柚皮素通过激活mTOR/p70S6K信号通路减轻胰岛素抵抗并改善H_2O_2诱导的SH-SY5Y细胞氧化应激损伤
|
摘要
目的:观察柚皮素(naringenin,NAR)对H_2O_2诱导的SH-SY5Y细胞氧化应激损伤的影响,并探索其机制。方法:通过CCK-8检测细胞存活率,筛选出H_2O_2的半数致死浓度,用该浓度H_2O_2同时加入20、40、80μmol/L柚皮素处理SH-SY5Y细胞4 h,通过CCK-8检测细胞存活率,选择适宜柚皮素浓度。按所选H_2O_2和柚皮素浓度进行后续实验。将实验分为3组。control组:细胞不进行任何干预处理;H_2O_2组:只加入600μmol/L H_2O_2;H_2O_2+NAR组:同时加入600μmol/L H_2O_2与40μmol/L柚皮素。4 h后,通过ANNEXIN V-FITC/PI凋亡检测试剂盒检测细胞凋亡率,DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species,ROS),Western blot检测哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、核糖体蛋白S6激酶(p70ribosomal S6 kinase,p70S6K),以及各自磷酸化形式蛋白[p-mTOR(Ser2448)、p-p70S6K(Thr389)],用胰岛素受体底物-1(insulin receptor substrate 1,IRS1)及其丝氨酸636+639位点磷酸化[p-IRS1(Ser636+Ser639)]蛋白的表达来观察胰岛素抵抗(insulin resistance,IR)。结果:H_2O_2降低SH-SY5Y细胞的存活率,呈剂量依赖性,600μmol/L浓度为半数致死剂量。柚皮素明显降低了H_2O_2诱导的SH-SY5Y细胞死亡,最适剂量为40μmol/L。与control组比,H_2O_2明显增加了细胞凋亡率、胞内ROS的产生,降低了p-mTOR(Ser2448)、p-p70S6K(Thr389)的表达,增加了p-IRS1(Ser636+Ser639)蛋白的表达(P<0.05);加入柚皮素后,相对于H_2O_2组,上述作用明显减弱(P<0.05)。结论:在体外氧化应激模型中,柚皮素能明显降低细胞内ROS的积累,提高细胞的存活率。其机制可能与激活mTOR/p70S6K信号通路、改善IR有关。
Objective:To investigate the effect of naringenin on SH-SY5 Y cells of neuroblastoma in oxidative stress induced by H_2O_2 and the possible mechanism. Methods:Firstly,a concentration of H_2O_2 that caused half cells death was chosen by CCK-8 assay.Three concentrations of naringenin(20,40,80 μmol/L)were added into H_2O_2-treated media,and cells survival rates were tested by CCK-8 assay after 4 hours. An optimal concentration of naringenin was chosen for the next experment in naringenin group. Next,three groups were chosen for the next experiment(control group:cells without any treatment;H_2O_2 group:cells with 600 μmol/L H_2O_2;H_2O_2+NAR group:cells with 600 μmol/L H_2O_2 and 40 μmol/L NAR simultaneously). The apoptosis rate was measured by Annexin V-FITC/PI Apoptosis Detection Kit,intercellular reactive oxygen species(ROS)was measured by fluorescent probe through flow cytometry,and m TOR/p70 S6 K signalling pathway-related protein[mTOR,p-mTOR(Ser2448),p70 S6 K,p-p70 S6 K(Thr389)] were tested by Western blot. Finally,insulin resistance-related protein insulin receptor substrate 1(IRS1)and phosphorylated IRS1 in serine 636 and serine 639[p-IRS1(Ser636+Ser639)] were detected by Western blot as well. Results:H_2O_2 significantly increased the SH-SY5 Y cells death,apoptosis,intercellular ROS and p-IRS1(Ser636+Ser639),and decreased the protein expression of p-mTOR(Ser2448)and p-p70 S6 K(Thr389)compared with control group(P<0.05). These effects were attenuated after NAR treatment,compared with H_2O_2 group(P<0.05). Conclusion:Naringenin reduced the ROS accumulation and the SH-SY5 Y cells death. The activation of mTOR/p70 S6 K signalling pathway and decreasing of insulin resistance may be involved in naringenin-induced antioxidative effects.
