| Objective: Acute kidney injury (AKI) is a common complication of critical illness. The development of AKI increases patient morbidity, predicts higher mortality, and consumes considerable health resources. The cause of AKI in critically ill patients is often multifactorial. Sepsis, a highly prevalent syndrome that prompts admission to intensive care, is a leading precipitant of AKI. Between 45 and 70% of all AKI is considered associated with sepsis, even up to 70.2%. In the past 20 years, despite many advances in therapeutic and research techniques (including the introduction of genomics and proteo- mics), fundamental changes in the outcome of patients with AKI have not occurred, and the mortality of sepsis is increasing. This is partly the result of our poor understanding of the pathogenesis of sepsis-induced renal dysfunc- tion, the lack of early biomarker of it and the absence of effective therapies.Recent research highlights a new possible and emerging concept for the pathogenesis of AKI: acute apoptosis. Several studies have demonstrated that broadspectrum/specific caspase inhibitor can decrease renal dysfunction in Ischemia/Reperfusion and intoxication AKI. In the study of sepsis-induced AKI found the main change was apoptosis, but many people questioned this viewpoint. Therefore, the role of apoptosis in sepsis-induced AKI is still controversial.Zymogens apoptosis can be triggered by a variety of stressors such as inflammatory cytokine, ischemia, oxidative stress, ultraviolet. Among the mul- tiple mediators of the complex process of apoptosis, the caspase enzyme cascade plays a central role.And caspase-3 is one of key enzymes in the cysteine proteases which exist as inactive zymogens. Activated caspase-3 res- ulting in degradation and deactivation of important protein of cytoplasmic nu- cleic and cellular skeleton. Ac-DEVD-CHO is an non-biological inhibitor by synthetic which can competitive inhibitor degradation of protein by combinati- on with caspase-3, and thereby inhibit caspase-3 mediated cell apoptosis.The aim of our research is to use cecal ligation and puncture mice model mimic sepsis-induced AKI. Detect the expression of apoptosis-related genes such as caspase-3, Bcl-2 and Bax with the immunohistochemisty staining and flow cytometry. To explore the protective effect of intervention caspase-3 inhibitor early in septic AKI mice and its possible mechanism.Methods: Seventy two healthy clean level of male C57BL/6 mice were subjected to cecal ligation and puncture (CLP, a model of polymicrobial sepsis) or sham operation. The animals were assigned into three equal groups (n=24) according to random number table. Sham group: Only laparotomy, flipping dilated bowel and pull the cecum then closed abdominal, not shut ligation and perforation cecum. Cecal Ligation and Puncture gourp(CLP group): subjected to cecal ligation and puncture. Caspase-3 inhibitor group(CI group): thirty minutes after CLP, Ac-DEVD-CHO (4μg/g) mixed with 1% DMSO was injected subcutaneously. Each postoperative give 37℃0.9%NaCl subcutane- ous injection, and give"Imipenem and Cilastatin Sodium"25mg/kg, abdomen hype 3 hours after operation, then 12 hours once lasts 2 days In the same doses. Each group collected blood in the 6th, 12th and 24th hour after operation by picking the eyeball. Take serum using centrifugation techniques store in -80℃, Used for testing the blood serum creatinine and urea nitrogen. Take the kidney is divided into three parts: the first part was fixed in 10% neutral formalin for 24 to 48 hours then embedded in paraffin, used for optical detection and pathological damage score; the second part embedded in paraffin after fixed in 4% paraformaldehyde for 24 to 48 hours, used to detect the expression of apoptosis related genes such as caspase-3, Bcl-2 and Bax; the last part embedded in paraffin after fixed in 70% alcohol for 24 to 48 hours, Used for check the renal cell apoptosis rate and the expressing of Bcl-2/Bax by flow cytometric.SPSS13.0 edition software package were used for statistic analysis, data were shown as mean±SD. Using Kruskal-Wallis H test for nonparameter test; and using completely random designed one-way ANOVA for compare differ- ences between multiple sample mean; adopts LSD-t-test or S-N-K inspection to analyze multiple mean differences between binary comparison, Heterogen- eity of variance using logarithmic transformation and Kruskal-Wallis H test, A Statistical significance was assumed at a P-value<0.05.Results:1 The changes of general situation and kidney general morphology in 3 groupsThe mice in sham group immediately waked up and activities were flexible after operation. Their fur were smooth and canthus secretions were less with normal eating and drinking. Celiac structure was clear, no abnormal ascites and odorous flavour, the surface of dilated bowel were smooth and moist. Compared with sham group, the changes of general situation were almost opposite in CLP group. Large light hemorrhagic ascites with stench, Celiac structure was not clear. The end of cecum were black. CI group mice were in between.Compare with sham group and CI group, the kidney of CLP group was hyperemia and edema obviously.2 The changes of kidney histopathology and tissue damage pathological score in 3 groupsHistopathological changes: Sham group: The glomeruli and renal tubules were nearly normal; CLP group: Glomerular and renal tubular were marked hyperemia, renal tubular epithelial cells volume increases, the cytoplasm have be