| BackgroundParkinson’s disease (PD) is a chronic progressive neurodegenerative movement disorder ranked as the second most common degenerative neurological disease, affecting more than 6 million people worldwide. The neuropathological hallmarks are characterized by a profound and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) with presence of Lewy bodies and dystrophic Lewy neuritis in surviving neurons. The clinical features of PD include motor impairments involving resting tremor, bradykinesia, postural instability and rigidity complicated with non-motoric symptoms such as autonomic, cognitive and psychiatric disorders. Although different pathogenic causes have been discovered, in the majority of cases, the critical mechanisms of PD remain unknown.It has been confirmed that mitochondrial dysfunction is one of the main pathogenesis of PD. DJ-1 is a relatively common genes associated with PD onset and its function is not very clear. It was mainly considered to play an important role in the fight against oxidative stress and apoptosis regulation, etc. Currently, there are already a large number of studies showing that DJ-1 play an important role in regulating mitochondrial function. In addition, the role of DJ-1 related to the regulation of PI3K/Akt pathway in the PD has been concerned, but their specific regulatory mechanisms are still not clear.At present, there is no ideal method in the treatment of PD. Traditional Chinese medicine has achieved certain effect for treatment of PD in clinical practice, which roughly attributed to the overall system control streatment principle, and has a unique advantage in the improvement of symptoms of PD as well as of the quality of life. Therefore, it’s very important for performing further research to explore the mechanism of PD so that more effective therapies can be discovered. This study on the basis of former research, taking DJ-1 as the core, to explore the effect of Da-Bu-Yin-Wan (DBYW) on the protection of mitochondrial function and the mechanisms based on PI3K/Akt signaling pathway.ObjectiveIn this study, DJ-1 overexpression plasmid was transfected into human neuroblastoma SH-SY5 cells and rat adrenal medullary pheochromocytoma PC 12 cells, to investigate how the DJ-1 on mitochondrial function and overexpression of DJ-1 protets the PI3K/Akt pathway. Then, PD cell model was established by 1-methyl-4-phenylpyridinium (MPP+) for investigating the molecular mechanism of protective effect of DBYW on mitochondrial function of PD based on PI3K/Akt signaling pathway.MethodsOur study includes 6 parts:1. DJ-1 overexpression plasmid transformation, amplification, extraction and transfection: Blank control plasmid and DJ-1 gene overexpression plasmid was transformed using heat shock method, then plasmid DNA was amplified, extracted and identified; the plasmids were transfected into SH-SY5Y and PC12 cells uding liposome mediated method. RT-PCR and Western blot techniques were used to detect the expression of DJ-1 mRNA and protein levels, for identifying the overexpression of DJ-1.2. DJ-1 overexpression on mitochondrial function of SH-SY5Y and PC12 cells:the cells were divided into three groups: ①control group:empty plasmid; ② vector group: Transfection of vector plasmid; ③ DJ-1 overexpression group:Transfection of DJ-1 overexpression plasmid. Laser-confocal microscopy was used to observe the mitochondrial activity; mitochondrial ATP level was detected using fluorescent enzyme method; mitochondrial Complex I activity dected with colorimetric analysis.3. DJ-1 overexpression on PI3K/Akt signaling pathway:protein expression of PI3K, Akt, GSK3β and Akt phosphorylation (Thr308/Ser473) was measured by Western blot.4. The effect of DBYW on survival rate of PD cell model:PD cell model was established by using the neurotoxin MPP+. Traditional Chinese medicine DBYW prepared by adaptive adjustable decocting method. Experimental groups: ①Control group:no transfection; ② Model group:1mM MPP+ in 48h; ③ overexpression of DJ-1 treatment group:DJ-1 overexpression plasmid transfection+1mM MPP+ in 48h;④ DBYW low-dose group: DJ-1 overexpression plasmid transfection + 1mM MPP+ in 48h+ low dose DBYW; ⑤ DBYW middle-dose group:DJ-1 overexpression plasmid transfection+1mM MPP+in 48h+ middle-dose DBYW; ⑥ DBYW high-dose group:DJ-1 overexpression plasmid transfection+1mM MPP+ in 48h+ high-dose DBYW. The effect of DBYW on survival rate of PD cell model detected by CCK-8.5. Effect of DBYW on mitochondrial function of PD cell model:Laser confocal microscopy was used to observe the mitochondrial activity, mitochondrial ATP content was detected by fluorescent enzyme method; mitochondrial Complex I activity was tested by colorimetric methodof traditional Chinese medicine, to explore the protective effect of DBYW and DJ-1 overexpression on mitochondrial function in PD cell model.6. Effect of DBYW on PI3K/Akt signal pathway that regulated by DJ-1 in PD cell model: protein expression of PI3K, Akt, GSKβ and Akt phosphorylation (Thr308/Ser473) of Akt was measured using Western blot.Results1. Amplification and extraction of DJ-1 overexpression plasmid were completed. Compared with the control group, mRNA and protein expressions of DJ-1 were significantly increased in DJ-1 overexpression group.2. Compared with control group, the mitochondrial activity, mitochondrial ATP levels and Complex I activity were enhanced by DJ-1 overexpression.3. Overexpression of DJ-1 can significantly improve Akt phosphorylation at Thr308 and the protein expression level of GSK3β The protein expressions of PI3K, Akt and its phosphorylation at Ser473 have no significant difference.4. Compared with normal control group, SH-SY5Y/PC12 cell damage is evidently observed in model group after MPP+ induction. With the increase of the concentration of MPP+, and as time progresses, the cell survival rate significantly declined. DJ-1 overexpression can enhance the survival rate of PD cell model, and DBYW can further improve the cell survival rate.5. Compared with normal control group, the activity mitochondria, the content of mitochondrial ATP and the activity Complex I were decreased in model group. The overexpression of DJ-1 can improve those damage. Compared with the DJ-1 overexpression model group, DBYW enhanced the effect of improve the mtochondrial dysfunction induced by MPP+, and it has a certain dose dependent.6. After MPP+ induction, the protein expression of DJ-1 of SH-SY5Y and PC12 cells were significantly decreased, DJ-1 protein level of DJ-1 overexpression model group of was significantly increased. Compared with DJ-1 overexpression model group, additional DBYW further enhanced the expression of DJ-1 protein. In the PI3K/Akt signaling pathway, DJ-1 overexpression upregulated Akt phosphorylation at Ser473 and Thr308 in the PD model cell. Compared with DJ-1 overexpression model group, DBYW further improved the two sites of levels of Akt phosphorylation. In addition, no significant change in protein expressions of PI3K and GSK3β between groups.Conclusions1. The overexpression of DJ-1 can enhance the SH-SY5Y/PC12 cell activity of mitochondria and mitochondrial Complex I, and increase the content of ATP in the cells. This suggests that overexpression of DJ-1 relates to the enhancement of the function of mitochondria in a certain extent.2. DJ-1 overexpression may improve mitochondrial function and the mechanism may be mediated by up-regulation of Akt phosphorylation at Thr308 and down-regulation protein expression of GSK3β.3. DBYW can improve the protective effect of DJ-1 overexpression on mitochondrial damage and cell death induced by MPP+, and its mechanism may involve the up-regulation of DJ-1 expression, which regulates PI3K/Akt signaling pathway in Akt phosphorylation at Ser473 and Thr308.
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