Andrographolide derivative AL-1 ameliorates TNBS-induced colitis in mice: involvement of NF-кB and PPAR-γ signaling pathways

Abstract

Andrographolide is a traditional herb medicine, widely used in Asia for conditions involving inflammation. The andrographlide-lipoic acid conjugate, AL-1, has been found being able to alleviate inflammation in our previous reports. Although the anti-inflammatory activity of AL-1 contributes to its cytoprotective effects, whether AL-1 can improve inflammatory bowel disease (IBD) and the underlying mechanisms of its action remain largely unknown. In this study, we investigated the anti-inflammatory effects of AL-1 in C57BL/6 mice with trinitrobenzenesulfonic acid (TNBS)-induced colitis. The body weight loss and length change of colon after TNBS instillation were more severe than those in normal mice. AL-1 treatment led to significant reductions in disease activity index (DAI), macroscopic score and colon mucosa damage index (CMDI) associated with TNBS administration. AL-1 inhibited the inflammatory response via lowering the level of inflammatory cytokines and myeloperoxidase (MPO) activity. AL-1 attenuated the expression of p-p65, p-IκBα and COX-2 in the colitis mice. The alleviation of colon injury by AL-1 treatment was also evidenced by the increased expression of PPAR-γ. These results indicated that AL-1 could protect intestinal tract from the injury induced by TNBS in mice, suggesting that AL-1 may have potential in treatment for IBD.

Figure 1. Structures of Andro, LA and AL-1.

Figure 2. Effects of AL-1 on colitis induced by TNBS instillation in C57BL/6.

(A) The time-course of body weight changes on day 3 after TNBS-induced colitis. (B) Effects of AL-1 and mesalazine on colon length of TNBS-induced colitis mice. (C) Disease activity index calculated as described in material and methods. (D) Representative photograph of colons from day 3 after the induction of TNBS-colitis. (E) Macroscopic score. Mice were challenged with solvent or TNBS at day 0 and treated with control, AL-1 (5, 15 and 45 mg/kg) or mesalazine (100 mg/kg) for days 0-3. Body weight was measured daily throughout the experiment. Values were shown as the means ± SEM, n = 8 for each group. **P < 0.01 vs control group, ^#P < 0.05 and ^##P < 0.01 vs mice treated with TNBS alone.

Figure 3. Effects of AL-1 on colonic histopathological changes.

(A) Histological appearance of mice colonic mucosa after haematoxylin and eosin (H&E) stain (original magnification 100×). (B) The score of colon mucosa damage index (CMDI) of mice. Mice were challenged with solvent or TNBS at day 0 and treated with control, AL-1 (5, 15 and 45 mg/kg) or mesalazine (100 mg/kg) for days 0–3. After sacrificed at day 3, sections of the colon were collected, embedded in paraffin, and cut into 4 μm of sections and stained with hematoxylin and eosin before examination. Values were shown as the means ± SEM, n = 8 for each group. **P < 0.01 vs control group, ##P < 0.05 vs mice treated with TNBS alone.

Figure 4. Effects of AL-1 on myeloperoxidase (MPO) activity in colonic tissues of mice.

Mice were challenged with solvent or TNBS at day 0 and treated with control, AL-1 (5, 15 and 45 mg/kg) or mesalazine (100 mg/kg) for days 0–3. Protein from colon tissue was extracted and MPO level was determined according to manufacturer’s instructions. Values were shown as the means ± SEM, n = 8 for each group. **P < 0.01 vs control group, ^#P < 0.05 and ^##P < 0.01 vs mice treated with TNBS alone.

Figure 5. Inhibitory effects of AL-1 on the pro-inflammatory cytokine production in serum level of mice.

The production of (A) TNF-α, (B) IL-1β and (C) IL-6, were assayed in colonic tissues of mice. Mice were challenged with solvent or TNBS at day 0 and treated with control, AL-1 (5, 15 and 45 mg/kg) or mesalazine (100 mg/kg) for days 0–3. The TNF-α, IL-1β and IL-6 concentration in the serum level were determined by ELISA. Values were shown as the means ± SEM, n = 8 for each group. **P < 0.01 vs control group, ^#P < 0.05 and ^##P < 0.01 vs mice treated with TNBS alone.

Figure 6. Effects of AL-1 on protein expression of COX-2, p-p65, p-IκBα and PPAR-γ in colon tissues.

(A–D) Mice were challenged with saline or TNBS at day 0 and treated with control, AL-1 (5, 15 and 45 mg/kg) or mesalazine (100 mg/kg) for days 0–3. Values were shown as the means ± SEM, n = 8 for each group. **P < 0.01 vs control group, ^##P < 0.01 vs mice treated with TNBS alone.