| Parkinson's disease(PD)is a common neurodegenerative disease,and there is no effective drug therapy.In recent years,more and more attention has been paid to the traditional Chinese medicine and monomer compoment of traditional Chinese medicine in the treatment of PD.Paeoniflorin(PF)is the main effective monomer component of traditional Chinese medicine paeoniflorin with neuroprotective effect.So the purpose of this study was to investigate the protective effect and mechanism of paeoniflorin for PD model damage,and to provide new ideas and methods for the treatment of PD.Firstly,the whole animal experiment was carried out to evaluate the neuroprotective effect of PF.If there is neuroprotective effect in the whole animal experiment,rotenone would be carried out on SH-SY5 Y cell line to induced PD cell model,further study the neuroprotection mechanism of PF.In vivo experiment,C57BL/6 mice were used in vivo experiment(male healthy C56BL/6 mice,weight 20±2 g).After fed and observed for one week,all the mice(n=43)were weighted up and randomly divided into3 groups firstly: normal control group(n=6),vehicle group(olive oil solution containing 2% DMSO,n=7)and model group(rotenone olive oil solution,n=30).Rotenone was given to C57BL/6 mice by contacting.On the 31,32,33,34,35,36 and 40 day,rotating rod experiment was carried out to evaluate the model.After 40 days,mice without modeling successfully were eliminated(n=4).The others(n=26)were randomly divided into 3 groups again: model group(treated with 7.5 mg rotenone,n=9),positive drug group(treated with 7.5 mg rotenone and 120 mg Madopar,n=8)and PF group(treated with 7.5 mg rotenone and 5 mg PF,n=9).During the 41 to 49 day,after administered with rotenone for 3 hours,mice were intragastric administrated once a day.Sterile water was given to normal control group,vehicle group,model group.Madopar and PF solution was given to positive drug group and PF group,once a day,for 9days.The changes of behavior in mice were observed by rotating rod test before and after drug intervention and graded by a rating scale.After the behavioral test,mice each group treated with 5% chloral hydrate,the anesthetized mice were quickly open the chest and perfused with physiological saline via the left ventricle.After the liver turning white,4%paraformaldehyde was used to fix the tissue.Then the brain tissue was removed and fixed by 4% paraformaldehyde for 24 hours.Then the brain tissue was gradiently dehydrated by alcohol(40 min with 50% ethanol,40 min with 70% ethanol,25 min with 90% ethanol,15 min with 100%ethanol,15 min with 100% ethanol),and make transparent(using xylene I for 15 min and xylene II for 15 min).After the wax impregnated 90 min,the tissue was embedded.Then the brain tissue was coronal section 5 ?m and HE stained.In vitro experiments,SH-SY5 Y damage model induced by rotenone was used as the extracelluar model of PD study.When the cell attachment growth reached 70%-80%,the cells were digested and divided into control group,0.0125%DMSO vehicle group,rotenone group,rotenone plus Madopar group,rotenone plus paeoniflorin group(the concentration of PF was set into three levels).The final concentration of rotenone was 0.25?mol·L-1,the final concentration of Madopar was 15 ?mol·L-1(based on L-dopa)and PF was 0.08 ?mol·L-1,0.4 ?mol·L-1 and 2 ?mol·L-1.After 24 h,the cell viability was measured by MTT,LDH viability in the cell supernatant was measured by trace enzyme standard method,the morphology by inverted microscope,the expression of ?-synuclein by western blotting and the formation of Lewy by HE straining.In the overall animal experiment,it showed no significant differencein latency time between the vehicle group and control group.And between rotenone group and vehicle group,the latency time of rotenone model group mice was significantly shorter than that of the veicle group(p<0.05).And the time of the positive drug group and the PF group was both longer than that of the model group.After HE staining,the substantia nigra compacts(SNC)of the control group and the vehicle group showed clear nucleus and nucleolus,as well as complete morphology of neurons.Compared with the vehicle group,the number of neurons in model group was less,the nucleus was unclear and cytoplasm concentrated dyeing.And the above changes could be reversed after given Madopar and PF.In vitro experiment,the cell viability and LDH viability both showed no significant difference between the vehicle group and control group(P>0.05).Compared with the vehicle group,the viability and number of cell was decreased,LDH viability increased significantly(P<0.05).the synapse became shorter and the shape became round(P<0.05),while the number of Lewy body and the expression of ?-synuclein were increased(P<0.05).After the addition of Madopar and PF,the changes were reversed.In summary,PF has neuroprotective effect on PD model damage induced by rotenone.And it may relate to reducing the formation of lewy body and decreasing the expression of ?-synuclein. |
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