Exendin-4通过激活Nrf2/HO-1通路减轻糖尿病小鼠的肝脏氧化应激及纤维化
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摘要
目的探讨Exendin-4对链脲佐菌素(STZ)诱导的糖尿病小鼠肝脏脂质代谢、氧化应激以及纤维化的作用以及其潜在的机制。方法将C57BL/6J小鼠随机分为对照组和糖尿病组(DM)。糖尿病组小鼠给予高脂饮食4周联合STZ造模,对照组小鼠普通饮食。DM小鼠造模成功后,再随机分为糖尿病对照组(DM-con)和糖尿病干预组(DM+E4)。DM+E4组小鼠给予Exendin-4每天1 nmol/kg体重,腹腔注射1次,共8周。监测体重、随机血糖,分析各组小鼠血脂水平,RT-PCR检测肝脏脂质代谢、纤维化和氧化应激指标,HE、天狼猩红以及油红O染色观察肝脏结构变化,Western blot检测肝脏TGF-β1、Nrf2以及HO-1的表达。结果DM组小鼠的血糖和体重较对照组明显升高(P<0.001),肝脏脂质沉积、纤维化以及氧化应激较对照组也显著增加,给予Exendin-4干预未显著改善肝脏的脂质沉积,但是Exendin-4治疗后糖尿病组小鼠血糖及TG明显降低(P<0.05),肝脏纤维化指标TGF-β、α-SMA及Col-I显著降低(P<0.05),抗氧化指标Nrf2、HO-1及GPX4显著升高(P<0.01)同时Nrf2和HO-1的蛋白表达水平也显著升高(P<0.01)。结论 Exendin-4在肝脏脂质沉积未明显减轻的情况下通过激活Nrf2/HO-1信号通路显著改善糖尿病小鼠的肝脏纤维化及氧化应激。
Objective To investigate the effects of exendin-4 on hepatic lipid metabolism, fibrosis and oxidative stress in mice with streptozotocin(STZ)-induced diabetes and explore the underlying mechanisms. Methods C57 BL/6 J mice were fed with high-fat diet(HFD) for 4 weeks and received intraperitoneal injections of 120 mg/kg STZ to induce diabetes. After successful modeling, the mice were randomized into diabetic control group and exendin-4 treatment group(DM + E4), and in the latter group, the mice were given a daily dose of 1 nmol/kg of exendin-4 for 8 weeks. The changes in the body weight(BW) and random blood glucose(RBG) in the mice were recorded. The mRNA expressions of the genes related with liver lipid metabolism, fibrosis and oxidative stress were analyzed using RT-PCR, and the structural changes of the liver tissues were observed with HE, Sirius red and oil red O staining; the expressions of TGF-β1, Nrf2 and HO-1 proteins in the liver tissues were detected using Western blotting. Results The diabetic mice showed significantly higher RBG levels and BW with obvious lipid deposition, fibrosis and oxidative stress in the liver as compared with the normal control mice(P<0.001). Exendin-4 treatment of the diabetic mice did not significantly lessened liver lipid deposition but obviously reduced the levels of RBG and TG(P<0.05), lowered the expression levels of liver fibrosis-related genes TGF-β, α-SMA and Col-I(P<0.05), increased the expression levels of the antioxidant genes Nrf2, HO-1 and GPX4(P<0.01), and enhanced the protein expressions of Nrf2 and HO-1 in the liver tissues(P<0.01). Conclusion Exendin-4 improves liver fibrosis and oxidative stress in diabetic mice by activating Nrf2/HO-1 pathway without significantly reducing liver lipid deposition.
