| 1.ObjectivesParkinson’s disease is currently the second most rapidly increasing neurodegenerative disease in terms of growth rate,disability and mortality,and there is no safe and effective treatment method.The therapeutic effect and potential mechanism of action of acupuncture,one of the commonly used treatments for Parkinson’s disease,have been controversial.The mechanism of action of the "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" acupoints has not yet been clarified.Many studies have suggested that Parkinson’s disease rats suffer from persistent endoplasmic reticulum stress and neuroinflammation.Therefore,we intend to address three questions through this experiment: 1.how to establish a screening model for Parkinson’s disease;2.whether the electroacupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" have therapeutic effects in Parkinson’s disease rats;3.Whether electroacupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" have therapeutic effects in Parkinson’s disease rats;3.Whether electroacupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" exert therapeutic effects by affecting SIRT1/ERS/NLRP3 signaling pathway.The results of this study are summarized below.The aim is to provide basic experimental evidence for the therapeutic effect and potential mechanism of action of electroacupuncture in the treatment of Parkinson’s disease,and to provide new ideas for the clinical treatment of Parkinson’s disease.2.Research method Experiment 1First,three common animal models of Parkinson’s disease were tested by establishing three common animal models of Parkinson’s disease,grouped as:rotenone control group,rotenone model group,6-OHDA control group,6-OHDA model group,MPTP control group,MPTP model group,respectively,using their respective classical behavioral assays(behavioral score for rotenone group,APO rotation method for 6-OHDA group,and absentee field experiment for MPTP group)comparison as well as TH,α-syn immunohistochemical analysis.Then,we performed interference validation for solvent and non-transverse non-acupuncture groups,with a total of five groups: Ctrl(control group),SPD(solvent group),PD(model group),SEA(non-transverse non-acupuncture group),and EA(electroacupuncture group).The SPD group was set up by interfering to exclude other toxic components in the screened rotenone solution;and the SEA group was set up to exclude the effect of non-menstrual non-acupuncture points;the solvent and nonmenstrual non-acupuncture groups were performed by behavioral(behavioral scoring,open field,hanging,and pole climbing),immunohistochemistry(α-syn),real-time fluorescence quantification(α-syn m RNA),and protein immunoblotting(α-syn)Interference term validation.Experiment 2First,to observe whether there is a sustained endoplasmic reticulum stress response in Parkinson’s disease rats,we set up two groups: Ctrl and PD.real-time fluorescence quantification(Bip,PERK,e IF2α,ATF6,IRE-1αm RNA)and protein immunoblotting(p-PERK,PERK,p-e IF2α,e IF2α,ATF6)were used for the Parkinson’s disease rat model endoplasmic reticulum stressrelated signaling pathways were examined.Then,we added the endoplasmic reticulum stress inhibitor 4-PBA to observe whether electroacupuncture could exert an inhibitory effect on sustained endoplasmic reticulum stress in Parkinson’s disease rats,setting up four groups: Ctrl,PD,EA,4-PBA.by behavioral(behavioral scoring,absentee field,suspension,pole climbing),transmission electron microscopy,real-time fluorescence quantification(α-syn,PERK,e IF2α,ATF6 m RNA)and protein immunoblotting(α-syn,pPERK,PERK,p-e IF2α,e IF2α)to observe the effects of electroacupuncture and 4-PBA on rats with Parkinson’s disease.Experiment 3First,to investigate whether endoplasmic reticulum stress activates downstream neuroinflammation,we further tested four groups of rats,Ctrl,PD,EA,and 4-PBA,and we detected downstream neuroinflammation by performing real-time fluorescence quantification(NLRP3,ASC,Caspase-1,IL-1β,IL-18,GSDMD m RNA)and protein immunoblotting(NLRP3,ASC,Caspase-1)for downstream neuroinflammation.Then,we added NLRP3 inhibitor MCC950 to observe whether electroacupuncture could exert its therapeutic effect by inhibiting neuroinflammation in Parkinson’s disease rats,setting up 4 groups: Ctrl,PD,EA,MCC950.by behavioral(behavioral score,open field,suspension,pole climbing),real-time fluorescence quantification(α-syn,PERK,e