| BackgroundParkinson's disease(PD)is a common progressive neurodegenerative disorder,affecting the population over 50 or 60 years.The main pathological hallmarks of PD are the progressive loss or degeneration of dopaminergic neurons from the midbrain substantia nigra pars(SNc)and the formation of Lewy body.The pathogenesis of Parkinson's disease is still obscure.Neuregulin1(NRG1)plays a key role in the dgrowth of nerves and neural function and has a close relation with the development of many kinds of neural disorders through activating ErbB receptor ErbB4,one of the tyrosine kinases,to transit a series of signals.Ginsenoside Rg1 has the function of anti-apoptosis and protection of neuron,but its effect on PD needs to be further investigated.ObjectiveTo explore the molecular mechanism of pathogenesis of PD induced by MPTP through investigating the effect of Ginsenoside Rg1 on expression and activity of NRG1 and ErbB4 in substantial nigra of mouse with PD and try to find a new target for the prevention and treatment of PD.Methods1.Experimental groups Fourty C57BL/6 mice were divided into control group,MPTP group,MPTP+Ginsenoside Rg1 group and MPTP+Ginsenoside Rg1+H89 group at random.2.The PD model established The mice of control group and MPTP model group had been treated with 0.9% normal saline solution(1ml/kg/d)by intraperitoneal injection,and the MPTP+Ginsenoside Rg1 group and the MPTP+Ginsenoside Rg1+H89 group had been injected with Ginsenoside Rg1(10mg/kg/d)intraperitoneally for 3 days.On the fourth day,the control group was still injected with normal saline solution(1ml/kg),but MPTP model group,MPTP+Ginsenoside Rg1 group and MPTP+Ginsenoside Rg1+H89 group were injected intraperitoneally with MPTP(20mg/kg)four times totally at interval of 2 hours.The mice of MPTP+Ginsenoside Rg1+H89 group were intraperitoneally injected with H89(1mg/kg)30 minutes earlier before they were treated with MPTP.3.Swimming test The behavioral change of the mice of all groups was determined by scoring the coordination ability of limbs of mice at different time points based on the scoring criteria of swimming test.4.Pathologic morphology The shape and quantity of neurons in the substantia nigra of mice were obversed through microscope.5.RT-PCR The expression of NRG1 and ErbB4 mRNA in substantial nigra of mice were detected by RT-PCR.6.Western blotting The expression level of ErbB4 protein and its phosphorylation protein were detected with Western blotting.Results1.The changes of general behavior After the second injection of MPTP,mice of the MPTP model group showed behavior changes at different degree,such as pilo-erection,sticking tails up,spinning etc within an hour.After the fourth injection of MPTP,mice showed muscle tremors,stiff tail,unsteady gait,reducing activities,and so on.The mentioned symptoms almost disappeared 24 hours later.The mice of Ginsenoside Rg1+H89 group also showed pilo-erection,stiff tail,muscle tremor,and unsteady gait at different degree after being injected H89 and MPTP.The mice of MPTP+Ginsenoside Rg1 group showed less symptoms compared to the two gropu mentioned above.The mice of control group didn't show any similar symptoms.2.Swimming Test The swimming test scores in the MPTP model group were significantly lower than those of the control group(P<0.05)at different time points.The swimming test scores of the MPTP+ginsenoside Rg1 group were obviously higher than those of the MPTP model group(P<0.05).The Values of swimming test of MPTP +ginsenoside Rg1+H89 group significantly decreased compared to the MPTP+ginsenoside Rg1 group(P<0.05).There is no difference about swimming test scores the MPTP model group and the MPTP+ginsenoside Rg1+H89 group(P>0.05).3.Pathologic morphological changes of dopaminergic neurons in the midbrain substantia nigra The axons of dopaminergic neurons became shorter and the quantity of dopaminergic neurons decreased significantly compared to the control group.The quantity of dopaminergic neurons of the MPTP model group(32.461±1.170)were obviously lower than that of the control group(40.334±0.845,t=-13.452,P=0.000).The quantity of dopaminergic neurons of the MPTP+ginsenoside Rg1group(35.925±0.681)is much more than that of the MPTP model group(t=-7.23,P=0.003).The quantity of dopaminergic neurons of the MPTP+ginsenoside Rg1+H89 group(33.648±0.966)significantly deceased compared to the MPTP+ginsenoside Rg1 group(t=3.230,P=0.008).There was no significant difference between the MPTP model group and MPTP+ginsenoside Rg1+H89 group(t=2.274,P=0.892).Variance analysis showed that there were significant differences among all groups(F=41.234,P=0.000).4.RT-PCR The expression level of Nrg1 mRNA of the model group(0.460±0.1704)is significantly lower than that of the control group(1.000±0.000,t=6.339,P=0.0079).The expression level of Nrg1 mRNA in MPTP+Ginsenoside Rg1 group(0.925±0.1681)was much higher than that of the MPTP model group(t=3.199,P=0.0494).Compared with MPTP+Ginsenoside Rg1 group,the expression level of Nrg1 mRNA of MPTP+ Ginsenoside Rg1+H89 group significantly decreased(t=1.916,P=0.0499).There was no significant difference between the MPTP model group and the MPTP+ginsenoside Rg1+ H89 group(t=1.134,P=0.3391).Variance analysis showed that there was statistical difference among different groups(F=8.0230,P=0.0211).The expression level of Nrg1-type I/II mRNA of the MPTP model group(0.376± 0.1201)was significantly lower than that of the control group(1.000±0.000,t=10.400,P=0.0019).The expression level of Nrg1-type I/II mRNAof the MPTP+Ginsenoside Rg1 group(0.766±0.1382)was obviously higher than that of the MPTP model group(t=3.377,P=0.0432).Compared to the MPTP+Ginsenoside Rg1 group,the expression level of Nrg1-type I/II mRNA in the MPTP+Ginsenoside Rg1+H89 group(0.310±0.2898)significantly deceasead(t=3.150,P=0.0513).There was no significant difference between the MPTP model group and the MPTP+ginsenoside Rg1+H89 group(t=0.3319,P=0.7618).Variance analysis showed that the difference between groups was statistically significant(F=12.46,P=0.0187).The expression level of Nrg1-type III mRNA of the MPTP+Ginsenoside Rg1 group(1.121±0.1812)was much higher than that of the MPTP model group(1.000±0.0000)(t=3.419,P=0.0419).5.Western blot Variance analysis showed that there was no significant difference on the expression level of ErbB4 protein among all groups(F=0.1578,P=0.8005).The expression level of phophorylation ErbB4 protein of the MPTP model group(0.339±0.133 9)was significantly lower than that of the control group(1.000±0.0000,t=8.548,P=0.0134).The expression level of phophorylation ErbB4 protein of the MPTP+ Ginsenoside Rg1 group(0.782±0.1369)was much higher than that of the MPTP model group(t=6.986,P=0.0199).The expression level of phophorylation ErbB4 protein of the MPTP+Ginsenoside Rg1+H89 group(0.365±0.2677)was obviously lower than that of the MPTP+Ginsenoside Rg1 group(t=4.407,P=0.0478).There was a significant difference among all groups analyzed by variance analysis(F=17.04,P=0.0282).Conclusions1.MPTP may induce PD through reducing the expression and activity of NRG1 and ErbB4 in the substantia nigra of C57BL/6 mice.2.Ginsenoside Rg1 may improve the symptoms of PD by activating NRG1/ErbB4 signal path in the substantia nigra of mice. |
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