Objective To investigate the effects of exendin-4 on hepatic lipid metabolism, fibrosis and oxidative stress in mice with streptozotocin(STZ)-induced diabetes and explore the underlying mechanisms. Methods C57 BL/6 J mice were fed with high-fat diet(HFD) for 4 weeks and received intraperitoneal injections of 120 mg/kg STZ to induce diabetes. After successful modeling, the mice were randomized into diabetic control group and exendin-4 treatment group(DM + E4), and in the latter group, the mice were given a daily dose of 1 nmol/kg of exendin-4 for 8 weeks. The changes in the body weight(BW) and random blood glucose(RBG) in the mice were recorded. The mRNA expressions of the genes related with liver lipid metabolism, fibrosis and oxidative stress were analyzed using RT-PCR, and the structural changes of the liver tissues were observed with HE, Sirius red and oil red O staining; the expressions of TGF-β1, Nrf2 and HO-1 proteins in the liver tissues were detected using Western blotting. Results The diabetic mice showed significantly higher RBG levels and BW with obvious lipid deposition, fibrosis and oxidative stress in the liver as compared with the normal control mice(P<0.001). Exendin-4 treatment of the diabetic mice did not significantly lessened liver lipid deposition but obviously reduced the levels of RBG and TG(P<0.05), lowered the expression levels of liver fibrosis-related genes TGF-β, α-SMA and Col-I(P<0.05), increased the expression levels of the antioxidant genes Nrf2, HO-1 and GPX4(P<0.01), and enhanced the protein expressions of Nrf2 and HO-1 in the liver tissues(P<0.01). Conclusion Exendin-4 improves liver fibrosis and oxidative stress in diabetic mice by activating Nrf2/HO-1 pathway without significantly reducing liver lipid deposition.
引文
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[4]Nishikawa K,Osawa Y,Kimura K.Wnt/β-Catenin signaling as a potential target for the treatment of liver cirrhosis using antifibrotic drugs[J].Int J Mol Sci,2018,19(10):3103.
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[6]Li J,Hu R,Xu SF,et al.Xiaochaihutang attenuates liver fibrosis by activation of Nrf2 pathway in rats[J].Biomed Pharmacot,2017,96(1):847-53.
[7]Meier JJ.GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus[J].Nat Rev Endocrinol,2012,8(12):728-42.
[8]Verspohl EJ.Novel therapeutics for type 2 diabetes:incretin hormone mimetics(glucagon-like peptide-1 receptor agonists)and dipeptidyl peptidase-4 inhibitors[J].Pharmacol Ther,2009,124(1):113-38.
[9]Bloomgarden ZT.Gut-derived incretin hormones and new therapeutic approaches[J].Diabetes Care,2004,27(10):2554-9.
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[11]Younce CW,Niu JL,Ayala J,et al.Exendin-4 improves cardiac function in mice overexpressing monocyte chemoattractant protein-1in cardiomyocytes[J].J Mol Cell Cardiol,2014,76:172-6.
[12]Sancar-Bas S,Gezginci-Oktayoglu S,Bolkent S.Exendin-4 attenuates renal tubular injury by decreasing oxidative stress and inflammation in streptozotocin-induced diabetic mice[J].Growth Factors,2015,33(5/6):419-29.
[13]Kusakabe T,Tanioka H,Ebihara K,et al.Beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and a high-fat diet[J].Diabetologia,2009,52(4):675-83.
[14]Chen Q,Yang W,Wang X,et al.TGF-β1 induces EMT in bovine mammary epithelial cells through the TGFβ1/Smad signaling pathway[J].Cell Physiol Biochem,2017,43(1):82-93.
[15]Tsuchida T,Friedman SL.Mechanisms of hepatic stellate cell activation[J].Nat Rev Gastroenterol Hepatol,2017,14(7):397-411.
[16]Drucker DJ,Nauck MA.The incretin system:glucagon-like peptide-1receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2diabetes[J].Lancet,2006,368(9548):1696-705.
[17]Tahrani AA,Barnett AH,Bailey CJ.Pharmacology and therapeutic implications of current drugs for type 2 diabetes mellitus[J].Nat Rev Endocrinol,2016,12(10):566-92.
