为探讨壳寡糖(chitosan oligosaccharide,COS)对H2O2诱导的人神经母细胞瘤SK-N-SH细胞损伤的保护作用及其机制,利用H2O2建立体外氧化损伤模型,MTT法检测细胞存活率,酶活性检测乳酸脱氢酶(lactate dehydrogenase,LDH)的释放情况,JC-10检测细胞线粒体膜电位的变化,ATP发光测定试剂盒检测细胞内ATP水平,蛋白质免疫印迹(Western Blot)检测细胞中Bcl-2、Bax、Nrf2和HO-1蛋白的表达。结果表明,与H2O2组相比,COS预处理可有效提高H2O2诱导的细胞存活率,有效抑制细胞内LDH的释放,明显恢复线粒体膜电位,提高细胞内ATP水平,促进Bcl-2、Nrf2和HO-1蛋白的表达,抑制Bax蛋白的表达。COS可保护SK-N-SH细胞免受H2O2诱导的氧化损伤,抑制细胞凋亡并促进抗氧化蛋白表达水平。
To investigate the protective effect of chitosan oligosaccharides (COS) on the cellular damage of SK-N-SH cells induced by H2O2, H2O2 was used to establish an in vitro oxidative damage model. The cell viability was detected by MTT, and the release of lactate dehydrogenase (LDH) was detected by kit. JC-10 was used to detect changes in cell mitochondrial membrane potential, ATP luminescence assay kit was used to detect intracellular ATP level, and the expression of Bcl-2, Bax, Nrf2 and HO-1 protein was detected by Western Blot. Results showed that compared with the H2O2 group, COS pretreatment could effectively increase the cell survival rate induced by H2O2, markedly inhibit the release of intracellular LDH, significantly restore mitochondrial membrane potential, increase intracellular ATP levels, and promote the expression of Bcl-2, Nrf2 and HO-1 protein, inhibit the expression of Bax protein. COS could protect SK-N-SH cells from oxidative damage induced by H2O2 via inhibiting cell apoptosis and promoting the expression of antioxidant proteins.
[1] JIA J P, WEI C B, CHEN S Q, et al.The cost of Alzheimer's disease in China and re-estimation of costs worldwide[J].Alzheimer's & Dementia, 2018, 14(4):483-491.
[2] CHEN Z C, ZHONG C J.Oxidative stress in Alzheimer's disease[J].Neuroscience Bulletin, 2014, 30(2):271-281.
[3] ALLAN BUTTERFIELD D, HALLIWELL B.Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease[J].Nature Reviews Neuroscience, 2019, 20(3):148-160.
[4] WANG C Y, HAO J, LIU X, et al.Isoforsythiaside attenuates Alzheimer's disease via regulating mitochondrial function through the PI3K/AKT pathway[J].International Journal of Molecular Sciences, 2020, 21(16):5 687.
[5] LIANG S, SUN Y X, DAI X L.A review of the preparation, analysis and biological functions of chitooligosaccharide[J].International Journal of Molecular Sciences, 2018, 19(8):2 197.
[6] DAI X L, HOU W Q, SUN Y X, et al.Chitosan oligosaccharides inhibit/disaggregate fibrils and attenuate amyloid β-mediated neurotoxicity[J].International Journal of Molecular Sciences, 2015, 16(5):10 526-10 536.
[7] DAI X L, CHANG P, ZHU Q Z, et al.Chitosan oligosaccharides protect rat primary hippocampal neurons from oligomeric β-amyloid 1-42-induced neurotoxicity[J].Neuroscience Letters, 2013, 554:64-69.
[8] JIA S L, LU Z, GAO Z L, et al.Chitosan oligosaccharides alleviate cognitive deficits in an amyloid-β1-42-induced rat model of Alzheimer's disease[J].International Journal of Biological Macromolecules, 2016, 83:416-425.
[9] FANG I M, YANG C M, YANG C H.Chitosan oligosaccharides prevented retinal ischemia and reperfusion injury via reduced oxidative stress and inflammation in rats[J].Experimental Eye Research, 2015, 130:38-50.
[10] PARK S, CHUN S, KIM D.Cold exposure lowers energy expenditure at the cellular level[J].Cell Biology International, 2013, 37(6):638-642.
[11] HE W L, CUI L L, ZHANG C, et al.Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress:Involving of mitochondria and energy metabolism[J].Experimental Cell Research, 2017, 350(1):83-90.
[12] ZHANG J X, CAI Q, JIANG M, et al.Mesencephalic astrocyte-derived neurotrophic factor alleviated 6-OHDA-induced cell damage via ROS-AMPK/mTOR mediated autophagic inhibition[J].Experimental Gerontology, 2017, 89:45-56.
[13] FINKEL T, HOLBROOK N J.Oxidants, oxidative stress and the biology of ageing[J].Nature, 2000, 408(6 809):239-247.
[14] WANG H R, ZHANG K Q, RUAN Z, et al.Probucol enhances the therapeutic efficiency of mesenchymal stem cells in the treatment of erectile dysfunction in diabetic rats by prolonging their survival time via Nrf2 pathway[J].Stem Cell Research & Therapy, 2020, 11(1):249-255.
[15] KIM Y, LIM H S, KIM Y, et al.Phytochemical quantification and the in vitro acetylcholinesterase inhibitory activity of Phellodendron chinense and its components[J].Molecules, 2017, 22(6):925.
[16] ZHAO Z Y, LUAN P, HUANG S X, et al.Edaravone protects HT22 neurons from H2O2-induced apoptosis by inhibiting the MAPK signaling pathway[J].CNS Neuroscience & Therapeutics, 2013, 19(3):163-169.
[17] LOBODA A, DAMULEWICZ M, PYZA E, et al.Role of Nrf2/HO-1 system in development, oxidative stress response and diseases:An evolutionarily conserved mechanism[J].Cellular and Molecular Life Sciences, 2016, 73(17):3 221-3 247.
[18] DE OLIVEIRA M R, DE BITTENCOURT BRASIL F, FÜRSTENAU C R.Inhibition of the Nrf2/HO-1 axis suppresses the mitochondria-related protection promoted by gastrodin in human neuroblastoma cells exposed to paraquat[J].Molecular Neurobiology, 2019, 56(3):2 174-2 184.
[19] ESTERAS N, DINKOVA-KOSTOVA A T, ABRAMOV A Y.Nrf2 activation in the treatment of neurodegenerative diseases:A focus on its role in mitochondrial bioenergetics and function[J].Biological Chemistry, 2016, 397(5):383-400.
[20] WANG Y M, XIONG Y L, ZHANG A P, et al.Oligosaccharide attenuates aging-related liver dysfunction by activating Nrf2 antioxidant signaling[J].Food Science & Nutrition, 2020, 8(7):3 872-3 881.
[21] TAO W J, SUN W J, LIU L J, et al.Chitosan oligosaccharide attenuates nonalcoholic fatty liver disease induced by high fat diet through reducing lipid accumulation, inflammation and oxidative stress in C57BL/6 mice[J].Marine Drugs, 2019, 17(11):645.