Objective:To investigate the effect of naringenin on SH-SY5 Y cells of neuroblastoma in oxidative stress induced by H_2O_2 and the possible mechanism. Methods:Firstly,a concentration of H_2O_2 that caused half cells death was chosen by CCK-8 assay.Three concentrations of naringenin(20,40,80 μmol/L)were added into H_2O_2-treated media,and cells survival rates were tested by CCK-8 assay after 4 hours. An optimal concentration of naringenin was chosen for the next experment in naringenin group. Next,three groups were chosen for the next experiment(control group:cells without any treatment;H_2O_2 group:cells with 600 μmol/L H_2O_2;H_2O_2+NAR group:cells with 600 μmol/L H_2O_2 and 40 μmol/L NAR simultaneously). The apoptosis rate was measured by Annexin V-FITC/PI Apoptosis Detection Kit,intercellular reactive oxygen species(ROS)was measured by fluorescent probe through flow cytometry,and m TOR/p70 S6 K signalling pathway-related protein[mTOR,p-mTOR(Ser2448),p70 S6 K,p-p70 S6 K(Thr389)] were tested by Western blot. Finally,insulin resistance-related protein insulin receptor substrate 1(IRS1)and phosphorylated IRS1 in serine 636 and serine 639[p-IRS1(Ser636+Ser639)] were detected by Western blot as well. Results:H_2O_2 significantly increased the SH-SY5 Y cells death,apoptosis,intercellular ROS and p-IRS1(Ser636+Ser639),and decreased the protein expression of p-mTOR(Ser2448)and p-p70 S6 K(Thr389)compared with control group(P<0.05). These effects were attenuated after NAR treatment,compared with H_2O_2 group(P<0.05). Conclusion:Naringenin reduced the ROS accumulation and the SH-SY5 Y cells death. The activation of mTOR/p70 S6 K signalling pathway and decreasing of insulin resistance may be involved in naringenin-induced antioxidative effects.
引文
[1]Blennow K,de Leon MJ,Zetterberg H.Alzheimer’s disease[J].Lancet,2006,368(9533):387-403.
[2]Ballard C,Gauthier S,Corbett A,et al.Alzheimer’s disease[J].Lancet,2011,377(9770):1019-1031.
[3]Fu QQ,Wei L,Sierra J,et al.Olfactory ensheathing cell-conditioned medium reverts Aβ25-35-induced oxidative damage in SH-SY5Y cells by modulating the mitochondria-mediated apoptotic pathway[J].Cell Mol Neurobiol,2017,37(6):1043-1054.
[4]Jayaraman A,Pike CJ.Alzheimer’s disease and type 2 diabetes:multiple mechanisms contribute to interactions[J].Curr Diab Rep,2014,14(4):476.
[5]Bomfim TR,Forny-Germano L,Sathler LB,et al.An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer’s disease-associated Aβoligomers[J].J Clin Invest.2012,122(4):1339-1353.
[6]Talbot K,Wang HY,Kazi H,et al.Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance,IRS-1 dysregulation,and cognitive decline[J].J Clin Invest,2012,122(4):1316-1338.
[7]Doronzo G,Viretto M,Russo I,et al.Nitric oxide activates PI3-Kand MAPK signalling pathways in human and rat vascular smooth muscle cells:influence of insulin resistance and oxidative stress[J].Atherosclerosis,2011,216(1):44-53.
[8]Zuo M,Rashid A,Churi C,et al.Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer[J].Br J Cancer,2015,112(6):1042-1051.
[9]Singh AK,Kashyap MP,Tripathi VK,et al.Neuroprotection through rapamycin-induced activation of autophagy and PI3K/Akt1/mTOR/CREB signaling against amyloid-beta-induced oxidative stress,synaptic/neurotransmission dysfunction,and neurodegeneration in adult rats[J].Mol Neurobiol,2017,54(8):5815-5828.
[10]Di Domenico F,Tramutola A,Foppoli C,et al.mTOR in Down syndrome:role in Aβand tau neuropathology and transition to Alzheimer disease-like dementia[J].Free Radic Biol Med,2018,114:94-101.
[11]Assini JM,Mulvihill EE,Burke AC,et al.Naringenin prevents obesity,hepatic steatosis,and glucose intolerance in male mice independent of fibroblast growth factor 21[J].Endocrinology,2015,156(6):2087-2102.
[12]Wang K,Chen Z,Huang J,et al.Naringenin prevents ischaemic stroke damage via anti-apoptotic and anti-oxidant effects[J].Clin Exp Pharmacol Physiol,2017,44(8):862-871.
[13]Yang W,Ma J,Liu Z,et al.Effect of naringenin on brain insulin signaling and cognitive functions in ICV-STZ induced dementia model of rats[J].Neurol Sci,2014,35(5):741-751.
