| Purpose: Parkinson's disease(PD)has become the second most common neurodegenerative disease after Alzheimer's disease.The prevalence and incidence of PD are increasing year by year.The main pathological characteristics of Parkinson's disease are gradual degeneration damage and loss of dopaminergic neurons in substantia nigra pars compacta,accompanied by the formation of Lewy bodies.The synapses of dopaminergic neurons with degeneration damage project into corpus striatum,which result in the decline of dopamine levels in the striatum and the imbalance of neurotransmitter homeostasis.Eventually,the patients with PD manifest motor and non-motor symptoms.At present,there is no intervention or drugs for modifing disease that can completely cure PD.Moreover,the pathogenesis of PD has not completely illuminated.The pathological changes of PD may be interactive mechanism,caused by the cascade pathogenesis,oxidative stress,mitochondrial dysfunction,accumulation of toxic proteins such as ?-synuclein,endoplasmic reticulum stress disorder,calcium homeostasis imbalance,decreased monoamine oxidase B activity,neuroinflammation,and glutamate excitatory neurotoxicity,etc,which may be involved with genetic background and external environmental factors.Trans-(-)-?-viniferin(?-viniferin)is a dehydrodimer of resveratrol with a five-membered oxygen ring.Although resveratrol has not been clinically proven to have a therapeutic effect on any specific disease respectively,these two compounds can regulate multiple neurobiological functions through different signal pathways.In terms of antioxidant and anti-inflammatory,?-viniferin is more biologically active than its monomer resveratrol.?-Viniferin can also promote the disaggregation of abnormal ?-amyloid and inhibit neuroinflammation.?-Viniferin has also been shown to activate autophagy through the AMPK pathway through activating SIRT3/LKB1/AMPK signal pathways,and act a neuroprotective role in Huntington's disease.These reported studies confirmed that ?-viniferin has potential multi-targeted therapy for neurodegenerative diseases.SIRT3(sirtuin 3)is an important mitochondrial deacetylase modifying enzyme.It has the characteristics of deacetylase,can regulate the activities of various enzymes and molecules in mitochondria,and can reduce oxidative stress damage by regulating mitochondrial-related signaling pathways.It regulates mitochondrial function and maintains the normal biological function of cells.As an important substrate for SIRT3,forkhead box O3(FOXO3)is a transcription factor that can activate multiple genes and mediate multiple signaling pathways,involving energy metabolism,oxidative stress,and protein homeostasis,apoptosis,cell development,differentiation and metabolic processes,autophagy and longevity.FOXO3 also plays a vital role in neurodegenerative diseases and is involved in the development of PD.FOXO3 is one of the key factors for the survival of dopaminergic neurons.It can mediate relevant signaling pathways and enhance the elimination of abnormal ?-synuclein aggregation.Our study assumed that ?-viniferin may have potential therapeutic effect for PD.This study firstly established an animal model of PD through 6-OHDA-mediated SD rats,and elucidate if ?-viniferin has neuroprotective effects,and determine if the neuroprotective function of ?-viniferin depend on antioxidative stress and promoting autophagy;after the therapeutic effect of ?-viniferin for PD animal model was confirmed,the rotenone-induced SH-SY5 Y cell model of PD was established to explore if ?-viniferin could enhance SIRT3 deacetylating FOXO3,promote nuclear relocalization of FOXO3 and regulate downstream protein expression and protective biological effect,including mediating mitochondrial homeostasis(mitochondrial biogenesis,fission and fusion,mitophagy),maintaining mitochondrial membrane potential,inhibiting apoptosis,and protecting neurons from oxidative stress.Methods: In the first section,the neuroprotective effect of ?-viniferin for 6-OHDA-mediated dopaminergic neuron damage in rat substantia nigra was evaluated.A 6-OHDA-mediated rat model of PD was established and divided into four groups.The treatment group was treated with ?-viniferin at different concentrations by intraperitoneal administration,and apomorphine was used to induce behavioral changes of PD in rats 4 weeks after the model established.Immunofluorescence TUNEL method was used to evaluate the apoptosis of dopaminergic neurons in substantia nigra.Western blot was used to evaluate the expression of SOD,BAX,BCL-2,and P62.In the second section,our study further explored the neuroprotective mechanism of ?