IF2α,NLRP3,ASC,caspase-1,IL-1β,IL-18,GSDMD m RNA)and protein immunoblotting(p-PERK,PERK,p-e IF2α,e IF2α,NLRP3,β-actin,ASC,Caspase-1)to observe the effects of electroacupuncture and MCC950 on rats with Parkinson’s disease.Experiment 4Since in the previous experiments,we only demonstrated that electroacupuncture can play the same role as endoplasmic reticulum stress inhibitor 4-PBA and NLRP3 inhibitor MCC950.But whether electroacupuncture acts via ERS/NLRP3 pathway remains to be further investigated.We found that SIRT1 protects the endoplasmic reticulum and maintains endoplasmic reticulum homeostasis in the upstream of ERS.Therefore,we inhibited the expression of SIRT1 using the SIRT1 inhibitor EX-527.First,we verified whether SIRT1 affects the behavior of rats by setting up Ctrl and EX-527(EX-527 single administration).We tested whether EX-527 would affect the behavior of rats by behavioral scoring,open field,water maze,suspension,and pole climbing experiments.Secondly,we set up 4groups: Ctrl,PD,PD + EX-527,PD + EA + EX-527,and observed whether electroacupuncture would still play a role in treating persistent endoplasmic reticulum stress in a rat model of Parkinson’s disease by behavioral(behavioral score,open field,suspension,pole climbing)and real-time fluorescence quantification(SIRT1,PERK,e IF2α m RNA)when EX-527 intervention.3.Results Experiment 11.The rotenone modeling method can simulate both DAergic neuronal damage and abnormal aggregation of α-syn in the Parkinson’s disease model.The 6-OHDA method: compared with the 6-OHDA control group,the mean optical density of TH and α-syn in the 6-OHDA model was statistically different(P < 0.01),but the mean optical density of α-syn was not statistically different(P > 0.05).MPTP method: Compared with the MPTP control group,the total distance of movement in the open field experiment,the total distance into the central area in the open field experiment,the number of upper limb elevation in the open field experiment,and the mean optical density of TH in the MPTP model group were statistically different(P < 0.01),but the mean optical density of α-syn was not statistically different(P > 0.05).2.The electroacupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" could improve the behavior of Parkinson’s disease and the abnormal aggregation of α-syn.Compared with the Ctrl group,there were no statistical differences in behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance traveled in the open field experiment,total distance into the central area in the open field experiment,number of upper limb elevations in the open field experiment,mean optical density of α-syn,and relative expression levels of α-syn m RNA in the substantia nigra of the brain in the SPD group(P>0.05),while there were no statistical differences in behavioral scores,hanging experiment scores,pole climbing experiment scores,and relative expression levels of α-syn m RNA in the PD group.In the PD group,there were no statistical differences in behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance traveled in the open field experiment,total distance into the central area in the open field experiment,number of upper limb elevations in the open field experiment,mean optical density of α-syn,relative expression level of α-syn m RNA in the substantia nigra,and α-syn protein content in the substantia nigra(P < 0.01);compared with the PD group,the SEA group had statistically different behavioral scores,hanging experiment scores,Compared with the PD group,there were no statistical differences in the behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance traveled in the open field experiment,total distance traveled into the central area in the open field experiment,number of upper limb elevation in the open field experiment,mean optical density of α-syn,and relative expression level of α-syn m RNA in the substantia nigra of the brain in the SEA group(P>0.05).There were statistical differences in behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance traveled in the open field experiment,total distance into the center of the open field experiment,number of upper limb elevations in the open field experiment,mean optical density of α-syn,relative expression level of α-syn m RNA in the brain substantia nigra,and α-syn protein content in the brain substantia nigra(P < 0.01).Experiment 21.Parkinson’s disease