Figure 1.
MPO activity in tissue homogenates at 2 hours after hemorrhagic shock. N = 5 rats per group. *p<0.05 by t-test vs. HS+SAL.
Figure 2.
Plasma and peritoneal fluid serine protease activity.
Trypsin-, chymotrypsin-, and elastase-like activities in (A) plasma and (B) peritoneal fluid expressed as equivalent activity to concentrations of pure trypsin, chymotrypsin, and elastase. Note the difference in scales of the ordinate. N = 6 rats per group. *p<0.05, paired t-test comparing pre vs. post HS+SAL or HS+ANGD; **p<0.05 by t-test vs. post HS+SAL; #p<0.05, paired Mann-Whitney test comparing pre vs. post HS+SAL or HS+ANGD.
Figure 3.
Plasma and peritoneal fluid caseinolytic activity.
Caseinolytic activity with and without addition of a serine protease inhibitor, 1 mM PMSF, of (A) plasma and (B) peritoneal fluid pre/post shock with saline or ANGD pre-treatment in the intestinal lumen. Protease activity was detectable, but low with this substrate. N = 5 rats per group. *p<0.05 by paired t-test vs. without PMSF; ≠p<0.05 by paired t-test comparing pre vs. post plasma or peritoneal fluid from HS+SAL or HS+ANGD animals.
Figure 4.
Plasma and peritoneal fluid gel zymography and trypsin levels.
(A) Gelatin gel zymography detection and quantification in Relative Intensity Units (RIU; bar graph beneath) of ∼20 kDa bands for plasma (right; N = 6, 6, 5 rats per group) and peritoneal fluid (left; N = 5 rats per group). Bands do not form or are greatly reduced when gels are renatured with ANGD or TLCK, respectively (not shown). (B) Immunoblot detection and quantification (bar graph beneath) of trypsin in plasma (left) and peritoneal fluid (right), revealing the 26 and 23 kDa isoforms of trypsin that are present. +p<0.05 by Mann-Whitney test vs. Pre-HS; ≠p<0.05 by t-test vs. Pre-HS; **p<0.05 by t-test HS+ANGD vs. HS+SAL; #p<0.05 pre vs. post HS+SAL or HS+ANGD by paired Mann-Whitney test; *p<0.05 by paired t-test comparing pre vs. post HS+SAL.
Figure 5.
Pancreatic trypsin levels in lung tissue.
(A) Two activity bands around 20 kDa were detected in lung homogenate by gel zymography. (B) Three isoforms of trypsin were detected in lung tissue homogenate using the femto ECL substrate. The 32 kDa band corresponds to the molecular weight of the trypsin precursor, trypsinogen, whereas the 23 and 26 kDa bands probably correspond to active forms of trypsin. N = 5, 6, 6 rats per group for No-HS, HS+SAL, and HS+ANGD, respectively. #p<0.05 vs. No-HS by Mann-Whitney test; ≠p<0.05 vs. No-HS by t-test.
Figure 6.
MMP-9 activity and levels in plasma and peritoneal fluid.
(A) Plate zymography with MMP-1/9 substrate, (B) gelatin gel zymography, and (C) immunoblots for MMP-9 in plasma (left) and peritoneal fluid (right). MMP-9 activity was elevated by both plate and gelatin gel zymography. There was no change in MMP-2. N = 6, 5, 5 rats per group for plasma and N = 6 rats per group for peritoneal fluid. Immunoblotting quantification is reported in RU with the pico ECL substrates. ≠p<0.05 vs. Pre-HS group by t-test; *p<0.05 by paired t-test comparing pre vs. post HS+SAL or HS+ANGD.
Figure 7.
MMP-9 activity and levels in vital organs.
(A) Gelatin gel zymography revealed increased levels of MMP-9 activity in the heart, liver, and lung (quantifications to the right) after hemorrhagic shock. There was no change in MMP-2. (B) Immunoblot detection of MMP-9 was upregulated in brain (bands detected with femto ECL substrate), heart, liver, and lung (bands detectable by pico ECL substrate). N = 5 rats per group, ≠p<0.05 by t-test vs. No-HS.