In Vitro And In Vivo Neuroprotective Effects Of Isoginkgetin(ISO) In Models Of Parkinson's Disease

Parkinson's disease(PD)is the second largest neurodegenerative disease after Alzheimer's disease in the world.The main pathological features of PD are the death and loss of dopaminergic neurons in the substantia nigra of the midbrain,the degeneration of the nigrostriatal dopaminergic system,and the formation of Lewy bodies in the eosinophilic inclusions in neurons.The causes of PD is still unclear.The existing clinical drugs can only improve,but can not cure the symptoms of Parkinson's disease.However the side effects are evere.In recent years,the hypothesis of inflammation and free radicals has raiised for PD research,which opened new directions for the development of effective drugs.Isoginkgetin is a natural compound that was first discovered in the traditional Chinese medicine Ginkgo.Due to the content is low in it,its pharmacological studies in PD area is rare.Previously,we found that the Yao folk medicine Zhubai contains high Isoginkgetin,which was widely used in the treatment of mental diseases.Therefore,this study mainly explored the effect of Isoginkgetin on PD related behavior and neuropathological changes in vitro and in vivo models,and studied involved mechanisms,Hopefully,the present study opens new avenues for the treatment of Parkinson's disease.Methods:1.Cellular model and Assay:SH-SY5Y cells were seeded in 96-well plate,differentiation by RA and TPA into dopaminergic neuron phenotype.After treatment with Isoginkgetin for 24 h,MPP~+was added for 24 h.The cell viability was detected by MTT assay;the release of ROS was detected by fluorescence spectrophotometer.The mRNA level of apoptosis and anti-oxidation related genes was detected by qPCR.The changes of apoptosis,anti-oxidation and neurotrophin protein levels were detected by Western Blot.Changes of synaptic length on primary neuronal cells were examined by immunofluorescence.2.Animal expreiment:The behavioral tests were used to evaluate the MPTP-induced mouse Parkinson's model.Flow cytometry was used to detect the expression of peripheral blood lymphocyte subsets and the autophagy ability of peritoneal macrophages.The ELISA kit was used to detect the activation of microglia and astrocyte of mouse striatum.PI3K/Akt/mTOR and MAPK pathway expression in mouse striatum by Western Blot analysis.Experimental results:1.Cellular tests:The activity of SH-SY5Y cells induced by MPP~+stimulation was significantly decreased,while the 1?g/mL Isoginkgetin significantly improved the activity of SH-SY5Y cells.The release of ROS from MPP~+-stimulated cells was significantly enhanced,Isoginkgetin could effectively inhibit the release of ROS.Furthermore,qPCR and Western Blot detection of apoptosis Bax,Bcl-2,Caspase3,oxidative stress Nrf2,HO-1,SOD,NQO-1 and other related genes and proteins,all indicate that MPP~+can cause cells apoptosis and oxidative stress,However,isoginkgetin effectively reversed apoptosis-related changes and activated the anti-oxidant system against oxidative stress induced by MPP~+.It also have an effect on the protrusion damage of neuronal cells induced by MPP~+in primary neuronal cell model of PD.2.Animal experimental results:Compared with model group induced by MPTP,the Isoginkgetin significantly attenuated behavioral impairments on pole test,rotarod and open field.In the peripheral system,Isoginkgetin also improved the decreased lymphocyte infiltration and disorder of autophagy in peritoneal macrophages and striatum.In the central system,Isoginkgetin attenuated MPTP-induced activation of central glial cells.Conclusions:1.In the cellular model of PD,MPP~+decreased cell viability,increased release ROS,enhanced expression of apoptosis-related genes and proteins,and reduction of neurotrophic factor secretion and other abnormal reactions in SH-SY5Y.Isoginkgetin can effectively improve the abnormal function of these aspects.suggesting that the Isoginkgetin may increase the secretion of neurotrophic factors to regulate the antioxidant system,and the interaction of apoptosis-related gene and proteins to improve the PD cell model induced by MPP~+.It is suggested that the Isoginkgetin improved the apoptosis-related gene proteins by regulating the body's antioxidant system and increasing the secretion of neurotrophic factors in SH-SY5Y cell induced by MPP~+.2.Isoginkgetin can improve MPTP-induced Parkinson's motor dysfunction by regulating the activation of central glial cells and peripheral lymphocytic infiltration and autophagy.