group(berberine treatment plus model),and Compound C and berberine group(Compound C and berberine treatment plus model). Western blotting was used to elevate the protein O-GlcNAcylation level and AMPK signal pathway activation. The reactive oxygen species(ROS)and mitochondrial membrane potential were tested by DHE and JC-1 staining,respectively. Results The protein O-GlcNAcylation and ROS level were significantly increased,and the phosphorylated AMPK level and mitochondrial membrane potential were reduced in HUVECs induced by high glucose stimulation. Berberine increased the phosphorylated AMPK level and mitochondrial membrane potential. Meanwhile,berberine decreased the protein O-GlcNAcylation and ROS level in the injured HUVECs. Moreover,the AMPK inhibitor attenuated the abovementioned protective effects of berberine.Conclusion Berberine might decrease the protein O-GlcNAcylation and oxidative stress of HUVECs injury induced by high glucose stimulation via AMPK signal pathway, thereby offering a potential therapeutic option for cardiovascular complications of diabetes.
Objective To investigate the effects of berberine on protein O-GlcNAcylation and oxidative stress in vascular endothelial cells injury induced by high-glucose,and explore the mechanisms involved in the progression. Methods Human umbilical vein endothelial cells(HUVECs)were divided into five groups:control group,high glucose-induced model group,Compound C group(AMPK inhibitor Compound C treatment plus model),berberine group(berberine treatment plus model),and Compound C and berberine group(Compound C and berberine treatment plus model). Western blotting was used to elevate the protein O-GlcNAcylation level and AMPK signal pathway activation. The reactive oxygen species(ROS)and mitochondrial membrane potential were tested by DHE and JC-1 staining,respectively. Results The protein O-GlcNAcylation and ROS level were significantly increased,and the phosphorylated AMPK level and mitochondrial membrane potential were reduced in HUVECs induced by high glucose stimulation. Berberine increased the phosphorylated AMPK level and mitochondrial membrane potential. Meanwhile,berberine decreased the protein O-GlcNAcylation and ROS level in the injured HUVECs. Moreover,the AMPK inhibitor attenuated the abovementioned protective effects of berberine.Conclusion Berberine might decrease the protein O-GlcNAcylation and oxidative stress of HUVECs injury induced by high glucose stimulation via AMPK signal pathway, thereby offering a potential therapeutic option for cardiovascular complications of diabetes.
引文
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[12]Yerra VG,Kalvala AK,Sherkhane B,et al.Adenosine monophosphate-activated protein kinase modulation by berberine attenuates mitochondrial deficits and redox imbalance in experimental diabetic neuropathy[J].Neuropharmacology,2018,131:256-270.
[13]Lan J,Zhao Y,Dong F,et al.Meta-analysis of the effect and safety of berberine in the treatment of type 2 diabetes mellitus,hyperlipemia and hypertension[J].J Ethnopharmacol,2015,161:69-81.
[14]Chang W,Chen L,Hatch GM.Berberine as a therapy for type 2diabetes and its complications:From mechanism of action to clinical studies[J].Biochem Cell Biol,2015,93(5):479-486.
[15]Chang W,Li K,Guan F,et al.Berberine Pretreatment Confers Cardioprotection Against Ischemia-Reperfusion Injury in a Rat Model of Type 2 Diabetes[J].J Cardiovasc Pharmacol Ther,2016,21(5):486-494.
[16]Bullen JW,Balsbaugh JL,Chanda D,et al.Cross-talk between two essential nutrient-sensitive enzymes:O-GlcNAc transferase(OGT)and AMP-activated protein kinase(AMPK)[J].J Biol Chem,2014,289(15):10592-10606.
[17]Xu Q,Yang C,Du Y,et al.AMPK regulates histone H2B O-GlcNAcylation[J].Nucleic Acids Res,2014,42(9):5594-5604.
[2]Peterson SB,Hart GW.New insights:A role for O-GlcNAcylation in diabetic complications[J].Crit Rev Biochem Mol Biol,2016,51(3):150-161.
[3]Wende AR.Post-translational modifications of the cardiac proteome in diabetes and heart failure[J].Proteomics Clin Appl,2016,10(1):25-38.
[4]Murata K,Morino K,Ida S,et al.Lack of O-GlcNAcylation enhances exercise-dependent glucose utilization potentially through AMP-activated protein kinase activation in skeletal muscle[J].Biochem Biophys Res Commun,2018,495(2):2098-2104.
[5]Chandirasegaran G,Elanchezhiyan C,Ghosh K,et al.Berberine chloride ameliorates oxidative stress,inflammation and apoptosis in the pancreas of Streptozotocin induced diabetic rats[J].Biomed Pharmacother,2017,95:175-185.
[6]Jeon BT,Heo RW,Jeong EA,et al.Effects of caloric restriction on O-GlcNAcylation,Ca(2+)signaling,and learning impairment in the hippocampus of ob/ob mice[J].Neurobiol Aging,2016,44:127-137.
[7]da Costa RM,da Silva JF,Alves JV,et al.Increased O-GlcNAcylation of Endothelial Nitric Oxide Synthase Compromises the Anti-contractile Properties of Perivascular Adipose Tissue in Metabolic Syndrome[J].Front Physiol,2018,9:341.
[8]Qin CX,Sleaby R,Davidoff AJ,et al.Insights into the role of maladaptive hexosamine biosynthesis and O-GlcNAcylation in development of diabetic cardiac complications[J].Pharmacol Res,2017,116:45-56.
[9]Sun H,Liu Q,Hu H,et al.Berberine ameliorates blockade of autophagic flux in the liver by regulating cholesterol metabolism and inhibiting COX2-prostaglandin synthesis[J].Cell Death Dis,2018,9(8):824.
[10]Wang K,Feng X,Chai L,et al.The metabolism of berberine and its contribution to the pharmacological effects[J].Drug Metab Rev,2017,49(2):139-157.
[11]Imenshahidi M,Hosseinzadeh H.Berberis Vulgaris and Berberine:An Update Review[J].Phytother Res,2016,30(11):1745-1764.
[12]Yerra VG,Kalvala AK,Sherkhane B,et al.Adenosine monophosphate-activated protein kinase modulation by berberine attenuates mitochondrial deficits and redox imbalance in experimental diabetic neuropathy[J].Neuropharmacology,2018,131:256-270.
[13]Lan J,Zhao Y,Dong F,et al.Meta-analysis of the effect and safety of berberine in the treatment of type 2 diabetes mellitus,hyperlipemia and hypertension[J].J Ethnopharmacol,2015,161:69-81.
[14]Chang W,Chen L,Hatch GM.Berberine as a therapy for type 2diabetes and its complications:From mechanism of action to clinical studies[J].Biochem Cell Biol,2015,93(5):479-486.
[15]Chang W,Li K,Guan F,et al.Berberine Pretreatment Confers Cardioprotection Against Ischemia-Reperfusion Injury in a Rat Model of Type 2 Diabetes[J].J Cardiovasc Pharmacol Ther,2016,21(5):486-494.
[16]Bullen JW,Balsbaugh JL,Chanda D,et al.Cross-talk between two essential nutrient-sensitive enzymes:O-GlcNAc transferase(OGT)and AMP-activated protein kinase(AMPK)[J].J Biol Chem,2014,289(15):10592-10606.
[17]Xu Q,Yang C,Du Y,et al.AMPK regulates histone H2B O-GlcNAcylation[J].Nucleic Acids Res,2014,42(9):5594-5604.