| Objective:Septic acute kidney injury(SAKI)is a serious disease in the intensive care unit,the morbidity and mortality is high,but the pathogenesis has not yet fully understood,The role of apoptosis in acute renal injury in sepsis has been paid more and more attention.A number of clinical and animal experiments show that sepsis occurs,the body is in relative adrenal insufficiency(relative adrenal insufficiency,RAI)state,low-dose glucocorticoid can protect the renal function of SAKI.In this study,we designed SAKI mouse model,using Ac-DEVD-CHO(Caspase-3 specific inhibitor)combined with low dose of methylprednisolone early intervention,The serum levels of TNF-a,acute renal injury markers NGAL(neutrophil gelatin-related apolipoprotein,apolipoprotein-2)were detected by ELISA,and the expression of Bax and Bcl-2 were detected by Western Blot,to investigate the protective effect and mechanism of the two interventions and joint interventions on the SAKI mouse model,for the clinical treatment of SAKI to provide new therapeutic targets and theoretical basis.Methods:72 male C57BL/6 mice were treated with Cecal Ligation and Puncture(CLP)method to construct SAKI model,were randomly divided into 6 groups(n = 12).? Sham group(sham group);? Cecal Ligation and Puncture group(CLP group);? DMSO control group(DMSO group);? Low-dose of methylprednisolone early intervention group(GC group);?Caspase-3 inhibitor early intervention group(CHO group);? Caspase-3 inhibitor combined with low-dose methylprednisolone group(GC+CHO).All mice were given 37 ? normal saline 0.3ml/10g after subcutaneous injection of fluid resuscitation,All mice were sacrificed at 6h,12h and 24h after operation.The levels of TNF-a and NGAL in serum were determined by ELISA,at the same time each group was treated with phosphate buffer perfusion to remove the mouse kidney,the expression of Bax and Bcl-2 in the kidney was measured by Western-Blot.ELISA analysis of the results using the microplate reader at 450nm wavelength measurement of the optical density(OD),Western-Blot stripe image gray value analysis using Gel-pro package analysis.Statistical analysis was performed using SPSS 20.0 software package(SPSS Company,Chicago,Illinois USA).Data were expressed as mean ± standard deviation(x± s).Statistical analysis was performed using the variance analysis of repeated measurements.P ? 0.05 The difference was statistically significant.Results:1.Serum TNF-a and serum NGAL in each group:There was no significant difference in serum TNF-a and serum NGAL between CLP group and DMSO control group at 6h,12h and 24h(P>0.05).The level of TNF-ain 6h CLP group and CHO group were significantly higher than those in Sham group(P<0.01).;Compared with CLP group,GC + CHO group had a lower trend than GC group,which was higher than that of Sham group,but there was no statistical difference(P>0.05).12h,24h Sham group serum TNF-a than 6h group showed an upward trend,but no significant difference(P>0.05),12h CLP group compared with 6h CLP group serum TNF-? was significantly increased,24 h CLP group than 12 h CLP group serum TNF-? was significantly increased(P<0.01).12h GC group was significantly lower than 12hCLP group and 12h GC + CHO group than 12h GC group was significantly reduced(P<0.01);12h GC group and GC + CHO group had an increasing trend compared with the 6h group,but there was no significant difference(P>0.05),24h GC group was significantly lower than 24h CLP group and 24h GC + CHO group was significantly lower than 24h GC group(P<0.01),24h GC group and GC + CHO group than the 12h group showed an upward trend,but no significant difference(P>0.05).The serum NGAL of the 12hCLP operation group was significantly higher than that of the 6hCLP operation group(P<0.01),The serum NGAL of 24hCLP operation group was significantly lower than that of 12hCLP operation group(P<0.01),but there was no significant difference compared with 6hCLP operation group(P>0.05);The CLP group in each time group was significantly higher than that in Sham group(P<0.01),GC group compared with CLP group serum NGAL decreased,but no significant difference(P>0.05),CHO group compared with CLP group serum NGAL significantly reduced(P<0.01),GC + CHO group had lower tendency than CHO group,but there was no significant difference(P>0.05).2.The expression of apoptosis-related proteins Bax and Bcl-2 in renal tissues of mice.6h Bax/actin ratio,CLP group was the highest,GC + CHO group was the lowest,which CLP group and sham group compared with the expression of Bax was statistically significant(P<0.05).Compared with CLP group,the expression of Bax in CHO group and GC + CHO group was significantly different(P<0.05).Compared with DMSO group,the expression of Bax in GC+CHO group was statistically significant(P<0.05).GC + CHO group had lower tendency than CHO group,but the difference was not statistically significant(P>0.05).12h Bax/P-actin ratio,CLP group was the highest,CHO group followed by GC+ CHO group lowest,which CLP group and sham group compared with the expression of Bax was statistically significant(P<0.05).Compared with CLP group,the expression of Bax in GC+CHO group was statistically significant(/P<0.05).Compared with DMSO group,the expression of Bax in GC+CHO group was statistically significant(P<0.05),GC+CHO group had lower tendency than CHO group,but the difference was not statistically significant(P>0.05).24h of the Bax/?-actin comparison,GC + CHO group was the highest,followed by CHO group,CLP group was the lowest,GC+CHO group compared with CLP group was statistically significant(P<0.05),CHO group compared with CLP group decreased trend,the other groups were no significant difference(P>0.05).6h Bcl-2/?-actin ratio,GC+CHO group was the highest,followed by CHO group,DMSO group was the lowest.The expression of Bcl-2 in CHO group and GC+CHO group and DMSO group was statistically significant(P<0.05),GC + CHO group had an increasing trend compared with CHO group,but the difference was not statistically significant(P>0.05).12h Bcl-2/?-actin ratio,GC + CHO group was the highest,CHO group followed,CLP group was the lowest.Compared with CLP group,the expression of Bcl-2 in CHO group and GC+CHO group was statistically significant(P<0.05),GC group and CLP group compared with Bcl-2 expression was statistically significant(P<0.05),Compared with CHO group,the expression of Bcl-2 in GC+CHO group was statistically significant(P<0.05).The ratio of Bcl-2/?-actin in 24h was the highest in GC+CHO group,followed by CHO group and CLP group.There was no significant difference between the groups(P>0.05),GC + CHO group had an increasing trend compared with CHO group,but the difference was not statistically significant(P>0.05).Conclusions:1.The use of low-dose methylprednisolone alone in the early intervention of sepsis-associated kidney injury in mice model can reduce the release of inflammatory cytokines TNF-? and alleviate mouse kidney injury;2.The use of Caspase-3 inhibitor Ac-DEVD-CHO alone in the early seizure of sepsis acute renal injury model can reduce the mouse kidney cell apoptosis,thereby reducing the mouse kidney injury;3.Caspase-3 inhibitor Ac-DEVD-CHO and low-dose methylprednisolone in combination,than the use of one of the drugs alone can more effectively reduce the mouse sepsis acute kidney injury model of renal cell apoptosis,inflammatory factor TNF-A release,reduce serum NGAL,can better reduce the mouse SAKI model of renal injury. |
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