| Objective:In this study,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinson’s disease(PD)model was used to observe the effects of electroacupuncture(EA)on motor function of PD mice,as well as the therapeutic effect of EA treatment on PD and its possible mechanism involved.We aimed to clarify whether EA treatment improved PD by inhibiting ferroptosis through activating Nrf2/GPX4 signaling pathway.Data collected in this study elucidated the mechanism of EA by activating nuclear factor erythroid-2-related factor(Nrf2),increasing the expression of glutathione peroxidase 4(GPX4),inhibiting the occurrence of lipid peroxidation,and reducing the ferroptosis of dopaminergic neurons in substantia nigra(SN),and all the above contributed to the improvement of motor function of PD,and provided new ideas exploring the mechanism of EA in PD treatment.Methods:1.The subacute PD model was established by intraperitoneal injection of MPTP(30 mg/kg,i.p.)in mice for 5 consecutive days,and the PD model was evaluated by rotarod test,traction test and open field test.EA treatment was given for 12 consecutive days(MPTP was given after EA pretreatment for 7 days).Behavioral tests were carried out to observe whether EA treatment ameliorated the motor function 12 hours after the end of the last EA treatment for PD mice.Levodopa(L-dopa)was given as the positive control group(L-dopa,20 mg/kg/d,i.g.,12 d),and the same volume of saline served as the control group.2.The number of tyrosine hydroxylase(TH)positive cells were observed by immunofluorescence staining in the SN,and the effect of EA treatment on the changes of TH~+cell number in the SN of PD mice was also determined.3.The expression changes of TH,and ferroptosis related proteins-Nrf2 and GPX4 were determined by Western blot(WB),as well as the contents of glutathione(GSH),malondialdehyde(MDA)and iron in the SN were detected by kits.The effects of EA treatment on the above changes were then analyzed.In order to further explore whether EA meliorated the motor function of PD mouse by inhibiting ferroptosis through activating Nrf2/GPX4 signaling pathway,alkaloid trigonelline(AT),an inhibitor of Nrf2,was administered orally(40 mg/kg/d,i.g.)to observe the expression changes of proteins in signaling pathway Nrf2/GPX4.Results:1.The behavioral tests results showed that the latency to fall in the MPTP(Model)group mice was shorter in the rotarod and traction test compared with the control group.In the open field test,the suspended and upright time,as well as the climbing and standing time were decreased in Model group,indicating that the of MPTP-induced subacute PD mouse model was established successfully,which could be used in the following experiments.Compared with the Model group,the latency to fall was longer in the Model+EA group in the rotarod and traction tests,as well as the suspended and upright time,and the climbing and standing time were increased in the open field test.The results were similar between the Model+EA and Model+L-dopa groups,and the difference was not statistically significant.The results above showed that EA treatment could improve the motor function of PD mouse.2.Compared with the Control group,the WB results showed that the expression levels of TH,and the proteins Nrf2 and GPX4 related to ferroptosis were significantly lower in the Model group,the content of ferroptosis-related factors GSH decreased significantly,while the contents of MDA and iron increased significantly in the SN of Model group mice after the kits detection.Compared with the Model group,the expression levels of TH,Nrf2 and GPX4 were significantly increased,as well as the content of GSH,while the contents of MDA and iron decreased in the SN from Model+EA group.The results collected above suggested that EA treatment could increase the expression levels of TH,Nrf2 and GPX4,and inhibit the cells ferroptosis in the SN of PD mice.3.The number of TH~+cells were significantly reduced in the SN of Model group mice using immunofluorescence staining compared with Control group.While,the number of TH~+cells were increased in Model+EA group compared with that of in Model group.The above results suggested that EA treatment could increase the number of TH~+cells and inhibit the loss of dopaminergic neuron in the SN of PD mice.4.The behavioral tests results showed that compared with the Model+EA group,the latency to fall was shorter of Model+EA+AT group mice in the rotarod test and traction test after oral administration of alkaloid trigonelline,an inhibitor for Nrf2.In the open field test,the suspended and upright time,as well as the climbing and standing time were significantly reduced of Model+EA+AT group mice.And the above results were statistically significant.Compared with Model+EA group,the WB results showed that the expression levels of TH,Nrf2 and GPX4 were decreased in Model+EA+AT group.The content of GSH decreased,while the contents of MDA and iron increased in the SN of Model+EA+AT group mice using Kit analysis.The number of TH~+cells was significantly reduced in the SN of Model+EA+AT group mice using immunofluorescence method,compared with Model+EA group.The collected data above suggested that EA treatment may inhibit the ferroptosis of dopaminergic neurons by activating signaling pathway Nrf2/GPX4,reduce the loss of dopaminergic neurons,and then improve the motor function of PD mice.Conclusions and significance:In this study,the subacute PD mouse model was established successfully by intraperitoneal injection of MPTP,and the motor function of PD mice was significantly improved after EA treatment.The mechanism study further indicated that EA treatment could inhibit the occurrence of ferroptosis in the SN of PD mice,and reduce the loss of dopaminergic neurons by activating signaling pathway Nrf2/GPX4.The results collected in this study suggested that EA treatment provided improvement for PD maybe due to the inhibition of ferroptosis and reduction of the loss of dopaminergic neurons by activating signaling pathway Nrf2/GPX4 in the SN,which provided a new idea for exploring the mechanisms of EA in PD treatment. |
