| Parkinson's disease(PD)is a neurodegenerative disease of central nervous system,caused by selective degeneration of substantia nigra of midbrain dopamine neurons,the decreased level of dopamine in the striatum.With the increasingly serious situation of aging population in the world,the incidence of the disease rate also increased.Although the pathological changes and clinical symptoms of PD have been well stated,the exact underlying mechanisms of dopaminergic cell death remain unknown.The current treatment of PD mainly concentrated on the treatment of symptoms but can not prevent the loss of midbrain substantia nigra dopaminergic neurons in PD patients.Therefore,it is significant to study the pathogenesis of the disease and to find new drug treatment,which is of great significance to delay the progression of PD.The pathogenesis of PD is complex.Aging,environmental factors,genetic factors,chronic inflammation and so on are closely linked with PD.At present,the pathogenesis of PD is not very clear,but it will cause the death of dopamine neurons in the substantia nigra.Find the pathway that prevent dopamine neuron death and to investigate PD pathogenesis and treatment is of great significance.In recent years,the role of Wnt signaling pathway in PD is gradually being recognized.Wnt signaling pathway regulates the development of the central nervous system,cell proliferation,differentiation,migration and other processes,which play a protective role in the development of dopaminergic neurons and the injury of dopaminergic neurons.There are three kinds of Wnt pathway,the most important one is the classical pathway,namely the Wnt/?-catenin pathway,the ?-catenin protein is the central link of classical Wnt signaling pathway,which regulates the expression of downstream genes by activated protein ?-catenin.Ginsenoside Rgl(Rgl)is one of the main active components of ginseng,which is extracted from the traditional Chinese herbal medicine.Previous studies of Rgl are mainly concentrated on the anti-inflammatory and immune regualtion.In recent years,some studies showed that Rgl in animal models of diseases of the central nervous system is beneficial,and researchers found that Rgl plays a protective role through anti-apoptosis and oxidative stress in PD models.But whether the protective effect of Rgl in PD model is related to the Wnt/?-catenin pathway is unknown.This study uses a MPTP neurotoxicity induced PD model in C57BL/6J mice,the effect of MPP+ to PC12 cells to establish the cell model of PD.We discussed the mechanism of Wnt/?-catenin pathway in PD and the protective effect of Rgl on PD is through the activation of the Wnt/p-catenin pathway,which provides a new method of drug therapy for the treatment of PD.Part ? Protective effects of ginsenoside Rgl on midbrain substantia nigra dopaminergic neurons in MPTP-induced PD mice modelObjective:To investigate the effects of ginsenoside Rgl on the behavior of MPTP-induced mice PD model,study the effects of ginsenoside Rg1 on the dopaminergic neurons in the substantia nigra of midbrain and striatal TH positive projecting fiber.Methods:Sixty healthy male C57BL/6J mice,were randomly divided into:(1)normal saline control group(control group);(2)Rgl high dose group(Rgl 20 mg/kg);(3)the MPTP model of PD group(MPTP 30 mg/kg);(4)MPTP+Rgl low dose treatment group(MPTP 30mg/kg+Rgl 5 mg/kg);(5)MPTP+Rgl medium dose group(MPTP 30 mg/kg+Rgl 10 mg/kg);(6)MPTP+Rgl high dose treatment group(MPTP 30 mg/kg+Rgl 20 mg/kg).We established mouse model of PD with the neurotoxicity of MPTP,MPTP with double distilled water is configured to 2mg/mL concentration,by intraperitoneal injection to male C57BL/6J mice 30mg/kg,the modeling time for 5 days.Rgl treatment groups were given 2 hours before the MPTP injection of the corresponding dose of Rgl,after the end of the MPTP model to give Rgl continuous treatment for another 10 days.Individual Rgl group was given intraperitoneal injection for 15 days.Saline control group was given intraperitoneal injection of normal saline for 15 days.After modeling,observe mouse behavior changes;with immunofluorescence,Western blot and RT-PCR to detect midbrain substantia nigra