Efficacy of Sishen Wan on dinitrobenzene sulfonic acid-induced ulcerative colitis and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB signal pathway

Abstract

Objective: To investigate the therapeutic effect of Sishen Wan (, SSW) on ulcerative colitis (UC) induced by dinitrobenzene sulfonic acid and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB (TLR2/IRAK4/NF-κB) sig-naling pathway in colonic tissue.

Methods: In this study, 120 Sprague-Dawley rats were randomly divided into blank and model groups. The experimental UC model in rats was established by subcutaneous injection of hydrocortisone + senna gavage for 21 d + dinitrobenzene sulfonic acid (DNBS)/ ethanol solution enema. The successful model rats were randomly divided into the model group; mesalazine (0.36 g/kg) group; and high-, medium-, and low- dose SSW (24, 12, and 6 g/kg) groups. The model and blank groups were gavaged with equal volumes of distilled water once a day for 21 d. The general condition of the rats was observed, and the body mass, fecal properties, and occult blood were recorded for calculating the disease activity index (DAI) score. The colonic tissue of the rats was collected, and its general morphology and pathological form were noted for obtaining the colonic mucosal injury index (CMDI) score. Hematoxylin-eosin staining was used to view the pathological changes of the colon tissue in each group, apoptosis of the cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and quantitative real-time polymerase chain reaction was used to measure the expressions of TLR2, myeloid differentiation primary response gene 88 (MyD88), IRAK4, and NF-κB p65 mRNA in the colon tissue. The expressions of TLR2, MyD88, IRAK4, and NF-κB p65 protein were detected using western blotting and immunohistochemistry assay, and the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the colon tissue were determined using enzyme linked immunosorbent assay.

Results: Compared with the blank group, the general condition of the model group was relatively poor. The DAI and CMDI scores of the model group increased significantly (< 0.01), the glands and intestinal mucosa disappeared partially, and several inflammatory cells infiltrated and gathered in the mucosal layer and base layer of the rats in the model group. Furthermore, the cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of rats in the model group increased significantly (< 0.01). The levels of IL-1β and TNF-α increased significantly in the colon tissue of rats in the model group (< 0.01). After treatment with SSW, compared with the model group, the general condition of the UC rats improved. Moreover, the DAI and CMDI scores of the UC rats decreased significantly (< 0.05), and the pathological changes in the colon tissue of the UC rats tended to be normal. The cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of the UC rats decreased gradually ( < 0.01), and the levels of IL-1β and TNF-α decreased significantly (< 0.01).

Conclusion: SSW can improve the general condition and alleviate the intestinal mucosal injury of UC model rats. Additionally, SSW can inhibit the TLR2/IRAK4/ NF-κB signaling pathway, but further studies are required to confirm it.

Keywords: NF-kappa B; Sishen Wan; colitis, ulcerative; interleukin-1 receptor-associated kinases; signal transduction; therapeutic uses; toll-like receptor 2.

Figure 1. Effects of SSW on body weight, DAI score and CMDI score of UC model rats

A: body weight in each groups; B: CMDI scores in each groups; C: DAI scores in each group. Blank: the normal rats administered with an equal volume of distilled water via oral gavage once a day for 21 d. Model: UC rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Mesalazine: UC rats administered with 0.36 g/kg mesalazine via oral gavage once a day for 21 d; High-, Medium-, Low-dose SSW: UC rats administered with SSW via oral gavage at the doses of 24, 12, and 6 g/kg, respectively. SSW: Sishen Wan; UC: ulcerative colitis; CMDI: colonic mucosal damage index; DAI: disease activity index. ^aP < 0.05 vs Blank group; ^bP < 0.05 vs Model group.

Figure 2. SSW improved the pathological lesions of colon tissue in UC model rats (hematoxylin-eosin staining)

A: the pathological lesions of colon tissue in each groups rats observed in under 40× light microscope; B: the pathological lesions of colon tissue in each groups rats observed in under 100× light microscope. The black arrow indicates infiltrating inflammatory cells and the red arrow indicates damaged intestinal mucosa. Blank: the normal rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Model: UC rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Mesalazine: UC rats administered with 0.36 g/kg mesalazine via oral gavage once a day for 21 d; High-, Medium-, Low-dose SSW: UC rats administered with SSW via oral gavage at the doses of 24, 12, and 6 g/kg, respectively. SSW: Sishen Wan; UC: ulcerative colitis.

