幼年大鼠内毒素血症时小肠上皮细胞凋亡及Caspase-3的表达

Intestinal epithelial apoptosis and Caspase-3 expression in rats with endotoxemia

摘要:

目的：儿科危重症中胃肠功能障碍的常见病因是严重感染,其发病机制与内毒素血症导致肠黏膜屏障功能破坏密切相关。该研究探讨幼年大鼠内毒素血症时肠黏膜屏障损伤的细胞凋亡情况及其可能的信号转导通路,为寻求新的治疗途径提供依据。方法：40只18日龄Wstar大鼠腹腔注射4mg/kg内毒素(大肠杆菌O55∶B5脂多糖1mg/mL)制成内毒素血症模型,对照组大鼠(n=40)腹腔注射1mL/kg生理盐水。两组分别在注射后2, 4, 6, 24, 72h处死8只大鼠,取4cm远端回肠,行苏木精伊红染色,光镜下观察小肠绒毛的病理变化,原位末端标记法(TUNEL)检测小肠上皮的细胞凋亡,免疫组织化学测定Caspase 3的表达。结果：光镜下对照组各时点小肠绒毛结构正常,内毒素组注射后4 ~ 72h可见少量炎症细胞浸润。注内毒素后2h上皮细胞凋亡和Caspase 3的表达明显增加,分别于24h和6h达高峰, 72h开始下降。内毒素组各时间点小肠绒毛上皮细胞凋亡指数和Caspase 3的表达均明显高于对照组,差异有显著性意义(P<0.01或P<0. 001)。结论：幼年大鼠内毒素血症时小肠上皮细胞凋亡增加,Caspase 3表达是其信号转导途径之一。细胞凋亡可能是严重感染时肠黏膜屏障破坏机制。

Abstract:

OBJECTIVE: Severe infection is a common cause of gastrointestinal dysfunction in children, the mechanism of which is closely related to endotoxemia and impairment of gut mucosal barrier function. The study was undertaken to investigate intestinal epithelial apoptosis and its possible signal-conducting pathway in rats with endotoxemia. METHODS: The model of endotoxemia was established by intraperitoneal injection of endotoxin (4 mg/kg of Escherichia coli O_ 55∶B_5 lipopolysaccharide) in 40 18-day-old rats (Endotoxin group). Another 40 rats, served as controls and received normal saline (1 mL/kg). Both groups of rats were sacrificed at 2, 4, 6, 24 and 72 hrs after injection. A segment, 4-cm-long, of lower ileum was then removed. The pathologic changes of small intestine villus were observed under an optical microscope (hematoxylin-eosin staining). Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Caspase-3 expression was measured by immunohistochemistry staining. RESULTS: The structure of small intestinal villi of the Control group remained normal at various time points, while inflammation cells infiltration was observed in the Endotoxin group 4-72 hrs after injection. The apoptotic cells significantly increased 2 hrs after endotoxin injection, and peaked at 24 hrs. The apoptotic index of the Endotoxin group at each time point was significantly higher than that of the Control group (P<0.001). Similarly, the Caspase-3 expression in the Endotoxin group at each time point was significantly higher than that of the Control group. The Caspase-3 expression increased 2 hrs after endotoxin injection and reached a peak at 6 hrs. CONCLUSIONS: Intestinal epithelial apoptosis increases in rats with endotoxemia. Caspase-3 expression is one of its signal-conducting pathways. Cellular apoptosis might be the underlying mechanism of gut mucosal barrier impairment during severe infection.