Caspase抑制剂z-VAD-fmk对大鼠烧伤后小肠组织损伤、细胞凋亡的影响及机制

Effects and mechanism of Caspase inhibitor z-VAD-fmk on small intestinal injury and mucosal apoptosis in rats after burn

目的观察广谱Caspase抑制剂z-VAD-fmk对大鼠烧伤后小肠组织损伤和细胞凋亡的影响,并探讨其机制。方法96只雄性Wistar大鼠随机分为对照组、致伤组、治疗组,每组各32只。致伤组和治疗组制作严重烧伤大鼠模型;治疗组造模后即刻经尾静脉注射3 mg/kg的z-VAD-fmk,12 h后再次注射1.5 mg/kg;致伤组用等量生理盐水干预。分别于造模后2、8、24、48 h处死大鼠并留取小肠组织,采用HE染色观察小肠组织病理改变并评分,采用TUNEL法测算小肠黏膜细胞凋亡指数(AI),荧光比色法检测小肠黏膜细胞Caspase-3活性,免疫组化法检测小肠组织中的热休克蛋白70(HSP70)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2基因相关X基因(Bax)蛋白。结果致伤组、治疗组造模后2、8、24、48 h小肠组织病理评分高于对照组,治疗组各时点评分均低于致伤组(P均<0.05)。致伤组、治疗组造模后2、8、24、48 h小肠黏膜细胞AI及Caspase-3活性高于对照组,治疗组各时点均低于致伤组(P均<0.05)。致伤组、治疗组造模后2、8、24、48 h小肠组织中HSP70、Bcl-2、Bax表达均高于对照组,治疗组各时点HSP70、Bcl-2表达高于对照组,Bax表达低于致伤组(P均<0.05)。结论早期应用z-VAD-fmk可以有效减轻大鼠烧伤后小肠组织损伤、减少细胞凋亡,其机制可能与HSP70、Caspase-3、Bcl-2和Bax的表达调节作用有关。

Objective To observe the effects and mechanism of Caspase inhibitor(z-VAD-fmk)on small intestinal injury and mucosal apoptosis in burned rats.Methods Ninety-six male Wistar rats were randomly assigned into the control group,injury group and treatment group,with thirty-two rats in each.Severe burn rat models were made in the injury group and the treatment group.In the treatment group,z-VAD-fmk of 3 mg/kg was injected through tail vein immediately after modeling,and 1.5 mg/kg was injected again 12 hours later.The rats in the injury group were treated with normal saline.Rats were killed at 2,8,24,and 48 h after modeling in each group and the small intestinal tissues were immediately removed at each time point mentioned above.HE staining was used to evaluate the pathological changes and grading,and TUNEL method for detecting the apoptotic index(AI)of small intestinal mucosal cells.The activity of Caspase-3 in the intestinal cells was measured with fluorescence colorimetry.Immunohistochemistry was used to detect the expression levels of heat stroke protein 70(HSP70),Bcl-2 and Bcl-2-associated X(Bax).Results The histomorphologic scores of the small intestine of the injury group and the treatment group at 2,8,24 and 48 h after modeling were higher than those of the control group,and the scores of the treatment group at all time points were lower than those of the injury group(all P<0.05).The AI and Caspase-3 activities of small intestinal mucosal cells in the injury group and the treatment group were higher than those in the control group at 2,8,24 and 48 h after modeling,while the activity of small intestinal mucosal cells in the treatment group was lower than that in the injury group at all time points(all P<0.05).The expression levels of HSP70,Bcl-2 and Bax in the small intestine tissues at 2,8,24 and 48 h after modeling in the injury group and the treatment group were higher than those of the control group.The expression levels of HSP70 and Bcl-2 in the treatment group were higher than those of the control group at each time point,and the expression of Bax was lower than that of the injury group(all P<0.05).Conclusion The early intervention of z-VAD-fmk could effectively alleviate small intestinal injury and decrease apoptosis,which may be related to the regulation of HSP70,Caspase-3,Bcl-2 and Bax.