Protection from experimental colitis by theaflavin-3,3'-digallate correlates with inhibition of IKK and NF-kappaB activation

Abstract

Background and purpose: Inflammatory bowel disease (IBD) is associated with activation of nuclear factor kappa B (NF-kappaB) involved in regulating the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokine genes. As theaflavin-3,3'-digallate (TFDG), the most potent anti-oxidant polyphenol of black tea, down-regulates NF-kappaB activation, we investigated if TFDG is beneficial in colonic inflammation by suppressing iNOS and proinflammatory cytokines.

Experimental approach: The in vivo efficacy of TFDG was assessed in mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Both mRNA and protein levels of proinflammatory cytokines and iNOS were analyzed in colon tissue treated with or without TFDG. NF-kappaB activation was determined by electrophoretic mobility shift assay and levels of NF-kappaB inhibitory protein (IkappaBalpha) were analyzed by Western blotting.

Key results: Oral administration of TFDG (5 mg kg(-1) daily i.g.) significantly improved TNBS-induced colitis associated with decreased mRNA and protein levels of TNF-alpha, IL-12, IFN-gamma and iNOS in colonic mucosa. DNA binding and Western blotting revealed increase in NF-kappaB activation and IkappaBalpha depletion in TNBS-treated mice from Day 2 through Day 8 with a maximum at Day 4, which resulted from increased phosphorylation of IkappaBalpha and higher activity of IkappaB kinase (IKK). Pretreatment with TFDG markedly inhibited TNBS-induced increases in nuclear localization of NF-kappaB, cytosolic IKK activity and preserved IkappaBalpha in colon tissue.

Conclusions and implications: TFDG exerts protective effects in experimental colitis and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-kappaB activation.

Figure 1

Time course of experimental schedules. Control and TFDG group received TFDG daily by oral gavage for a total of 18 days. Both TNBS and TFDG group received TNBS by intracolonic administration at day 10 whereas the control group received 30% ethanol at day 10. Groups of mice were killed (Sample) at 12, 14 or 18 days after the start of the study.

Figure 2

Effect of TFDG on NO production. (a) Peritoneal macrophages (10⁶ cells ml⁻¹) were cotreated with LPS (100 ng ml⁻¹) and various concentrations of TFDG for 24 h at 37°C. Cell viability was assessed by an MTT assay. (b) Macrophages (10⁶ cells ml⁻¹) were incubated with LPS (100 ng ml⁻¹) either alone or in the presence of 40 μM each of TFDG, TFD1, TFD2 or TR. Each bar shows the mean±s.d. of three independent experiments. (c) The nature of iNOS expression by various agents was also determined by RT-PCR of its mRNA transcript.

Figure 3

Effect of TFDG treatment on established colitis. Mice were treated with 0.1 ml of TNBS (60 mg ml⁻¹) intracolonically and assessed at various times (2–8 days) after treatment. (a) Colonic damage was scored by blinded assessment based on validated scoring system as described in Methods. Evaluation of body weights (b), spleen (c) and colon (d) expressed as a percentage of the original weight on day 0. A significant weight gain was observed at 4 days after TNBS administration in spleen and colon. TFDG pretreatment (5 and 10 mg kg⁻¹) significantly prevented the loss in body weight (b) as well as reduced the organ weight (c and d). (e) Histological scoring was performed semi-quantitatively in hematoxylin and eosin-stained sections. (f) Assessment of myeloperoxidase activity in TNBS-treated mice in comparison to control. Values are means ± s.d. of 10 mice for each group. Results for the TFDG treatment group were compared against those for the TNBS-treated group with a two-sided Wilcoxon rank-sum test. ^*P<0.01, ^**P<0.05 vs TNBS. Histological appearance of mice colonic mucosa after hematoxylin and eosin stain, treated with 30% ethanol in PBS (g), treated with TNBS (60 mg ml⁻¹) in 30% ethanol (h) and pretreated with TFDG (5 mg kg⁻¹) (i). TNBS-induced colonic inflammation (at 4 days) was mostly associated with loss of goblet cells, crypt damage, mucosal ulceration and accompanying edema of the submucosa. Pretreatment with TFDG significantly attenuated the damages in morphology associated with TNBS treatment. Original magnification: × 250.

Figure 4

Proinflammatory cytokine expression in colonic mucosa of mice with TNBS-induced colitis. Oral pretreatment with TFDG (5 mg kg⁻¹) showed a significant decrease in the expression levels of IFN-γ, IL-12 p40 and TNF-α in the colon of TNBS-treated mice both at mRNA (a) and protein (b) level as analyzed by RT-PCR and ELISA, respectively. RT-PCR products were visualized by ethidium staining. RNA samples were obtained from six mice in each group. Results are representative of three separate samples. β-actin expression levels were used as controls for RNA content and integrity. Band intensities quantified by densitometry are shown as bar diagrams.

Figure 5

A time–course pattern of iNOS expression. iNOS expression by RT-PCR (a) and Western blot (b) in colonic mucosa of colitic mice and TFDG-pretreated colitic mice on day 2, 4 and 8 after TNBS administration. RT-PCR products were visualized by ethidium bromide staining. RNA samples were obtained from six mice in each group. Results are representative of duplicate samples of three separate experiments and the densitometric evaluations are means of three independent experiments. ^*P<0.01; ^**P<0.001 vs control. β-actin expression levels were used as controls for RNA content and integrity.

Figure 6

Effects of TFDG pretreatment (5 mg kg⁻¹) on the kinetics of NF-κB activation in colon tissue samples of mice with TNBS-induced colitis. (a) Nuclear extracts were obtained from mouse colon at the indicated time period and analyzed for NF-κB activity by EMSA. Each lane represents colon nuclear extracts from the control, TNBS or TFDG group. UP indicates 50 × molar excess of unlabeled probe. Oral administration of TFDG consistently decreased the NF-κB-binding activity, which reached maximum at day 4 after TNBS administration and then started decreasing. (b) For supershift assay, nuclear extracts from colonic mucosa of mice at 4 day after TNBS administration were incubated with antibodies against individual components of NF-κB complex for 30 min before to EMSA analysis. (c) Western blot analysis of nuclear protein fraction shows a time-dependent migration of p50 and p65 into the nucleus of colon tissues in TNBS-induced colitis. Oral administration of TFDG (5 mg kg⁻¹ day⁻¹) markedly suppressed the TNBS-induced nuclear transmigration. The blots were analyzed densitometrically and are normalized to β-actin. The results are representative of duplicate samples of three separate experiments and the densitometric evaluations are means of three independent experiments. ^*P<0.01; ^**P<0.001 vs control.

Figure 7

Effect of TFDG pretreatment on IκBα protein expression and IKK activation in the colon tissue of mice with TNBS-induced colitis. (a) Western blot analysis shows a time dependent increased preservation of IκBα protein in TFDG (5 mg kg⁻¹ day⁻¹) pretreated groups as opposed to depletion in TNBS groups. The blots were analyzed densitometrically and the values are normalized to β-actin. Results are representative of one of three independent experiments. (b) Whole cell extracts were prepared and immunoprecipitated with antibodies against IKKβ and IKKα. Activity of immunoprecipitated IKK was measured using GST-IκBα as substrate and GST phosphorylated-IκBα was visualized by autoradiography. Relative amounts of IKKα and IKKβ in the whole cell extracts were determined by Western blots.