[14]马晶,杨文青,查何,等.柚皮素对阿尔茨海默病模型大鼠学习记忆能力的影响[J].中药材,2013,36(2):271-276.
[15]Zbarsky V,Datla KP,Parkar S,et al.Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson’s disease[J].Free Radic Res,2005,39(10):1119-1125.
[16]Khan MB,Khan MM,Khan A,et al.Naringenin ameliorates Alzheimer’s disease(AD)-type neurodegeneration with cognitive impairment(AD-TNDCI)caused by the intracerebroventricular-streptozotocin in rat model[J].Neurochem Int,2012,61(7):1081-1093.
[17]Zbarsky V,Datla KP,Parkar S,et al.Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson’s disease[J].Free Radic Res,2005,39(10):1119-1125.
[18]Abaza MS,Orabi KY,Al-Quattan E,et al.Growth inhibitory and chemo-sensitization effects of naringenin,a natural flavanone purified from Thymus vulgaris,on human breast and colorectal cancer[J].Cancer Cell Int,2015,15:46.
[19]Liao AC,Kuo CC,Huang YC,et al.Naringenin inhibits migration of bladder cancer cells through downregulation of AKT and MMP2[J].Mol Med Rep,2014,10(3):1531-1536.
[20]Chong ZZ,Shang YC,Zhang L,et al.Mammalian target of rapamycin:hitting the bull’s-eye for neurological disorders[J].Oxid Med Cell Longev,2010,3(6):374-391.
[21]Maiese K,Chong ZZ,Shang YC,et al.mTOR:on target for novel therapeutic strategies in the nervous system[J].Trends Mol Med,2013,19(1):51-60.
[22]Taga M,Mouton-Liger F,Paquet C,et al.Modulation of oxidative stress and tau phosphorylation by the m TOR activator phosphatidic acid in SH-SY5Y cells[J].FEBS Lett,2011,585(12):1801-1806.
[23]Copps KD,White MF.Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1and IRS2[J].Diabetologia,2012,55(10):2565-2582.
[24]Singh I,Carey AL,Watson N,et al.Oxidative stress-induced insulin resistance in skeletal muscle cells is ameliorated by gamma-tocopherol treatment[J].Eur J Nutr,2008,47(7):387-392.
[25]Doronzo G,Viretto M,Russo I,et al.Nitric oxide activates PI3-Kand MAPK signalling pathways in human and rat vascular smooth muscle cells:influence of insulin resistance and oxidative stress[J].Atherosclerosis,2011,216(1):44-53.
[26]Jayaraman A,Pike CJ.Alzheimer’s disease and type 2 diabetes:multiple mechanisms contribute to interactions[J].Curr Diab Rep,2014,14(4):476.
[27]Chen Z,Zhong C.Decoding Alzheimer’s disease from perturbed cerebral glucose metabolism:implications for diagnostic and therapeutic strategies[J].Prog Neurobiol,2013,108:21-43.
[28]Steen E,Terry BM,Rivera EJ,et al.Impaired insulin and insulinlike growth factor expression and signaling mechanisms in Alzheimer’s disease-is this type 3 diabetes?[J].J Alzheimers Dis,2005,7(1):63-80.
[2]Ballard C,Gauthier S,Corbett A,et al.Alzheimer’s disease[J].Lancet,2011,377(9770):1019-1031.
[3]Fu QQ,Wei L,Sierra J,et al.Olfactory ensheathing cell-conditioned medium reverts Aβ25-35-induced oxidative damage in SH-SY5Y cells by modulating the mitochondria-mediated apoptotic pathway[J].Cell Mol Neurobiol,2017,37(6):1043-1054.
[4]Jayaraman A,Pike CJ.Alzheimer’s disease and type 2 diabetes:multiple mechanisms contribute to interactions[J].Curr Diab Rep,2014,14(4):476.
[5]Bomfim TR,Forny-Germano L,Sathler LB,et al.An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer’s disease-associated Aβoligomers[J].J Clin Invest.2012,122(4):1339-1353.
[6]Talbot K,Wang HY,Kazi H,et al.Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance,IRS-1 dysregulation,and cognitive decline[J].J Clin Invest,2012,122(4):1316-1338.
[7]Doronzo G,Viretto M,Russo I,et al.Nitric oxide activates PI3-Kand MAPK signalling pathways in human and rat vascular smooth muscle cells:influence of insulin resistance and oxidative stress[J].Atherosclerosis,2011,216(1):44-53.
[8]Zuo M,Rashid A,Churi C,et al.Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer[J].Br J Cancer,2015,112(6):1042-1051.
