Synergetic Neuroprotect Effect And Mechanism Of DHA And ASA In Parkinson's Disease

Parkinson's disease(PD)is the most common age-related disease,the treatment of PD with conventional drugs carries serious adverse reactions and cannot fix the root cause of PD,the degeneration of dopaminergic neurons,which limits conventional drug usage in clinical practice.In recent years,research on the pathogenesis of PD and its clinical manifestations has led to the discovery of an increasing number of novel targets in PD,including several small molecule targeted compoundsDHA is quantitatively the most important omega-3 PUFA in the brain,and consequently the most studied,whereas the availability of high purity DPA preparations has been extremely limited until recently,limiting research into its effects.DHA enhances neurite outgrowth and promotes synaptogenesis and synaptic expression of synapsin,and glutamate receptors in rat hippocampal neurons.In vitro DHA has consistently been shown to promote the differentiation of neural stem cells into neurons.Therefore,we hypothesized that DHA had protective effect on PD.In addition,some non-steroidal anti-inflammatory drugs(NSAID)could specifically bind to and activate PPAR.Aspirin(ASA)is the most common NSAID drug used for treating pyrexia,pain,inflammation and antiplatelet.It also possesses some newly discovered benefits,including neuroprotective and cancer-inhibiting effects.Aspirin could upregulate and activate PPAR in macrophages.However,whether aspirin modulates PPAR in neurons is still being explored.Micro-RNAs(miRNAs)are small non-coding,single stranded RNA molecules consisting of 22 nucleotides.miRNAs control gene expression by base pairing to mRNA and trigger translation repression.Abnormal miRNA expression has been associated with various neurological disorders,such as AD,PD,Huntington's disease,amyotrophic lateral sclerosis,schizophrenia,and autism.Certain miRNAs are highly elevated in brain tissues of patients with PD,such as miR-21,which actively participates in the autophagy pathway carried out by chaperone.In the first part of this study,6-OHDA was injected into the left substantia nigra of rats to establish the PD rat model.And by intraperitoneal injection of apomorphine,open field test and tail suspension test behavior detection model of success;in addition,the expression of Tyrosine hydroxylase by immunohistochemical method compared with rats injected with ipsilateral and contralateral substantia nigra and striatum of brain regions,for the purpose of establishing a stable and reliable animal model for subsequent experiments.In the second part,we used PD rat model,PD patients and neuroblastoma SH-SY5Y cell line as the research object.Morphology detected by Nissl staining and FJB staining,QRT-PCR,Western blot and other molecular detection means further revealed the expression of miR-21,PPAR alpha and RXR alpha,and the possible relationships between the three,to provide experimental basis for the study on the molecular mechanism of the follow-up;the third part mainly selects the first SH-SY5Y cell lines.Through the bioinformatics prediction of miR-21 and PPAR 'alpha 3 potential UTR binding sites,and detected by fluorescence reporter gene technology to clarify the miR-21 PPAR alpha and RXR expression regulation mechanism.Then,through the SH-SY5Y cell line screening high expression of PPAR alpha,inflammatory pathway related protein detection(NF-?Bp65,phosphorylated ERK1/2 and COX-2)and the signal pathway of apoptosis related proteins(PTEN,AKT and Caspase-3 phosphorylation),neurotrophic factor cells(PSD-95,BDNF and GDNF)expression of multiple proteins miR-21-PPAR,to further clarify the regulatory mechanism for alpha/RXR alpha neurons.Finally,and ASA were used to detect the activity of miR-21,RXR and PPAR,and the expression levels of mRNA and protein were detected by a variety of molecular methods by using DHA and/or drugs.Furthermore,the expression levels of inflammatory pathway,apoptosis pathway and neurotrophic factor were further detected,and the possible mechanism of DHA and/ASA in neurons and its future application were revealed.Part ? Establishment and