The Protective Effect Of Ginsenoside Rg3on Dopaminergic Neurons In Rotenone-Induced PD Model And Its Possible Mechanism

Parkinson’s disease (PD) is one of major neurodegenerative disorders, which frequently occurs in older adults. PD is characterized by the progressive degeneration of dopaminergic neurons and the formation of Lewy body (LB). These result in a decreasement of dopamine (DA) in the nigrostriatal pathway, which ultimately leads to functional changes throughout the basal ganglia loops. Patients’ main features are bradykinesia, muscle rigidity, tremor and ataxia. The pathogenesis of PD is complex, but it is believed that it may be associated with oxidative stress, mitochondrial injury, nervous inflammation and neural excitotoxicity.Ginsenoside Rg3is one of the main components of traditional Chinese herbal medicine ginseng, which can prevent and cure cancer effectively, improve cardiovascular function, prevent platelet aggregation, protect brain cells and improve immunity. Our previous study showed that ginsenoside Rg3could relieve the movement disorders of PD rats that were induced by rotenone loaded nano lipid carrier (R-NLC), and effectively inhibit apoptosis of DA neurons in substantia nigra. Further studies showed that its role was related to the weakened oxidative stress, the raised ratio of Bcl-2and Bax and the inhibition of caspase pathway.As a continuation of the work above, PD rats induced by R-NLC and PC12cell injury model induced by rotenone were prepared. We strived to find further conclusive evidence of the protective effect of Rg3on rotenone-induced dopaminergic neuronal damage in vivo and vitro, focusing on the role of p38MAPK-iNOS-NO pathway. Many methods were carried out to explore the molecular mechanisms of protection on DA neurons in PD model.The main results were as follows:1. The protective effect of ginsenoside Rg3on DA neurons in rotenone-induced PD models.Methods:PC12cell groups:(1) control group:0.1%DMSO;(2) model group:1μM rotenone;(3)1μM rotenone+0.3125μM Rg3;(4)1μM rotenone+0.625μM Rg3;(5)1μM rotenone+1.25μM Rg3;(6)1μM rotenone+2.5μM Rg3;(7)1μM rotenone+5μM Rg3;Rg3solutions with different concentration were added4hours before rotenone. After24hours of incubation, the optical density(OD) value was determined by MTT, and the cell survival rate of each group was calculated. Apoptosis rate was measured by flow cytometry..The rats were divided into6groups:(1) control group:sc blank NLC+ig0.5%CMC-Na;(2) model group:sc R-NLC+ig0.5%CMC-Na;(3) the positive drug group:sc R-NLC+ig selegiline(11mg/kg);(4) the Rg3group at low dose:sc R-NLC+ig Rg3(3mg/kg);(5) the Rg3group at middle dose:sc R-NLC+ig Rg3(6mg/kg);(6) the Rg3group at high dose:sc R-NLC+ig Rg3(12mg/kg)Rats were subcutaneously injected with R-NLC every other day:the first time0.5mg/kg, the second time0.8mg/kg, and then1mg/kg every time. Rats were injected28days totally.3days in advance of injection, other drugs were administered orally once a day for31days.24hours after the last injection, the substantia nigra and striatum were separated rapidly. The appearance and behavior changes of rats, the cell morphology of substantia nigra(SN) and the levels of striatal DA were investigated.Results:(1) Injury in PC12cells:the OD value of model group was0.340±0.007and the survival rate was69.5%, which was significantly lower than that of control group (P<0.01); after the administration of0.3125、0.625、1.25、2.5、5μM Rg3, the OD value were0.354±0.011(P <0.05),0.388±0.018(P<0.01),0.423±0.009(P<0.01),0.441±0.007(P<0.01) and0.416±0.007(P<0.01), respectively. The survival rates were72.4%、79.3%、86.5%、90.3%和85.1%, respectively, which were higher compared that of with model group, significantly.(2) Apoptosis rates of cells:The early and late apoptotic or dead cells was significantly increased by the administration of rotenone, and the apoptosis rate reached to71.1%. After pretreatment with1.25and2.5μM Rg3, the apoptotic rate was47.9%and28.1%, which were significantly lower compared with that of the model group (P<0.01). This showed that Rg3could inhibit rotenone-induced apoptosis.(3)The Impact on the behavior and the DA neurons of PD rats:①Behavioral score; Scores of Rg3at low, medium and high dose groups were1.57±0.98,1.57±0.98,1.43±1.13, respectively, which were lower compared with that of model group (3.83±3.13) significantly (P<0.05).