Excessive N-acetyl-p-benzoquinone imine(NAPQI)formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen(APAP)overdose caused acute liver failure(ALF).S-glutathion...Excessive N-acetyl-p-benzoquinone imine(NAPQI)formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen(APAP)overdose caused acute liver failure(ALF).S-glutathionylation is a reversible redox post-translational modification and a prospective mechanism of APAP hepatotoxicity.Glutaredoxin-1(Glrx1),a glutathione-specific thioltransferase,is a primary enzyme to catalyze deglutathionylation.The objective of this study was to explored whether and how Glrx1 is associated with the development of ALF induced by APAP.The Glrx1 knockout mice(Glrx1^(-/-))and liver-specific overexpression of Glrx1(AAV8-Glrx1)mice were produced and underwent APAPinduced ALF.Pirfenidone(PFD),a potential inducer of Glrx1,was administrated preceding APAP to assess its protective effects.Our results revealed that the hepatic total protein S-glutathionylation(PSSG)increased and the Glrx1 level reduced in mice after APAP toxicity.Glrx1^(-/-)mice were more sensitive to APAP overdose,with higher oxidative stress and more toxic metabolites of APAP.This was attributed to Glrx1 deficiency increasing the total hepatic PSSG and the S-glutathionylation of cytochrome p4503a11(Cyp3a11),which likely increased the activity of Cyp3a11.Conversely,AAV8-Glrx1 mice were defended against liver damage caused by APAP overdose by inhibiting the S-glutathionylation and activity of Cyp3a11,which reduced the toxic metabolites of APAP and oxidative stress.PFD precede administration upregulated Glrx1 expression and alleviated APAP-induced ALF by decreasing oxidative stress.We have identified the function of Glrx1 mediated PSSG in liver injury caused by APAP overdose.Increasing Glrx1 expression may be investigated for the medical treatment of APAP-caused hepatic injury.展开更多
目的:观察谷氧还蛋白1(glutaredoxin1,Grx1)在高糖诱导的血管内皮细胞凋亡中的作用及机制.方法:在高糖环境中培养人脐静脉内皮细胞,制备人脐静脉内皮细胞损伤模型.细胞分为正常对照组、损伤组、Grx1预保护组.倒置显微镜下观察细胞形态....目的:观察谷氧还蛋白1(glutaredoxin1,Grx1)在高糖诱导的血管内皮细胞凋亡中的作用及机制.方法:在高糖环境中培养人脐静脉内皮细胞,制备人脐静脉内皮细胞损伤模型.细胞分为正常对照组、损伤组、Grx1预保护组.倒置显微镜下观察细胞形态.MTT比色法检测细胞活力以初步观察Grx1对细胞损伤的作用;采用Annexin VFITC/PI双染法,流式细胞仪检测Grx1对人脐静脉内皮细胞凋亡的影响;利用Western blot法分析Grx1对Akt、JNK蛋白水平表达的影响.结果:Grx1预保护组与损伤组比较,细胞状态明显改善.与高糖损伤组比较,Grx1预保护组的细胞存活率显著提高(78%±3% vs 59%±2%,P<0.05),早期凋亡率(0.2360%±0.0156% vs 0.4156%±0.0374%,P<0.05)和晚期凋亡率(0.2433%±0.0278% vs 0.3689%±0.0083%,P<0.05)均明显降低.与正常对照组相比,高糖组p-JNK相对含量明显增多(0.64±0.07 vs 0.48±0.03,P<0.05),而高糖组p-Akt水平较正常对照组显著降低(1.29±0.035 vs 0.69±0.11,P<0.01).同时,hGrx1保护后p-JNK蛋白水平较高糖组显著降低(0.39±0.05 vs 0.64±0.07,P<0.05);而p-Akt蛋白水平较高糖组显著增高(0.69±0.11 vs 1.07±0.13,P<0.01).结论:Grx1可通过抑制JNK激活及激活Akt通路来拮抗高糖诱导的内皮细胞凋亡.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.:82025007,81930020,and 82170874)China Postdoctoral Science Foundation(Grant No.:2022M710099).