identification of PD rat model induced by 6-OHDAObjective:Animal models were important for the development of PD drugs and new treatment strategies.In this part of experiments,the left brain striatum of SD rats were injected with 6-OHDA,and the model was successfully identified by a series behavioral and pathological methods.PD rat model induced by 6-OHDA was established to provide a reliable model for subsequent molecular mechanism studies and drug therapy.Methods:1)Quantitative 6-OHDA solution was injected into the left brain striatum with a stereotaxic apparatus to induce the PD model.2)After 4 weeks of modelling,0.5 mg/kg apomorphine was injected intraperitoneally.Rats were observed for 30 min to identify if the PD model was successful by counting inward rotation number.Through the open field experiment and the tail test,a series of behavioral changes in rats were detected.Expression of tyrosine hydroxylase(TH)in injected substantia nigra(left)and no injection(right)of the brain was measured by immunohistochemistry.Results:1)6-OHDA was injected into the left substantia nigra brain region of rats by stereotaxic apparatus.Intraperitoneal injection of apomorphine could significantly induce rats to end-to-end turn round to the healthy side,indicating that the model was successful;2)In the open field test,the rats of the model group cross grid frequency was reduced,while sitting time was increased.And all the model rats showed dyskinesia.3)Tail suspension immobility experimental results showed that the resting time of rats in model group increased.4)In the model group,the expression of TH in the substantia nigra and striatum of the rats was significantly lower in 6-OHDA injection part than that in the non injected side.Conclusion:The 6-OHDA could induce rat PD model which shared many characteristics of human PD in the pathological and pathophysiological features.It is easy to operate and is a reliable method to prepare PD model by injecting 6-OHDA into rat's brain substantia nigra.Part ? Study for the expression of related genes in rat model and patients with PDObjective:Expressions of miR-21,PPAR and alpha RXR alpha in rat model of PD,PD patients and SH-SY5Y cell line were detected by Western blot and real-time quantitative polymerase chain reaction(QRT-PCR).The correlation in the three meculars was determined,which provided the experimental basis for subsequent molecular mechanism research.Methods:The experiment was divided into two parts:in vivo and in vitro.Firstly,we determined the damage effect of 6-OHDA on neuronal cells in vitro(Nissl staining and FJB staining).Secondly,expressions of miR-21,PPARa and RXRa in the brain of PD rats were analysed.Then,expressions of miR-21,PPAR alpha and RXR alpha were further determined in SH-Y5Y cells and PD patients serum.By comparing the levels of miR-21,PPAR and RXR in PD patients and normal subjects,the correlation between them was studied to provide a basis for the further study of molecular mechanisms.Results:1)Nissl staining showed that SH-SY5Y cells showed obvious purple before 6-OHDA treatment(from 0 to 8 hours).While positive purple was decreased after 6-OHDA treating for 12 hours,and disappeared after 6-OHDA treating for 24 hours.2)The results of FJB staining showed that fluorescence intensity was weak at 0,4,8 hours after 6-OHDA(1 g/ml)treatment.However,fluorescence increased gradually at 12 and 24 hours.These results indicate that 6-OHDA can cause obvious damage to the cells,leading to neuronal degeneration.3)QPCR results showed that expression of miR-21 was significantly lower in prefrontal lobe and thalamus of injected brain region than that in the contralateral side.In the other three brain regions,the level of miR-21 in the injected brain region was higher than that in the contralateral side.miR-21 expression in substantia nigra was 4.32 times more than that in the contralateral side.Expression of PPAR alpha in ST and SN of injection side are lower than that of the contralateral side,while it was increased in CB.RXRa changed only in two brain regions,OB and CB,it was decreased in OB and increased in CB.4)In the peripheral blood of PD patients,genetic testing