天冬氨酸特异性半胱氨酸蛋白酶3抑制剂对腹腔感染脓毒症急性肾损伤小鼠血清炎症细胞因子的影响

The effect of caspase-3 inhibitor on the concentrations of serum inflammatory cytokines in sepsis related acute kidney injury induced by peritoneal cavity infection in mice

目的 观察天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）抑制剂Ac-DEVD-CHO对腹腔感染脓毒症急性肾损伤（AKI）小鼠血清炎症细胞因子的影响. 方法 将102只C57BL/6小鼠按随机数字表法均分成假手术组、模型组和caspase-3抑制剂组（CI组）3组.采用盲肠结扎穿孔术（CLP）制备脓毒症AKI小鼠模型;CI组在CLP术前给予Ac-DEVD-CHO 4 μg/g腹部皮下注射进行干预.各组均于术后6、12、24 h检测血尿素氮（BUN）、肌酐（Cr）浓度;用酶联免疫吸附法（ELISA）检测血清肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-6、IL-10）浓度;用流式细胞仪检测肾组织细胞凋亡率;用实时荧光定量聚合酶链反应（PCR）检测肾组织caspase-3 mRNA表达;并观察小鼠4 d和7 d的存活率. 结果 与假手术组比较,模型组各时间点血清BUN、TNF-α、IL-6、IL-10及肾组织细胞凋亡率、caspase-3 mRNA表达均显著升高,术后6 h Cr显著升高,4 d、7 d存活率显著下降.与模型组比较,CI组各时间点BUN显著下降;术后6 h Cr显著下降,12 h和24 h恢复至假手术组水平;各时间点血清TNF-α、IL-6水平降低,IL-10水平升高[TNF-α（μg/L）6 h：436.2±64.2比653.6±8.9,12 h：233.4±85.4比579.7±137.1,24 h：151.0±90.3比551.0±119.8;IL-6（μg/L） 6 h：1 033.2±345.8比1 595.3±159.4,12 h：366.3±68.3比1 330.7±249.8,24 h：241.2±208.4比815.3±572.7;IL-10（μg/L）6 h：33.6±10.4比26.6±4.5,12 h：37.2±5.0比24.5±4.3,24 h：38.3±5.5比18.2±1.6,均P〈0.05];肾组织细胞凋亡率及caspase-3 mRNA表达显著下降[凋亡率6 h：（13.9±3.2）%比（18.3±1.4）%,12 h：（10.5±3.6）%比（15.9±3.5）%,24 h：（8.4±1.8）%比（12.5±2.1）%;caspase-3 mRNA 6 h：1.95±0.16比3.84±0.35,12 h：1.89±0.19比3.97±0.73,24 h：2.01±0.20比4.97±0.24,均P〈0.05],并提高了小鼠4 d存活率（80%比20%）,7 d存活率无变化（20%比20%）. 结论 caspase-3抑制剂对腹腔感染脓毒症AKI小鼠血清促炎/抗炎细胞因子水平的影响可能与降低肾组织细胞凋亡有关.

Objective To observe the effects of caspase-3 inhibitor Ac-DEVD-CHO on the concen-trations of serum inflammatory cytokines in sepsis related acute kidney injury induced by peritoneal cavity infection in mice. Methods One hundred and two 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=34） according to random number table：sham group,model group,and caspase-3 inhibitor（CI） group.Thirty minutes before CLP,Ac-DEVD-CHO（4 μg/g） was injected subcutaneously in CI group.The levels of blood urea nitrogen（BUN） and creatinine（Cr） were determined,and the concentrations of tumor necrosis factor-α（TNF-α）,interleukins（IL-6 and IL-10） were measured by enzyme linked immunosorbent assay （ELISA）,the renal cell apoptosis rate was determined by flow cytometry and the expression of caspase-3 mRNA was determined by real time reverse transcription-polymerase chain reaction（RT-PCR） at 6,12 and 24 hours after operation.The 4-day and 7-day survival rates of three groups of mice were observed. Results Compared with sham group,the concentrations of serum BUN,TNF-α,IL-6,IL-10 and the renal cell apoptosis rates,the caspase-3 mRNA expression were increased significantly at all time points after CLP,the concentrations of serum Cr were increased significantly at 6 hours,with the 4-day and 7-day survival rates were decreased significantly.Compared with model group,in CI group,the concentrations of serum BUN were decreased significantly at all time points after operation and those of Cr were decreased significantly at 6 hours,then restored to those of the sham group at 12 hours and 24 hours;the concentrations of serum TNF-α,IL-6 were decreased and those of IL-10 elevated significantly at all time points[TNF-α（μg/L） 6 hours：436.2±64.2 vs.653.6±8.9,12 hours：233.4±85.4 vs.579.7±137.1,24 hours：151.0±90.3 vs.551.0±119.8;IL-6（μg/L） 6 hours：1 033.2±345.8 vs.1 595.3±159.4,12 hours：366.3±68.3 vs.1 330.7±249.8,24 hours：241.2±208.4 vs.815.3±572.7;IL-10（μg/L） 6 hours：33.6±10.4 vs.26.6±4.5,12 hours：37.2±5.0 vs.24.5±4.3,24 hours：38.3±5.5 vs.18.2±1.6,all P〈0.05];the renal cell apoptosis rate and the expression of caspase-3 mRNA were decreased significantly at all time points[apoptosis rates 6 hours：（13.9±3.2）% vs.（18.3±1.4）%,12 hours：（10.5±3.6）% vs.（15.9±3.5）%,24 hours：（8.4±1.8）% vs.（12.5±2.1）%;caspase-3 mRNA 6 hours：1.95±0.16 vs.3.84±0.35,12 hours：1.89±0.19 vs.3.97±0.73,24 hours：2.01±0.20 vs.4.97±0.24,all P〈0.05].The 4-day survival rate of CI group was improved （80% vs.20%）,but that of 7-day did not change （20% vs.20%）. Conclusion The modulation of caspase-3 inhibitor on the concentrations of serum inflammatory cytokines in sepsis related acute kidney injury induced by peritoneal cavity infection may be associated with a decrease in renal cell apoptosis by Ac-DEVD-CHO.