Mechanism Studies Of The Capsaicin Alleviates Apoptosis In The Cell Model With 6-OHDA-induced Parkinson's Disease

Parkinson's disease(PD),as the third largest killer that threatens the health of the elderly seriously,has become a common disease in the elderly.PD is associated with the age factor,and the incidence increases with human age.Epidemiological survey studies showed that the global prevalence of PD is about 0.3%,2%over 65 and 5%over 85.PD brings a great burden on the patients' family and society.At present,the existing drugs and surgical treatments are very limited,which can only partially improve the clinical symptoms and can not delay the progress of PD disease.The PD symptoms will aggravate gradually until the loss of motor function,and eventually the patient dies due to many complications.PD is a progressive neurodegenerative disease characterized by the selective death and degeneration of black dopaminergic neurons,its occurrence mechanism is very complex,the etiology of PD is unclear,the molecular mechanism leading to neuronal death has not been fully clarified,but scientific studies have found multiple pathways related to PD pathology,which the main pathogenic factors include:?-Synuclein aggregation,mitochondrial dysfunction,oxidative stress and autophagy,etc.Thus one major objective of PD research is to explore potential disease-modifying drugs that slow or stop the underlying neurodegenerative process.However,no ideal neuroprotective drugs or treatments have been identified.So finding new therapeutic targets for PD is particularly important.In previous research,our lab found that capsaicin protects against oxidative insults and alleviates behavioral deficits in rats with 6-OHDA-induced Parkinson's disease via activation of TRPV1.In this study,we found that in a 6-OHDA-induced rat model of PD,the expression levels of TH in the SN and striatum were significantly decreased.Consistent with this result,rats presented motor deficits,including apomorphine-induced rotations and abnormal behaviors in the open-field test.In addition,the antioxidant system was also involved in the ipsilateral hemisphere,which included the downregulation of SOD and CAT and a higher MDA level.Interestingly,if capsaicin,an agonist of the TRPV1 receptor,was applied immediately after injection with 6-OHDA,rats presented less severe behavioral deficits.Higher levels of TH and TRPV1 than in 6-OHDA lesioned rats were also observed.Moreover,the antioxidant system was activated in the ipsilateral hemisphere.It is suggested that capsaicin may be a new neuroprotective drug for Parkinson's disease(PD).While TRPV1 may be a target of action for capsaicin.Capsaicin is an alkaloid found primarily in the fruit of the Capsicum genus and is the classical agonist of a nonselective cation channel,transient receptor potential vanilloid subfamily member 1(TRPV1).Traditionally,TRPV1 has been considered to be involved in a broad range of diseases.Recently,more and more studies have found that TRPV1 is not only highly expressed in sensory neurons but also present in various regions of the brain.Moreover,experiments have suggested that TRPV1 can significantly reduce deficits in motor and cognitive functions.The preliminary experiments of our lab showed that capsaicin is mainly involved in rescuing nigral neuron survival by inhibiting oxidative stress on the ipsilateral side,protecting against DA neuron loss.Therefore,TRPV1 might be an effective neuroprotective target for PD.Furthermore,the purpose of this study is to reveal the possible molecular mechanism which capsaicin can reduced the behavioral defects of 6-OHDA Parkinson's disease rats from activating TRPV 1.Our experiments showed that 108 differential genes were found comparing with the control group,and Actgl expression was down-regulated and Gsta2 expression was up-regulated after capsaicin intervention;Actins are a family of highly conserved cytoskeletal proteins that play fundamental roles in nearly all aspects of eukaryotic cell biology.The ability of a cell to divide,move,endocytose,generate contractile force,and maintain shape is reliant upon functional actin-based structures.The Actgl gene provides instructions for making a protein called gamma(y)-actin,which is part of the actin protein family.Proteins in this family are organized into a network of fibers called the actin cytoskeleton,which makes up the structural framework inside cells.These proteins play important roles in determining cell shape and controlling cell movement(motility).Actgl is involved in actin production,and actin is closely related to autophagy,which connects Actgl with autophagy and participates in the regulation of autophagy indirectly.Current studies have shown that inactivation of autophagy signal transduction pathway plays an important role in many