| BackgroundPakinson’ Disease (PD) is a progressive neurodegenerative disorder characterized by motor abnormalities, including resting tremor, rigidity, slow execution of movement, postural instability, and the pathological mechanisms of PD have not been demonstrated. Eph receptor tyrosine kinases and their ligands are members of tyrosine kinases family. EphA5/ephrin-A5can promote neurite outgrowth of dopaminergic neurons and regulate the formation of mesostriatal pathway. But it has been less known about its effect during pathogenesis of PD. Cell division cycle42(Cdc42) may be involved in neuronal survival by promoting formation the actin cytoskeleton. Ginsenoside Rgl may be useful in improving and treating PD, but its exact mechanism has not been clarified.ObjectiveTo explore new targets for prevention and treatment of PD through investigating the EphA5expressions in the Substantia nigra and ephrinA5, Cdc42in the striatum of mice with PD induced by MPTP, as well as the influence of ginsenoside Rgl on their expressions.MethodsC57BL/6mice were randomly divided into three groups, including the model group, ginsenoside Rgl group and control group. The mice of control group were treated with0.9%saline (1ml/kg, i.p.); the mice of model group were injected with MPTP (20mg/kg, i.p.); the mice of ginsenoside Rgl group were pretreated with ginsenoside Rgl(10mg/kg, once a day for3days, i.p.) before the MPTP was injected. The mice will be treated with MPTP on the day when the ginsenoside Rgl was given the mice for the last time, and MPTP was injected2hs later. The behavioral changes of mice were observed every day. The experimental materials were connected on the7th day after mice were treated with MPTP according to different experiments.(1) Behavioral change To observe the behavioral features and their swimming styles of mice. The swimming scores were recorded.(2) Pathomorphological observation To observe the pathomorphological changes of dopaminergic neurons under microscopy after the tissues was stained with HE.(3) RT-PCR To detect the expressions of tyrosine hydroxylase (TH), EphA5mRNA in SN of the midbrain and ephrinA5mRNA in striatum by RT-PCR.(4) Immunohistochemistry To investigate the expressions of TH protein in SN and striatum with immunohistochemistry.(5) Western blot analysis To detect the expressions of TH, EphA5protein in SN, ephrinA5, Cdc42protein in striatum by western blotting.Results(1) Behavioral features The mice of model group showed motor abnormalities, including resting tremor, rigidity and bradykinesia after the third injection of MPTP. Comparing with the model group, the mice of ginsenoside Rgl treated group showed less behavioral symptoms, and the mice of control group did not have any behavioral changes. During the same testing time, the mice of model group showed poorly concordant limbs. Most of the mice floated in water for a long time. The mice of ginsenoside Rgl treated group floated in water by chance. The mice of control group swimmed actively. Swim-scores of model group were significantly lower than those of the control group and ginsenoside Rg1group (P<0.05).(2) Pathomorphological observation The number of dopaminergic neurons of model group was reduced significantly comparing with the ginsenoside Rgl treated group and control group. (3) RT-PCR①The level of TH mRNA expression in the model group (0.84±0.09) was significantly lower than that of the control group (0.96±0.07,P<0.05).②The level of EphA5mRNA expression (0.53±0.06) in the model group was significantly lower than those of the control group (0.85±0.01, P<0.01) and ginsenoside Rgl group (0.81±0.01, P<0.01). There were statistical significance among groups (F=318.72, P<0.01).③The level of EphrinA5mRNA expression (0.75±0.01) in the model group was significantly higher than those of the control group (0.38±0.01, P<0.01) and the ginsenoside Rgl group (0.33±0.40, P<0.01). There were statistical significance among groups (F=6.088, P<0.01).(4) Immunohistochemistry analysis①The number of TH-positive neurons in the SNc of the model group (6.33±4.53) was significantly lower than those of the control group (16.80±6.63, P<0.01) and the ginsenoside Rgl group (13.73±4.76, P<0.05). There were statistical significance among groups (F=14.97, P<0.01).②The TH-IR fiber densities in the model group (13.75%±6.67) was significantly lower than that of the control group (51.40%±9.81,P<0.00)and ginsenoside Rgl group(23.34%±5.91, P<0.05). There were statistical significance among groups (F=45.75,P<0.01).(5)Western blot analysis①The level of TH protein expression in the model group (0.33±0.01) was significantly lower than those of the control group (0.64±0.04, P<0.01) and ginsenoside Rgl group (0.51±0.05, P<0.05). There were statistical significance among groups (F=119.39, P<0.01).②The level of EphA5protein expression in the model group (0.02±0.01) was significantly lower than those of the control group (0.19±0.02, P<0.01) and ginsenoside Rgl group (0.09±0.01,P<0.01). There were statistical significance among groups (F=343.90, P<0.01).③The level of ephrinA5protein expression (0.19±0.01) in the model group was significantly higher than those of the control group (0.17±0.01,P<0.01) and Ginsenoside Rgl group (0.07±0.01, P<0.01). There were statistical significance among groups(F=799.99, P<0.01).④The level of Cdc42protein expression in the model group (0.70±0.08) was significantly higher than those of the control group (0.43±0.06,P<0.01) and ginsenoside Rgl group (0.37±0.08, P<0.01). There were statistical significance among groups (F=31.047, P<0.01).Conclusions(1) MPTP may induce PD of mice by reducing EphA5expression in SN of midbrain and promoting expression of ephrinA5and Cdc42in striatum.(2) Ginsenoside Rgl may improve the symptoms of mice with PD induced by MPTP through increasing EphA5expression in SN of midbrain, decreasing the expressions of ephrinA5, and Cdc42in striatum. |
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