| BackgroundParkinson’s disease(PD)is the second most common neurodegenerative disease in the world.Its clinical manifestations include motor symptoms such as motor retardation,postural and gait disorders,and a series of non-motor symptoms,including constipation.The main pathological change of PD is the progressive loss of dopaminergic neurons in the pars compacta of substantia nigra accompanied by a decrease in the level of dopamine in the striatum.Previous studies have shown that the significant decrease in dopamine content in the striatum is closely related to the occurrence of motor symptoms of PD.On the other hand,the exact pathogenesis of PD is not clear,which may involve mitochondrial dysfunction,aging,and the interaction between genetic and environmental factors.Mitochondrial biogenesis is a mechanism to maintain mitochondrial homeostasis under pathophysiological conditions.This process maintains normal mitochondrial function by increasing the copy number of mitochondrial DNA(mtDNA)and increasing the production of ATP.A recent study showed that in Parkin-deficient human dopaminergic neurons,mitochondrial dysfunction was mainly the result of mitochondrial biogenic defects,although the neurons have obvious mitophagy defects.Therefore,targeting mitochondrial biogenesis may become a new strategy for the prevention and treatment of PD.SIRT1/PGC-1α/TFAM axis plays an important role in mitochondrial biogenesis.The silent information regulator 1(SIRT1)is a histone/non-histone deacetylase dependent on nicotinamide adenine dinucleotide(NAD+).With the increase of NAD+/NADH ratio,SIRT1 deacetylation activates downstream peroxisome proliferator y coactivator-1α(PGC-1α),resulting in activation of mitochondrial transcription factor A(TFAM)precursor protein synthesis.After a series of transport and processing,the precursor protein is combined with mtDNA to regulate its replication and transcription and promote mitochondrial biogenesis.Therefore,SIRT1/PGC-1α/TFAM pathway plays a central role in mitochondrial biogenesis.Urolithin A(UA)is the main intestinal metabolite of foods that are rich in ellagic tannins and ellagic acids,such as pomegranate,berries,and nuts.It has a variety of pharmacological effects,such as anti-inflammation,anti-oxidation,anti-aging,maintaining,and improving mitochondrial function.It has been shown that UA can play a neuroprotective effect on Alzheimer’s disease(AD)by activating mitophagy.Another study also revealed that UA can up-regulate the expression of SIRT1 in BV2 microglia.However,whether UA plays a protective role in PD is unknown.Therefore,in the present study,we investigated whether UA is protective in the experimental model of PD by promoting mitochondrial biogenesis.The results of this study will be helpful for finding potential therapeutic drugs and drug targets for PDObjectives1.To determine the neuroprotective effect of UA on PD.2.To elucidate the molecular mechanism that UA promotes mitochondrial biogenesis and plays a protective role of PD.Methods1.In vivo experiments1.1 To determine the neuroprotective effect of UA on 6-OHDA-induced PD mice model:After intraperitoneal injection of UA 10 mg/kg for 7 days in C57BL/6 mice,the PD mouse model was established by stereotactic injection of 6-OHDA.Weight measurement,fecal weight measurement,and behavioral experiments were performed at 1 week,2 weeks,and 3 weeks after 6-OHDA injection,and the mice brain tissues were taken for frozen section 3 weeks after 6-OHDA injection.Nissl staining and TH immunohistochemical experiments were performed to detect the protective effect of UA on dopaminergic neurons.GFAP immunofluorescence was carried out to detect the effect of UA on the activation of astrocytes.Besides,the substantial nigra and striatum were taken at the same time,and the effect of UA on the expression of TH protein was detected by western blot assay.1.2 To determine the effect of UA on the level of ROS and the morphology of mitochondria in the brain of PD mice induced by 6-OHDA:DCFH-DA test was used to measure the production of ROS under pathological conditions;western blot analysis was performed to detect the expression of 4-HNE;transmission electron microscope experiment was carried out to detect the effect of UA on dopaminergic neurons mitochondrial morphology in the substantia nigra.1.3 To explore the effect of UA on the SIRT1-mediated PGC-1α/TFAM signaling pathway in vivo:western blot assay was used to detect the effect of UA on SIRT1,PGC-1α,and TFAM protein expression in the substantia nigra of PD mice.2.In vitro experiments2.1 To determine the protective effect of UA on PC-12 cells:CCK-8 test was used to detect the effect of different concentrations of UA on cell viability after exposure to 6-OHDA;TUNEL staining and FITC Annexin V/PI flow double staining