②HE staining of substantia nigra:Compared with the control group, the neurons in model group showed condensated nuclear, shrinked volume and reduced intracellular the Nissl substance. After the administration of Rg3, the symptoms above could be significantly improved.③Levels of striatal DA:Level of DA of striatal in model group was0.57±0.11nmol/g. Compared with control group (16.80±11.36nmol/g), the contents of DA was significantly decreased (P<0.01). Contents of DA at low, medium and high dose of Rg3were1.38±0.79nmol/g,1.15±0.34nmol/g and9.49±6.93nmol/g, respectively, which were higher compared with that of model group significantly (P<0.05)Conclusion:Rg3could inhibit rotenone-induced injury of DA neurons in vivo and in vitro.. It could also inhibit rotenone-induced apoptosis in PC12cells.2. The effects of Rg3on the iNOS vitality and NO content of dopaminer-gic neurons in rotenone-induced PD models.Methods:PC12cell groups:(1) control group:0.1%DMSO;(2) model group:1μM rotenone;(3)1μM rotenone+1.25μM Rg3;(4)1μM rotenone+2.5μM Rg3. The rats were divided and administrated as the same as Part One. The iNOS vitality and NO content were measured by colorimetry.Results:(1) The iNOS vitality and NO content in cells:The iNOS vitality and NO content in model group were0.692±0.030U/mL and7.67±1.45μM, while0.431±0.051U/mL and2.90±0.53μM in control group (P<0.01). Groups of1.25and2.5μM Rg3showed different iNOS vitality as0.636±0.031U/mL and0.643±0.009U/mL and different NO content as4.84±1.20μM and4.91±1.72μM,which were significantly decreased (P<0.05).(2) The iNOS vitality and NO content in rats:Those in model group were0.223±0.044U/mgprot and8.06±2.35μmol/gprot, while0.142±0.016U/mgprot and4.57±1.30μmol/gprot in control group (P<0.01). Groups of low, medium and high doses of Rg3showed different iNOS vitality as0.140±0.033U/mgprot,0.132±0.023U/mgprot and0.145±0.035U/mgprot, and different NO content as5.26±1.18μmol/gprot,4.41±1.12μmol/gprot and5.40±1.03μmol/gprot, which were significantly decreased (P<0.05in groups of low and high doses, and P<0.01in group of medium dose).Conclusion:Rg3could inhibit the increasement of iNOS vitality and NO content significantly in vivo and in vitro, which suggests that Rg3may protect neurons by inhibiting the generation of NO.3. The re I at i onsh i p of p38MAPK and NO of DA neurons i n rotenone-i nduced PD models.Methods:(1) Experiments in vitro:(1) control group:0.1%DMSO;(2) model group:1μM rotenone;(3)1μM rotenone+10μM SB203580. The cell viability was determined by MTT. The iNOS vitality and NO content were measured by colorimetry.(2)Experiments in vivo:The rats were divided and administrated as the same as Part One.24hours after the last injection, the expression of p-p38MAPK in SN was measured by Western blotting. The percentage of p-p38MAPK and iNOS positive cells were observed by immunohistochemistry, and their non-linear correlation analysis was carried out.Results:(1) The protective effect of SB203580on the survival rate, the iNOS vitality and NO content of PC12cells:Compared with model group(69.0%), the survival rate of SB203580group was81.0%, which was significantly increased (P<0.05). The iNOS vitality and NO content in model group were0.692±0.030U/mL and7.67±1.45μM, Pretreatment with SB203580showed different iNOS vitality and NO content as0.634±0.014U/mL and5.27μ1.66μM, which were significantly decreased (P<0.05). These prompted that mechanism of rotenone-caused PD may be related to p38MAPK, and p38MAPK pathway may regulate the vitality of iNOS and NO content.(2) The content of p-p38MAPK:Compared with the control group, the expression of p-p38MAPK in model group was significantly enhanced, which was significantly reduced after pretreatment with Rg3. All above prompt that the resistance to apoptosis of Rg3may be related to the lower level of p-p38MAPK.(3) Correlation of p-p38MAPK and iNOS:Compared with control group, the percentage of p-p38MAPK and iNOS positive cells in model group were increased significantly. Pretreatment with Rg3could significantly reverse this situation. Statistical analysis revealed that the expressions of these two proteins were positively correlated, which suggested Rg3reduced the expression of iNOS through inhibiting the phosphorylation of p38MAPK, probably.Conclusion:Rg3may inhibit phosphorylation of p38MAPK, reduce the activation of iNOS, decrease the expression of NO, and play an important role of anti-PD eventually.