硫辛酸对脂多糖诱导帕金森病细胞模型的保护作用及机制
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摘要
背景:近年越来越多的研究证实神经免疫和炎症反应参与了帕金森病的发病,硫辛酸目前被证实具有抗炎和神经保护作用。目的:探讨硫辛酸对脂多糖诱导PC12细胞制备的帕金森病细胞模型的保护作用及相关可能机制。方法:将PC12细胞分为正常细胞组、脂多糖-帕金森病组和硫辛酸+脂多糖-帕金森病组。硫辛酸预处理PC12细胞2 h后,再用脂多糖干预PC12细胞。用CCK8法筛选最佳帕金森细胞模型制备的脂多糖浓度以及硫辛酸最佳干预浓度;细胞免疫组织化学法检测各组细胞中酪氨酸羟化酶的表达;ELISA法检测各组中细胞上清液中白细胞介素6和肿瘤坏死因子α水平;Western blot检测各组细胞中NF-κBp65、酪氨酸羟化酶的蛋白表达情况。结果与结论:①建造帕金森细胞模型最佳脂多糖的质量浓度为600μg/L,最佳硫辛酸干预浓度为25μmol/L;②硫辛酸干预后可以促进酪氨酸羟化酶阳性细胞表达,减少帕金森病细胞模型的炎症因子释放,并且可以抑制NF-κBp65的产生,提高酪氨酸羟化酶含量;③结果提示,硫辛酸对帕金森病细胞模型的作用可能通过抑制NF-κB信号通路,减轻炎症状态,从而起到对神经元的保护作用。
BACKGROUND: Neuroimmune and inflammatory reaction have been shown to participate in the occurrence of Parkinson's disease. Lipoic acid possesses anti-inflammatory and neuroprotective roles. OBJECTIVE: To investigate the protective effects of lipoic acid on PC12 cell models of Parkinson's disease induced by lipopolysaccharide and the underlying mechanism. METHODS: PC12 cells were divided into blank control, model(Parkinson's disease model induced by lipopolysaccharide) and treatment(Parkinson's disease model treated by lipoic acid) groups. PC12 cells were pretreated with lipoic acid for 2 hours, and then stimulated with lipopolysaccharide. The optimal concentration of lipopolysaccharide and lipoic acid was determined by cell counting-kit 8 assay. The expression of tyrosine hydroxylase was detected by immunohistochemistry. The levels of tumor necrosis factor α and interleukin 6 were detected by ELISA kit. The protein expression levels of nuclear factor-κBp65 and tyrosine hydroxylase were detected by western blot assay. RESULTS AND CONCLUSION: The optimal concentration of lipopolysaccharide was 600 μg/L for constructing cell model of Parkinson disease, and 25 μmol/L lipoic acid was the optimal intervention concentration. Lipoic acid could increase the number of tyrosine hydroxylase positive cells, reduce the release of inflammatory factors in cell model of Parkinson's disease, inhibit the production of nuclear factor-κBp65 and increase tyrosine hydroxylase content. Therefore, lipoic acid exhibits its effects on cell model of Parkinson's disease through inhibiting the nuclear factor-κB signaling pathway and reducing inflammation, thereby exerting neuroprotection.