experiment were used to test the effect of different concentrations of UA on cell apoptosis after exposure to 6-OHDA.2.2 To determine the effect of UA on ROS level and mitochondrial activity and morphology of PC-12 cells:Cellular ROS level was assessed using DCFH-DA;mitochondrial activity staining test was used to detect the effect of different concentrations of UA on mitochondrial activity;transmission electron microscope was used to observe the effect of different concentrations of UA on mitochondrial morphology.2.3 To detect the protective effect of UA on 6-OHDA-induced mitochondrial dysfunction in PC-12 cells:the effects of different concentrations of UA on mitochondrial membrane proteins were detected by Western blot analysis;the effects of different concentrations of UA on mitochondrial membrane potential were detected by JC-1 flow cytometry analysis and fluorescence staining,and the effects of different concentrations of UA on cellular ATP level were detected.qPCR was used to detect the effect of different concentrations of UA on mitochondrial DNA content.2.4 To determine whether UA promotes mitochondrial biogenesis and alleviates 6-OHDA-induced mitochondrial dysfunction in PC-12 cells by activating the SIRT1-mediated PGC-1α/TFAM signaling pathway:western blot assay was used to detect the effects of different concentrations of UA on the expression of SIRT1,PGC-1α,and TEAM protein levels.EX527 was used to inhibit the activity of SIRT1,and the effects of UA on cell survival rate,apoptosis rate,mitochondrial membrane potential,and mitochondrial DNA were detected as previously described,to determine whether UA protects PC-12 cells by promoting mitochondrial biogenesis through SIRT1/PGC-1α/TFAM signal pathway.3.BioinformaticsBioinformatics was used to analyze the public database to clarify the correlation between the pathogenesis of PD and mitochondrial dysfunction and to provide a theoretical basis for SIRT1 as a drug target for UA.Results1.UA could alleviate the weight loss and the constipation of PD mice caused by 6-OHDA;UA could alleviate 6-OHDA-induced behavioral disorder and dopaminergic neuronal loss in PD mice;UA could alleviate the neuronal inflammation in the brain caused by 6-OHDA.2.UA could reduce the 6-OHDA-induced increase of ROS level in the brain of PD mice,and maintain the normal morphology of mitochondria under the pathological condition of 6-OHDA.3.UA could activate the SIRT1/PGC-1α/TFAM signal pathway in vivo.4.UA could protect PC-12 cells against 6-OHDA-induced cell death.5.UA could reduce the increase of total ROS level in PC-12 cells induced by 6-OHDA,and ameliorate the morphological abnormality induced by 6-OHDA in PC-12 cells.6.UA could alleviate 6-OHDA-induced mitochondrial dysfunction in PC-12 cells:UA could increase the levels of the mitochondrial outer membrane and intimal marker protein in 6-OHDA-treated PC-12 cells,decrease 6-OHDA-induced mitochondrial membrane potential depolarization,increase ATP production and mtDNA copy number in PC-12 cells.7.UA could increase the protein expression of SIRT1,PGC-1α,and TFAM and promote mitochondrial biogenesis in PC-12 cells exposed to 6-OHDA.The protective effect of UA on PC-12 cells was partly abolished in the presence of EX527,a specific inhibitor of SIRT1,and the protective effect of UA on mitochondrial membrane potential and the increase in the copy number of mtDNA caused by UA in PC-12 cells were also attenuated by EX527.EX527 also inhibited the activation of SIRT1/PGC-1 a/TFAM signal pathway induced by UA.8.The results of bioinformatics analysis further support that SIRT1 may be the target of UA to exhibit its neuroprotective effects in PDConclusions1.UA protects PC-12 cells against 6-OHDA-induced injury and attenuates dopaminergic neurotoxicity in the 6-OHDA-induced PD mice model.2.UA exhibits its neuroprotective effects in PD by promoting mitochondrial biogenesis through activating the SIRT1-mediated PGC-1α/TFAM signal pathway.Innovations and limitations1.The neuroprotective effect of UA on PD is clarified for the first time in our study.The results of our study provide a new strategy for finding potential therapeutic drugs for PD.2.Our study revealed for the first time that UA mediates the protective effect of mitochondrial biogenesis on PD by activating the SIRT1/PGC-1α/TFAM signal pathway,which provides a theoretical basis for finding potential drug targets of PD.3.The active site of UA binding to SIRT1 was not found.4.Aging is currently one of the hottest fields in medical sciences and SIRT1 has been widely reported as a "senescence gene" while PD is also an aging neurodegenerative disease.This study connects SIRT1 and PD through UA,but does not fully clarify the role of UA and SIRT1 in the process of "aging". |
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