The Mechanism Of SIRT1 Attenuates Sepsis-Induced Acute Kidney Injury By Deacetylate HMGB1

Sepsis refers to the body's response to infection and lead to life-threatening organ dysfunction.Although the true incidence of sepsis remains unknown,a conservative estimate suggests that sepsis is a major cause of death and critical illness worldwide.The treatment of sepsis is costly,and data from the United States in 2011 show that more than $ 20 billion(5.2%of US hospital clinical costs).Acute kidney injury(AKI)is one of the most common complications in the development of sepsis.The incidence of AKI increases with the severity of sepsis,and patients with sepsis associated acute kidney injury(SA-AKI)when the mortality rate doubled,significantly higher than other factors led to AKI.SA-AKI is characterized by acute renal failure,characterized by inadequate blood filtration,water,electrolyte regulation,and urine.Because the pathogenesis of SA-AKI has not been fully elucidated,the clinical treatment of SA-AKI patients with symptomatic support for the main,so the pathogenesis of SA-AKI and as a target for drug research and development has important theoretical and clinical significance.HMGB1 is a highly conserved and ubiquitous DNAbinding protein which functions as a structural protein of chromatin.It localizes in the nuclei of almost all cell types and participates in the maintenance of nucleosome structure and DNA replication.Extracellular HMGB1 functions as a damage-associated molecular pattern(DAMP)molecule and activates proimflammatory signaling pathways through TLR2,TLR4,the receptor for advanced glycation end products(RAGE)and chemokine(C-X-C motif)receptor 4(CXCR4).HMGB1 translocates from the nucleus to the cytoplasm upon the activation of monocytes by inflammatory signals through the hyper-acetylation of two major clusters of lysine residues within two nuclear localization sequence(NLS)sites.However,the mechanism of HMGB1 deacetylation is not clear and HMGB1-mediated inflammatory response is involved in the development of acute renal injury in sepsis has not been reported.Renal tubular epithelial cell(RTEC)is the main cell of renal tubules,which is involved in renal tubular reabsorption,secretion and excretion and other functions.Ischemia,infection and toxic effects caused by RTEC damage is a major factor in renal tubular dysfunction,they accelerated the development of AKI.However,the specific mechanism of RTEC in SA-AKI is not clear.Sirtuins are a highly conserved family of proteins.Encoding by silent information regulator(SIR)gene.Sirtuins belong to class III histone deacetylase family of enzymes.SIRT1,the mammalian ortholog of yeast Sir2,is the most studied mammalian sirtuin.SIRT1 plays important roles in embryonic development and skeletal muscle differentiation.It has diverse functions in the cells ranging from chromatin modification and epigenetics to roles in metabolic pathways,inflammation and stress response.It interacts with and deacetylates histones and a number of non-histone substrates.The non-histone protein substrates of SIRT1 include but not limited to tumor suppressor p53,NF-kB,PGC-1?,FOXO,liver X receptor(LXR),PARP,Ku70 and HIF-la.Our recent study found that SIRT1 protein expression and activity decreased in mice kidney tissue and renal tubular epithelial cells after sepsis,while SIRT1 activation attenuated SA-AKI.And recent study have revealed thatHMGB1 is one of the target protein by SIRTI and activation of SIRT1 improves survival in a mouse model of endotoxemia.These results suggest that the mechanism by which SIRT1 relieves SA-AKI may be associated with SIRT1 deacetylation of HMGB1.Through the literature review,we hypothesized that sepsis challenge resulted in decreased expression and activity of SIRT1 protein in renal tubular epithelial cells,increased HMGB1 nuclear translocation,increased release of HMGB1 into cytoplasm,increased systemic inflammatory response after sepsis The Therefore,we propose to construct SA-AKI-related animal and cell models:1,observe the protein expression and nuclear translocation of HMGB1 in each treatment group,the changes of various inflammatory indexes,the renal function and the survival time of sepsis mice;2,to explore the