BACKGROUND: Neuroimmune and inflammatory reaction have been shown to participate in the occurrence of Parkinson's disease. Lipoic acid possesses anti-inflammatory and neuroprotective roles. OBJECTIVE: To investigate the protective effects of lipoic acid on PC12 cell models of Parkinson's disease induced by lipopolysaccharide and the underlying mechanism. METHODS: PC12 cells were divided into blank control, model(Parkinson's disease model induced by lipopolysaccharide) and treatment(Parkinson's disease model treated by lipoic acid) groups. PC12 cells were pretreated with lipoic acid for 2 hours, and then stimulated with lipopolysaccharide. The optimal concentration of lipopolysaccharide and lipoic acid was determined by cell counting-kit 8 assay. The expression of tyrosine hydroxylase was detected by immunohistochemistry. The levels of tumor necrosis factor α and interleukin 6 were detected by ELISA kit. The protein expression levels of nuclear factor-κBp65 and tyrosine hydroxylase were detected by western blot assay. RESULTS AND CONCLUSION: The optimal concentration of lipopolysaccharide was 600 μg/L for constructing cell model of Parkinson disease, and 25 μmol/L lipoic acid was the optimal intervention concentration. Lipoic acid could increase the number of tyrosine hydroxylase positive cells, reduce the release of inflammatory factors in cell model of Parkinson's disease, inhibit the production of nuclear factor-κBp65 and increase tyrosine hydroxylase content. Therefore, lipoic acid exhibits its effects on cell model of Parkinson's disease through inhibiting the nuclear factor-κB signaling pathway and reducing inflammation, thereby exerting neuroprotection.
引文
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[13]和青.LPS经鼻小鼠帕金森病模型的建立及Rho激酶靶点干预探讨[D].上海:复旦大学,2013.
[14]Smith AR,Shenvi SV,Widlansky M,et al.Lipoicacid as a potential therapy for chronicdisease associated with oxidative stress.Curr Med Chem.2004;11(9):1135-1146.
[15]李艳花,和青,尉杰忠,等.硫辛酸对脂多糖诱导的帕金森病小鼠黑质多巴胺能神经元损伤的影响[J].中国病理生理杂志,2015,31(2):201-206.
[16]Zaitone SA,Abo-Eimatty DM,Shaalan AA.Acety1-L-carnitine and a-lipoicacid affect rotenone-induced damage in nigral dopaminergic neurons of rat brain implication for Parkinson’s disease therapy.Pharmacol Biochem Behav.2012;100(3):347-360.
[17]Müller U,Krieglstein J.Prolonged pretreatment with alpha-lipoic acid protects cultured neurons against hypoxic,glutamate-,or iron-induced injury.J Cereb Blood Flow Metab.1995;15(4):624-630.
[18]Perez-Matos MC,Morales-Alvarez MC,Mendivil CO.Lipids:ASuitable Therapeutic Target in Diabetic Neuropathy?.J Diabetes Res.2017;2017:6943851.
[19]Dong Y,Wang H,Chen Z.Alpha-Lipoic Acid Attenuates Cerebral Ischemia and Reperfusion Injury via Insulin Receptor and PI3K/Akt-Dependent Inhibition of NADPH Oxidase.Int JEndocrinol.2015;2015,903186.
[20]Kamarudin MN,Mohd Raflee NA,Hussein SS,et al.(R)-(+)-α-lipoic acid protected NG108-15 cells against H?O?-induced cell death through PI3K-Akt/GSK-3βpathway and suppression of NF-κβ-cytokines.Drug Des Devel Ther.2014;8:1765-1780.
[21]Kones R.Mitochondfial therapy for Parkinson’s disease:neuroprotective pharmaconutrition may be disease-modifying.Clin Pharmacol.2010;2:185-198.
[22]马玲,周勇,王莉,等.硫辛酸减轻高尿酸血症大鼠氧化应激损伤[J].基础医学与临床,2015,35(8):1037-1041.
[23]Rochette L,Ghibu S,Richard C,et al.Direct and indirect antioxidant properties of alpha-lipoic acid and therapeutic potential.Mol Nutr Food Res.2013;57(1):114-125.
[24]Kleinkauf-Rocha J,Bobermin LD,Machado PM,et al.Lipoicacid increases glutamate uptake,glutamine symhetase activity and glutathione content in C6 astrocyte cell line.Int J Dev Neurosci.2013;31(3):165-170.
[25][Papanas N,Ziegler D.Efficacy ofα-lipoic acid in diabetic neuropathy.Expert Opin Pharmacother.2014;15(18):2721-2731.
