Pancreatic digestive enzyme blockade in the intestine increases survival after experimental shock

Abstract

Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors--6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate, tranexamic acid, or aprotinin--into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multiorgan failure, which can be treated with protease inhibitors that are currently available for use in the clinic.

Fig. 1

Serine protease activity in the intestinal wall and in femoral plasma. Enzyme activity was measured as a function of liberated tyrosine (Tyr) at 40 individual segments of the wall of the rats’ small intestines along from the duodenum (left position on the abscissa) to the cecum (right position). (A) Protease activity in the intestinal lumen of nonischemic sham controls. Data are averages ± SD. (B) Protease activity in all shock models. The intestines in hemorrhagic shock were collected at 1 hour after the hypotensive period, in peritonitis shock at 4 hours after placement of cecal material, and in endotoxic shock at 4 hours after endotoxin administration. Data are averages ± SD with the number of rats (n) indicated. (C) Protease activity values in the plasma of nonischemic sham controls, and in hemorrhagic, peritonitis, and endotoxic shock rats without (black bars) and with (hashed bars) protease inhibitor ANGD in the lumen. Samples were collected at the same periods after shock as the intestine in (A). Data are averages ± SD, with the number of plasma samples in each group indicated in each column and one plasma sample per rat. In (B) and (C), *P < 0.05 versus respective untreated group by analysis of variance (ANOVA).

Fig. 2

Enteral protease blockade attenuates acute organ damage. Representative macroscale view of small intestine, heart [right ventricle (rv)], and lung in sham control (n = 5) and at 2 hours after the hypotensive period of hemorrhagic shock without (n = 3) and with the protease inhibitor ANGD (n = 3). Characteristic lesion sites due to escape of red blood cells from microvessels (Fig. 3) are marked by arrows.

Fig. 3

Enteral protease blockade prevents intestinal tissue damage and microhemorrhages in heart and lung. Representative cross sections of the small intestine, heart [right ventricle (rv)], and lung in nonischemic sham controls (n = 3) and in hemorrhagic shock without (n = 3) and with (n = 3) ANGD. Sections were labeled with toluidine blue, and the intestine was also labeled with mucin stain (second row, red). The tissues were collected at 1 hour after hypotension. Cleavage of intestinal villus tips (arrowheads) is prominent in untreated shock. Insets show extravasation of red blood cells into the cardiac interstitium (arrows) and into the alveolar space of the lung (arrows) in the untreated cases.

Fig. 4

Villus length and fraction of villi with intact epithelial lining after enteral tranexamic acid treatment. (A) Villus lengths. (B) Fraction of villi with intact epithelial lining. Data are averages ± SD; n = 3 rats in each group with 30 to 40 villi per rat derived from three sections at three equally spaced locations between duodenum and proximal ileum. Sham control is the same for all three shock models. *P < 0.05 compared to respective treated group, ANOVA.

Fig. 5

Plasma cardiac troponin levels and lung weight without and with enteral tranexamic acid treatment. (A) Plasma cardiac troponin I levels at 4 hours after induction of shock. (B) Lung wet/dry weight ratios at 4 hours after induction of shock. Data are averages ± SD (n = 3 rats per group). *P < 0.05 versus untreated, ANOVA; ^‡P < 0.05 compared to control, ANOVA.