specific mechanism of SIRT1 activation of deacetylated HMGB1.Through the above study,we can provide new ideas for the pathogenesis and treatment of SA-AKI in clinic.Part One The expression and nucleocytoplasmic translocation of HMGB1 in CLP-induced septic mice kidneyMethodMice were underwent cecal ligation and puncture(CLP)to mimic sepsis.All animals were divided into 11 groups according to the time after CLP.Detecting serum HMGB1 by using ELISA,the expression of HMGB1 and SIRT1 in kidney,nucleocytoplasmic translocation and degree of acetylation of HMGB1 8h after CLP by using Western blot.According to our previous study,renal tubular epithelial cells play an important role in the carcinogenesis of SA-AKI,so we constructed the SA-AKI-related cell model by LPS-stimulated human kidney 2 cell(HK-2)The According to the time of LPS stimulation,divided into different observation points,the detection of indicators and methods and animal models the same.Result1.The serum HMGB1 content in CLP-induced sepsis mice was increased and the content of HMGB1 in renal tissue was decreased.2.After CLP,the HMGB1 nucleus and the acetylation level were increased.Part Two The effect of SIRT1 on protein expression,nuclear translocation and acetylation of HMGB1MethodUse CLP-induced sepsis mouse model and LPS-stimulated HK-2 cell model.SIRT1 agonism and inhibition were specific agonist SRT1720 and specific inhibitor Ex527,respectively.Animal experiments were divided into four groups:sham group(sham group),CLP + Vehicle group,CLP + SRT1720 group and CLP + EX527 group.Cell experiments have been confirmed by SIRT1 chemical agonists and inhibitors,as well as SIRTI siRNA.The control group(LPS + Vehicle group,LPS +SRT1720 group and CLP + EX527 group)were divided into control group,chemical control group and control group.The interference conditions for SIRT1 were con-siRNA group,con-siRNA +LPS group,SIRT1siRNA group and SIRT1siRNA+ LPS group respectively.The expression of SIRT1 and HMGB1 was detected by Western Blot.The interaction between SIRT1 and HMGB1 was detected by immunofluorescence assay.Co-Immunoprecipitation(Co-IP)was used to investigate the interaction between SIRT1 and HMGB1,The changes of lysine sites in SIRT1 agonized HMGB1 were determined by Western blotting.Result1.Excitatory SIRT1 reduced CLP-induced sepsis mouse kidney HMGB1 acetylation levels,reduced nuclear translocation.SIRT1 inhibition is the opposite.2.Cell model confirms that agitation and inhibition(down-regulation)SIRT1 results are consistent with animal experiments.3.The interaction between SIRT1 and HMGB1 and the lysine sites of SIRT1 deacetylated HMGB1 were mainly K29,K28 and K30 Part Three To investigate the effect of SIRT1 deacetylation of HMGBl on inflammatory response,renal pathology and overall survival in septic mice.MethodThe CLP-induced sepsis mice model was divided into four groups according to the different treatment conditions:sham group,CLP + Vehicle group,CLP +SRT1720 group and CLP + EX527 group.The expressions of TLR2,TLR4 and RAGE receptors were detected by Western Blot.The levels of serum proinflammatory cytokines TNF-a,IL-6 and IL-1? were detected by ELISA.The levels of serum BUN and Cr were measured by routine method.Pathology and apoptosis of renal tulbular cells.The survival time of mice was observed and the survival rate was calculated.Result1.Excitatory SIRT1 deacetylation of HMGB1 can inhibit the inflammatory response of sepsis mice2.SIRT1 deacetylation of HMGB1 can reduce the apoptosis of renal tubular epithelial cells in septic mice,improve renal function,prolong survival time and increase the survival rate.Conclusions1.HMGB1 acetylation increased in the renal tubular epithelial cells of SA-AKI-related animal and cell models,HMGBl nuclear translocation and extracellular secretion increased.2.In SA-AKI-related animal and cellular models of renal tubular epithelial cells,SIRT1 agonization can down-regulate HMGBl acetylation levels,nuclear translocation.3.The main lysine sites of SACT1 deacetylated HMGB1 were K29,K28 and K30,respectively.4.Excitatory SIRT1 can reduce the inflammatory response of CLP-induced sepsis mice,inhibit apoptosis,improve renal function,increase the overall survival time of mice.