-viniferin in a rotenone-induced cell model of PD and emphasizing the role of SIRT3 deacetylating FOXO3 pathway.For rotenone-induced SH-SY5 Y neurotoxicity damage,neuroprotective property of ?-viniferin on mitochondrial damage and oxidative stress were investigated in present study by focusing on SIRT3 deacetylating FOXO3 pathway.The shRNA was used to inhibit the expression of SIRT3 and FOXO3,and the efficiency of gene silencing of SIRT3 and FOXO3 was identified by Western blot.MTT was used to determine the optimal treatment concentration and time of rotenone and ?-viniferin,and 6 different treatment groups were designed.After grouping,cell viability of different groups was investigated by MTT method;intracellular reactive oxygen species of different groups was investigated by flow cytometry;fluorescence and flow cytometry were used to detect mitochondrial membrane potential and intracellular ATP levels was detected;flow cytometry was used to explore the apoptosis level by Annexin V-FITC.Western blot was used to detect expression levels of SIRT3,FOXO3,PGC-1?,TFAM,Drp1,Fis1,Mfn2,Parkin,Nix,Bnip3 and P62.Immunoprecipitation assessed deacetylated FOXO3 level.Subcellular fractions were extracted and the expression of FOXO3 in the nucleus,cytoplasm and mitochondria was assessed by Western blot.Immunofluorescence was used to evaluate the level of SIRT3 expression.The mitochondrial morphological changes and mitochondrial autophagy were observed by transmission electron microscope.Results: In the first section,it has been shown that ?-viniferin can improve the behavior change of 6-OHDA-mediated rat model of PD.Compared with the model group,TUNEL staining showed that ?-viniferin can inhibit 6-OHDA-mediated apoptosis level of dopaminergic neurons in substantia nigra,promote the expression of antioxidant SOD2,and down-regulate the expression of pro-apoptotic protein BAX,and up-regulate the expression of anti-apoptotic protein BCL-2.Simultaneously,?-viniferin can also increase the expression of autophagy-related markers LC3 and P62(P <0.05),suggesting that the neuroprotective effect of ?-viniferin on 6-OHDA-mediated dopaminergic neurons in substantia nigra is mainly involved with antioxidative stress and promote autophagy.In the second section,our study revealed that ?-viniferin treatment upregulated SIRT3 expression,the latter promoted FOXO3 deacetylation and nuclear localization,where its expression was negatively correlated with the decrease of ATP production and reactive oxygen species(ROS)production.?-Viniferin treatment alleviates rotenone-induced mitochondrial depolarization and mitigated loss of cell viability.However,when cells were pre-transfected with SIRT3 or FOXO3 shRNA prior to ?-viniferin treatment,the increase of ATP production was reversed.?-viniferin drives the expression of mitochondrial homeostasis-related proteins and enhanced the initiation of mitophagy by strengthening SIRT3 to FOXO3 deacetylation,thereby significantly up-regulate and promote mitochondrial biogenesis proteins(PGC-1? and TFAM),and promotes mitochondrial fusion and fission proteins(Drp1,Fis1 and Mfn2)and regulating mitochondrial autophagy-related proteins(Nix,Bnip3,Parkin and P62).Transmission electron microscopy was used to observe the formation of autophagosomes and autophagy compartments in the ?-viniferin treatment group,and mitochondria were significantly elongated(indicating fission or fusion).However,in the model group,the mitochondrial intimal surface was enlarged,and the mitochondrial ridges were thickened and osmotic swelling,suggesting an appearance of pre-apoptosis of mitochondria.Conclusion: Our findings provide evidence that ?-viniferin has potent effect and activity of anti-oxidative stress and maintains mitochondrial homeostasis by regaining SIRT3 deacetylating FOXO3 in PD model,mainly including regulating mitochondrial biogenesis by promoting the expression of TFAM and PGC-1?;promoting Mfn2,Fis1 and Drp1 to maintain mitochondrial division and fusion balance;up-regulating the expression of Parkin,Bnip3 and Nix to induce tmitochondrial autophagy.Thereby through the above pathway,?-viniferin could maintain mitochondrial homeostasis,suppresses neurotoxicity induced by the rotenone and 6-OHDA,alleviate oxidative stress damage and maintain mitochondrial function.?-Viniferin triggered overexpression of SIRT3 and FOXO3 deacetylation mediated by SIRT3 may represent new therapeutic modalities for PD. |
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