[9]Singh AK,Kashyap MP,Tripathi VK,et al.Neuroprotection through rapamycin-induced activation of autophagy and PI3K/Akt1/mTOR/CREB signaling against amyloid-beta-induced oxidative stress,synaptic/neurotransmission dysfunction,and neurodegeneration in adult rats[J].Mol Neurobiol,2017,54(8):5815-5828.
[10]Di Domenico F,Tramutola A,Foppoli C,et al.mTOR in Down syndrome:role in Aβand tau neuropathology and transition to Alzheimer disease-like dementia[J].Free Radic Biol Med,2018,114:94-101.
[11]Assini JM,Mulvihill EE,Burke AC,et al.Naringenin prevents obesity,hepatic steatosis,and glucose intolerance in male mice independent of fibroblast growth factor 21[J].Endocrinology,2015,156(6):2087-2102.
[12]Wang K,Chen Z,Huang J,et al.Naringenin prevents ischaemic stroke damage via anti-apoptotic and anti-oxidant effects[J].Clin Exp Pharmacol Physiol,2017,44(8):862-871.
[13]Yang W,Ma J,Liu Z,et al.Effect of naringenin on brain insulin signaling and cognitive functions in ICV-STZ induced dementia model of rats[J].Neurol Sci,2014,35(5):741-751.
[14]马晶,杨文青,查何,等.柚皮素对阿尔茨海默病模型大鼠学习记忆能力的影响[J].中药材,2013,36(2):271-276.
[15]Zbarsky V,Datla KP,Parkar S,et al.Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson’s disease[J].Free Radic Res,2005,39(10):1119-1125.
[16]Khan MB,Khan MM,Khan A,et al.Naringenin ameliorates Alzheimer’s disease(AD)-type neurodegeneration with cognitive impairment(AD-TNDCI)caused by the intracerebroventricular-streptozotocin in rat model[J].Neurochem Int,2012,61(7):1081-1093.
[17]Zbarsky V,Datla KP,Parkar S,et al.Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson’s disease[J].Free Radic Res,2005,39(10):1119-1125.
[18]Abaza MS,Orabi KY,Al-Quattan E,et al.Growth inhibitory and chemo-sensitization effects of naringenin,a natural flavanone purified from Thymus vulgaris,on human breast and colorectal cancer[J].Cancer Cell Int,2015,15:46.
[19]Liao AC,Kuo CC,Huang YC,et al.Naringenin inhibits migration of bladder cancer cells through downregulation of AKT and MMP2[J].Mol Med Rep,2014,10(3):1531-1536.
[20]Chong ZZ,Shang YC,Zhang L,et al.Mammalian target of rapamycin:hitting the bull’s-eye for neurological disorders[J].Oxid Med Cell Longev,2010,3(6):374-391.
[21]Maiese K,Chong ZZ,Shang YC,et al.mTOR:on target for novel therapeutic strategies in the nervous system[J].Trends Mol Med,2013,19(1):51-60.
[22]Taga M,Mouton-Liger F,Paquet C,et al.Modulation of oxidative stress and tau phosphorylation by the m TOR activator phosphatidic acid in SH-SY5Y cells[J].FEBS Lett,2011,585(12):1801-1806.
[23]Copps KD,White MF.Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1and IRS2[J].Diabetologia,2012,55(10):2565-2582.
[24]Singh I,Carey AL,Watson N,et al.Oxidative stress-induced insulin resistance in skeletal muscle cells is ameliorated by gamma-tocopherol treatment[J].Eur J Nutr,2008,47(7):387-392.
[25]Doronzo G,Viretto M,Russo I,et al.Nitric oxide activates PI3-Kand MAPK signalling pathways in human and rat vascular smooth muscle cells:influence of insulin resistance and oxidative stress[J].Atherosclerosis,2011,216(1):44-53.
[26]Jayaraman A,Pike CJ.Alzheimer’s disease and type 2 diabetes:multiple mechanisms contribute to interactions[J].Curr Diab Rep,2014,14(4):476.
[27]Chen Z,Zhong C.Decoding Alzheimer’s disease from perturbed cerebral glucose metabolism:implications for diagnostic and therapeutic strategies[J].Prog Neurobiol,2013,108:21-43.
[28]Steen E,Terry BM,Rivera EJ,et al.Impaired insulin and insulinlike growth factor expression and signaling mechanisms in Alzheimer’s disease-is this type 3 diabetes?[J].J Alzheimers Dis,2005,7(1):63-80.