Fig 1.
Electrophysiological responses and FITC-LPS flux quantification of ischemia-injured porcine jejunal mucosa to treatment with single doses of LA at various concentrations.
LA was added to the apical bathing solution after a 30-minute equilibration period to allow for stabilization of baseline electrical measurements (indicated by the arrow at 30-minutes). Statistical significance (* or **) is shown between ischemia-injured jejunal mucosa treated with 1 μM LA and untreated ischemia-injured jejunal mucosa. TER Values are means ± SEM; n ≥ 7 except for LA (0.1 μM) where n = 3. Total LPS flux values are means ± SEM; n ≥ 9.
Fig 2.
Histological examination of porcine jejunal mucosa subjected to ischemia and ex vivo recovery.
A: Unrecovered non-ischemic (uninjured) porcine jejunum. B: unrecovered ischemia-injured porcine jejunum. C: Recovered ischemia-injured porcine jejunum. D: Recovered ischemia-injured porcine jejunum treated with LA (1 μM). Ischemic injury resulted in the detachment of the epithelial monolayer from the basement membrane and sloughing of epithelium into the intestinal lumen (2B). After 4 hours of recovery, there were no histological differences between ischemia-injured jejunum treated with 1 μM LA (2D) versus untreated ischemia-injured jejunum (2C). Villous height appeared to be similar between groups, and no denuded surface was observed in either group. Hematoxylin and eosin stain, 100 μM scale bar.
Table 1.
Histomorphometric assessment of unrecovered and recovered ischemia-injured porcine jejunum.
Fig 3.
Immunofluorescent analysis of claudin-4 of ischemia-injured porcine jejunum after LA (1 μM) treatment.
Arrows indicate where claudin-4 is well localized within the epithelium. Scale bar represents 100 μM.
Fig 4.
Expression of tight junction proteins in LA-treated ischemia-injured porcine jejunum.
A: Representative western blot images from cytoplasmic and membrane fractions from ischemia-injured porcine jejunum. Lanes for panel A are as follows: 1: non-ischemic (unrecovered), 2: ischemic (unrecovered), 3: non-ischemic (recovered), 4: ischemic (recovered), 5: ischemic + 1 μM LA (recovered) B: Densitometry analysis of western blot bands for claudin-4 from cytoplasmic and membrane protein fractions of represented tissues. Claudin-4 expected band size using this monoclonal antibody is ~18 kDa. β-actin loading control band size is expected at ~42 kDa. Statistical significance (**) is between ischemic + LA (1 μM) (recovered) tissues vs untreated ischemic (recovered) tissues. Values are means ± SEM; n = 3 for each group.
Fig 5.
In vitro degradation of LA by AM.
Two separate runs (A & B) are shown. Each graph represents data from samples collected from LA, MgCl2, and AM solution mixtures. Control data from samples not containing AM are not shown. C: Western blot analysis of uninjured and injured (unrecovered) porcine jejunum for AM expression.
Fig 6.
Mass spectrometry analysis of Ringers solution collected from the Ussing chambers (N = 4) during ex vivo recovery in the presence of LA.
A: LA fragments F1 and F2 are generated during the 240-minute ex vivo recovery. Additionally, two more LA fragments, Fragment #3 (F3) and Fragment #4 (F4) were also detected upon MS analysis of the same Ringers solution samples.
Fig 7.
Electrophysiological responses of ischemia-injured porcine jejunal mucosa to treatment with A: LA fragments F1 alone or with LA, B: F2 alone or with LA, and C: F3 alone or with LA.
Values are means ± SEM; n ≥ 3. F1/F2 alone or with LA was added to the apical bathing solution after a 30-minute equilibration period to allow for stabilization of baseline electrical measurements. A: Statistical significance (*) is shown between F1 (10 μM) alone vs LA (1 μM). B: Statistical significance (* or **) is shown between F2 (10 μM) + LA (1 μM) vs LA (1 μM). Additional statistical significance (#) is shown between F2 (10 μM) alone vs LA (1 μM). C: No statistical significance was detected between groups at specific timepoints. Values are means ± SEM; n ≥ 3.
Fig 8.
Electrophysiological responses and histomorphology of ischemia-injured porcine jejunal mucosa to treatment with single doses of the chirally-altered LA molecule A6 at various concentrations.
Statistical significance is between either ischemic tissue treated with A6 vs untreated ischemic tissue (*) or ischemic tissue treated with LA vs untreated ischemic tissue (#) during the 240-minute recovery period. Values are means ± SEM; n ≥ 4.
Fig 9.
Mass spectrometry analysis of Ringers solution collected from the Ussing chambers (N = 4) during ex vivo recovery in the presence of A6 or LA.
A: Degradation of either LA or A6 during ex vivo recovery. The half-life of LA and A6 in ischemic injured jejunum tissue is 35 minutes and 78 minutes, respectively. B: A6 fragments A6F1, A6F2, and A6F4 are generated during the 240-minute ex vivo recovery. Values are means ± SEM; n = 4.
Fig 10.
Electrophysiological responses of ischemia-injured porcine jejunal mucosa to treatment with A: A6F1 alone or with A6 and B: A6F2 alone or with A6.
Statistical significance is between A6F1 (10 μM) + A6 (0.1 μM) vs A6 (0.1 μM) alone. Values are means ± SEM; n ≥ 4.