[18]Patel V,Joharapurkar A,Kshirsagar S,et al.Coagonist of GLP-1 and glucagon receptor ameliorates development of Non-Alcoholic fatty liver disease[J].Cardiovasc Hematol Agents Med Chem,2018,16(1):35-43.
[19]Oztay F,Sancar-Bas S,Gezginci-Oktayoglu SA,et al.Exendin-4partly ameliorates-hyperglycemia-mediated tissue damage in lungs of streptozotocin-induced diabetic mice[J].Peptides,2018,99:99-107.
[20]Kim J,Park S,You Y,et al.Suppression of ROS production by exendin-4 in PSC attenuates the high glucose-induced islet fibrosis[J].PLoS One,2016,11(12):e163187.
[21]Crosas-Molist E,Fabregat I.Role of NADPH oxidases in the redox biology of liver fibrosis[J].Redox Biol,2015,6:106-11.
[22]Richter K,Kietzmann T.Reactive Oxygen species and fibrosis:further evidence of a significant liaison[J].Cell Tissue Res,2016,365(3):591-605.
[23]Gezginci-Oktayoglu S,Sacan O,Yanardag R,et al.Exendin-4improves hepatocyte injury by decreasing proliferation through blocking NGF/TrkA in diabetic mice[J].Peptides,2011,32(2):223-31.
[24]Wang ZX,Hou L,Huang LH,et al.Exenatide improves liver mitochondrial dysfunction and insulin resistance by reducing oxidative stress in high fat diet-induced obese mice[J].Biochem Biophys Res Commun,2017,486(1):116-23.
[25]Mangmool S,Hemplueksa P,Parichatikanond W,et al.Epac is required for GLP-1R-mediated inhibition of oxidative stress and apoptosis in cardiomyocytes[J].Mol Endocrinol,2015,29(4):583-96.
[26]Chen YT,Tsai TH,Yang CC,et al.Exendin-4 and sitagliptin protect kidney from ischemia-reperfusion injury through suppressing oxidative stress and inflammatory reaction[J].J Transl Med,2013,11:270.
[27]Loboda A,Damulewicz M,Pyza EA,et al.Role of Nrf2/HO-1 system in development,oxidative stress response and diseases:an evolutionarily conserved mechanism[J].Cell Mol Life Sci,2016,73(17):3221-47.
[28]Oh YS,Jun HS.Effects of Glucagon-Like peptide-1 on oxidative stress and Nrf2 signaling[J].Int J Mol Sci,2018,19(1):26.
[29]Mahmoud AM,Hozayen WG,Ramadan SM.Berberine ameliorates methotrexate-induced liver injury by activating Nrf2/HO-1 pathway and PPARγ,and suppressing oxidative stress and apoptosis in rats[J].Biomed Pharmacother,2017,94:280-91.
[30]Ma X,Luo Q,Zhu H,et al.Aldehyde dehydrogenase 2 activation ameliorates CCl4-induced chronic liver fibrosis in mice by upregulating Nrf2/HO-1 antioxidant pathway[J].J Cell Mol Med,2018,22(8):3965-78.
[31]Kim MH,Kim EH,Jung HS,et al.EX4 stabilizes and activates Nrf2via PKC delta,contributing to the prevention of oxidative stress-induced pancreatic beta cell damage[J].Toxicol Appl Pharmacol,2017,315:60-9.
[32]Zhou TF,Zhang MQ,Zhao L,et al.Activation of Nrf2 contributes to the protective effect of Exendin-4 against angiotensin II-induced vascular smooth muscle cell senescence[J].Am J Physiol Cell Physiol,2016,311(4):C572-82.
[33]Zhao SM,Gao HL,Wang YL,et al.Attenuation of high GlucoseInduced rat cardiomyocyte apoptosis by exendin-4 via intervention of HO-1/Nrf-2 and the PI3K/AKT signaling pathway[J].Chin JPhysiol,2017,60(2):89-96.