[26]Zhao L,Hu FX.Alpha-Lipoic acid treatment of aged type 2diabetes mellitus complicated with acute cerebral infarction.Eur Rev Med Pharmacol Sci.2014;18(23):3715-3719.
[27]Saygin M,Asci H,Cankara FN,et al.The impact of high fructose on cardiovascular system:Role of alpha-lipoicacid.Hum Exp Toxicol.2015;35(2):194-204.
[28]Hegazy SK,Tolba OA,Mostafa TM,et al.Alpha·lipoicacid improves subclinical left ventricular dysfunction in asymptomatie patients with type 1 diabetes.Rev Diabet Stud.2013;10(1):58-67.
[29]Koriyama Y,Nakayama Y,Matsugo S,et al.Anti-inflammatory effects of lipoic acid through inhibition of GSK-3βin lipopolysaccharide-induced BV-2 microglial cells.Neurosci Res.2013;77(1-2):87-96.
[30]Jiang S,Zhu W,Li C,et al.α-Lipoic acid attenuates脂多糖-induced cardiac dysfunction through a PI3K/Akt-dependent mechanism.Int Immunopharmacol.2013;16(1):100-107.
[31]Ying Z,Xie X,Chen M,et al.Alpha-lipoic acid activates eNOSthrough activation of PI3-kinase/Akt signaling pathway.Vascul Pharmacol.2015;64:28-35.
[32]Liu SF,Malik AB.NF-kappa B activation as a pathological mechanism of septic shock and inflammation.Am J Physiol,Cell Mol Physiol.2006;290(1):L622-L645.
[33]Wu DC,Teismann P,Tieu K,et al.NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson′s disease.Proc Natl Acad Sci USA.2003;100(10):6145-6150.
[34]Zhang F,Qian L,Flood PM,et al.Inhibition of IkappaBkinase-beta protectsdopamine neurons against lipopolysaccharide-induced neurotoxicity.J Pharmacol Exp Ther.2010;333(3):822-833.
[35]Li Y,Ma QG,Zhao LH,et al.Effects of lipoic acid on immune function,the antioxidant defense system,and inflammation-related genes expression of broiler chickens fed aflatoxin contaminated diets.Int J Mol Sci.2014;15(4):5649-5662.
[36]Kim HS,Kim HJ,Park KG,et al.Alpha-lipoic acid inhibits matrix metallop-roteinase-9 expression by inhibiting NF-kappaBtranscriptional activity.Exp Mol Med.2001;39(1):106-113.
[37]Li G,Fu J,Zhao Y,Ji K,et al.Alpha-lipoic acid exerts anti-inflammatory effects on lipopolysaccharide-stimulated rat mesangial cells via inhibition of nuclear factor kappa b(NF-kappab)signaling pathway.Inflammation.2015;38(2):510-519.
[38]Koriyama Y,Nakayama Y,Matsugo S.et al Anti-inflammatory effects of lipoic acid through inhibition of GSK-3βin lipopolysaccharide-induced BV-2 microglial cells.Neurosci Res.2013;77(1/2):87-96.
[39]Li YH,He Q,Yu JZ,et al.Lipoic acid protects dopaminergic neurons in lps-induced parkinson′s disease model.Metab Brain Dis.2015;30(5):1-10.
[2]Jha MK,Suk K,G1ia.based biomarkers and their functional role in the CNS.Expert Rev Proteomics.2013;10:43-63.
[3]Gonz61ez H,Elgueta D,Montoya A,et al.Neuroimmune regulation of mieroglial activity involved in neuro-inflammation and neurodegenerative diseases.J Neuroimmunol.2014;274:1-13.
[4]Sveinbjornsdottir S.The clinical symptoms of Parkinson’s disease.J Neurochem.2016;139:318.
[5]He H,Wang S,Tian J,et al.Protective effects of 2,3,5,4-tetra-hydroxystilbene-2-O-B-dglucoside in the MPTP-induced mouse model of Parkinson’s disease:Involvement of reactive of oxygen species mediated JNK,P38 and mitochondrial pathways.Eur J Pharmacol.2015;767:175-182.
