Protective Effects of Taurine Chloramine on Experimentally Induced Colitis: NFκB, STAT3, and Nrf2 as Potential Targets

Abstract

Taurine chloramine (TauCl) is an endogenous anti-inflammatory substance which is derived from taurine, a semi-essential sulfur-containing β-amino acid found in some foods including meat, fish, eggs and milk. In general, TauCl as well as its parent compound taurine downregulates production of tissue-damaging proinflammatory mediators, such as chemokines and cytokines in many different types of cells. In the present study, we investigated the protective effects of TauCl on experimentally induced colon inflammation. Oral administration of TauCl protected against mouse colitis caused by 2,4,6-trinitrobenzene sulfonic acid (TNBS). TauCl administration attenuated apoptosis in the colonic mucosa of TNBS-treated mice. This was accompanied by reduced expression of an oxidative stress marker, 4-hydroxy-2-nonenal and proinflammatory molecules including tumor necrosis factor-α, interleukin-6 and cyclooxygenase-2 in mouse colon. TauCl also inhibited activation of NFκB and STAT3, two key transcription factors mediating proinflammatory signaling. Notably, the protective effect of TauCl on oxidative stress and inflammation in the colon of TNBS-treated mice was associated with elevated activation of Nrf2 and upregulation of its target genes encoding heme oxygenase-1, NAD(P)H:quinone oxidoreductase, glutamate cysteine ligase catalytic subunit, and glutathione S-transferase. Taken together, these results suggest that TauCl exerts the protective effect against colitis through upregulation of Nrf2-dependent cytoprotective gene expression while blocking the proinflammatory signaling mediated by NFκB and STAT3.

Keywords: 2,4,6-trinitrobenzene sulfonic acid; NFκB; Nrf2; STAT3; colitis; heme oxygenase-1; taurine; taurine chloramine.

Figure 1

Effects of TauCl on inflammatory damage in the colon of TNBS-treated mice. (A) TauCl (20 mg/kg) was given on daily basis by gavage for 10 days before and for 3 days after intrarectal administration of 2.5% TNBS in EtOH. The comparison of body weight change (B), DAI (C), and colon length (D). Data are expressed as means ± SD (n = 3 for each group). *^, **^, *** Significantly different between groups compared (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2

Attenuation of TNBS-induced colonic cell death, MPO activity, and oxidative damage by administration of TauCl. (A) Microscopic examination of H&E stained colonic mucosa from control mice and those treated with TNBS alone for 3 days, TNBS plus TauCl and TauCl alone. Immunohistochemical detection of TUNEL-positive cells and 4-HNE-modified proteins (brown spots) in mouse colon. Magnifications, ×100; Scale bar, 200 μm. (B) MPO activity in colonic mucosa. The enzyme assay was conducted as described in Materials and Methods. ** Significantly different between groups compared (** p < 0.01). (C) Western blot analysis of 4-HNE-modified protein expression in the colon strips of mice. Actin was used as an equal loading control. * p < 0.05. (D) Cleavage of caspase-3 was assessed by Western blot analysis. * p < 0.05.

Figure 3

Effects of TauCl administration on expression of proinflammatory cytokines and COX-2 in mouse colonic mucosa. (A) RT-PCR analysis of mRNA expression of proinflammatory cyokines (tnf-α, il-6) and cox-2. (B,C) Colonic expression of cox-2 and COX-2 expression was assessed by RT-PCR and Western blot analysis, respectively. Data are expressed as means ± SD (n = 3 for each group). *^, **^, *** Significantly different between groups compared (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Inhibitory effects of TauCl on TNBS-induced p65 phosphorylation and nuclear localization of NFκB. (A) Expression of the phosphorylated NFκB p65 subunit was determined by Western blot analysis using nuclear extracts. (B) Accumulation of the phosphorylated as well as the total form of p65 was measured by immunofluorescence staining. The same tissue sections were stained with H&E. Scale bar, 200 μm. Results are presented as means ± SD. * p < 0.05; ** p < 0.01.

Figure 5

Inhibitory effects of TauCl on phosphorylation of STAT3 and its target protein expression in the colonic tissue of TNBS-treated mice. (A) Expression of P-STAT3 and cyclin D1 was measured by Western blot analysis. Actin was used as an equal loading control. *^, ** Significantly different between groups compared (* p < 0.05; ** p < 0.01). (B) Phosphorylated as well as total STAT3 was detected by immunofluorescence staining. The same tissue sections were stained with H&E. Scale bar, 200 μm.

Figure 6

Upregulation of antioxidant signaling molecules induced by TauCl administration in mouse colon. The expression of ho-1 (A) and its protein product (B) was determined by RT-PCR and Western blot analysis, respectively. (C) Expression of other antioxidant genes and nrf2 was measured by RT-PCR. *^, **^, *** Significantly different between groups compared (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 7

Induction of Keap1 degradation and nuclear translocation of Nrf2 by TauCl in mouse colon. Cytoplasmic levels of Nrf2 and Keap1 (A) and nuclear accumulation of Nrf2 (B) were measured by Western blot analysis. Nuclear localization of Nrf2 was verified by immunofluorescence staining (C). *^, ** Significantly different between groups compared (* p < 0.05; ** p < 0.01). Scale bar, 200 μm.

Figure 8

Molecular mechanisms by which TauCl protects against experimentally induced colitis. Abbreviations: MPO, myeloperoxidase; HOCl, hypochlorous acid.