Pancreatic enzymes generate cytotoxic mediators in the intestine
Pancreatic enzymes generate cytotoxic mediators in the intestine
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DOI：
10.1097/01.shk.0000235139.20775.7f
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发表时间：
2007-03-01
期刊：
影响因子：
3.1
通讯作者：
Schmid-Schoenbein, Geert W.
中科院分区：
文献类型：
--
作者：
Penn, Alexander H.;Hugli, Tony E.;Schmid-Schoenbein, Geert W.
最近的证据表明，休克伴随着肠粘膜屏障的失效和胰腺消化酶进入肠壁。为了研究肠道酶消化产生的细胞毒性介质的形成，我们将大鼠小肠壁匀浆应用于人中性粒细胞，并使用流式细胞术测量碘化丙啶摄取来确定细胞毒性。我们表明，缺血后的小肠匀浆闭塞的上级肠系膜和腹腔动脉3小时，但不是没有缺血，是细胞毒性的。用纯化的胰蛋白酶、糜蛋白酶或弹性蛋白酶消化非缺血肠壁匀浆，产生细胞毒性介质。在细胞死亡之前，我们看到了以水泡形成形式的细胞损伤和由于水泡形成而导致的细胞大小变化的流式细胞术测量。细胞毒性被阻止丝氨酸蛋白酶抑制与苯甲基磺酰氟（PMSF）之前，但不是后壁匀浆的蛋白水解消化，表明蛋白酶的酶促作用的匀浆是必要的细胞毒性。从肠腔收集的液体中的酶消化的壁匀浆的细胞毒性大于由单个纯化的蛋白酶消化的细胞毒性。如果在加入壁匀浆前，用酶抑制剂PMSF和6-脒基-2-萘基对胍基苯甲酸二甲磺酸盐联合抑制腔液中的消化酶，则无法检测到细胞毒性。消化的肠壁匀浆通过疏水玻璃纤维过滤器降低细胞毒性。此外，我们发现腔液本身可能是细胞毒性的，可能是因为消化摄入的食物。为了测试消化的食物是否具有细胞毒性，我们将大鼠食物均质化，并在体外用胰凝乳蛋白酶或腔液中的内源性酶消化。与腔液或未消化食物相比，腔液消化食物后细胞毒性显著增加。这些结果表明休克中存在一种以前未知的出血性坏死机制。
Recent evidence indicates that shock is accompanied by a failure of the mucosal barrier in the intestine and entry of pancreatic digestive enzymes into the wall of the intestine. To investigate the formation of cytotoxic mediators produced by enzymatic digestion of the intestine, we applied homogenates of rat small intestinal wall to human neutrophils and used flow cytometry measurements of propidium iodide uptake to determine cytotoxicity. We show that homogenates of the small intestine after ischemia by occlusion of the superior mesenteric and celiac arteries for 3 h, but not without ischemia, are cytotoxic. Digestion of homogenates of nonischemic intestinal wall with purified trypsin, chymotrypsin, or elastase, proteases normally present in the intestinal lumen, yielded cytotoxic mediators. Before cell death, we saw cell damage in the form of bleb formation and flow cytometry measurements of cell size changes due to blebbing. Cytotoxicity was prevented by serine protease inhibition with phenylmethylsulfonyl fluoride (PMSF) before, but not after proteolytic digestion of the wall homogenates, indicating that enzymatic action of proteases on the homogenate is necessary for cytotoxicity. Cytotoxicity of wall homogenates digested by enzymes in the fluid collected from the lumen of the intestine was greater than digests by the individual purified proteases. Cytotoxicity is undetectable if digestive enzymes in the luminal fluid are inhibited with a combination of enzyme inhibitors PMSF and 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate before addition of wall homogenates. Passage of digested intestinal wall homogenates across a hydrophobic glass-fiber filter reduced cytotoxicity. Furthermore, we found that luminal fluid itself may be cytotoxic, possibly because of digestion of ingested food. To test whether digested food can be cytotoxic, we homogenized rat food and digested it in vitro with chymotrypsin or endogenous enzymes in luminal fluid. Cytotoxicity was significantly increased after digestion of food by luminal fluid compared with luminal fluid or undigested food. These results indicate the presence of a previously unknown mechanism for hemorrhagic necrosis in shock.