人参皂苷Rg1对LRRK2突变致帕金森病果蝇的治疗作用探讨
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摘要
探讨人参皂苷Rg1对LRRK2突变致帕金森病果蝇模型的脑神经保护作用及其机制。使用TH-GAL4;UASLRRK2-1915T品系果蝇(简称1915T)进行实验,使用1915T品系果蝇作为病理模型组,TH-GAL4品系果蝇作为阴性对照组。给予不同浓度的人参皂苷Rg1,饲养一定时间后,将帕金森综合征(parkinson's syndrome,PD)病理模型组与对照组进行比较,利用果蝇寿命实验,检测生命周期,筛选最佳药物作用浓度。利用该浓度进一步检测神经元凋亡,同时取果蝇头部提取蛋白质,采用Western blot方法检测P-LRRK2、Nrf2、GCLC、P-P38MAPK、ERK、TH等蛋白表达的变化。结果表明:人参皂苷Rg1最佳药物浓度确定为0.4 mmol/L,将0.4 mmol/L人参皂苷Rg1于果蝇幼虫发育时期和成虫发育时期同时给与药物干预后,可以显著挽救LRRK2突变导致的果蝇存活率降低、爬行能力降低以及多巴胺含量的降低。人参皂苷Rg1能有效降低PD模型果蝇的病理症状,作用途径可能是通过其作用于P-38信号通路以保护PD中的多巴胺能神经元。
The neuroprotective effect and mechanism of ginsenoside Rg1 on LRRK2 mutation-induced drosophila model of drosophila melanogaster was studied.TH-GAL4;UAS-LRRK2-1915T strain drosophila(abbreviated as 1915T)was used for the experiment. The 1915T strain drosophila was used as the pathological model group,and the TH-GAL4 strain drosophila was used as the negative control group. Different concentrations of ginsenoside Rg1 were administered. After a certain period of time,the pathological model group of parkinson's syndrome(PD)was compared with the control group,and the life cycle was tested by drosophila life test to select the optimal drug concentration.Neuronal apoptosis was further detected by screening the optimal concentration of the drug. At the same time,the protein was extracted from the head of drosophila,and the expression of P-LRRK2,Nrf2,GCLC,P-P38MAPK,ERK,TH and other proteins were detected by Western blot.The optimal drug concentration of ginsenoside Rg1 was determined to be 0.4 mmol/L.Simultaneous treatment of drosophila during larval development and adult development with 0.4 mmol/L ginsenoside Rg1 significantly reduced the survival rate of drosophila,decreased crawling ability and decreased dopamine content caused by LRRK2 mutation.Ginsenoside Rg1 may protect dopaminergic neurons in PD by P-38 signaling pathway to reduce pathological symptoms of PD model drosophila.
The neuroprotective effect and mechanism of ginsenoside Rg1 on LRRK2 mutation-induced drosophila model of drosophila melanogaster was studied.TH-GAL4;UAS-LRRK2-1915T strain drosophila(abbreviated as 1915T)was used for the experiment. The 1915T strain drosophila was used as the pathological model group,and the TH-GAL4 strain drosophila was used as the negative control group. Different concentrations of ginsenoside Rg1 were administered. After a certain period of time,the pathological model group of parkinson's syndrome(PD)was compared with the control group,and the life cycle was tested by drosophila life test to select the optimal drug concentration.Neuronal apoptosis was further detected by screening the optimal concentration of the drug. At the same time,the protein was extracted from the head of drosophila,and the expression of P-LRRK2,Nrf2,GCLC,P-P38MAPK,ERK,TH and other proteins were detected by Western blot.The optimal drug concentration of ginsenoside Rg1 was determined to be 0.4 mmol/L.Simultaneous treatment of drosophila during larval development and adult development with 0.4 mmol/L ginsenoside Rg1 significantly reduced the survival rate of drosophila,decreased crawling ability and decreased dopamine content caused by LRRK2 mutation.Ginsenoside Rg1 may protect dopaminergic neurons in PD by P-38 signaling pathway to reduce pathological symptoms of PD model drosophila.