[34]Deng CH,Cao J,Han JQ,et al.Liraglutide activates the Nrf2/HO-1antioxidant pathway and protects brain nerve cells against cerebral ischemia in diabetic rats[J].Comput Intell Neurosci,2018,1-7.
[2]Lonardo A,Nascimbeni F,Mantovani A,et al.Hypertension,diabetes,atherosclerosis and NASH:Cause or consequence[J]?J Hepatol,2018,68(2):335-52.
[3]Mcpherson S,Hardy T,Henderson E,et al.Evidence of NAFLDprogression from steatosis to fibrosing-steatohepatitis using paired biopsies:Implications for prognosis and clinical management[J].JHepatol,2015,62(5):1148-55.
[4]Nishikawa K,Osawa Y,Kimura K.Wnt/β-Catenin signaling as a potential target for the treatment of liver cirrhosis using antifibrotic drugs[J].Int J Mol Sci,2018,19(10):3103.
[5]Wang R,Wang J,Song FX,et al.Tanshinol ameliorates CCL4-induced liver fibrosis in rats through the regulation of Nrf2/HO-I and NF-kappa B/I kappa B alpha signaling pathway[J].Drug Des Devel Ther,2018,12(5):1281-92.
[6]Li J,Hu R,Xu SF,et al.Xiaochaihutang attenuates liver fibrosis by activation of Nrf2 pathway in rats[J].Biomed Pharmacot,2017,96(1):847-53.
[7]Meier JJ.GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus[J].Nat Rev Endocrinol,2012,8(12):728-42.
[8]Verspohl EJ.Novel therapeutics for type 2 diabetes:incretin hormone mimetics(glucagon-like peptide-1 receptor agonists)and dipeptidyl peptidase-4 inhibitors[J].Pharmacol Ther,2009,124(1):113-38.
[9]Bloomgarden ZT.Gut-derived incretin hormones and new therapeutic approaches[J].Diabetes Care,2004,27(10):2554-9.
[10]Kim S,Jung J,Kim H,et al.Exendin-4 improves nonalcoholic fatty liver disease by regulating glucose transporter 4 expression in ob/ob mice[J].Korean Journal of Physiology&Pharmacology,2014,18(4):333-9.
[11]Younce CW,Niu JL,Ayala J,et al.Exendin-4 improves cardiac function in mice overexpressing monocyte chemoattractant protein-1in cardiomyocytes[J].J Mol Cell Cardiol,2014,76:172-6.
[12]Sancar-Bas S,Gezginci-Oktayoglu S,Bolkent S.Exendin-4 attenuates renal tubular injury by decreasing oxidative stress and inflammation in streptozotocin-induced diabetic mice[J].Growth Factors,2015,33(5/6):419-29.
[13]Kusakabe T,Tanioka H,Ebihara K,et al.Beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and a high-fat diet[J].Diabetologia,2009,52(4):675-83.
[14]Chen Q,Yang W,Wang X,et al.TGF-β1 induces EMT in bovine mammary epithelial cells through the TGFβ1/Smad signaling pathway[J].Cell Physiol Biochem,2017,43(1):82-93.
[15]Tsuchida T,Friedman SL.Mechanisms of hepatic stellate cell activation[J].Nat Rev Gastroenterol Hepatol,2017,14(7):397-411.
[16]Drucker DJ,Nauck MA.The incretin system:glucagon-like peptide-1receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2diabetes[J].Lancet,2006,368(9548):1696-705.
[17]Tahrani AA,Barnett AH,Bailey CJ.Pharmacology and therapeutic implications of current drugs for type 2 diabetes mellitus[J].Nat Rev Endocrinol,2016,12(10):566-92.
[18]Patel V,Joharapurkar A,Kshirsagar S,et al.Coagonist of GLP-1 and glucagon receptor ameliorates development of Non-Alcoholic fatty liver disease[J].Cardiovasc Hematol Agents Med Chem,2018,16(1):35-43.
