Research On The Therapeutic Effect And Mechanism Of Wuzi Yanzong Pill On Parkinson’s Disease Model Mice

Objective:In this study,Wuzi Yanzong Pill(WYP)was used to interfere with MPTP induced Parkinson’s disease(PD)model in mice.The therapeutic effect of WYP on PD and its possible mechanism were observed,providing scientific theoretical basis for the treatment of PD with WYP in the future.Methods:C57BL/6 male mice were randomly divided into Normal group,Model group and WYP treatment group.One week of intraperitoneal injection of MPTP(d1:15mg/ kg,d2:20mg/kg,d3-d7:30mg/kg)was used to establish the classical PD model of mice.At the same time,the Normal and Model group were given 50mL/kg/d sodium chloride solution,and the WYP treatment group was given 16g/kg/d WYP solution,twice a day for two weeks.The general behavior of animals in each group was observed after each injection of MPTP.On the 15 th day of the experimental cycle,gait test,climbing pole test,adhesive tape removal test and open field test were used to evaluate the movement function,exploration behavior and anxiety of animals.TH neurons in substantia nigra of midbrain were observed by immunohistochemistry and TH positive cells were counted.The levels of GSH,MDA in the brain was detected by microenzyme labeling.Western blot was used to detect the expressions of CAT,SOD,p-PERK,p-eIF2α,ATF4,p-IRE1α,XBP1,ATF6,and the levels of CHOP,ASK1,p-JNK,Caspase-12,Caspase-9 and Caspase-3.Results:1.The effect of WYP on the general physical signs and behavior of PD mice1.1 The effect of WYP on the general physical signs of PD miceWhen MPTP was injected for 10-20 minutes for the first time,acute reactions such as limb tremor,bristle,arched back,vertical tail and so on appeared in mice at different degrees.The above symptoms gradually reduced after 30-60 minutes,accompanied by mild activity reduction,slow response and so on.With the increase of dosage and times of administration,the acute response gradually became stable,but the symptoms of Model were more prominent than WYP treatment,such as movement reduction,gait instability,and movement retardation.No behavioral changes were observed in normal animals.1.2 The effect of WYP on the behavior of PD miceIn gait analysis,compared with the Normal,the paw area and hindlimb stance width of the Model was significantly increased(P<0.01),the angle of RH and LH was obviously increased(P<0.05),the angle of LF was significantly reduced(P<0.05),the difference of RF angle was not statistically significant(P>0.05);compared with the Model,the paw area and hindelimb stance width of the WYP treatment was reduced(P<0.05,P<0.01),the angle of LF was increased(P<0.05),and the angle of RF,RH and LH was not statistically different(P>0.05).In the climbing pole test,compared with the Normal,the turning and climbing pole time in the model were significantly prolonged(P<0.01);compared with the Model,the time used in the WYP treatment was significantly reduced(P<0.05).In the adhesive removal test,compared with the Normal,the removal time of the Model was significantly increased(P<0.001);compared with the Model,the time of WYP treatment was significantly shortened(P<0.01).In the open field test,the activity routes of Normal mice were complex and varied,and the tracks were all over the bottom of the box;in the Model,the action routes were simple and mostly moved along the wall around the bottom of the open box,the movement in the central area was relatively less,and the anxiety was obvious;after WYP treatment,the activity increased,the tracks were evenly distributed,the behaviors in the central area increased significantly,and the anxiety was relieved.Compared with the Normal,the number of dressing in the Model decreased,the average speed slowed down,the rest time prolonged(P<0.05),and the total distance of movement shortened(P<0.01);compared with the Model,the number of dressing in the WYP treatment increased,the average speed increased,the rest time shortened(P<0.05),and the total distance of movement increased(P<0.01).2.The effect of WYP on TH expression in the substantia nigra of PD miceIn the immunohistochemistry analysis,TH positive neurons in the substantia nigra of normal brain tissue were closely arranged,with deep cytoplasm staining and uniform color.Neurons in the Model were scattered with different cytoplasm colors.WYP treatment effectively improved this phenomenon.Compared with the Normal,TH positive cells in the Model were significantly reduced(P<0.001);compared with the Model,TH positive cells in the WYP treatment were significantly increased(P<0.01).3.The effect of WYP on oxidative stress in brain of PD miceCompared with the Normal,the GSH content in the Model decreased significantly(P<0.001),MDA increased(P<0.001),CAT and SOD protein expression decreased obviously(P<0.01);compared with Model,the GSH content in the brain of the WYP treatment increased significantly(P<0.001),MDA content decreased(P<0.001),SOD and CAT expression increased obviously(P<0.01).4.The effect of WYP on endoplasmic reticulum stress in PD mice4.1 The effect of WYP on UPR mediated signaling pathway in PD miceThe expression of BIP protein in the Model was significantly increased than Normal(P<0.001),and in the WYP treatment was significantly decreased than Model(P<0.01).Compared with the Normal,the protein levels of p-PERK,p-eIF2α,ATF4,p-IRE1α and XBP1 in Model were significantly increased(P<0.001,P<0.01);compared with the Model,the relative expression levels of p-PERK,p-eIF2α,XBP1(P<0.01)and ATF4,p-IRE1α(P<0.05)in the WYP treatment were significantly decreased.There was no significant change of ATF6 content in the brain of the three groups(P>0.05).4.2 The effect of WYP on ERS mediated apoptosis in PD miceThe expression of apoptotic factor CHOP in Model was significantly increased than Normal(P<0.01),while that in WYP treatment was decreased(P<0.05).Compared with the Normal,ASK1 and Caspase-12 protein level increased(P<0.01),Caspase-9 and Caspase-3 content increased(P<0.05);compared with the Model,Caspase-12 and Caspase-9 level decreased(P<0.05),ASK1 and Caspase-3 content decreased in WYP treatment,but there was no statistical significance(P>0.05).There was no significant change in p-JNK protein levels between the three groups(P>0.05).Conclusion:1.WYP can effectively improve the general performance and motor symptoms of PD mice,relieve anxiety and delay the loss of DA neurons in the substantia nigra of the midbrain.2.WYP can alleviate oxidative stress in brain by up-regulating the expressions of SOD and CAT,increasing the content of GSH and reducing MDA.3.WYP can regulate UPR mediated PERK/eIF2α/ATF4 and IRE1α/XBP1 signaling pathways,inhibit the phosphorylation and dimerization of transmembrane proteins,reduce the expression of downstream transcription factors,reduce ERS response and alleviate cell damage.4.WYP can regulate ERS mediated apoptosis,inhibit the expression of apoptotic factor CHOP,inhibit the levels of Caspase-12,Caspase-3 and Caspase-9,and then reduce neuron apoptosis.