[6]Benskey MJ,Perez RG,Manfredsson FP.The contribution of alpha synuclein to neuronal survival and function-implications for parkinson′s disease.J Neurochem.2016;137(3):331-359.
[7]Gallagher DA,Schapira AH.Etiopathogenesis and treatment of Parkinson′s disease.Curr Top Med Chem.2009;9(10):860-868.
[8]Giordano S,Darley-Usmar V,Zhang J.Autophagy as an essential cellular antioxidant pathway in neurodegenerative disease.Redox Biol.2014;2(1):82-90.
[9]Labandeira-Garcia JL,Rodriguez-Pallares J,Dominguez-Meijide A,et al.opamine-angiotensin interactions in the basal ganglia and their relevance for Parkinson’s disease.Mov Disord.2013;28(10):1337-1342.
[10]Borrajo A,Rodriguez-Perez AI,Diaz-Ruiz C,et al.Microglial TNF-alpha mediates enhancement of dopaminergic degeneration by brain angiotensin.Glia.2014;62(1):145-157.
[11]Hirsch EC,Vyas S,Hunot S.Neuroinflanunation in Parkinson's Disease.Parkinsonism Relat Disord.2011;18:85-64.
[12]杨丽娟,董军,陆大祥,等.姜黄素对脂多糖激活小胶质细胞条件培养基诱导损伤的海马神经元保护作用及机制[J].中国病理生理杂志,2010,26(4):742-747.
[13]和青.LPS经鼻小鼠帕金森病模型的建立及Rho激酶靶点干预探讨[D].上海:复旦大学,2013.
[14]Smith AR,Shenvi SV,Widlansky M,et al.Lipoicacid as a potential therapy for chronicdisease associated with oxidative stress.Curr Med Chem.2004;11(9):1135-1146.
[15]李艳花,和青,尉杰忠,等.硫辛酸对脂多糖诱导的帕金森病小鼠黑质多巴胺能神经元损伤的影响[J].中国病理生理杂志,2015,31(2):201-206.
[16]Zaitone SA,Abo-Eimatty DM,Shaalan AA.Acety1-L-carnitine and a-lipoicacid affect rotenone-induced damage in nigral dopaminergic neurons of rat brain implication for Parkinson’s disease therapy.Pharmacol Biochem Behav.2012;100(3):347-360.
[17]Müller U,Krieglstein J.Prolonged pretreatment with alpha-lipoic acid protects cultured neurons against hypoxic,glutamate-,or iron-induced injury.J Cereb Blood Flow Metab.1995;15(4):624-630.
[18]Perez-Matos MC,Morales-Alvarez MC,Mendivil CO.Lipids:ASuitable Therapeutic Target in Diabetic Neuropathy?.J Diabetes Res.2017;2017:6943851.
[19]Dong Y,Wang H,Chen Z.Alpha-Lipoic Acid Attenuates Cerebral Ischemia and Reperfusion Injury via Insulin Receptor and PI3K/Akt-Dependent Inhibition of NADPH Oxidase.Int JEndocrinol.2015;2015,903186.
[20]Kamarudin MN,Mohd Raflee NA,Hussein SS,et al.(R)-(+)-α-lipoic acid protected NG108-15 cells against H?O?-induced cell death through PI3K-Akt/GSK-3βpathway and suppression of NF-κβ-cytokines.Drug Des Devel Ther.2014;8:1765-1780.
[21]Kones R.Mitochondfial therapy for Parkinson’s disease:neuroprotective pharmaconutrition may be disease-modifying.Clin Pharmacol.2010;2:185-198.
[22]马玲,周勇,王莉,等.硫辛酸减轻高尿酸血症大鼠氧化应激损伤[J].基础医学与临床,2015,35(8):1037-1041.