Figure 3. SSW alleviated the apoptosis of colonic cells in UC model rats

A: apoptotic cells (Green) in colon tissue were detected through TUNNEL staining (× 200); B: statistically analyzed by Image J software and GraphPad Prism 7.0. Blank: The normal rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Model: UC rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Mesalazine: UC rats administered with 0.36 g/kg mesalazine via oral gavage once a day for 21 d; High-, Medium-, Low-dose SSW: UC rats administered with SSW via oral gavage at the doses of 24, 12, and 6 g/kg, respectively. SSW: Sishen Wan; UC: ulcerative colitis, TUNNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. aP < 0.05 vs Blank group; bP < 0.05 vs Model group.

Figure 4. SSW reduced the level of IL-1 and TNF-α in colon tissues of UC model rats (ELISA assay)

Blank: The normal rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Model: UC rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Mesalazine: UC rats administered with 0.36 g/kg mesalazine via oral gavage once a day for 21 d; High-, Medium-, Low-dose SSW: UC rats administered with SSW via oral gavage at the doses of 24, 12, and 6 g/kg, respectively. SSW: Sishen Wan; UC: ulcerative colitis, IL-1β: interleukin-1β; TNF-α: tumor necrosis factor-α. ^aP < 0.05 vs Blank group; ^bP < 0.05 vs Model group.

Figure 5. SSW reduced the level of TLR2, MyD88, IRAK4 and NF-κB p65 mRNAs in colon tissue of UC model rats, which were detected by qPCR

Blank: the normal rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Model: UC rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Mesalazine: UC rats administered with 0.36 g/kg mesalazine via oral gavage once a day for 21 d; High-, Medium-, Low-dose SSW: UC rats administered with SSW via oral gavage at the doses of 24, 12, and 6 g/kg, respectively. SSW: Sishen Wan; UC: ulcerative colitis; qPCR: quantitative real-time polymerase chain reaction; TLR2: toll-like receptor 2; MyD88: myeloid differentiation primary response gene 88; IRAK4: interleukin-1 receptor-associated kinase-4; NF-κB p65: nuclear factor-κB p65. ^aP < 0.05 vs Blank group; ^bP < 0.05 vs Model group.

Figure 6. SSW reduced the level of TLR2, MyD88, IRAK4 and NF- κB p65 proteins in colon tissue of UC model rats

Immunohistochemistry (A, × 200) and Western Blotting assay (C) were used to detect the protein expression in colon tissue, respectively. The immunohistochemical images and Western Bloting bands were semi-quantitatively analyzed by Image J software (C, D). Blank: The normal rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Model: UC rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Mesalazine: UC rats administered with 0.36 g/kg mesalazine via oral gavage once a day for 21 d; High-, Medium-, Low-dose SSW: UC rats administered with SSW via oral gavage at the doses of 24, 12, and 6 g/kg, respectively. SSW: Sishen Wan; UC: ulcerative colitis; TLR2: toll-like receptor 2; MyD88: myeloid differentiation primary response gene 88; IRAK4: interleukin-1 receptor-associated kinase-4; NF-κB p65: nuclear factor-κB p65. ^aP < 0.05 vs Blank group; ^bP < 0.05 vs Model group.

Figure 7. SSW reduced the level of NF-κB p65 protein in the nucleus of colon tissue of UC model rats

Western Blotting assay (A) were used to detect the protein expression, and the intensities of Western Bloting bands were semi-quantitatively analyzed by Image J software (B). Blank: The normal rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Model: UC rats administered with an equal volume of distilled water via oral gavage once a day for 21 d; Mesalazine: UC rats administered with 0.36 g/kg mesalazine via oral gavage once a day for 21 d; High-, Medium-, Low-dose SSW: UC rats administered with SSW via oral gavage at the doses of 24, 12, and 6 g/kg, respectively. SSW: Sishen Wan; UC: ulcerative colitis; NF-κB p65: nuclear factor-κB p65; p-NF-κB: phosphorylated NF-kB. ^aP < 0.05 vs Blank group; ^bP < 0.05 vs Model group.