| Objectives Observation of butyl phthalide effect on the improvement of the Parkinson’s disease with depression mice behavior and neuron injury protection,area of the substantia nigra striatum NLRP3 inflammatory corpuscle,microglia and related the impact of the expression of inflammatory cytokines,explore butyl phthalide to treat Parkinson’s disease with depression treatment effect and mechanism of action,provide new ideas for the treatment of Parkinson’s disease with depression.Methods Forty adult male C57 mice were randomly divided into normal control group,Parkinson’s disease with depression model group,medoba treatment group,butylphthalide combined medoba treatment group.Mice models of Parkinson’s disease were established by intraperitoneal injection of MPTP(30mg/kg×7d),and 2-3 mice were screened by paralysis tremor score.Chronic unpredictable mild stress(21 days)was used to prepare mice models of Parkinson’s disease with depression.Medoba treatment group,medoba6.25mg/kg,once a day,for 7 days;Butylphthalide combined with medoba treatment group,medoba 6.25mg/kg and butylphthalide 120mg/kg,once a day,for 7 days.Parkinson-like behavior of mice was tested by rotating rod test,climbing rod test and hanging test.Depressive-like behavior was evaluated by forced swimming test,tail-hanging test,and sugar water preference test.The Ionized calcium binding adaptor of NLRP3 inflammasome and microglia in substantia nigrostriatum was detected by western-blotting Iba-1 and the protein expression levels of related inflammatory factors,such as endogenous proinflammatory factors(IL-1β,TNF-α)and anti-inflammatory factors(IL-10,ARG-1).The positive expression of NLRP3 and a-synuclein(α-synuclein α-syn)in the substantia nigra striatum was qualitatively analyzed by immunohistochemistry.The abnormal activation of microglia and the damage of dopamine neurons were observed by immunofluorescence.The m RNA relative expression levels of pro-inflammatory factors(IL-1β,TNF-α)and anti-inflammatory factors(IL-10,ARG-1)in the substantia nigra striatum were detected by real-time PCR.Data were processed by SPSS23.0,and measures with normal distribution or approximate normal distribution were expressed as mean ±standard deviation((?)±).One-way an OVA was used for comparison between groups,and LSD was used for pial comparison.0.05,indicating statistical difference.Results 1.Behavioral test results:(1)PD-like behavior test results: Compared with the normal control group,the experiment time of rod rotation and suspension was significantly reduced in the model group,while the experiment time of rod climbing was significantly increased,and the difference was statistically significant(P<0.05).Compared with the model group,the rotating rod test time and hanging test time of the metobar treatment group and butylphthalide combined with metobar treatment group were significantly increased,and the time of climbing bar test was significantly shortened,and the time of butylphthalide combined with metobar treatment group was more obvious,the difference was statistically significant(P<0.05).(2)Depression-like behavior test results: Compared with the normal control group,the model group had longer forced swimming immobility time and tail suspension immobility time,and lower ratio of sugar water preference experiment,the difference was statistically significant(P<0.05);Compared with the model group,the forced swimming immobility time and tail suspension time in the metobar treatment group and butylphthalide combined metobar treatment group decreased,and the ratio of sugar water preference experiment increased,especially in the butylphthalide combined metobar treatment group,the difference was statistically significant(P<0.05).2.Changes of dopaminergic neurons: Compared with normal control group,the number of dopamine neurons in the model group was significantly reduced,and TH positive expression was decreased;Compared with model group,the number of dopamine neurons in butylphthalide combined with medoba treatment group increased,and TH positive expression level increased.3.α-SYN expression in substantia nigra striatum of each group:compared with normal control group,the number of α-SYN positive cells in model group was significantly increased and the staining was darker,and the difference was statistically significant(P<0.05);Compared with model group,the number of α-SYN positive cells in butylphthalide combined with metopar treatment group was significantly reduced,and the staining was lighter,the difference was statistically significant(P<0.05),and there was no significant difference between the Parkinson’s disease treatment group and the model group(P=0.18).4.Results of NLRP3 protein expression in the substantia nigra striatum:Compared with the normal control group,the NLRP3 protein expression level and the number of positive cells in the model group increased significantly,and the absorbance value increased,with statistically significant differences(P<0.05);Compared with the model group,the expression level of NLRP3 protein,the number of positive cells and the absorbance value of butylphthalide combined with medoba treatment group decreased,and the differences were statistically significant(P<0.05);Compared with model group,there was no significant change in metopar group(P=0.62).5.Expression level of microglia marker IBA-1 protein in the substantia nigra striatum and abnormal activation of microglia were observed: Compared with the normal control group,the expression level of IBA-1protein in the model group increased,the number of activated microglias increased significantly,the morphological cell body became thicker,and the axon decreased.Compared with the model group,the expression level of IBA-1 protein in the butylphthalide combined with metopar treatment group increased,and the number of activated microglias decreased significantly.The morphology recovered slightly,and decreased significantly compared with the metopar group.6.Protein expression levels and m RNA relative expression levels of M1 pro-inflammatory factors(IL-1β,TNF-α)and M2anti-inflammatory factors(ARG-1,IL-10)in microglia: Compared with normal control group,the expression of M1 pro-inflammatory factor in model group was significantly increased,while the expression of M2 anti-inflammatory factor was decreased,with statistically significant differences(P<0.05);Compared with the model group,the expression level of M1 pro-inflammatory factor decreased in butylphthalide combined with metobar,while the expression level of M2 anti-inflammatory factor increased,which decreased significantly compared with metobar alone,with statistically significant difference(P<0.05).Conclusions Butylphthalide can improve PD-and depression-like symptoms of Parkinson’s disease with depression model mice,and has protective effect on neurons.The mechanism may be related to inhibiting the activation of microglia and inflammasome,inhibiting the expression of inflammatory factors and alleviating inflammatory response.Figure 9;Table 6;Reference 89. |
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