Neuroprotective Effect And Mechanism Of Catalpol On MPTP Mouse Model Of Parkinson's Disease

Objective: Parkinson's disease(PD),also known as tremor paralysis,is a common degenerative disease of the aged nervous system,which seriously affects the physical and mental health of patients.With the acceleration of global aging,the incidence of PD is increasing year by year.The main pathological change of PD was significant degeneration and death of dopaminergic(DA)neurons in the compact part of substantia nigra of the midbrain.The pathogenesis of PD is still unclear.It is generally accepted that apoptosis,inflammatory reaction and oxidative stress of DA neurons are the main causes of PD,in which inflammatory response plays an important role in the pathogenesis of PD.Anti-inflammation may reduce the risk of PD and delay the progression of the disease.Catalpol is an iridoid terpene extracted from Rehmannia glutinosa.Studies have shown that catalpol has a variety of pharmacological activities,such as anti-apoptosis,anti-oxidation and anti-inflammation,and plays a neuroprotective role by reducing the release of inflammatory factors.However,whether catalpol has a preventive effect on PD and its mechanism has not been reported.Therefore,in this paper,we used PD mouse model to study the protective effect of catalpol on DA neurons and its related mechanism.Methods: In this study,C57BL/6 mice were randomly divided into three groups: control group,model group and catalpol group.The mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),and catalpol was injected intraperitoneally for 2weeks.The motor ability and exploratory behavior of the three groups of mice were detected by open field,rod climbing and rotating rod tests,and the expression of tyrosine hydroxylase(TH)positive cells in substantia nigra and striatum of the three groups were detected by immunohistochemical method.Western blot was used to detect the expression of TH and ?-Synuclein in substantia nigra,TH and DA transporter(DAT)in striatum,apoptosis-related proteins Caspase-9,Cleaved-Caspase-9,Caspase-3,Cleaved-Caspase-3,Bcl2 and Bax,MKK4/JNK/c-Jun pathway related proteins in substantia nigra,antioxidant enzymes SOD1,NLRX1 and GPX4,.The protein expression levels of inflammatory cytokines GFAP,Iba1,IL-1 ?,TNF-? and NLRP3,neurotrophicfactor growth associated protein 4(GAP43)and vascular endothelial growth factor(VEGF).The activity of SOD1 and ROS was determined,and the expression of TH,GFAP and Ibal labeled positive cells in substantia nigra were analyzed by immunofluorescence confocal laser scanning microscope.Results:1.The results of open-field behavior experiment showed that compared with the control group,the MPTP-induced Parkinson model mice reduced the distance and times of walking in the central area of the model mice,and the distance and times of walking into the central area of mice were improved after treatment with catalpol,which indicated that catalpol improved the exploratory behavior of MPTP-induced Parkinson model mice,while there was no significant change in the three groups of rod climbing test and rotating rod test.2.The results of immunohistochemistry and Western blot showed that compared with the control group,the number of TH positive cells in substantia nigra and striatum in MPTP group decreased,the expression level of TH protein in substantia nigra decreased and the expression level of ?-Synuclein protein increased,while the expression level of TH and DAT protein decreased in striatum.After the model mice were treated with catalpol,the above indexes were inhibited or restored,indicating that the PD model was successfully induced.3.The results of Western blot showed that compared with the control group,the expression of apoptosis-related proteins Cleaved-Caspase-9 and Cleaved-Caspase-3 in the substantia nigra of MPTP group increased.After treatment with catalpol,the above indexes were inhibited and the ratio of Bcl2/Bax decreased,although there was no significant change in Caspase-9,Caspase-3,indicating that catalpol reduced the apoptosis of DA neurons induced by MPTP.4.The results of Western blot showed that compared with the control group,the expression of p-MKK4,p-JNK and p-c-Jun in substantia nigra of MPTP group increased.Compared with MPTP group,the protein expression of p-MKK4,p-JNK and p-c-Jun decreased in the model mice treated with catalpol,indicating that catalpol may inhibit the apoptosis of DA neurons by regulating MKK4/JNK/c-Jun signal pathway.5.Compared with the control group,the,Western blot results showed that the expression level of SOD1 and GPX4 protein in substantia nigra decreased and the expression level of NLRX1 protein increased in MPTP group.Compared with MPTP group,the expression level of SOD1 and GPX4 protein increased and the expression level of NLRX1 protein decreased after catalpol treatment.In addition,catalpol significantly restored the activity level of SOD1 and inhibited the activity level of ROS.It is suggested that catalpol has antioxidant effect on DA neurons in MPTP.6.The results of immunofluorescence confocal microscope showed that compared with the control group,the expression of GFAP and Iba1 in substantia nigra of MPTP group increased,and the expression of GFAP and Iba1 decreased after catalpol treatment,indicating that the activation of astrocytes and microglia could be inhibited by catalpol.The results of Western blot were consistent with the results of immunofluorescence confocal.Compared with the control group,the expression levels of inflammatory cytokines in substantia nigra and inflammatory corpuscles GFAP,Iba1,IL-1 ?,TNF-?and NLRP3 in MPTP group were increased,and the above indexes were inhibited after catalpol treatment.It is suggested that catalpol can alleviate the inflammatory reaction caused by MPTP.7.The results of Western blot showed that compared with the control group,the expression of GAP 43 and VEGF protein in substantia nigra of MPTP group decreased,while the expression of GAP 43 and VEGF protein increased after catalpol treatment.It shows that catalpol can promote nerve regeneration.Conclusion: catalpol has the effects of anti-apoptosis,antioxidation,inhibiting inflammation and promoting nerve regeneration on DA neurons in MPTP-induced mouse model of Parkinson's disease.The mechanism may be through the regulation of MKK4/JNK/c-Jun signal pathway to play a neuroprotective effect.