| Background and ObjectiveParkinson's disease(PD)is a progressive degenerative disease of the central nervous system.The loss of dopaminergic neurons and the decrease of dopamine content in the striatum may result in tremor,motor retardation,unstable posture and other clinical symptoms due to the damage of the pathway in the substantia nigra.Environmental factors such as neurotoxins may accelerate the degenerative death of dopaminergic neurons.Many factors,such as oxidative stress,apoptosis,neuroinflammation,protein misfolding and accumulation,were found to play an important role in the pathogenesis of PD.However,its etiology and pathogenesis are still not completely clear.The current drug therapy for PD is mainly symptomatic and does not prevent the death and progression of dopaminergic neurons.Therefore,it is important for the prevention and treatment of PD to seek new safe and effective neuroprotective drugs.Curcumin is one of the main active polyphenols in rhizomes of turmeric,it has been used as food additive and traditional Chinese medicine.It has many pharmacological effects,such as anti-oxidation,anti-cancer,anti-inflammation,neuroprotection and so on.The dysfunction of the antioxidation system in the substantia nigra leads to the increase of oxidative stress,the production of excessive free radicals,the destruction of the cell membrane and the production of lipid peroxidation,leading to the apoptosis of dopaminergic neurons in the substantia nigra.It may be an important cause of PD.The results showed that curcumin could inhibit the activity of dopaminergic neurons by scavenging reactive oxygen free radicals,increasing the activity of antioxidant enzymes,maintaining the stability of mitochondrial membrane potential and inhibiting the activity of Caspase3 enzyme.Wnt signaling pathway is involved in regulating cell proliferation,differentiation,stress,apoptosis and other cell biological behaviors.In the classical Wnt signaling pathway,signal molecules such as Wntl?Wnt3a?Wnt8 bind to its transmembrane receptor Frizzled protein and transmit the signal to ?-catenin,which accumulates in cells and enters the nucleus with the T cytokine / lymphocyte enhancement factor(T-cell factor/lymphoid enhancer factor,TCF/LEF),regulating its downstream target gene expression,the effect is closely related to cell apoptosis and proliferation and differentiation.At present,the research on this signal pathway in Parkinson's disease is rare in the literature at home and abroad.The purpose of this study was to investigate the protective effect of curcumin on the 6-OHDA induced Parkinson's disease cell model and its mechanism to provide the theoretical basis for prevention and treatment of PD.Materials and MethodsPrimary culture of embryonic mesencephalic cells from 15 day pregnant rats.On the 5th day of culture,the curcumin group was divided into three subgroups with different concentrations of curcumin(10 and 15 umol/L)for 5 days PD cell model in vitro was established by adding 6-OHDA of 100umol/L for 2 h on the 10 th day.6-OHDA group was primary cultured cells plus 100 umol/L 6-OHDA.The control group was primary cultured cells and collected cells for 10 days.Cell activity was detected by four methazo azolate(MTT)method.Detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling(Tunel).Detection of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)content in primary mesencephalic cells by chemical colorimetry.Western-blot assay was used to detect the expression of wnt3 a,?-catenin in mesencephalic primary cells.The expression of wnt3 a,?-cateninine c-myc and cylinD1 mRNA were detected by qRT-PCR.Results1.MTT :Determined by MTT :5umol/L curcumin had no protective effect on 6-OHDA induced cell injury,and the cell survival rate was not significantly different from that of 6-OHDA group(P > 0.05)and curcumin(15umol/L)had protective effect on 6-OHDA induced cell injury,and the cell survival rate was higher than that in 6-OHDA group.The difference was significant(P < 0.05),and the effect of 10umol/L was the strongest.2.Detection of apoptosis by TUNEL:The apoptosis rate of 6-OHDA group was higher than that of control group(P < 0.05).In the apoptosis rate of 5umol/L curcumin group no significant difference compared with that of the 6-OHDA group(P> 0.05).The apoptotic rate of 10 umol/L and 15 umol/L curcumin group was significantly lower than that of the control group(P < 0.05).The decrease of curcumin in 10umol/L group was more obvious.3.Determination of GSH-Px activity and MDA content in SODH-Px by chemical colorimetry:Compared with the control group,the activity of SOD and GSH-Px in curcumin subgroup(5?10?15umol/L)decreased significantly(P < 0.05).5 umol/L curcumin was no significant difference compared with the 6-OHDA group.The activities of SOD and GSH-Px in 10 umol/L and 15umol/L curcumin groups increased and decreased(P < 0.05).4.Detection of protein expression by Western blot:The expression of wnt3 a and ?-catenin in 5umol/L curcumin group and 6-OHDA group was significantly lower than that in control group(P < 0.05),however,the expression of wnt3 a and ?-catenin protein in 10 and 15umol/L curcumin group was significantly higher(P < 0.05),especially in 10umol/L curcumin group(P < 0.05).There was no significant difference in the expression of wnt3 a and ?-catenin protein in 5umol/L curcumin group compared with 6-OHDA group(P > 0.05),but the expression of wnt3 a and ?-catenin protein in 10 and 15umol/L curcumin group were increased(P < 0.05).5.Detection of Wnt3 a,?-cateninine c-myc and cylinD1 mRNA by qRT-PCR:Compared with the control group,the expression of wnt3 a,?-catenin c-myc and cylinD1 mRNA in 5umol/L curcumin group and 6-OHDA group was significantly lower than that in control group(P < 0.05)and the expression of wnt3 a,?-catenin c-myc and cylinD1 mRNA in 10 and 15umol/L curcumin group was significantly higher(P < 0.05),especially in 10umol/L curcumin group(P < 0.05);the expression of wnt3 a,?-cateninine c-myc and cylinD1 mRNA in 5umol/L curcumin group was no significant difference compared with the 6-OHDA group.(P >0.05),the expression of wnt3 a,?-cateninine c-myc and cylinD1 mRNA in 10 and 15umol/L curcumin group were higher(P < 0.05).ConclusionCurcumin can attenuate the damage induced by 6-OHDA in primary mesencephalic cells.The mechanism is that may active Wnt/ ?-catenin signaling pathway,increase of cell viability,the decrease of apoptosis and the anti-oxidation effect of curcumin. |
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