obviously vacuoles degeneration and glass sample degeneration, and accompanied by inflammatory cell infiltration; CI group: Compare with CLP group the damage to renal tubular epithelial was obviously ligtening, a few cells mild swelling, fall off and empty bubble degeneration, and the renal tubular shape remained relatively good.Renal tubular damage pathological score: The pathologyical score of pos- toperative 6h CLP group were significant increased than sham group and CI group(P<0.01), the latter two groups had no statistical differences(P>0.05). Postoperative 12h and 24h had the similar results. Sham group,CLP group and CI group mice all declined with time with no statistical differences(P>0.05).3 The changes of serum creatinine and urea nitrogen (BUN) levels in 3 groupsThe levels of postoperative 6h creatinine in CLP group were significantly higher than the sham group(P<0.01), CI group obviously decreased the serum creatinine which CLP group increased(P<0.01), but compare to sham group there were no statistical differences(P>0.05). The creatinine of postoperative 12h and 24h in three groups had no statistical differences(P>0.05).The levels of creatinine in sham group and CLP group both declined with time at 6h, 12h, 24h postoperation, but had no statistical differences(P>0.05). And the levels of creatinine in CI group also declined with time, and decreased significantly at 24h compare to 6h point (P<0.05).The levels of BUN in CLP group have the same trend with creatinine results. The levels of BUN in CLP group at 12h of CLP group mice were higher than the sham group(P<0.05), the remaining two group without statistical differences(P>0.05). The levels of BUN at 24h points had no statistical differences between the arbitrary two groups(P>0.05).The levels of BUN in sham group declined with time at 6h, 12h, 24h postoperation, but were no statistical differences(P>0.05), and that in CLP group at 12h were higher than 6h and 24h , but have no statistical differenc- es(P>0.05). CI group and CLP group mice had the similar results.4 The changes of renal apoptosis rates in 3 groupsThe levels of postoperative 6h apoptosis rate of renal tubular epithelial cell in CLP group were significantly increased than the sham group(P<0.01), CI group obviously decreased the apoptosis rate which CLP group increased(P<0.01), and compare to sham group there were also had obviously statistical differences(P<0.01). The levels of postoperative 12h and 24h had the similar results.The levels of apoptosis rate in sham group declined with time at 6h, 12h, 24h postoperation, but had no statistical differences(P>0.05). And the levels of apoptosis rate in CLP group also declined with time, and decreased signific- antly at 24h compare to 6h point (P<0.01). The levels of postoperative in CI group had the similar results, and had significantly statistical differentces bet- ween the arbitrary two groups(P<0.01).5 The changes of renal apoptosis-related Bcl-2 and Bax in 3 groupsThe levels of postoperative 6h Bcl-2 CLP group and CI group were higher than sham group, but had no statistical differences(P>0.05). The levels of postoperative 12h and 24h CLP group was obviously higher than sham group (P<0.01), and CI group was the highest one(P<0.01).The levels of Bcl-2 in sham group postoperative 6h, 12h, 24h had no stat- istical difference(P>0.05). CLP intraclass the levels of Bcl-2 in postoperative 12h, 24h obviously higher than 6h and had statistical significance(P<0.01), 24h relatively 12h had no statistical difference(P>0.05). CI group had the si- milar results.Compare to sham group the levels of Bax in CLP group were increased, but have no statistical differences (P>0.05), CI group exactly decreased the levels of Bax in CLP group(P<0.01); postoperative 12h and 24h the levels of Bax in CLP group was obviously higher than sham group(P<0.01), other results were as well as in 6h group.The levels of Bax in sham group had no statistical difference with time at postoperative 6h, 12h 24h (P>0.05). CLP intraclass the levels of Bax in postoperative12h, 24h obviously higher than 6h, and had statistical significan- ce(P<0.01), compare to 12h, the levels of Bax in 24h were obviously increas- ed(P<0.01). CI group had the similar results. 6 The changes of renal caspase-3, Bax, and Bcl-2 immunohistochemical in 3 groupsThe expression of capase-3 in sham group were little; higher expression in endochylema in the CLP group(P<0.05);CI group obviously decreased the caspase-3 expression which CLP group increased(P<0.05). Sham group co- mpare to CI group had no statistical difference(P>0.05). Bcl-2 was little expression in the renal tissue at postoperative 24h in sham group; and higher expression in endochylema in the CLP group(P<0.05); highest in the CI group(P<0.05), three is increasing trend.Bax was little expression in the renal tissue at postoperative 24h in sham group; higher expression in endochylema in the CLP group(P<0.05); CI group obviously decreased the levels of Bax which CLP group were increased(P<0.05), Sham group and CI group had no significantly difference (P>0.05).Conclusions:1 Apoptosis is an important mechanism in septic acute kidney injury.2 s the key enzyme in the apoptosis way, caspase-3 plays an important role in sepsis-induced acute kidney injury.3 The protective effect of Ac-DEVD-CHO is attributed to inhibiting the expre- ssion of caspase-3 which in turn reduces apoptosis of renal tubular epithelial cell realized. |
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