The Effect Of Endogenous H₂S On Renal Cell Apoptosis During Urine Derived Sepsis

Purpose By copying the rabbit model of sepsis and generating inhibitors(PAG) with the application of endogenous hydrogen sulfide, the formation of hydrogen sulfide and reduction of the effect onsepsis treatment are clarified, the mechanism of hydrogen sulfide in the urine of sepsis and its effect on cell apoptosis in renal injury and the NF-κB will be evaluated.Method:1. Ligating one side of the distal ureteral of the New Zealand rabbit and injecting Escherichia coli(ATCC 25922) on the upper side, which produces sepsis acute rabbit model caused by acute urinary tract obstruction.2. 32 healthy rabbits(1.8-2.2 Kg/only) were randomly divided into four groups: the control group(group A); the sham group(group B); the sepsis group(group C); the PAG group(group D), each group of eight.3.Each group was divided into 5 points in time: preoperative and postoperative 12 h, 24 h,36h, 48 h. Anal temperature(RT), respiratory(RR) and heart rate(HR) and others were observed at different time points.4.The blood was collected, peripheral blood leukocyte counted and analyzed, and renal function(Cr, BUN) detected.5.Measuring h2 s concentration after the operation in 48 hours.6.The kidney tissues HE staining were ticked out after the execution of each rabbit, and then the changes of the structure form were observed. The PCR method was to detect the Bcl- 2, Bax and CSE protein of each rabbit; tunel method to detect the renal tissue cell apoptosis; immunohistochemical method to detect the expression of NF- B Κ in each rabbit organization.Result:1.Comparing each rabbit’s preoperative and postoperative body temperature, breathing and heart rate. There was no obvious difference of group A and group B(P>0.05). Group C was higher than that of group A and B(P<0.05). Group D was higher than that of group C(P<0.05).2.The blood results: there was no obvious difference in white blood cells, neutrophils, preoperative serum creatinine(CR), blood urea nitrogen(BUN) of each group, no statistical significance. The CR, BUN results of the postoperative blood leukocytes and neutrophils: Group A compared with group B, no significant difference(P > 0.05); Group C higher than that of A, B two groups(P < 0.05); Group D, compared with group C, all the indicators were on the rise, with statistical significance(P < 0.05).3.The detection of plasma H2 S concentration in 48h: no obvious difference was found between group A and group B, no statistical significance(P > 0.05). Compared with A, B, the plasma H2 S concentration of group C, D in the two groups was significantly decreased. The difference was statistically significant(P < 0.05). The plasma H2 S content in group D was significantly lower in group C,(P < 0.05).4. The pathological morphology:The pathological morphological was changed and HE dyed. The structural form of kidney morphology in A, B two groups of rabbit was roughly same and in normal form. The abscess was visible in kidney tissues in Group C, with a large number of inflammatory cell infiltration, renal pelvis and calyces mucosa falling off. Glomerular and renal capsule come to change and renal tubular was altered.Group D was more serious than that in group C, with a nuclear fragmentation dissolved.5. RT-PCR method: the expression of Bax, Bcl- 2 and CSE protein in kidney tissue: the difference of group A and group B were small; The Bax protein expression was strongest in group D. On the contrary, the poorest in the Bcl- 2 and CSE protein expression. Compared with group C, the Bax protein expression was significantly enhanced in group D, while the Bcl- 2 and CSE protein expression decreased significantly.6. TUNEL method: kidney cell apoptosis: a small renal tubular epithelial cell apoptosis could be seen in A, B two groups. In group C, ribbon small cell apoptosis and apoptosis body was forming. Compared with A, B two groups, P < 0.05. Group D was more serious in cell apoptosis showing broad shape(P < 0.05).7. Immunohistochemical method: there was no obvious difference in NF-κB between group A and group B(P > 0.05); Compared with A, B group, clear result was found in NF- κB proteinexpression of group C after 48 h(P < 0.05) with statistical significance; In group D, compared with group C, after 48 h, NF- κB protein detection had also enhanced(P < 0.05). It was statistically significant.Conclusion: 1. A urine-derived sepsis rabbits model was successfully established in this study.2. The application of PAG can inhibit the formation of rabbit of urosepsis endogenous H2 S, aggravate of urosepsis renal injury;Endogenous H2 S is able to decrease the expression of NF-κB and Bax as well as enhance Bcl-2 expression.