Sieving characteristics of cytokine- and peroxide-induced epithelial barrier leak: Inhibition by berberine

Table 1 The effect of tumor necrosis factor-α + interferon-γ + interleukin-1β and hydrogen peroxide on transepithelial flux of ¹⁴C-polyethylene glycol and ¹²⁵I-epidermal growth factor

	14C-PEG flux		125I-EGF flux
	cpm/min per square centimeter	pmol/min per square centimeter	cpm/min per square centimeter	fmol/min per square centimeter
Control	3.74 ± 0.07	7.54 ± 0.20	43.7 ± 2.0	0.034 ± 0.005
Cytomix	3.40 ± 0.12	6.87 ± 0.27	30.3 ± 1.4	0.015 ± 0.001
Cytomix/H2O2	9.80 ± 0.28a	20.97 ± 0.64a	192.0 ± 4.0c	1.21 ± 0.029c

^aP < 0.05 vs control;

^cP < 0.05 vs Cytomix-only condition (one-way ANOVA followed by Tukey’s post hoc testing). CACO-2 cell layers on Millipore polycarbonate filters were refed in control medium or medium containing the combination of 50 ng/mL tumor necrosis factor-α, 100 ng/mL interferon-γ, and 50 ng/mL interleukin-1β (apical and basal-lateral compartments) for 48 h. On the day of radiotracer flux studies, the cell layers were exposed to control saline or saline containing 1 mmol/L hydrogen peroxide for 3 h. These studies were performed using 0.1 mmol/L, 0.025 μCi/mL

¹⁴C-polyethylene glycol (MW 4000) and 10 nmol/L, 0.5 μCi/mL

¹²⁵I-EGF (MW 6100), as described in Materials and Methods. Total ¹²⁵I flux rates (as cpm/min per square centimeter) were adjusted, based upon the percent of intact ¹²⁵I-EGF in the basal-lateral compartment (using column chromatography), and expressed finally as fmol/min per square centimeter. Similar gel chromatography analyses were performed for ¹⁴C-PEG experiments, but here all isotopes that diffused across the epithelial cell layer was found to be 4000 MW PEG. Data shown represent the mean standard error for an n = 8 in all cases. PEG: Polyethylene glycol; EGF: Epidermal growth factor.

Table 2 Summary of the magnitude of effect of combinations of cytokines and hydrogen peroxide on transepithelial electrical resistance, mannitol leak, lactulose leak and polyethylene glycol leak

	Transepithelial electrical resistance decrease	Mannitol leak increase	Lactulose leak increase	Polyethylene glycol leak increase
TNF	++	1	0	ND
TNF/IFN	+	1	0	ND
TNF/IFN/IL1β	+	++	0	0
TNF/IFN/IL1β + hydrogen peroxide	++++	++++	++++	++++

CACO-2 cell layers having been exposed to the above combinations of cytokines and hydrogen peroxide were evaluated for type (salts, mannitol, lactulose, PEG) and magnitude of leak produced. All treatments increased leakage of salts as seen by a decrease in TER, but large molecule leak was seen only in the presence of peroxide alone or both cytokines and hydrogen peroxide, never in the presence of cytokines alone.

¹Indicates that the observed effect did not routinely achieve statistical significance;

⁰Indicates that an effect was measured but no significant change was observed. ND: Not determined; IFN-γ: Interferon-γ; IL1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; PEG: Polyethylene glycol.