Effect Of H₂S On Expression Of P38MAPK And Caspase3 And Apoptosis Of Renal Cells In Urinary Sepsis

Objective To establish New Zealand rabbit model of urine-derived sepsis intervened with NaHS and PAG.To observe the expression of p38MAPK and Caspase3 in kidney tissues of New Zealand rabbit and their changes of renal cell apoptosis.To explore the effects of H₂S on the expression of p38MAPK and Caspase3 in renal tissues with urine-derived sepsis and the apoptosis of renal cells,thus,the possible mechanism of H₂S antagonism to renal damage and renal cell apoptosis of urinary sepsis was revealed.Methods1、The 40 male New Zealand rabbits were divided into groups A,B,C,D and E with 8 rabbits in each group.2、Group A was a blank control group.Group B was the model control group,and the left ureter was dissociated and treated with abdominal closure.In group C,the distal left ureter was ligated and the proximal end of the ligated ureter was injected with escherichia coli（ATCC25922）to establish a New Zealand rabbit model of urinary sepsis associated with upper urinary tract obstruction and infection.Group D was treated with NaHS solution（NaHS8.4umol/kg）via the ear vein of New Zealand rabbits on the basis of group C.Group E was intraperitoneal injected with dl-propargyl glycine solution（PAG solution37.5mg/kg）on the basis of group C.3、Anal temperature（RT）,heart rate（HR）,respiratory rate（RR）,peripheral blood white blood cell count（WBC）,neutrophil count（N）,creatinine（Cr）,urea nitrogen（BUN）,and c-reactive protein（CRP）were measured at four time points before surgery,24h after surgery,48h after surgery,and 72h after surgery.4、HE staining was performed on the left kidney of New Zealand rabbits 72h after the operation,and the morphological and structural changes of renal tissue cells were observed under an optical microscope.5、Apoptosis of renal cells was detected by Tunel method at 72 h after operation.6、The expression levels of cysteine synthase（CBS）,p38mitogen-activated protein kinase（p38MAPK）and cysteine proteinase 3（Caspase3）in renal tissue were detected by ELISA at 72h after the operation.7、The relative expression of Caspase3 mRNA in renal tissue was detected by real-time fluorescence quantitative PCR（RT-qPCR）at 72h after surgery.Results1、Comparison of vital signs:there was no significant difference in preoperative RT,HR and RR among the five groups of New Zealand rabbits（P>0.05）.RT,HR and RR in group B were higher at 24h after operation than in group A at the same time point（P<0.05）.RT,HR and RR of group B were basically normal at 48h and 72h after operation,and there was no statistical significance in comparison with group A at the same time point（P>0.05）.RT,HR and RR were significantly higher in group C at 24h,48h and 72h than those in group A and B at the same time point（P<0.05）.RT,HR and RR in group D at 24h,48h and 72h after surgery were lower than those in group C at the same time point（P<0.05）,but still higher than those in group A and B at the same time point（P<0.05）.RT,HR and RR in group E were higher at 24h,48h and 72h than those in group C at the same time point（P<0.05）.2、Comparison of WBC,N and CRP values in peripheral blood:there was no significant difference in preoperative WBC,N and CRP values among the five groups of New Zealand rabbits（P>0.05）.WBC,N and CRP values in group B increased at 24h after surgery compared with group A at the same time point（P<0.05）.The values of WBC,N and CRP of group B were normal at 48h and 72h after surgery,and there was no significant difference between group B and group A at the same time point（P>0.05）.WBC,N and CRP values of group C at 24h,48h and 72h after surgery were significantly higher than those of group A and group B at the same time point（P<0.05）.WBC,N and CRP values at the three postoperative time points in group D were higher than those in group A and group B at the same time point（P<0.05）,but lower than those in group C at the same time point（P<0.05）.The values of WBC,N and CRP in group E at three postoperative time points were significantly higher than those in group C at the same time point（P<0.05）.3、Renal function comparison:there was no significant difference in preoperative Cr and BUN between the five groups（P>0.05）.There was no significant difference in Cr and BUN between group B and group A at three postoperative time points（P>0.05）.Cr and BUN in group C were higher at each postoperative time point than those in group A and B at the same time point（P<0.05）.Cr and BUN in group D at each postoperative time point