model can induce persistent endoplasmic reticulum stress.Compared with the Ctrl group,the relative expression levels of PERK,e IF2α and ATF6 m RNA in the PD group were statistically different(P < 0.05);the p-PERK,p-e IF2α and ATF6 protein levels in the PD group were statistically different(P < 0.01);the relative expression levels of Bip m RNA and IRE-1α m RNA in the PD group were not statistically different(P < 0.05).expression levels were not statistically different(P > 0.05).2.Electroacupuncture could inhibit the persistent endoplasmic reticulum stress response in the rat model of Parkinson’s disease.Compared with Ctrl,there were statistical differences in behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance traveled in the open field experiment,total distance into the central area in the open field experiment,and the number of upper limb elevation in the open field experiment in the PD group(P < 0.01);the relative expression levels of α-syn,PERK,e IF2α,and ATF6 m RNA in the substantia nigra of rats in the PD group were statistically Compared with the PD group,there were statistical differences in the behavioral scores,suspension test scores,pole climbing test scores,total distance travelled in the open field test,total distance into the central area in the open field test,and the number of upper limb lifts in the open field test in the EA group(P < 0.01).The relative expression levels of α-syn,PERK,and e IF2α m RNA in the substantia nigra of rats in the EA group were statistically different(P < 0.01);the protein contents of α-syn,p-PERK,and p-e IF2α in the substantia nigra of rats in the EA group were statistically different(P < 0.05);the behavioral scores,suspension test scores,and pole climbing test scores of rats in the 4-PBA group were statistically different(P <0.05);the behavioral scores,suspension test scores,and pole climbing test scores of rats in the 4-PBA group were statistically different.experiment,pole climbing experiment,total distance traveled in the open field experiment,total distance into the central area in the open field experiment,and the number of upper limb elevation in the open field experiment were statistically different(P < 0.05);the relative expression levels of α-syn,PERK,e IF2α,and ATF6 m RNA in the brain substantia nigra of rats in the 4-PBA group were statistically different(P < 0.05);the relative expression levels of α-syn,PERK,e IF2α,and ATF6 m RNA in the brain substantia nigra of rats in the 4-PBA group were statistically different The protein levels of α-syn,p-PERK and pe IF2α in the substantia nigra of the brain were statistically different(P < 0.01).Experiment 31.Endoplasmic reticulum stress mediates NLRP3 inflammatory vesicle pathway in Parkinson’s disease model.Compared with Ctrl,the relative expression levels of NLRP3,ASC,Caspase-1,IL-1β,IL-18,and GSDMD m RNA in rat brain substantia nigra of PD group were statistically different(P< 0.05);the protein contents of NLRP3,ASC,and Caspase-1 in rat brain substantia nigra of PD group were statistically different(P < 0.01);compared with Compared with the PD group,the relative expression levels of NLRP3,ASC,Caspase-1,IL-1β,IL-18,and GSDMD m RNA in the substantia nigra of rats in the EA and 4-PBA groups were statistically different(P < 0.05);the protein contents of NLRP3,ASC,and Caspase-1 in the substantia nigra of rats in the EA and 4-PBA groups were statistically different(P < 0.05);the protein contents of NLRP3,ASC,and Caspase-1 in the substantia nigra of rats in the EA and 4-PBA groups were(P < 0.05).2.Electroacupuncture can inhibit the activation of ERS/NLRP3 signaling pathway in Parkinson’s disease.Compared with Ctrl,there were statistical differences in behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance of movement in the open field experiment,total distance into the central area in the open field experiment,and the number of upper limb elevation in the open field experiment in the PD group rats(P < 0.01);brain substantia nigra α-syn,PERK,e IF2α,NLRP3,ASC,caspase-1,The relative expression levels of IL-18 and GSDMD m RNA were statistically different in the PD group(P < 0.05);the protein contents of pPERK,p-e IF2α,NLRP3,ASC and caspase-1 in the PD group were statistically different(P < 0.01);compared with the PD group,the behavioral scores,hanging experiment scores,pole climbing experiment,total distance traveled in the EA