Activation SIRT1 Attenuates LPS-induced Acute Kidney Injury By Regulation Of Mitochondrial Injury And PGC-1a/TFAM Signaling

Despite the rapid development of medical level and various advanced life support technologies,there is still a lack of effective treatment drugs for sepsis.Up to now,the therapeutic effect of sepsis is not ideal and its complex pathogenesis has not been clarified.It has become an important public health problem because of its high morbidity,high mortality and high treatment cost.Sepsis is the most common cause of severe acute kidney injury(AKI)in critically ill patients.Moreover,sepsis-related AKI is an independent risk factor for death.Although there are various therapeutic schedules for sepsis-related AK1,the patient’s mortality still remains high.However,little is known about the pathophysiological theory of the development of AKI in sepsis.Therefore,it is very important to elucidate the pathogenesis of AKI in sepsis and look for potential therapeutic drugs to reduce the mortality of sepsis patients.Studies has confirmed that the protection of renal function can reduce the mortality in different sepsis model mice,suggesting that the intervention of renal function protection may bring new ideas for sepsis treatment.Kidney is a highly metabolized and mitochondria rich organ,so its function depends on the normal structure and stable function of mitochondria.When the cell oxidative stress is unbalanced,the mitochondrial permeability transition pores(mPTP)opens abnormally.Then a large amount of water and ions enter into the mitochondrial matrix,causing mitochondrial swelling,mitochondrial outer membrane rupture,decreased membrane potential,and increased production of oxygen-free radicals.A large number of reactive oxygen(ROS)damages mitochondrial protein,membrane integrity and DNA,increases membrane permeability,decreases membrane potential,and decreases mitochondrial DNA(mtDNA)copy number.Then ATP synthesis is reduced,mitochondrial biogenesis is reduced,and energy consumption is exhausted.All of these eventually lead to mitochondrial dysfunction.When oxygen-free radicals and ROS hard to be eliminated,this process may lead to vicious cycle,turther lead to cell dysfunction,and eventually lead to cell death.In recent years,many clinical studies have shown that mitochondrial dysfunction is significantly related to the severity and prognosis of sepsis,while improving mitochondrial biogenesis may increase the survival rate of patients.Even researches on mitochondrial dysfunction in sepsis shows a good application prospect.Sirtuin 1(SIRT1)is a homologous family protein of yeast chromatin silencing information regulator 2(Sir2),which is widely expressed in almost all mammalian organs.Sirtuin 1 is an important regulatory protein in aging,inflammatory response,oxidative stress,mitochondrial function protection,metabolic regulation and tumor formation.SIRT1 is mainly located in the nucleus and participates in the regulation of cell metabolism,stress response,aging and apoptosis through deacetylation.Some studies have shown that activation of SIRT1 can improve the survival ability of cells against oxidative stress,and play a role in organ function protection in neurodegenerative diseases,diabetes,myocardial damage,renal ischemia-reperfusion,and so on.The previous study of our research group showed that SIRT1 played a protective role of kidney in sepsis rats model.Therefore,we used human renal tubular epithelial cells(HK-2 cells)to build an in vitro sepsis kidney injury model to further study the protective role of SIRT1 in sepsis-related AKI.Peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α)is encoded by nuclear genes and is the main regulator of oxidative metabolism and mitochondrial biogenesis.Studies show that SIRT1 activation can improve transcription activity,reduce ROS production and promote the synthesis of ATP,to play a protective role of cells by deacetylating PGC-1α.Therefore,SIRT1 may play a protective role in AKI by regulating PGC-1α.Mitochondrial transcription factor A(TFAM)is a nuclear encoded protein,synthesized in the cytoplasm and introduced into mitochondria,which plays a protective role in mitochondria.TFAM promotes the replication and transcription of mtDNA and regulates the copy number of mtDNA.TFAM can also bind to mitochondrial DNA nonspecifically,keep its structure stable by forming nuclear-like structure.PGC-1α can promote the expression of TFAM by regulating the activity of nuclear respiratory factor(NRF).Therefore,researches above show that PGC-1α may play a protective role in sepsis through TFAM.Based on the above literature review and previous studies,we can propose the following hypothesis:SIRT1 has a protective effect on LPS-induced renal tubular epithelial cell injury in sepsis,and this protective effect may depend on the activation of SIRT1.Activation of SIRT1 enhances the expression of PGC-1α and TFAM,and thus plays a protective role in cell function.SIRT1/PGC-1α/TFAM can protect cell function by protecting