引文
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[3]Covy J P,Giasson B I.α-Synuclein,leucine-rich repeat kinase-2,and manganese in the pathogenesis of Parkinson disease[J].Neurotoxicology,2011,32(5):622-629
[4]Esteves A R,Swerdlow R H,Cardoso S M.LRRK2,a puzzling protein:insights into Parkinson's disease pathogenesis[J].Exp Neurol,2014,261:206-216
[5]MacLeod D,Dowman J,Hammond R,et al.The familial Parkinsonism gene LRRK2 regulates neurite process morphology[J].Neuron,2006,52(4):587-593
[6]Imai Y,Gehrke S,Wang H Q,et al.Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila[J].EMBO J,2008,27(18):2432-2443
[7]Jeong G R,Jang E H,Bae J R,et al.Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration[J].Mol Neurodegener,2018,13(2):1-8
[8]Chen X C,Zhu Y G,Zhu L A,et al.Ginsenoside Rg1 attenuates dopamine induced apoptosis in PC12 cells by suppressing oxidative stress[J].Eur J Pharmacol,2003,473(1):1-7
[9]Shi C,Zhang Y,Zhang Z,et al.Effect of phosphorylated-ERK1/2 on inducible nitric oxide synthase expression in the substantia nigra of mice with MPTP-induced Parkinson disease[J].South Med Univ,2009,29(1):60-63
[10]Ge K L,Chen W F,Xie J X,et al.Ginsenoside Rg1 protects against6-OHDA-induced toxicity in MES23.5 cells via Akt and ERK signaling pathways[J].Ethnopharmacol,2010,127(1):118-123
[11]Liu Q,Kou J P,Yu B Y.Ginsenoside Rg1 protects against hydrogen peroxide-induced cell death in PC12 cells via inhibiting NF-κBactivation[J].Neurochem Int,2011,58(1):119-125
[12]Wang Q,Zheng H,Zhang Z F,et al.Ginsenoside Rg1 modulates COX-2 expression in the substantia nigra of mice with MPTP-induced Parkinson disease through the P38 signaling pathway[J].Nan Fang Yi Ke Da Xue Xue Bao,2008,28(9):1594-1598
[13]Wang J,Xu H M,Yang H D,et al.Rg1 reduces nigral iron levels of MPTP-treated C57BL6 mice by regulating certain iron transport proteins[J].Neurochem Int,2009,54(1):43-48
[14]Wang G,Pan J,Chen S D.Kinases and kinase signaling pathways:potential therapeutic targets in Parkinson's disease[J].Prog Neurobiol,2012,98(2):207-221
[15]Liao J,Wu C X,Burlak C,et al.Parkinson disease-associated mutation R1441H in LRRK2 prolongs the"active state"of its GTPase domain[J].Proc Natl Acad Sci U S A,2014,111(11):4055-4060
[16]Nakaso K,Nakamura C,Sato H,et al.Novel cytoprotective mechanism of anti-parkinsonian drug deprenyl:PI3K and Nrf2-derived induction of antioxidative proteins[J].Biochemical&Biophysical Research Communications,2006,339(3):915-922
[17]Nguyen T,Sherratt P J,Nioi P,et al.Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleo cytoplasmic shuttling by Keap1[J].Journal of Biological Chemistry,2005,280(37):32485-32492
[18]Chen X C,Chen Y,Zhu Y G,et al.Protective effect of ginsenoside Rg1 against MPTP-induced apoptosis in mouse substantia nigra neurons[J].Acta Pharmacol Sin,2002,23:829-834
[19]Chen X C,Zhou Y C,Chen Y,et al.Ginsenoside Rg1 reduces MPTP-induced substantia nigra neuron loss by suppressing oxidative stress[J].Acta Pharmacol Sin,2005,26:56-62