Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis

Abstract

Activation of the TNF-α receptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Expression of TNFR1 and TNFR2 was measured by quantitative RT-PCR and western blotting. The effect of PFB on colitis was evaluated by examining the inflammatory response and intestinal epithelial barrier function. Our results showed that both TNFR1 and TNFR2 expression were significantly increased in a colitis model, and the increase was significantly reversed by PFB. Colitis symptoms, including infiltration of inflammatory cells, cytokine profiles, epithelial cell apoptosis, and epithelial tight junction barrier dysfunction were significantly ameliorated by PFB. Compared with fruit bromelain and stem bromelain complex, the inhibition of TNFR2 induced by PFB was stronger than that exhibited on TNFR1. These results indicate that PFB showed a stronger selective inhibitory effect on TNFR2 than TNFR1. In other words, purification of fruit bromelain increases its selectivity on TNFR2 inhibition. High expression of epithelial TNFRs in colitis was significantly counteracted by PFB, and PFB-induced TNFR inhibition ameliorated colitis symptoms. These results supply novel insights into potential IBD treatment by PFB.

Keywords: TNF-α receptor; bromelain; cytokines; inflammation; inflammatory bowel disease; myosin light chain kinase; purification; tight junction.

Figure 1

Toxicity of purified fruit bromelain (PFB) in normal cells and rats. (A) Cytotoxicity of PFB was studied by MTT assay in the intestinal epithelial cell line IEC-6. PFB was administered to rats by gavage once daily for 14 consecutive days (n = 6). Expression levels of the colonic cytokines TNF-α (B), IL-1β (C), and IL-8 (D) were examined by enzyme-linked immunosorbent assay, and expression levels of mRNA and protein of TNFR1 and TNFR2 (E,F) were examined by quantitative real-time PCR and western blotting, respectively (n = 6). Data are expressed as the mean ± SD. Values in the normal control (NC) group are set to 100%, and other values are given relative to the NC group.

Figure 2

Purified fruit bromelain (PFB) alleviated colitis symptoms. Colitis symptoms were examined after 14 days of PFB therapy. (A) Hematoxylin and eosin -staining of rat colonic tissue (20×, scale bar is 100 µm). Effects of PFB on (B) body weight, (C) food intake, (D) macroscopically visible damage, and (E) colon weight-to-length ratio in colitis rats. Data in the sham group are set to a relative value of 100% and expressed as the mean ± SD. Other data are the relative values compared with sham: **P < 0.01 compared with sham group (n = 6 rats); ^##P < 0.01 compared with TNBS control group (n = 6 rats). SASP, sulfasalazine.

Figure 3

Purified fruit bromelain (PFB) attenuated neutrophil infiltration and cytokine profiles. Effects of PFB on (A) myeloperoxidase (MPO), (B) TNF-α, (C) IL-1β, and (D) IL-8 in rat colon tissue determined by enzyme-linked immunosorbent assay. Data are expressed as the mean ± SD; **P < 0.01 compared with sham group; ^##P < 0.01 compared with TNBS control group (n = 6 rats). SASP, sulfasalazine.

Figure 4

Purified fruit bromelain (PFB) reversed the increased intestinal permeability. After 14 days of PFB therapy, the intestinal permeability in each group was examined through testing serum recovery of FD-4 in vivo (A) and transepithelial electrical resistance (TER) in Caco-2 cells (B) (n = 6). Cells were incubated with 100 ng/mL lipopolysaccharide (LPS) for 24 h in the presence or absence of PFB. Data in the sham group or normal control (NC) cell group are set to a relative value of 100% and expressed as the mean ± SD. Other data are the relative values compared with sham or NC: **P < 0.01 compared with sham or NC group; ^##P < 0.01 compared with TNBS control or LPS control group.

Figure 5

Potential mechanisms underlying purified fruit bromelain (PFB) induced therapeutic effects on colitis. Colonic segments were isolated after 14 days of PFB treatment. Quantitative real-time PCR analysis of TNF-α receptor (TNFR) mRNA (A); Western blotting analysis of TNFR protein (B–D), inflammation marker NF-κB (B,E), tight junction barrier related protein myosin light chain kinase (MLCK) (B,F) and (B,G), apoptosis-related proteins Bax (B,H) and Bcl-2 (B,I). Data are expressed as the mean ± SD. Values in the sham group are set to 100% and other values are given relative to those in the sham group: **P < 0.01 compared with sham group; ^#P < 0.05 and ^##P < 0.01 compared with TNBS control group (n = 6 rats).

Figure 6

Downregulation of TNFR2 in purified fruit bromelain (PFB)-induced inhibition of inflammation and epithelial barrier dysfunction. Effects of PFB on expression of TNFR2, NF-κB and MLCK in the presence of siRNA targeting TNFR2. (A) Representative Western blotting image for effects of PFB on expression of TNFR2, NF-κB and MLCK; statistical analysis of PFB on the expression of TNFR2 (B), NF-κB (C), and MLCK (D). Representative Western blotting image for effects of PFB on NF-κB and MLCK. Data are expressed as the mean± SD. Values are given relative to normal control (NC) in si-control (100%) and other values are given relative to the NC group: **P < 0.01 compared with NC; ^##P < 0.01 compared with corresponding LPS control.

Figure 7

Downregulation of TNFR1 in purified fruit bromelain (PFB)-induced inhibition of inflammation and epithelial barrier dysfunction. Effects of PFB on expression of NF-κB and myosin light chain kinase (MLCK) in the presence of small interfering RNA targeting TNFR1. Data are expressed as the mean ± SD. Values are given relative to LPS-treated group (control, 100%) and other values are given relative to the control: **P < 0.01 compared with LPS control.

Figure 8

Comparative study of purified fruit bromelain (PFB) with bromelain complex. Western blotting analysis of the effects of purified PFB (A), stem bromelain complex (B), and fruit bromelain complex (C) on the protein expression of TNFR1 and TNFR2. Data are expressed as the mean ± SD. Values in normal control (NC) group are set to 100% and other values are given relative to those in the NC group: **P < 0.01 compared with the NC group; ^#P < 0.05 and ^##P < 0.01 compared with LPS control group; ^&&P < 0.01 compared with as indicated (n = 6 experiments).