dopamine neurons of tyrosine hydroxylase(TH)and dopamine transporter(DAT)protein and mRNA level changes;immunofluorescence method to detect striatal TH positive projecting fiber.Results:1.The behavioral test(pole test,traction test,swimming test)showed that the motor function was impaired in MPTP groups rats,and the motor function of Rgl treatment groups were significantly better than that of MPTP group.2.The number of TH positive neurons in MPTP group was significantly lower than that in control group,while the number of TH positive neurons in Rg1 treatment groups were significantly higher than that in MPTP group.3.The protein and mRNA levels of TH and DAT in MPTP group were lower than that in control group,but in Rg1 treatment groups,the protein and mRNA levels of DAT and TH were higher than the MPTP model group.4.Compared with the control group,the TH positive projection fibers in the striatum in the MPTP group were significantly decreased,and the TH positive projection fibers in the striatum of the Rg1 treatment groups were significantly increased compared with that of the MPTP group.Conclusion:Rg1 can improve the behavioral damage of MPTP-induced PD mice model,reduce the loss of dopaminergic neurons in the substantia nigra of midbrain and increase the TH positive projection fibers in the striatum.Part ? Neuroprotecive effects of ginsenoside Rg1 in MPTP-induced PD mice model through the Wnt/?-catenin signaling pathwayObjective:To investigate the mechanism of Wnt/?-catenin signaling pathway in MPTP-induced PD mice model,to clarify whether the ginsenoside Rg1 can exert protective effects through regulation of Wnt/?-catenin signaling pathway in MPTP-induced mice model of PD.Methods:Sixty healthy male C57BL/6J mice,were randomly divided into:(1)normal saline control group(control group);(2)Rg1 high dose group(Rg1 20 mg/kg);(3)MPTP model of PD group(30 mg/kg);(4)MPTP+Rg1 low dose treatment group(MPTP 30 mg/kg+Rg1 5 mg/kg);(5)MPTP+Rg1 medium dose group(MPTP 30 mg/kg+Rgl 10 mg/kg);(6)MPTP+Rgl high dose treatment group(MPTP 30 mg/kg+Rgl 20 mg/kg).We adopt MPTP-induced mice model of Parkinson's disease,MPTP with double distilled water is configured to 2mg/mL concentration,by intraperitoneal injection to male C57BL/6J mice 30mg/kg,modeling time for 5 days.Rgl treatment groups were given 2 hours before the MPTP injection of the corresponding dose of Rgl,after the end of the MPTP model to give Rg1 continuous treatment for another 10 days.Individual Rgl group was given intraperitoneal injection for 15 days in a row for 15 days.Saline control group was given intraperitoneal injection of normal saline for 15 days.We used TUNEL apoptosis assay detected the mesencephalic substantia nigra apoptosis changes;western blot detected the protein level of apoptotic protein caspase-3 and anti-apoptotic protein Bcl-xL changes in mesencephalic substantia nigra;immunofluorescence,western blot and RT-PCR were used to detect the protein and mRNA level changes of Wnt/?-catenin signaling pathway marked molecules in midbrain substantia nigra dopaminergic neurons.Results:1.Compared with the control group,the number of substantia nigra cells apoptosis increased significantly in the MPTP group,while the number of substantia nigra cells apoptosis in the Rgl treatment group was significantly decreased compared with the MPTP group.2.MPTP treated mesencephalic substantia nigra apoptotic protein caspase-3 protein levels was significantly higher compared with control group,the anti-apoptotic protein Bcl-xL protein levels decreased significantly compared with control group;however the protein level of caspase-3 in the Rgl treatment group was significantly lower than that of the MPTP group,and the Bcl-xL protein level was significantly higher than that of the MPTP group.3.Compared with the control group,the protein levels of Wntl and ?-catenin in MPTP group decreased,the protein levels of GSK-3? and p-GSK-3? elevated;however,the protein levels of Wntl and ?-catenin in Rgl treatment group elevated compared with MPTP group,the protein levels of GSK-3p and p-GSK-3? decreased compared with MPTP group.4.Compared with the control group,the mRNA levels of Wntl and ?