Research On The Role Of Cpy2e1 Gene In MPTP-induced Mouse Model Of Parkinson’s Disease

Parkinson’s Disease（PD）is a common neurodegenerative disease of the nervous system.Its main motor symptoms are static tremor,myotonia,bradykinesia and postural and gait abnormalities.The main pathological features of PD are the loss of dopaminergic neurons in Substantia Nigra Pars Compacta（SNpc）and the formation of Lewy Bodies（LBs）and Lewy Neurites（LNs）composed of misfolded α-synuclein（α-syn）.When PD patients were first diagnosed,a large number of dopaminergic neurons（DA-ergic）in SNpc have already lost.Currently,there is no effective treatment for PD,mostly based on dopamine replacement therapy.Although the current treatment can better control motor symptoms,it cannot prevent the progression of neurodegeneration,and the increasingly serious sports injury.Therefore,the study on the pathogenesis of PD is very important for its early prevention and intervention.Oxidative stress is an important approach to cause the death of neurons and the pathogenesis of PD,oxidation damage is also an important research direction of the current PD,however,remains to be further discussed causes oxidative stress and oxidative stress is closely related to intracellular metabolic process,Cytochrome oxidase P450 2E1（Cytochrome P450 2E1,CYP2E1）in drug metabolism and play an important role in the process of biodegradation of toxic compounds,CYP2E1 was found in DA neurons in the substantia nigra and striate body expression,Multiple studies have suggested that CYP2E1 may play a certain promoting role in the progression of PD,but the relevant mechanism has not been clarified.Based on neural toxins 1-Methyl-4-phenyl 1,2,3,6 four hydrogen pyridine（Methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP）transformation process in the brain,guess CYP2E1 may participate in this study the toxicity of MPTP in glial cells in the MPP+ form transformation process,causing glial cell activation,oxidative stress and MAPK pathways activated and a series of downstream cascade,eventually lead to nerve damage,including can DA-ergic neurons.In order to explore the role of CYP2E1 in the etiology and development of PD and its related mechanisms,MPTP-induced PD animal models were constructed using normal mice and Cyp2e1 knockout mice.In addition,epigallocatechin-3-gallate（EGCG）was selected as a CYP2E1 inhibitor for simultaneous studies based on its natural and safe properties,easy to penetrate BBB,anti-inflammatory,anti-oxidative and anti-apoptotic properties,and studies have shown that EGCG has a good inhibitory effect on CYP2E1 activity in vitro.In this study,it was found that in MPTP-induced PD mouse model,Cyp2e1 gene knockout and intrapitoneal preconditioning with EGCG significantly improved PDlike behavioral disorders,pathological protein p-α-syn accumulation,nerve cell apoptosis,including DA-ergic neurons,and glial activation triggered by MPTP exposure.The inhibitory regulation of CYP2E1 may inhibit p-α-syn accumulation by regulating the expression of LC3 B in the autophagosomal degradation pathway.Inhibition of CYP2E1 can reduce oxidative damage and improve the living environment of DA-ergic neurons by detecting oxidative stress indexes in the striatum brain homogenate.Further exploration revealed that Cyp2e1 gene knockout and EGCG intervention may improve PD-like symptoms induced by MPTP by regulating JNK and ERK signaling pathways in the MAPK pathway.Methods1.PD mouse model was established by intraperitoneal injection of MPTP and the model was evaluated by behavioristicsPD mouse model was established by intraperitoneal injection of MPTP,pretreatment of intraperitoneal injection of EGCG was used to prevent nerve damage in mice.Open field experiment was used to evaluate the autonomous activity ability of mice to reflect the psychological changes of anxiety.Hanging test and rod rotation test were used to evaluate the coordination and motor balance ability of mice.2.The detection of pathological damage of dopaminergic neurons in the substantia nigra and striatum of miceThe activity of tyrosine hydroxylase and the accumulation of p-α-syn in the substantia nigra and striatum of mice were detected by immunohistochemistry and immunofluorescence histochemistry to reflect the survival status of dopaminergic neurons.3.The detection of protein degradation pathway changesImmunofluorescence histochemistry was used to detect autophagy lysosomal and ubiquitin-proteasome degradation pathway LC3 B and Ubiquitin in substantia nigra and striatum.4.The detection of oxidative stress level and nerve cells apoptosis in the substantia nigra and striatumThe spectrophotometer was used to detect the changes of MDA,H₂O₂,CAT and GPx level in the brain homogenate of the striatum to evaluate the antioxidant damage ability.TUNEL staining was applied to evaluate the apoptosis of nerve cells in the substantia nigra and the striatum of mice.5.The activation of microglia and astrocytes in the substantia nigra and striatum of mice were detectedImmunofluorescence was used to detect the protein expression of microglia Iba1 and GFAP in the substantia nigra and striatum of mice to evaluate the degree of the activation of microglia and astrocytes.6.The activation of MAPK pathway in the substantia nigra and striatum was detectedJessTM protein analysis instrument was used to detect the protein expression changes of JNK,p-JNK,ERK,p-ERK,p38,p-p38 in MAPK pathway of substantia nigra and striatum.Results1.Intraperitoneal injection of MPTP can significantly damage the spontaneous motor status and motor balance and coordination ability of mice,Cyp2e1 knockout and EGCG pretreatment can effectively improve the behavioral injury of mice.2.Immunohistochemical experiments showed that Cyp2e1 knockout and EGCG pretreatment could significantly reduced the damage of DA-ergic neurons induced by MPTP and effectively reduced the accumulation of pathological protein p-α-syn.3.Cyp2e1 knockout and EGCG pretreatment could activate the autophagy lysosomal protein degradation pathway to clear the accumulation of pathological protein p-α-syn.4.Cyp2e1 knockout and EGCG pretreatment could enhance the antioxidant damage ability in the striatum and inhibit the apoptosis of nervous cell in the substantia nigra and striatum.5.Cyp2e1 knockout and EGCG pretreatment could alleviated microglia and astrocytes activation of the substantia nigra and striatum of mice.6.Cyp2e1 knockout and EGCG pretreatment could improve PD symptoms by regulating MAPK signaling pathways.ConclusionCyp2e1 knockout and EGCG pretreatment can improve the nerve injury caused by MPTP to a certain extent,which is related to the activation of autophagy lysosomal pathway,the enhancement of antioxidant capacity,the inhibition of microglial cell and astrocytes activation,and the regulation of MAPK pathway to inhibit the apoptosis of dopaminergic neurons.CYP2E1 may play an important role in the pathological development of PD.