decreased compared with those in group C at the same time point（P<0.05）,but increased compared with those in group A and group B at the same time point（P<0.05）.Cr and BUN in group E at three postoperative time points were higher than those in group C at the same time point（P<0.05）.4、The morphology of renal tissues in each group was compared under the optical microscope 72h after surgery:the tubules and glomerular structures of group A and group B were normal.In group C,the glomeruli were obviously atrophic,the lumen was obviously enlarged,and the renal tubules edema was obvious.Necrotic tissue remained in the renal tubules,and inflammatory cell infiltration was observed in the renal interstitium.Pathological changes in group D were significantly reduced compared with those in group C.The pathological changes in group E were significantly worse than those in group C and D,and the glomerular structure was destroyed,so the renal capsule lumen could not be distinguished.5、Apoptotic cells were scattered in the renal tissue of rabbits in group A and group B,and there was no significant difference in the apoptotic index between the two groups（P>0.05）.Apoptotic cells in the renal tissue of group C were concentrated into clusters,and apoptotic cells covered the whole field of vision.The apoptotic index of the renal tissue of group C was significantly higher than that of group A and group B（P<0.05）.The apoptosis index of rabbit kidney tissue in group D was significantly lower than that in group C（P<0.05）.The apoptosis index of group D was still significantly increased compared with that of group A and group B（P<0.05）.However,there was no apoptotic cell cluster in group D.Apoptotic cells in group E were more concentrated than those in group C,and the apoptotic index in group E was significantly higher than that in group C and group D（P<0.05）.6、Postoperative 72h renal tissue CBS activity detection:there was no significant difference between group A and group B in the activity of CBS（P>0.05）.The activity of CBS in renal tissues of group C,group D and group E was compared in pairs,and the difference was statistically significant（P<0.05）.The activity of CBS in renal tissues of group C and group E was significantly lower than that of group D（P<0.05）.The activity of CBS in renal tissue of group E was significantly lower than that of group C（P<0.05）.The activity of CBS in renal tissues of group A and group B was significantly higher than that of group C,group D and group E（P<0.05）.7、Expression levels of p38MAPK and Caspase3 proteins in renal tissues at 72 hours after surgery:the expression levels of p38MAPK and Caspase3 in group A were not significantly different from those in group B（P>0.05）.The expression levels of p38MAPK and Caspase3 in the renal tissues of group C,group D and group E were significantly higher than those of group A and group B（P<0.05）.P38MAPK and Caspase3expression levels in renal tissues of group C,group D and group E were compared in pairs,and the difference was statistically significant（P<0.05）.The expression levels of p38MAPK and Caspase3 in the renal tissues of group C and group E were significantly higher than those of group D（P<0.05）.The expression levels of p38MAPK and caspase-3 in the renal tissues of group E were significantly higher than those of group C（P<0.05）.8、Relative expression of Caspase3 mRNA in renal tissue at 72h after surgery:there was no significant difference in Caspase3 mRNA expression between group A and group B（P>0.05）.The expression level of Caspase3 mRNA in group C,D and E was significantly higher than that of group A and B（P<0.05）.The expression of Caspase3 mRNA in renal tissues of group C,group D and group E was compared in pairs,and the difference was statistically significant（P<0.05）.The expression level of Caspase3 mRNA in renal tissues of group C and group E was significantly higher than that of group D（P<0.05）.The expression level of Caspase3 mRNA in renal tissue of group E was significantly higher than that of group C（P<0.05）.Conclusions1、The expressions of p38MAPK,Caspase3 and Caspase3 mRNA in renal tissues of urinary sepsis rised and the apoptosis index of renal tissues rised.2、H₂S can lower the expression of p38MAPK,caspase3 and Caspase3 mRNA in renal tissues of urinary sepsis,and can reduce the apoptosis index of renal tissues.3、H₂S can reduce renal damage in urogenic sepsis,the mechanism may be related to the inhibition of the expression of p38MAPK and Caspase3 by H₂S in renal tissues to reduce the apoptosis of renal cells.