diseases,including neurodegenerative diseases.Parkinson's disease is a neurodegenerative disease.Some current studies have shown that autophagy may be one of the mechanisms of Parkinson's disease.Glutathione S-transferase 2(Gsta2)is a member of the glutathione S-transferase(GSTs)superfamily,which encodes multifunctional enzymes important in the detoxification of electrophilic molecules,including carcinogens,mutagens,and several therapeutic drugs,by conjugation with glutathione.Gsta2 is one of the functional antioxidant response elements(AREs),which play a role in defense against oxidative stress.Recently,one study demonstrated that upregulating Gsta may serve as a compensatory mechanism against elevated oxidative stress,which accompanies obesity.On the other hand,downregulating Gsta can impair the defense against oxidative stress.Therefore,in our study,Actgl and Gsta2 were used as target genes to explore the possible molecular mechanism of activating TRPV1 by capsaicin which reduce the behavioral defects induced by 6-OHDA in Parkinson's disease rats.ObjectiveThe present study aims to explore the possible molecular mechanisms of TRPV1 activation using the PD cell model induced by 6-OHDA in terms of oxidative stress,cytoskeletal and autophagy to reduce the behavioral defects of 6-OHDA-PD rats.To provide the theoretical supports and new ideas for the treatments of Parkinson's disease.MethodsWe used capsaicin-treated and paraffin-embedded wax blocks containing substantia nigra tissue from 6-OHDA-induced Parkinson's disease rats to analyze transcriptional changes using Affymetrix GeneChip Whole Transcript Expression Arrays.A total of 108 genes were differentially expressed in response to capsaicin treatment.Because of mouse origin,the matched human genes were searched in NCBI and compared with the human genes in the gene bank,and then the genes shared by human and mouse sources were selected.According to the function of these common differentially genes,7 interesting genes of them were selected for further analysis:Olr724,COX1,Gsta2,Rab5a,Potef,Actg1,and Acadsb,of which Rab5a,Potef,Actgl,and Acadsb were downregulated and Olr724,COX1,Gsta2 were upregulated.The seven genes may be closely related to PD.The mRNA sequences of the seven genes were searched and downloaded in NCBI,and then the matching human gene information was found by using Protein-Protein BLAST function in NCBI.The PD cell model induced by 6-OHDA was established.SH-SY5Y cells were stimulated with different concentrations of 6-OHDA,the proliferation of SH-SY5Y cells was detected by CCK-8,and the apoptosis of PD cells was analyzed by flow cytometry.We used gene overexpression technique to transfer cells.Seven genes were cloned into the same eukaryotic expression vector pcDNA3.1 to construct eukaryotic expression plasmid,and the eukaryotic expression plasmid was transformed into SH-SY5Y cell model and PD cell model,respectively.His-tag and HA-tag were used for WB detection.Detection of apoptosis by flow cytometry,and the statistical method was used to determine that whether this effect is statistically significant.ResultsPD cell model was successfully established.we applied 50 ?mol/ml of 6-OHDA to well-grown SH-SY5Y cells and finally successfully established the PD cell model.Successful construction of the overexpression vector.We constructed the overexpression vectors of seven target genes and transformed them into SH-SY5Y cells.In addition,seven kinds of overexpression vectors were transfected into SH-SY5Y cells and the proteins were detected relatively quantitatively.The protein expression was detected by immunofluorescence.Only 2 of the 7 genes were detected by Gsta2 and Actg 1.Gsta2 and Actg 1 were successfully overexpressed in SH-SY5Y cells.Apoptosis detection resultsWe transfected the Gsta2 and Actgl overexpression to PD model cells and then detected apoptosis with the CCK8 apoptosis kit and flow cytometry.The CCK8 kit can directly detect cell apoptosis,whereas flow cytometry is used to detect the cell survival rate,to which the apoptosis rate is inversely proportional.The results showed a significant difference in apoptosis between cells overexpressing Gsta2 and the vitamin C control group(p=0.0008<0.05)and between cells overexpressing Actgl and the vitamin C control group(P<0.0001<0.05).The data using SPSS 16.0 software.ConclusionWe demonstrated that capsaicin targets TRPV1 through downregulation of Actg1 and upregulation of Gsta2 in PD rats,which could reduce apoptosis and protect cells by regulating the autophagy pathway and oxidative stress pathway.Consequently it is the possible mechanism that capsaicin alleviates apoptosis in a cell model of 6-OHDA-induced Parkinson's disease from regulation of Actgl and Gsta2.TRPV1 is promising therapeutic targets for alleviating the progression of PD.