辅助腹腔复苏对大鼠失血性休克早期肠损伤的动态影响

Dynamic effects of adjunct peritoneal resuscitation on the early intestinal injury of rats after hemorrhagic shock

目的探讨辅助腹腔复苏（APR）对大鼠失血性休克早期肠损伤的动态影响及可能机制。方法将96只雄性Wistar大鼠按随机数字表法分成对照组、休克组、林格液APR组和透析液APR组4组，每组再分为复苏后1、2、6h3个时间点，每个时间点8只大鼠。复制失血性休克动物模型。休克组采用常规静脉复苏（回输失血＋2倍失血量的生理盐水）；林格液APR组和透析液APR组在常规静脉复苏基础上，分别于开始液体复苏时一次性腹腔注射100mL/kg林格液或2．5％葡萄糖腹膜透析液。各组于相应时间点活杀动物，用紫外比色法检测血浆二胺氧化酶（DAO）活性，镜下观察小肠组织病理改变并进行肠黏膜损伤评分，用反转录一聚合酶链反应（RT—PCR）检测小肠组织核转录因子-κB（NF—κB）、热休克蛋白70（HSP70）的mRNA表达，取剩余肠组织计算湿／干质量（W／D）比值。结果与对照组比较，休克组复苏后1h，血浆DAO活性、小肠黏膜损伤评分、小肠组织NF—κB和HSP70的mRNA表达、肠W／D比值即明显升高，除NF—κB mRNA于复苏后2h达高峰外，余各指标均随时间逐渐升高。与休克组比较，透析液APR组复苏后1、2、6h各时间点血浆DAO活性（U／L：8．78±0．76比13．30±1．37，9．67±0．92比16．56±1．61，10．53±0．84比18．31±1．72，均P〈0．05）、小肠黏膜损伤评分（分：1．36±0．31比3．5l±0．66，2．28±0．40比4．02±0．76，2．47±0．38比4．76±0．77，均P〈0．05）、肠组织NF—κB mRNA表达（灰度值：0．658±0．062比0．765±0．067，0．945±0．077比1．158±0．096，0．761±0．062比0．912±0．082，均P〈0．05）、肠W／D比值（3．91±0．26比4．34±0．33，3．96±0．32比4．51±0．40，4．37±0．33比4．99±0．43，均P〈O．05）明显降低，肠组织HSP70 mRNA表达显著增强（灰度值：0．717±0．054比0．635±0．056，0．853±0．068比0．745±0．071，1．258±0．111比1．108±0．105，均P〈0．05）。林格液APR组仅复苏后1h血浆DAO活性、肠黏膜损伤评分显著低于休克组，其余各指标变化与休克组比较差异无统计学意义。结论2．5％葡萄糖腹膜透析液APR可减轻失血性休克早期肠损伤，其作用机制可能与增强肠组织HSP70mRNA表达、抑制NF—κB mRNA表达有关。

Objective To investigate the dynamic effects of adjunct peritoneal resuscitation （APR） on the early intestinal injury of rats with hemorrhagic shock, and explore its possible mechanisms. Methods According to random number table, 96 male Wistar rats were assigned randomly into control group, shock group, Ringer solution APR group and dialysate APR group. Each group was further divided into three time points, namely 1, 2, and 6 hours after resuscitation, with 8 rats for each time point. The model of hemorrhagic shock was reproduced. The rats in the shock group received shed blood plus twice amount of normal saline as conventional intravenous resuscitation （CIR）. Besides CIR, at the beginning of resuscitation, those rats in Ringer solution APR and dialysate APR groups were given 100 mL/kg of Ringer solution or 2.5% glucose-based peritoneal dialysis solution intravenously, respectively. The rats in four groups were sacrificed at 1, 2 and 6 hours after resuscitation, respectively. Plasma activity of diamine oxidase （DAO） was detected by ultraviolet spectrophotometry colorimetry. Intestinal pathological changes were observed under light microscope, and the grading of the intestinal mucosal damage （ GIMD ） score was estimated. The expressions of nuclear factor-κB （NF-κB） and heat shock protein 70 （HSP70） mRNA in the intestinal tissue were determined with reverse transcription-polymerase chain reaction （RT-PCR）, and the wet/dry weight ratios （W/D） of the remaining intestinal tissue were measured. Results Compared with the control group, plasma activity of DAO, GIMD score, expressions of NF-κB and HSP70 mRNA and W/D ratios of intestinal tissue at 1 hour after resuscitation were increased significantly in the shock group. Except the expression of NF-κB mRNA reached the peak level at 2 hours after resuscitation, all the other parameters increased with time. Compared with the shock group, plasma activity of DAO （U/L： 8.78 ± 0.76 vs. 13.30± 1.37, 9.67 ± 0.92 vs. 16.56±1.61, 10.53 ±0.84 vs. 18.31 ± 1.72, all P 〈 0.05）, GIMD score （1.36 ± 0.31 vs. 3.51 ±0.66, 2.28 ±0.40 vs. 4.02 ±0.76, 2.47±0.38 vs. 4.76 ±0.77, all P 〈 0.05 ）, the expressions of NF-κB mRNA in the intestine tissue （ gray value： 0.658 ± 0.062 vs. 0.765 ±.067, 0.945 ±0.077 vs. 1.158 ± 0.096, 0.761 ± 0.062 vs. 0.912±0.082, all P 〈 0.05）, and W/D ratios （3.91 ±0.26 vs. 4.34±0.33, 3.96±0.32 vs. 4.51 ±0.40, 4.37±0.33 vs. 4.99 ± 0.43, all P 〈 0.05 ） at 1, 2, 6 hours after resuscitation in the dialysate APR group were decreased significantly, but the expressions of HSP70 mRNA were increased significantly （gray value： 0.717±.054 vs. 0.635±0.056, 0.853 ±0.068 vs. 0.745 ±0.071, 1.258 ±0.111 vs. 1.108 ±0.105, all P 〈 0.05 ）. The activity of DAO and GMID score at 1 hour after resuscitation in the Ringer solution APR group were decreased significantly compared with the shock group, while the differences of the other parameters between the Ringer solution APR group and shock group showed no statistical significance. Conclusion APR with 2.5% glucose-based peritoneal dialysis solution can significantly mitigate the early intestinal injury secondary to hemorrhagic shock, and the mechanism may be associated with enhancement of the expressions of HSP70 mRNA and inhibition of the expressions of NF-κB mRNA.