| BackgroundParkinson’s disease (PD) is a common degenerative disease of the central nervous system in the elderly, characterized by the pathological features of degeneration and progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and declining levels of dopamine in the striatum. Clinical symptoms of PD include of resting tremor, muscle rigidity, bradykinesia, abnormal gait and postural instability. The pathogenesis of PD is inadequately understood. Both Eph receptors and their ligand ephrins belong to Ephrin family protein and constitute the largest family of tyrosine kinase receptors. It has been shown that EphB6and EphrinB1are related to Alzheimer’s disease and motor nerve function, but the role of EphB6and EphrinB1in PD pathogenesis is still poorly understood. Ginsenoside Rgl may improve and treat PD through adjusting apoptosis, but its exact mechanism is still not elucidated.ObjectiveTo investigate the molecular mechanisms of PD induced by1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and explore new targets for prevention and treatment of PD through determining expressions of EphB6and ephrinBl in substantia nigra as well as the regulation of Ginsenoside Rgl on their expressions.MethodThe C57BL/6mice were randomly divided into control group, model group, ginsenoside Rgl group, MPTP+ginsenoside Rgl+H-89group. The control group received intraperitoneal injection of saline (1ml/kg) and were injected totally four times at intervals of2hrs; model group were given intraperitoneal injection of MPTP (20mg/kg) and were injected tatally four times at intervals of2hrs; ginsenoside Rgl group were given intraperitoneal injection of ginsenoside Rgl (10mg/kg/d) for three days, on the third day the mouse were injected intraperitoneally MPTP (20mg/kg) and were injected tatally four times at intervals of2hrs, and the first injection of MPTP was followed by intraperitoneal injection of ginsenoside Rgl2hrs later; the mice of MPTP+ginsenoside Rgl+H-89group were injected ginsenoside Rgl and MPTP with the same way as the group of ginsenoside Rg1, and the intraperitoneal injection of H-89(2mg/kg) was followed by firstly injection of MPTP30mins later. On the seventh day after the last injection of MPTP, the experimental materials were collected according to different experimental requirements. After the last injection of MPTP, the behavior changes of mice were observed by swimming; the pathological morphology changes of dopaminergic neurons in substantia nigra were observed through microscopy after the tissues were stained with HE; the expressions of EphB6ephrinB1mRNA in the substantia nigra were determined with RT-PCR; the expressions of EphB6and ephrinB1protein in the substantia nigra were investigate by Western blotting.Result1.Behavioral changes in mice:The mice of model group were with tremor, piloerection, circling and cocky tail at different degrees after the third intraperitoneal injection of MPTP in10mins. After the fourth injection of MPTP, the mice of model group showed further abnormal gait, reducing activity, bradykinesia, and these symptoms almost disappeared24hrs later. The mice of MPTP+ginsenoside Rgl+H-89group also showed the symptoms mentioned above after intraperitoneal injection of MPTP and H-89within10mins. Compared to model group and MPTP+ginsenoside Rgl+H-89group, the symptoms mentioned above of the mice of ginsenoside Rgl appeared much later and less. The control group did not show the behavioral changes mentioned above.The swimming values of model group and MPTP+ginsenoside Rgl+H-89group were much less than those of the control group and ginsenoside Rgl group (P<0.05).2. the pathological morphological changes of dopaminergic neurons in the substantia nigra of mice:Compared with the control group, the number of dopaminergic neurons in the substantia nigra of model group decreased significantly and the axon of dopaminergic neurons became shorter. The number of dopaminergic neurons in the ginsenoside Rgl group increased a little more than that of model group. The number of dopaminergic neurons in the MPTP+ginsenoside Rgl+H-89group was less than that of ginsenoside Rg group.3. RT-PCR:RT-PCR results showed the expression of EphB6mRNA of model group (0.87±0.08) was significantly higher than those of the control group (0.08±0.01, P<0.01) and ginsenoside Rgl group (0.45±0.05, P<0.01), respectively. The expression of EphB6mRNA of model group was obviously lower than that of MPTP+ginsenoside Rgl+H-89group (1.01±.06, P<0.01). The expression of EphB6mRNA of MPTP+ginsenoside Rgl+H-89group was much higher than that of ginsenoside Rgl group (P<0.01). There was a significant difference between the groups with analysis of variance (F=173.71, P <0.01). The expression of EphrinB1mRNA in the substantia negra of model group (15.11±1.67) was significantly higher than those of the control group (0.87±0.01, P <0.01) and ginsenoside Rgl group (1.21±0.06, P<0.01), respectively. The expression of EphrinB1mRNA in the substantia negra of model group was much lower than that of MPTP ginsenoside Rg1+H-89group (17.18±0.94, P<0.01). The expression of EphrinB1mRNA in the substantia negra of MPTP+ginsenoside Rgl+H-89group was significantly higher than that of ginsenoside Rgl group (P<0.01). There is a statistically significant difference between groups through analysis of variance (F=5.39, P<0.05).4. Western blotting:Western blotting results showed the expression of EphB6protein in the substantia nigra of model group (1.02±0.06) was significantly higher than that of the control group (0.58±0.06, P<0.01), and was much lower than that of MPTP+ginsenoside Rgl+H-89group (1.75±0.48, P<0.05). The expression of EphB6 protein in the MPTP+ginsenoside Rgl+H-89group was significantly higher than that of ginsenoside Rgl group (P<0.01). Analysis of variance showed a significantly statistical difference between groups (F=292.17, P<0.01). The expression of EphrinBl protein in the substantia nigra of model group (1.03±0.24) was significantly higher than those of the control group (0.48±0.02, P<0.05) and ginsenoside Rgl group (0.97±0.04, P<0.01), respectively. The expression of EphrinB1protein in the substantia nigra of model group was obviously lower than that of MPTP+ginsenoside Rgl+H-89group (1.59±0.21, P <0.05). The expression of EphrinBl protein in the MPTP+ginsenoside Rgl+H-89group was significantly higher than that of ginsenoside Rgl group (P<0.05). Analysis of variance showed there was a significant diference between groups (F=11.44, P<0.05).Conclusion1. MPTP may induce PD through promoting the expressions of EphB6/ephrinBl in the substantia nigra of mice.2. Ginsenoside Rgl may improve the symptoms of PD by reducing the expressions of EphB6/ephrinBl in the substantia nigra of mice induced by MPTP through protein Kinase A signaling pathway. |
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