[23]Rochette L,Ghibu S,Richard C,et al.Direct and indirect antioxidant properties of alpha-lipoic acid and therapeutic potential.Mol Nutr Food Res.2013;57(1):114-125.
[24]Kleinkauf-Rocha J,Bobermin LD,Machado PM,et al.Lipoicacid increases glutamate uptake,glutamine symhetase activity and glutathione content in C6 astrocyte cell line.Int J Dev Neurosci.2013;31(3):165-170.
[25][Papanas N,Ziegler D.Efficacy ofα-lipoic acid in diabetic neuropathy.Expert Opin Pharmacother.2014;15(18):2721-2731.
[26]Zhao L,Hu FX.Alpha-Lipoic acid treatment of aged type 2diabetes mellitus complicated with acute cerebral infarction.Eur Rev Med Pharmacol Sci.2014;18(23):3715-3719.
[27]Saygin M,Asci H,Cankara FN,et al.The impact of high fructose on cardiovascular system:Role of alpha-lipoicacid.Hum Exp Toxicol.2015;35(2):194-204.
[28]Hegazy SK,Tolba OA,Mostafa TM,et al.Alpha·lipoicacid improves subclinical left ventricular dysfunction in asymptomatie patients with type 1 diabetes.Rev Diabet Stud.2013;10(1):58-67.
[29]Koriyama Y,Nakayama Y,Matsugo S,et al.Anti-inflammatory effects of lipoic acid through inhibition of GSK-3βin lipopolysaccharide-induced BV-2 microglial cells.Neurosci Res.2013;77(1-2):87-96.
[30]Jiang S,Zhu W,Li C,et al.α-Lipoic acid attenuates脂多糖-induced cardiac dysfunction through a PI3K/Akt-dependent mechanism.Int Immunopharmacol.2013;16(1):100-107.
[31]Ying Z,Xie X,Chen M,et al.Alpha-lipoic acid activates eNOSthrough activation of PI3-kinase/Akt signaling pathway.Vascul Pharmacol.2015;64:28-35.
[32]Liu SF,Malik AB.NF-kappa B activation as a pathological mechanism of septic shock and inflammation.Am J Physiol,Cell Mol Physiol.2006;290(1):L622-L645.
[33]Wu DC,Teismann P,Tieu K,et al.NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson′s disease.Proc Natl Acad Sci USA.2003;100(10):6145-6150.
[34]Zhang F,Qian L,Flood PM,et al.Inhibition of IkappaBkinase-beta protectsdopamine neurons against lipopolysaccharide-induced neurotoxicity.J Pharmacol Exp Ther.2010;333(3):822-833.
[35]Li Y,Ma QG,Zhao LH,et al.Effects of lipoic acid on immune function,the antioxidant defense system,and inflammation-related genes expression of broiler chickens fed aflatoxin contaminated diets.Int J Mol Sci.2014;15(4):5649-5662.
[36]Kim HS,Kim HJ,Park KG,et al.Alpha-lipoic acid inhibits matrix metallop-roteinase-9 expression by inhibiting NF-kappaBtranscriptional activity.Exp Mol Med.2001;39(1):106-113.
[37]Li G,Fu J,Zhao Y,Ji K,et al.Alpha-lipoic acid exerts anti-inflammatory effects on lipopolysaccharide-stimulated rat mesangial cells via inhibition of nuclear factor kappa b(NF-kappab)signaling pathway.Inflammation.2015;38(2):510-519.
[38]Koriyama Y,Nakayama Y,Matsugo S.et al Anti-inflammatory effects of lipoic acid through inhibition of GSK-3βin lipopolysaccharide-induced BV-2 microglial cells.Neurosci Res.2013;77(1/2):87-96.
[39]Li YH,He Q,Yu JZ,et al.Lipoic acid protects dopaminergic neurons in lps-induced parkinson′s disease model.Metab Brain Dis.2015;30(5):1-10.