results also showed that:compared with the normal group,miR-21 in peripheral blood of PD patients was significantly increased,PPARa was decreased and the expression level of RXR alpha,in two groups show no significant differences.In addition,the correlation analysis showed that miR-21 was negatively correlated with PPARa in peripheral blood of PD patients,but not related with RXRa.Conclusion:1)The above results confirmed that miR-21 was highly expressed in PD rat models,PD patients and cell lin.And the expression of PPARa was inhibited.2)In addition,miR-21 was negatively correlated with PPARa.Although the expression of RXRa was not affected,and there is no correlation,but based on important roles of RXR alpha in modulating gene expression,it was believed that miR-21 could regulate PPAR alpha/RXR alpha heterodimers formation and further involved in the occurrence and development of PD by certain mechanism.Part ? Molecular mechanism of synergetic neuroprotective effect through regulation of related genes by DHA and asprinObjective:In this experiment,we intend to detect the mechanism that miR-21 regulates PPARa by bioinformatics and molecular detection.In addition,this study attempts to elucidate the intrinsic mechanism of DHA and ASA protectiing neurons by cell experiments.Methods:1)To identify the relationship of miR-21 and PPARa,miR-21 mimics or miR-21 inhibitors were used to transfecte SH-Y5Y cells.Western blot and QRT-PCR were performed to detect the protein level of PPARa.2)To identify whether PPARa was directly modulated by miR-21,plasmid PGL3-PPARa-3'UTR containing 3'UTR of PPARa was co-transfected with miR-21 mimics or miR-21 inhibitors into SH-Y5Y cells and the activation of PGL3-PPARa-3'UTR was detected.3)To explore the targets of DHA and ASA,a Lantha Screen TR-FRET PPAR alpha Coactivator Assay kit and a Lantha Screen TR-FRET RXR alpha Coactivator Assay kit were used in this study.4)SH-Y5Y cells were treated with DHA for 48 hours and miR-21 expression was examined by qRT-PCR.5)SH-Y5Y cells were transfected with pcDNA3.1-PPARa.And western blotting was performed to determine the expression of PTEN,pAKT,Caspase-3,NF-KB p65,pERKl/2,COX-2,PSD-95,BDNF and GDNF.Results:1)Luciferase assay demonstrated that,compared with control group,miR-21 mimics effectively decreased the luciferase activity,while miR-21 inhibitors increased the luciferase activity,suggesting that miR-21 might target 3'UTR of PPARa.2)Western blot results indicated that miR-21 reduced the expression PPARa but miR-21 inhibitor enhanced the expression PPARa.3)DHA and ASA were agonists of RXR? and PPARa,respectively.The EC50 of DHA was 10.75?M for RXRa,and the EC50 of ASA was 3.04 ?M for PPARa.4)To further support this result,luciferase activity was detected.DHA could enhance RXRa binding to RXRE.Similar results were obtained in ASA.ASA increased PPARa binding to PPRE in a dose-dependent manner.Additionally,PPARa protein expression could be elevated by DHA.However,ASA could not affect PPARa protein levels,suggesting that ASA increased the activity of PPARa but not the protein quantity.5)qRT-PCR result suggested that DHA inhibited miR-21 in a dose-dependent manner.These results showed that DHA not only enhanced PPARa activity but also increases PPARa protein expression by inhibiting miR-21.6)Meanwhile,miR-21 mimics inhibited the heterodimers formation of PPARa and RXRa.However,DHA but not ASA increased the heterodimer formation of PPARa and RXRa.7)The western blot results suggested that PPARa was able to down-regulate expression of PTEN,pAKT,Caspase-3,NF-KB p65,pERKl/2 and COX-2 and up-regulate expression of PSD-95,BDNF and GDNF.Conclusion:These data revealed that the combination of DHA and ASA could improve PD by multiple aspects.The specific mechanism was that DHA activated RXRa and inhibited miR-21.However,ASA could activate of PPARa.Inhibition of miR-21 could not exert suppressed function on PPARa.Simultaneously,activated PPARa and RXRa formed heterodimer and entered the nucleus easily.After entering the nucleus,the heterodimers exhibited regulating functions in proteins associated with apoptosis,inflammation and neurotrophy in neuron.