Regulating Effect And Mechanism Of Echinacoside On Dopaminergic Neuron-microglia Network

Objective1.To elucidate the temporal correlation between microglia activation in substantia nigra compact area and DA neuron injury in PD mice,the co-localization in space,the damage of dopamine neuron-microglia survival microenvironment,and the behavioral changes resulting therefrom.2.On the basis of defining the pathological changes of substantia nigra compact part in PD mice,the effects of echinacoside,an active component of Cistanche deserticola Ma,on the survival microenvironment of dopamine neurons-microglia and neurobehavioral changes were observed.The intervention effects of active components of Cistanche deserticola Ma on PD mice were further elucidated.3.To investigate the effect of neutralizing antibody Anti-CX3CR1 on the intervention of active ingredients of Cistanche deserticola Ma on PD model,and to confirm the hypothesis that active ingredients of Cistanche deserticola Ma may regulate the microenvironment of dopamine neuron-microglia survival by inhibiting fractalkine/CX3CL1,and partly to reveal the mechanism of intervention of active ingredients of Cistanche deserticola Ma on PD model.MethodsIn this experiment,C57BL/6 male mice aged 9-12 weeks were used as experimental animals to establish PD mice model.The mice in the experimental group required daily intraperitoneal injection of MPTP for a dose of 30 mg/kg for 7 consecutive days.The minocycline group and the echinacoside group need to be pre-protected by intraperitoneal injection of minocycline(30mg/kg)and echinacoside(20mg/kg)in the first 3 days of establishing the MPTP model,and then observed the effects of minocycline and echinacoside administration on motor function,microglia activation and DA neuron cell survival.One hour before MPTP administration,the lateral ventricle was injected with AntiX3CR1(5ul)for 7 days.Observed behavioral differences,immunofluorescence intensity of microglia markers,neuronal survival rate,and expression level of CX3CR1 in the brain and investigated the regulating effect of echinacoside on dopaminergic neuron-microglia network and its mechanism.Results1.After MPTP was administered intraperitoneally,the behavioral abnormalities of mice were progressively aggravated,microglia were activated,the immunofluorescence intensity of OX-42 marker was increased,the number of dopamine neurons was greatly decreased,and progressive degeneration died.The results showed that the higher the survival rate of neurons,the lower the behavioral score of PD mice,and there was a negative correlation between them.2.After the intraperitoneal injection of minocycline and echinacoside,the immunofluorescence expression of OX-42 was decreased,which also significantly reduced the activation of microglia,increased the number of TH positive cells and the survival rate of DA neurons as well as improving behavioral disorders in PD mice.At the same time,the WB test results also showed that the expression level of CX3CR1 in the brain of mice treated with echinacoside was significantly decreased.3.WB results showed that the activation of microglia was reduced after injection of antibody Anti-CX3CR1 into the lateral ventricle,and the expression level of CX3CR1 in the brain of PD mice was decreased.Conclusion1.After MPTP administration,the neurobehavioral abnormalities occurred in the mice,the microglia was activated,and the number of dopaminergic neurons decreased,indicating that the PD model was successfully replicated.2.Intraperitoneal administration of minocycline and echinacoside can inhibit the activation of microglia in PD mouse model,increased the survival rate of dopamine neurons,and alleviated neurobehavioral abnormalities.It was speculated that minocycline and echinacoside may play a certain neuroprotective effect on PD mice.3.Intracerebroventricular injection(icv)of receptor antibody Anti-CX3CR1 can inhibite the activation of microglia,increase the survival4.Rate of dopamine neurons and alleviate neurobehavioral abnormalities in PD mice.WB results showed that the expression level of CX3CR1 in the brain of Anti-CX3CR1 group was consistent with that in the echinacoside group.It was suggested that echinacoside,an active component of Cistanche deserticola Ma,may further improve the dopaminergic neurological function of MPTP mice by inhibiting the expression of CX3CR1,thereby maintaining the steady state of neuron-microglia survival microenvironment.