group were statistically different(P < 0.01).The relative expression levels of α-syn,PERK,e IF2α,NLRP3,ASC,caspase-1,IL-1β,IL-18,and GSDMD m RNA in the substantia nigra of rats in the EA group were statistically different from those in the PD group(P < 0.05).statistical differences(P < 0.05);the brain substantia nigra p-PERK,p-e IF2α,NLRP3,ASC,and caspase-1 protein levels in EA group rats were statistically different(P < 0.05);the behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance traveled for locomotion in the open field experiment,total distance into the central area in the open field experiment,and The relative expression levels of e IF2α,NLRP3,ASC,caspase-1,IL-1β,and GSDMD m RNA in the substantia nigra of rats in the MCC950 group were statistically different(P < 0.01);the relative expression levels of NLRP3,ASC,and GSDMD in the substantia nigra of rats in the MCC950 group were statistically different(P < 0.01);the relative expression levels of NLRP3,ASC,and GSDMD in the substantia nigra of rats in the MCC950 group were statistically different(P < 0.01),Caspase-1 protein levels were statistically different in the MCC950 group(P < 0.01).Experiment 41.SIRT1 inhibitor EX-527 does not cause neurological disorders in rats.Compared with Ctrl,there were no statistical differences in behavioral scores,suspension experiment scores,pole climbing experiment scores,total distance traveled for movement in the open field experiment,total distance into the central area in the open field experiment,number of upper limb raises in the open field experiment,mean escape latency in the water maze,and exploration time in the original platform quadrant in the EX-527 group(P > 0.05);2.Electrodes had a modulatory effect on the SIRT1/ERS/NLRP3 signaling pathway.Compared with the Ctrl group,the behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance of movement in the open field experiment,total distance into the central area in the open field experiment,and the number of upper limb elevation in the open field experiment of rats in the PD group were statistically different(P < 0.01);the relative expression levels of α-syn,SIRT1,e IF2α,and NLRP3 m RNA in the brain substantia nigra of rats in the PD group were statistical differences(P< 0.05).The relative expression levels of SIRT1 and PERK m RNA in the substantia nigra of rats in the PD+EX-527 group were statistically different from those in the PD group(P<0.05).Compared with PD + EX-527 group,there were no statistical differences in behavioral scores,hanging experiment scores,pole climbing experiment scores,total distance of movement in the open field experiment,total distance into the central area in the open field experiment,and the number of upper limb elevation in the open field experiment in the PD + EA + EX-527 group(P > 0.05);brain substantia nigraα-syn,SIRT1 PERK,e IF2α,and NLRP3 m RNA relative expression levels were not statistically different(P > 0.05).4 ConclusionThe above results have led us to the following conclusions:(1)The ichthyodene-modeling method can simulate both DAergic neuronal damage and abnormal α-syn aggregation in the Parkinson’s disease model,and electroacupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" can improve the behavior of Parkinson’s disease and abnormal α-syn aggregation.The electroacupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" can improve the behavior of Parkinson’s disease and the abnormal aggregation of α-syn.(2)The rat model of Parkinson’s disease can induce persistent endoplasmic reticulum stress,and electroacupuncture may inhibit the persistent endoplasmic reticulum stress response in the rat model of Parkinson’s disease.(3)Endoplasmic reticulum stress mediates the NLRP3 inflammatory vesicle pathway in the Parkinson’s disease model,and electroacupuncture may inhibit the activation of the ERS/NLRP3 signaling pathway in Parkinson’s disease.(4)Electroacupuncture has a regulatory effect on the SIRT1/ERS signaling pathway.This study further revealed that electroacupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" may mediate the activation of the SIRT1/ERS/NLRP3 signaling pathway through the regulation mechanism of the SIRT1/ERS/NLRP3 signaling pathway.The therapeutic effects of the acupuncture points "Fengfu(GV16)","Taichong(LR3)" and "Zusanli(ST36)" were further revealed. |
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