mitochondrial function.Objective:It has been confirmed that SIRT1 can protect renal function in sepsis acute kidney injury by early animal experiments of the research group.In this study,we intend to conduct molecular mechanism discussion on the basis of early research.Therefore,we intend to build a cell model of sepsis acute kidney injury,and comprehensively use various molecular biological techniques to explore whether activation of SIRT1 attenuates LPS-induced acute kidney injury by regulation of mitochondrial injury and PGC-1α/TEAM signaling.The study findings will provide new ideas for the pathogenesis of sepsis and new strategies for the treatment of sepsis.Methods:HK-2 cells were used to establish LPS-induced sepsis cell model.Using SIRT1 specific agonist(SRT1720)and inhibitor(EX527)to investigate SIRT1 activation can play a protective role in renal tubular epithelial cells and mitochondria function.Furthermore,the expression of PGC-1α and TFAM was detected to verify the protective effect of SIRT1-PGC-1α-TFAM pathway on renal tubular epithelial cells in sepsis.Mitochondrial function detection:JC-1 was used to detect the mitochondrial membrane potential.TMRM was used to detect the opening of mitochondrial permeability transition pore.CellTiter-Glo(?)Luminescent Cell Viability Assay was used to detect the level of ATP in cells.Real-time fluorescent quantitative PCR was used to detect the copy number of mtDNA.ROS level detection:Intracellular ROS level was detected by DCFH-DA probe.MitoSOXTM Red probe was used to detect the level of superoxide in mitochondria.Results:1.SRT1720 alleviates LPS-induced decrease of SIRT1 activityLPS stimulation reduced SIRT1 activity,SRT1720 could alleviate the inhibition of LPS(P<0.05)and restore SIRT1 activity.However,EX527 increased the inhibition of LPS and inhibited SIRT1 activity(P<0.05).2.SRT1720 alleviates LPS-induced decrease in SIRT1 protein expression in HK-2 cellsLPS reduced the expression of SIRT1,SRT1720 could alleviate the decrease of SIRT1 expression in HK-2 cells caused by LPS and effectively inhibit the effect of LPS(P<0.05),while EX527 increased the effect of LPS inhibition(P<0.05).3.SRT1720 affectes the expression of PGC-1α via SIRT1 activationSRT1720 promoted the expression of PGC-1a(P<0.05),while EX527 and EX527+LPS inhibited the expression of PGC-1α(P<0.05).6.SRT1720 affectes the expression of TFAM via SIRT1 activationSRT1720 promoted the expression of TFAM(P<0.05),while EX527 and EX527+LPS inhibited the expression of TFAM(P<0.05).5.SIRT1 activation inhibits LPS-induced decrease in mitochondrial membrane potentialCompared with the LPS group,the number of monomer forms of JC-1 decreased,and the poly/monomer ratio of JC-1 increased after SRT1720 treatment(P<0.05).In contrast,EX527 caused the number of monomer forms of JC-1 increased,and the poly/monomer ratio of JC-1 decreased(P<0.05).6.SIRT1 activation inhibits LPS-induced mPTP openingThe results showed that in the LPS-treated group,the red fluorescence was weakened.SRT1720 preconditioning suppressed the effect of LPS(P<0.05),while the red fluorescence was reduced by EX527(P<0.05).7.SIRT1 activation attenuates LPS-induced decrease in mitochondrial ATP levelCompared with the Control group,intracellular ATP level was lower than that in the LPS group.In SRT1720 pretreatment group,the ATP level was higher than that in the LPS group(P<0.05).In contrast,after pretreatment with EX527,ATP level decreased compared with the LPS group(P<0.05).8.SIRT1 activation attenuates LPS-induced decrease in mtDNA copy numberCompared with Control group,the relative expression level of ND1 decreased significantly in LPS-treated group(P<0.05)SRT1720 preconditioning by activating SIRT1 increased the expression of ND1 compared to LPS-treated group(P<0.05).In contrast,the expression of ND1 was reduced after pretreatment with EX527 by inhibition of SIRT(P<0.05).9.SIRT1 activation inhibits LPS-induced intracellular ROS increaseIn the intracellular ROS level test,the results showed that ROS production was increased in the LPS group compared with the Control group(P<0.05).Compared with the LPS group,SRT1720 reduced ROS production(P<0.05),while EX527 promoted ROS increase(P<0.05).In the detection of superoxide in mitochondria,the results showed that compared with the Control group,the production of superoxide was increased in the LPS group(P<0.05).Compared with the LPS group,SRT1720 reduced the production of superoxide(P<0.05),while EX527 increased the production of superoxide(P<0.05).Conclusions:1.LPS induces a decrease in SIRT1 activity and protein expression in HK-2 cells.2.SIRT1 activation can promote the expression of PGC-1α and TFAM proteins.3.SIRT1 activation can inhibit LPS-induced mitochondrial dysfunction.4.SIRT1 activation can inhibit LPS-induced ROS increase in HK-2 cells.5.SIRT 1 plays a protective role on mitochondrial and cellular functions by promoting the expression of PGC-1α and TFAM protein and reducing oxidative stress,so as to play a protective role in sepsis related AKI.