Figure 1.
Inhibitory profile of acarbose and nafamostat mesilate.
Lineweaver-Burk Plot with inset Michaelis-Menten plot of amylase activity with different concentrations of acarbose (A) and trypsin activity with different concentrations of nafamostat mesilate (B). Enzyme kinetic parameters, maximum enzyme velocity (Vmax, RFU/min.µg), Michaelis-Menten constant (Km, mM or µM) for amylase or trypsin with different concentrations of inhibitors calculated after non-linear regression (C).
Figure 2.
Amylase and trypsin activity in intestine homogenates is increased during SAO.
Enzyme activity of amylase (A) and trypsin (B) in intestine homogenates of SHAM animals or animals subjected to SAO protocol with luminal inhibition using acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without (NI). Activity of luminal contents of SHAM intestines for each enzyme is shown at the end of the graphs. Western blot for amylase, trypsin and β-actin in intestine homogenates of groups described above (C) with corresponding density levels measurements (D, E). Values are mean±SEM (n = 4)/group **P<0.001 compared to SHAM, †††P<0.0001 compared to SAO30, ‡P<0.05 and ‡‡P<0.001 compared to ACA, §§§ P<0.0001 compared to all the other groups.
Figure 3.
In situ zymography for trypsin in jejunal sections.
Representative micrographs of trypsin activity as observed by fluorescence of specific substrate (blue), nuclei counterstaining with propidum iodine (red) in SHAM animals or animals subjected to SAO protocol with luminal inhibition with acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without inhibitors (SAO30).
Figure 4.
Tissue localization of mucin 2 and trypsin on jejunal sections.
Representative immunostaining of mucin 2 (green), trypsin (red), with nuclei counterstaining (blue) for jejunal sections corresponding to SHAM animals or animals subjected to SAO protocol with luminal inhibition with acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without inhibitors (SAO30).
Figure 5.
Mucin isoforms are degraded and MUC2 mRNA is up-regulated during intestinal ischemia.
Western blot for mucin 2 and mucin 13 in jejunal homogenates of SHAM animals or animals subjected to SAO protocol with luminal inhibition with acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without (NI) (A). Density levels measurement of mucin 2 (B) and mRNA expression of MUC2 (C). Mucin 13 density levels of different fragments (D). Values are mean±SEM (n = 4)/group **P<0.001 compared to SHAM, †††P<0.0001 compared to SAO30, ‡P<0.05 and ‡‡P<0.001 compared to ACA.
Figure 6.
Epithelial barrier is disrupted during intestinal ischemia.
Western blot for intra- and extra-cellular domains of E-cadherin in jejunal homogenates of SHAM animals or animals subjected to SAO protocol with luminal inhibition with acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without (NI) (A) with corresponding density measurements. Values are mean±SEM (n = 4)/group * P<0.01 compared to sham, †† P<0.001 as compared to ACA, ‡ P<0.01 as compared to TA (B). Representative micrographs of jejunal sections for groups described above, showing localization of FITC-dextran (green) with nuclei counterstaining (blue) (C).
Figure 7.
TLR4 is degraded during intestinal ischemia.
Western blot for intra- and extra-cellular domains of TLR4 in jejunal homogenates of SHAM animals or animals subjected to SAO protocol with luminal inhibition with acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without (NI) (A) with corresponding density level measurements (B). Immunohistochemistry of intra- and extra-cellular domains of TLR4 (brown) and nuclei counterstaining with hematoxylin (blue) (C). Values are mean±SEM (n = 4)/group *P<0.05 compared to SHAM.
Figure 8.
E-cadherin localization in intestinal epithelial cells after trypsin exposure.
Representative micrographs of IEC-18 cells immunostained for the intra-cellular domain of E-cadherin (green), actin (red), and nuclei (blue) after exposure to serum free medium (SFM) with different trypsin concentrations.
Figure 9.
Mucin layer protects E-cadherin on intestinal epithelial cells from trypsin-mediated degradation.
Western blot for intra- and extra-cellular domains of E-cadherin of IEC-18 cells without a mucin layer after exposure with serum free without and with trypsin (SFM and SFM+Try respectively) and IEC-18 cell with a mucin layer after exposure to serum free media with or without trypsin (SFM+Mucin and SFM+Mucin +Try) (A) with corresponding density levels measurements (B). Values are mean±SEM (n = 4)/group, **P<0.001 and ***P<0.0001 compared to SFM; ††P<0.001, †††P<0.0001 compared to SFM+Mucin; and ‡‡P<0.001, ‡‡‡P<0.0001 compared to SFM+Mucin+Try.
Figure 10.
Mucin layer protects TLR4 on intestinal epithelial cells from trypsin-mediated degradation.
Western blot for intra- and extra-cellular domains of E-cadherin of TLR4 cells without a mucin layer after exposure with serum free without and with trypsin (SFM and SFM+Try respectively) and IEC-18 cell with a mucin layer after exposure to serum free media with or without trypsin (SFM+Mucin and SFM+Mucin +Try) (A) with corresponding density levels measurements (B). Values are mean±SEM (n = 4)/group, **P<0.001 compared to SFM, ††P<0.001 compared to SFM+Mucin, and ‡P<0.05compared to SFM+Mucin+Try.
Figure 11.
Mucin layer reduces FITC-dextran diffusion across the intestinal epithelial cells monolayer.
Rate of diffusion of FITC-dextran across mucin alone, IEC-18 cell monolayer alone, or IEC-18 cell monolayer with a mucin layer after exposure to serum free media alone (SFM) or serum free media with trypsin (SFM+Try). Values are mean ± SEM (n = 4), **P<0.001 compared to SFM cells alone, †††P<0.001 compared to SFM+tryp cells alone, §§§P<0.0001 compared the other groups. (One-way Anova fallowed by Bonferroni’s post-hoc).