文摘Excessive N-acetyl-p-benzoquinone imine(NAPQI)formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen(APAP)overdose caused acute liver failure(ALF).S-glutathionylation is a reversible redox post-translational modification and a prospective mechanism of APAP hepatotoxicity.Glutaredoxin-1(Glrx1),a glutathione-specific thioltransferase,is a primary enzyme to catalyze deglutathionylation.The objective of this study was to explored whether and how Glrx1 is associated with the development of ALF induced by APAP.The Glrx1 knockout mice(Glrx1^(-/-))and liver-specific overexpression of Glrx1(AAV8-Glrx1)mice were produced and underwent APAPinduced ALF.Pirfenidone(PFD),a potential inducer of Glrx1,was administrated preceding APAP to assess its protective effects.Our results revealed that the hepatic total protein S-glutathionylation(PSSG)increased and the Glrx1 level reduced in mice after APAP toxicity.Glrx1^(-/-)mice were more sensitive to APAP overdose,with higher oxidative stress and more toxic metabolites of APAP.This was attributed to Glrx1 deficiency increasing the total hepatic PSSG and the S-glutathionylation of cytochrome p4503a11(Cyp3a11),which likely increased the activity of Cyp3a11.Conversely,AAV8-Glrx1 mice were defended against liver damage caused by APAP overdose by inhibiting the S-glutathionylation and activity of Cyp3a11,which reduced the toxic metabolites of APAP and oxidative stress.PFD precede administration upregulated Glrx1 expression and alleviated APAP-induced ALF by decreasing oxidative stress.We have identified the function of Glrx1 mediated PSSG in liver injury caused by APAP overdose.Increasing Glrx1 expression may be investigated for the medical treatment of APAP-caused hepatic injury.
基金Supported by Postdoctoral Researcher of Heilongjiang Postdoctoral Management Office(No.LBH-Q16225)Qiqihar Medical University(No.QY2016LX-01 and QY2016B-36)~~
文摘目的:观察谷氧还蛋白1(glutaredoxin1,Grx1)在高糖诱导的血管内皮细胞凋亡中的作用及机制.方法:在高糖环境中培养人脐静脉内皮细胞,制备人脐静脉内皮细胞损伤模型.细胞分为正常对照组、损伤组、Grx1预保护组.倒置显微镜下观察细胞形态.MTT比色法检测细胞活力以初步观察Grx1对细胞损伤的作用;采用Annexin VFITC/PI双染法,流式细胞仪检测Grx1对人脐静脉内皮细胞凋亡的影响;利用Western blot法分析Grx1对Akt、JNK蛋白水平表达的影响.结果:Grx1预保护组与损伤组比较,细胞状态明显改善.与高糖损伤组比较,Grx1预保护组的细胞存活率显著提高(78%±3% vs 59%±2%,P<0.05),早期凋亡率(0.2360%±0.0156% vs 0.4156%±0.0374%,P<0.05)和晚期凋亡率(0.2433%±0.0278% vs 0.3689%±0.0083%,P<0.05)均明显降低.与正常对照组相比,高糖组p-JNK相对含量明显增多(0.64±0.07 vs 0.48±0.03,P<0.05),而高糖组p-Akt水平较正常对照组显著降低(1.29±0.035 vs 0.69±0.11,P<0.01).同时,hGrx1保护后p-JNK蛋白水平较高糖组显著降低(0.39±0.05 vs 0.64±0.07,P<0.05);而p-Akt蛋白水平较高糖组显著增高(0.69±0.11 vs 1.07±0.13,P<0.01).结论:Grx1可通过抑制JNK激活及激活Akt通路来拮抗高糖诱导的内皮细胞凋亡.
