Protective Effect Of Resveratrol On Zebrafish Dopamine Neurons And Its Mechanism

Parkinson is a common central neurodegenerative disease,The pathogenesis is related to the selective and progressive death of dopaminergic neurons in substantia nigra,the exact mechanism is unclear.It has been reported that oxidative stress plays an important role in the development of Parkinson's disease.Resveratrol is a natural substance that exists in a variety of plants,it has anti-oxidation and anti-inflammatory effect,Whether resveratrol has a preventive effect on Parkinson's disease is not yet clear.Therefore,the aim of this study was to investigate the protective effect of resveratrol on neurons of PD zebrafish induced by MPTP and explore the possible mechanism of resveratrol.Objectives To study the effect of resveratrol on dopaminergic neurons in PD zebrafish and to explore its mechanism.Resveratrol is a natural polyphenolic compound found in grapes,peanuts,mulberries and polygonum cuspidatum,which has cardiovascular,anti-tumor,anti-inflammatory,anti-oxidation and anti-ischemic biological characteristics.PD is a neurodegenerative disease that occurs in the middle-aged and elderly.It is caused by dopaminergic neuron degeneration and necrosis in the substantia nigra,resulting in reduction of dopamine in the striatum.MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)has been shown to decrease the number of dopamine in striatum and cause Parkinson's disease,it also can decrease dopaminergic neurons and PDrelated gene expression,makes zebrafish tail bending,swimming behavior changes that similar to the performance of Parkinson's disease,plant-derived monomer on PD zebrafish impact arouse our attention.In this study,zebrafish treated with MPTP was used as an animal model for Parkinson's disease,to study the effect of resveratrol on PD zebrafish and its possible mechanism.methods Zebrafish breeding using large-scale automatic cycle breeding system,automatic light-dark ratio of 14 h / 10 h,Experiments were carried out using wild-type AB strain zebrafish and Tg(vmat2: GFP)transgenic zebrafish,Daily feeding three times.In the evening after feeding to choose brood mating,The next day the embryos were collected and cultured in an incubator at 28,5°C,The zebrafish was transferred to the six-well plate after the zebrafish was cultured for 72 hours.The experiment is divided into five groups,1)blank control group(con group),2)200 u M / m L MPTP treatment group(MPTP group),3)60 u M / m L RE treatment group(RE group),4)pre-MPTP and RE(M1R group),5)pre-RE and RE(MR1 group).Laser confocal microscopy was used to observe the expression of Tg(vmat2: GFP)transgenic zebrafish dopamine neurons and wild-type AB strains of zebrafish TH immunofluorescence.The expression of parkin,pink1,dj-1 and apoptosis-related genes bax,p21,bcl2 and antioxidant gene ho-1 were detected by real-time fluorescence quantitative PCR.TUNEL staining was used to observe the apoptosis of brain cells in zebrafish.The expression of NRF2 was detected by immunoblotting.results1.After treatment of MPTP,the zebrafish showed tail bending,swimming behavior changes,including swim speed slowed down,turning more,aggregation in the edge of the six-well plate,and the heart rate slowed to 40 times per minute.after adding resveratrol,Tail bending has been alleviated,swimming behavior,speed,etc.have been partly restored,heart rate has rebounded.2.In Tg(vmat2: GFP)transgenic zebrafish,after MPTP treatment,the number of dopamine neurons decreased,fluorescence intensity of dopamine neurons decreased,in M1 R and MR1 group the degree of reduction was lighter than that of MPTP group,and the fluorescence intensity of resveratrol was also increased.3.In the wild-type zebrafish,TH immunohistochemistry results showed that the administration of MPTP can lead to the decrease of TH-positive neurons.in M1 R and MR1 group,the number of TH-positive neurons was higher than MPTP group,and the fluorescence intensity also increased.The order of drug addition had no significant difference on TH positive neurons.4.Real-time quantitative PCR results showed that MPTP treatment,PD-related genes,especially parkin and dj-1 decreased significantly,pink1 expression decreased, but increased after resveratrol treatment,indicating that resveratrol can adjust PD gene,The expression of cat and sod and ho-1 decreased in MPTP group and increased after adding resveratrol,which indicated that resveratrol could protect dopaminergic neurons from oxidative stress in vivo.5.TUNEL staining showed that apoptotic cells in zebrafish were significantly increased after MPTP treatment,and the number of apoptotic cells decreased with the addition of resveratrol.There was no significant difference between M1 R and MR1 group.and also no significant difference between the control and resveratrol groups6.The expression of NRF2 in MPTP-treated group was significantly lower than that in control group(P <0.05).The expression of NRF2 increased with the treatment of resveratrol,the difference has statistically significant.Resveratrol alone also activates NRF2 expression.There was no significant difference in the expression of M1 R and MR1.conclusion1.MPTP-induced PD zebrafish model was constructed successfully,and resveratrol reduced MPTP toxicity while it did not cause damage to zebrafish2.Resveratrol can protect zebrafish dopamine neurons3.Resveratrol regulates the expression of PD-related genes,apoptosis-related genes and antioxidant-related genes to protect dopaminergic neurons,suggesting that the protective effect of resveratrol can be achieved by inhibiting oxidative stress4.Resveratrol reduces apoptosis due to MPTP5.Resveratrol-protected dopaminergic neurons can be achieved by affecting the NRF2 / ARE signaling pathway.