-catenin in MPTP group decreased,the mRNA level of GSK-3? elevated;however,the mRNA levels of Wntl and ?-catenin in Rgl treatment group elevated compared with MPTP group,the mRNA level of GSK-3?decreased compared with MPTP group.5.The immunofluorecence staining levels of?-catenin and GSK-3? in dopaminergic neurons were consistent with the protein and mRNA levels.Conclusion:Wnt/?-catenin signaling pathway was impaired in midbrain substantia nigra dopaminergic neurons of MPTP-induced Parkinson's disease mice model,Rgl in MPTP-induced Parkinson's disease mice model can activate the Wnt/p-catenin signaling pathway.Part ? Neuroprotecive effects of ginsenoside Rgl in MPP+-induced PC12 cells PD model through the Wnt/p-catenin signaling pathwayObjective:To establish MPP+-induced PC 12 cells in vitro PD model,to explore the the exact mechanism of Wnt/?-catenin signaling pathway in MPP+-induced PC 12 cells model,to clarify whether Rgl could play a protective role through the regulation of Wnt/?-catenin signaling pathway in MPP+-induced PC 12 cells.Methods:When investigated the Wnt/?-catenin signaling pathway,cells were divided into five groups:(1)negative control group(control group):with cells,without the addition of MPP+,Rgl and DKK1;(2)MPP+ group(500 pM);(3)MPP++Rgl(20?M)group;(4)MPP++Rgl+DKK1 group;(5)MPP++DKK1 group.We used CKK8 to detect cell viability and screened Rgl optimum concentration and action time;TUNEL apoptosis detection method for detecting apoptosis in PC 12 cell changes;western blot detection of PC 12 cells apoptotic protein caspase-3 and anti-apoptotic protein Bcl-xL protein levels changes;immunofluorescence,western blot and RT-PCR were used to detect the protein and mRNA levels changes of Wnt/?-catenin signaling pathway marked molecules in PC 12 cells.Results:1.CKK-8 was used to screen out the effect of 20 ?M Rgl for 24 hours as the best concentration and time of detection of Wnt/?-catenin signaling pathway.2.The number of apoptosis of PC12 cells in MPP+ group increased significantly than in the control group;and the apoptosis of PC 12 cells decreased significantly in Rgl treatment group compared with the MPP+ group;DKK1 in the intervention group were given at the same time,Rgl reduce apoptosis effect is not obvious.3.The PC 12 cells apoptotic protein caspase-3 protein level in MPP+ group was significantly higher than that of control group,and anti-apoptotic Bcl-xL protein level in MPP+ group decreased significantly than that of control group;however the protein level of caspase-3 in the Rg1 treatment group was significantly lower than that of the MPP+ group,and the Bcl-xL protein level was significantly higher than that of the MPP+ group;DKK1 in the intervention group were given at the same time,Rgl on the level of apoptotic protein reduction effect is not obvious.4.Compared with the control group,the protein levels of Wntl and ?-catenin in MPP+ group decreased,the protein levels of GSK-3? and p-GSK-3(3 elevated;however,the protein levels of Wntl and p-catenin in Rgl treatment group elevated compared with MPP+ group,the protein levels of GSK-3? and p-GSK-3? decreased compared with MPP+ group;DKK1 in the intervention group were given at the same time,the change effect of Rgl on the protein levels of these markers of Wnt/?-catenin signaling pathway is not obvious.5.Compared with the control group,the mRNA levels of Wntl and ?-catenin in MPP+ group decreased,the mRNA level of GSK-3? elevated;however,the mRNA levels of Wntl and ?-catenin in Rg1 treatment group elevated compared with MPP+ group,the mRNA level of GSK-3? decreased compared with MPP+ group;DKK1 in the intervention group were given at the same time,the change effect of Rgl on the mRNA levels of these markers of Wnt/?-catenin signaling pathway is not obvious.6.The immunofluorecence staining levels of?-catenin and GSK-3? in PC 12 cells were consistent with the protein and mRNA levels.Conclusion:Wnt/?-catenin signaling pathway was impaired in MPP+-induced PC12 cells PD model,Rgl can exert neuroprotective effects in MPP+-induced PC12 cells PD model through the Wnt/p-catenin signaling pathway. |
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