Study On The Neuroprotective Effect And Mechanism Of Dl-3-N-Butylphthalide In Cell Models Of Parkinson’s Disease

[Background and Objective]The pathogenesis of Parkinson’s disease(Parkinson’s Disease,PD)is inconclusive.Studies have shown that neuroinflammation,apoptosis,mitochondrial dysfunction and oxidative stress play key roles in the pathogenesis of PD.Dl-3-n-butylphthalide(NBP)is a new drug independently developed in China.There was evidence to support its potential pharmacological effects including anti-inflammatory response,inhibition of neuronal apoptosis,reduction of oxidative stress injury,improvement of mitochondrial dysfunction and so on.At present,NBP is mainly used in clinical treatment of stroke,but its application in neurodegenerative diseases is still lack of reliable evidence.Through previous research and reading the literature,we found that the target of NBP is consistent with the main pathogenesis of PD,so it is speculated that NBP may have a potential protective effect on PD.Therefore,the purpose of this study is to study the neuroprotective effect of NBP on PD and explore its possible mechanism so as to provide a theoretical basis for the clinical application of NBP in PD.[Methods]1.After SH-SY5Y cells were induced by different concentrations of 6-OHDA for 24 hours,the cell viability was detected by CCK8 method,and the optimum concentration of 6-OHDA was determined to construct PD cell model.2.After pretreatment with different concentrations of NBP for 6 hours,the optimal concentration of 6-OHDA was added to induce 24 hours.The CCK8 method was used to detect the cell viability and determine the optimal protective concentration of NBP.3.Flow cytometry and fluorescence microscopy were used to detect the effect of NBP on the apoptosis rate and intracellular ROS level of PD cells induced by 6-OHDA.4.Detecting the expression of TH and Nurrlby Western Blot(WB)and Immunofluorescence(IF)exqeriments.5.Detecting the expression of inflammatory factors and NLRP3 inflammasome-related proteins in each group of cells by RT-qPCR and WB experiments.6.Detecting the expression of α-Syn,p-α-Syn and Parthanatos pathway-related proteins such as PAR,PARP1,AIF and so on by WB and IF experiments.7.Detecting the expression of mitochondrial oxidative stress,dysfunction,and autophagy-related proteins such as ClpP/SOD2,Parkin/PINK1,p-AMPK/AMPK,LC3II/LC3I and so on by Western Blot experiment.8.Detecting the expression of vesicle transport related proteins such as NSF and SYNJ1 in each group by Western Blot experiment.[Results]1.The optimal concentration of 6-OHDA was 100uM.NBP could increase the cell viability in a dose dependent manner,in which 5uM could significantly inhibit apoptosis and ROS production,and increase the expression of TH and Nurrl.5uM NBP was selected as the therapeutic dose in the follow-up study.2.NBP pretreatment can reduce the expression of inflammatory cytokines and NLRP3 inflammasome bodies induced by 6-OHD A.3.Compared with 6-OHDA group,the expression of α-Syn,p-α-Syn PAR,PARP1 and nuclear transfer of AIF protein decreased in 6-OHDA+NBP group.4.Compared with 6-OHDA group,the expression of ClpP,SOD2,Parkin,PINK1 in 6-OHDA+NBP group increased,while the ratio of p-AMPK/AMPK to LC3Ⅱ/LC3Ⅰdecreased.5.Compared with 6-OHDA group,the expression of NSF and SYNJ1 proteins in 6-OHDA+NBP group was higher.[Conclusions]Our results show that NBP can significantly improve the cell viability of 6-OHDA-induced cells,reduce cell apoptosis,and inhibit ROS production,and enhance the expression of TH and Nurrl.It has obvious neuroprotective effects in Parkinson’s disease.Moreover,NBP does not target a single molecule in the treatment of Parkinson’s disease,but may protect damaged cells by inhibiting neuroinflammation,blocking PARP1/PAR/a-Syn feedforward circulation,reducing oxidative stress damage of mitochondria,protecting mitochondrial function,regulating autophagy,and regulating the release and reuptake of neurotransmitters.