The Effect Of Extracellular Histones On Apoptosis Of Renal Tubular Epithelial Cells In Sepsis

BackgroundAcute renal injury(AKI)is one of the most common and serious complications in the development of sepsis.Different from non-sepsis AKI,apoptosis of renal tubular epithelial cells is the main pathogenic factor of acute renal injury caused by sepsis.Studies have shown that extracellular histones involved in the pathogenesis of AKI,however,studies on sepsis indicate that extracellular histones were a major factor leading to death in patients with sepsis.Therefore,this study will explore the mechanism of AKI caused by extracellular histones in sepsis on the basis of previous experiments and literatures.Objective1.To explore whether extracellular histones can induce tubular epithelial cell apoptosis;2.To explore whether extracellular histones can induce apoptosis of renal tubular epithelial cells through mitochondrial pathway.MethodsIn vitro experiments:1.The induction of extracellular histones on apoptosis of HK-2 cells50μg/ml histones(equal volume culture medium as a control,the same)were co-cultured with human kidney-2 cell lines(HK-2).The apoptotic rate of HK-2 cells was measured by flow cytometry at 2h,4h and 6h,respectively,to determine the optimal culture time of extracellular histones.Then,different concentrations(25μg/ml,50μg/ml,100μg/ml)of extracellular histones were co-cultured with HK-2 cells with the optimal culture time.Apoptosis was detected by flow cytometry,clear whether the effect of extracellular histones were concentration dependent.2.Extracellular histones induced apoptosis of HK-2 cells through mitochondrial pathwayDifferent concentrations(25μg/ml,50μg/ml,100μg/ml)of extracellular histones were co-cultured with HK-2 cells with the optimal culture time,then stained with Rhodamin123 for flow cytometry to detect changes of mitochondrial membrane potential;Different concentrations(25μg/ml,50μg/ml,100p,g/ml)of extracellular histones were co-cultured with HK-2 cells with the optimal culture time,then extracted cell protein.The expression of mitochondrial membrane protein Bcl-2,MAPK p38 phosphorylated protein(p-p38),protein Caspase-9 and Caspase-3 was detected by western blot.In vivo experiments:Different concentrations(50mg/kg,100mg/kg)of extracellular histones were injected via tail vein into BALB/c mice(equal volume saline as a control,the same).Survival rate was observed within 6 hours.After 6 hours,collected blood with the method of removing the eyeball,then killed the mice.Removed the two kidneys from the mice and removed the kidney pedicle as well as kidney capsule at the same time.Part of renal tissue was grinded to make cell suspension,apoptosis was detected by flow cytometry;part of renal tissue was made into paraffin sections and the apoptosis was detected by TUNEL staining.Took some of the renal tissue to extract the protein and the expression of mitochondrial membrane protein Bcl-2 and MAPK p38 phosphorylated protein was detected by western blot.Isolated serum from the blood of mice for the detection of creatinine,urea nitrogen and cystatin.Statistical AnalysisSPSS 20.0 software was used for statistical analysis,the analysis results were expressed as mean ± standard deviation(X±S).The one-way ANOVA was used to compare the significance of the mean differences among the groups;comparisons between the two groups were expressed by Student’s t test;survival analysis was performed by Kaplan-Meier method,P value of less than 0.05 was considered statistically significant.Results1.In vitro resultsThe apoptotic rate of HK-2 cells in different histone concentrations is significantly higher than that of the control group(P<0.01).Compared with the control group,the expression of MAPK p38 phosphorylated protein,protein Caspase-9 and Caspase-3 is increased and the expression of Bcl-2 protein is decreased,the difference is statistically significant(P<0.05).The mitochondrial membrane potential of different histone concentrations is significantly lower than that of the control group(P<0.01).2.In vivo resultsCompared with the control group,the cumulative survival rate of the histone 100mg/kg group is significantly lower(P = 0.001),but the cumulative survival rate of the 50mg/kg group has no significant difference(P = 0.055).The apoptotic rate of mouse kidney cells and the expression of MAPK p38 phosphorylated protein in different histone concentrations are higher than that of the control group,however,the expression of Bcl-2 protein is decreased(P<0.05).Compared with the control group,the serum creatinine value of histone 100mg/kg group is increased,the difference is statistically significant(P<0.01).In addition,the number of apoptotic kidney cells detected by TUNEL staining are increasedConclusionThe results of in vivo and in vitro experiments show that the extracellular histones can activate MAPK p38 pathway and act on mitochondrial pathway to induce renal tubular epithelial cells apoptosis during sepsis,and then lead to acute renal injury.