[19]Oztay F,Sancar-Bas S,Gezginci-Oktayoglu SA,et al.Exendin-4partly ameliorates-hyperglycemia-mediated tissue damage in lungs of streptozotocin-induced diabetic mice[J].Peptides,2018,99:99-107.
[20]Kim J,Park S,You Y,et al.Suppression of ROS production by exendin-4 in PSC attenuates the high glucose-induced islet fibrosis[J].PLoS One,2016,11(12):e163187.
[21]Crosas-Molist E,Fabregat I.Role of NADPH oxidases in the redox biology of liver fibrosis[J].Redox Biol,2015,6:106-11.
[22]Richter K,Kietzmann T.Reactive Oxygen species and fibrosis:further evidence of a significant liaison[J].Cell Tissue Res,2016,365(3):591-605.
[23]Gezginci-Oktayoglu S,Sacan O,Yanardag R,et al.Exendin-4improves hepatocyte injury by decreasing proliferation through blocking NGF/TrkA in diabetic mice[J].Peptides,2011,32(2):223-31.
[24]Wang ZX,Hou L,Huang LH,et al.Exenatide improves liver mitochondrial dysfunction and insulin resistance by reducing oxidative stress in high fat diet-induced obese mice[J].Biochem Biophys Res Commun,2017,486(1):116-23.
[25]Mangmool S,Hemplueksa P,Parichatikanond W,et al.Epac is required for GLP-1R-mediated inhibition of oxidative stress and apoptosis in cardiomyocytes[J].Mol Endocrinol,2015,29(4):583-96.
[26]Chen YT,Tsai TH,Yang CC,et al.Exendin-4 and sitagliptin protect kidney from ischemia-reperfusion injury through suppressing oxidative stress and inflammatory reaction[J].J Transl Med,2013,11:270.
[27]Loboda A,Damulewicz M,Pyza EA,et al.Role of Nrf2/HO-1 system in development,oxidative stress response and diseases:an evolutionarily conserved mechanism[J].Cell Mol Life Sci,2016,73(17):3221-47.
[28]Oh YS,Jun HS.Effects of Glucagon-Like peptide-1 on oxidative stress and Nrf2 signaling[J].Int J Mol Sci,2018,19(1):26.
[29]Mahmoud AM,Hozayen WG,Ramadan SM.Berberine ameliorates methotrexate-induced liver injury by activating Nrf2/HO-1 pathway and PPARγ,and suppressing oxidative stress and apoptosis in rats[J].Biomed Pharmacother,2017,94:280-91.
[30]Ma X,Luo Q,Zhu H,et al.Aldehyde dehydrogenase 2 activation ameliorates CCl4-induced chronic liver fibrosis in mice by upregulating Nrf2/HO-1 antioxidant pathway[J].J Cell Mol Med,2018,22(8):3965-78.
[31]Kim MH,Kim EH,Jung HS,et al.EX4 stabilizes and activates Nrf2via PKC delta,contributing to the prevention of oxidative stress-induced pancreatic beta cell damage[J].Toxicol Appl Pharmacol,2017,315:60-9.
[32]Zhou TF,Zhang MQ,Zhao L,et al.Activation of Nrf2 contributes to the protective effect of Exendin-4 against angiotensin II-induced vascular smooth muscle cell senescence[J].Am J Physiol Cell Physiol,2016,311(4):C572-82.
[33]Zhao SM,Gao HL,Wang YL,et al.Attenuation of high GlucoseInduced rat cardiomyocyte apoptosis by exendin-4 via intervention of HO-1/Nrf-2 and the PI3K/AKT signaling pathway[J].Chin JPhysiol,2017,60(2):89-96.
[34]Deng CH,Cao J,Han JQ,et al.Liraglutide activates the Nrf2/HO-1antioxidant pathway and protects brain nerve cells against cerebral ischemia in diabetic rats[J].Comput Intell Neurosci,2018,1-7.