Protective effect of naringenin on acetic acid-induced ulcerative colitis in rats

Abstract

Aim: To evaluate the ameliorative effect of naringenin (NG) during ulcerative colitis (UC) in rats.

Methods: Rats were treated with three different doses (25, 50 and 100 mg/kg per day) of NG and a single dose of mesalazine (MES, 300 mg/kg per day) for seven days prior to ulcerative colitis induction by 4% acetic acid (AA). Twenty four hours after AA rectal administration, animals were scarified and the colonic tissues were dissected. Colonic mucus content was estimated using Alcian blue dye binding technique. In colon tissues, levels of total glutathione sulphadryls (T-GSH), non-protein sulphadryls (NP-SH) and thiobarbituric acid reactive substances (TBARS) were evaluated. The activities of the antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD) were measured. Concentrations of nucleic acids (DNA and RNA) and total protein were also estimated in colon tissues. Colonic levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and nitric oxide (NO) were estimated. In cross section of colitis tissue the histopathological changes were observed.

Results: Colonic mucus content was decreased in AA compared to controls (587.09 ± 65.59 mg/kg vs 941.78 ± 68.41 mg/kg, P < 0.001). AA administration markedly reduced T-GSH (5.25 ± 0.37 nmol/L vs 3.04 ± 0.24 nmol/L, P < 0.01), NP-SH (3.16 ± 0.04 nmol/L vs 2.16 ± 0.30 nmol/L, P < 0.01), CAT (6.77 ± 0.40 U/mg vs 3.04 ± 0.2 U/mg, P < 0.01) and SOD (3.10 ± 0.11 U/mg vs 1.77 ± 0.18 U/mg, P < 0.01) while TBARS, TNF-α, IL-1β, IL-6, PGE2 and NO levels (15.09 ± 3.84 nmol/L vs 59.90 ± 16.34 nmol/L, P < 0.01; 113.56 ± 1.91 pg/mg vs 134.24 ± 4.77 pg/mg, P < 0.01; 209.20 ± 36.38 pg/mg vs 422.19 ± 31.47 pg/mg, P < 0.01; 250.83 ± 25.09 pg/mg vs 638.58 ± 115.9 pg/mg, P < 0.01; 248.19 ± 36.98 pg/mg vs 541.74 ± 58.34 pg/mg, P < 0.01 and 81.26 ± 2.98 mmol/g vs 101.90 ± 10.73 mmol/g, P < 0.001) were increased in colon of rats with UC compared controls respectively.Naringenin supplementation, significantly and dose dependently increased the colonic mucus content. The elevated TBARS levels were significantly decreased (39.35 ± 5.86 nmol/L, P < 0.05; 26.74 ± 3.17 nmol/L, P < 0.01 nmol/L and 17.74 ± 2.69 nmol/L, P < 0.01) compared to AA (59.90 ± 16.34 nmol/L) group while the decreased levels of T-GSH and NP-SH and activities of CAT and SOD found increased by NG treatments in dose dependent manner. The decreased values of nucleic acids and total protein in AA group were also significantly (P < 0.01) increased in all three NG supplemented groups respectively. NG pretreatment inhibited the TNF-α levels (123.76 ± 3.76 pg/mg, 122.62 ± 3.41 pg/mg and 121.51 ± 2.61 pg/mg vs 134.24 ± 4.78 pg/mg, P < 0.05) compared to AA group, respectively. Interleukins, IL-1β and IL-6 levels were also decreased in NG50 + AA (314.37 ± 16.31 pg/mg and 292.58 ± 23.68 pg/mg, P < 0.05) and NG100 + AA (416.72 ± 49.62 pg/mg and 407.96 ± 43.87 pg/mg, P < 0.05) when compared to AA (352.46 ± 8.58 pg/mg and 638.58 ± 115.98 pg/mg) group. Similar decrease (P < 0.05) was seen in PGE2 and NO values when compared to AA group. The group pretreated with MES, as a reference drug, showed significant (P < 0.01) protection against the changes induced in colon tissue by AA administration respectively.

Conclusion: In present study, NG produced antioxidant and anti-inflammatory effects demonstrating protective effect in inflammatory bowel disease.

Keywords: Inflammatory bowel disease; Naringenin; Oxidative stress; Ulcerative colitis.

Figure 1

Effect of naringenin on colon weight/length (A), mucus concentration (B) and induction of ulceration and its protection by treatments in colonic tissue of rats with acetic acid-induced ulcerative colitis (C) (n = 6). Values in (A) and (B) are expressed as mean ± SE and analyzed using one way analysis of variance followed by Newman-Keuls post hoc test. ^bP < 0.01 control vs acetic acid (AA); ^cP < 0.05, ^dP < 0.01 AA vs naringenin (NG) 25 + AA, NG50 + AA, NG100 + AA or mesalazine (MES) + AA groups. Groups in (C) are arranged as follows: control (1), AA (2), NG25 + AA (3), NG50 + AA (4), NG100 + AA (5) and MES + AA (6).

Figure 2

Histopathological changes with their intensity are presented. A-1 and 2: Histopathological colonic sections showing normal benign looking mucosa; B-1 and 2: Diffused active colitis with superficial erosions, stormal edema, dense acute and chronic inflammatory cells infiltrate with widely ulcerating mucosa; C-1 and 2: Reparative epithelial changes with little ulcer healing and inflammatory cells infiltrate; D-1 and 2: Reparative epithelial changes and healing ulcer with lymphoid follicle form; E-1 and 2: Healing ulcer and reparative epithelial changes; F-1 and 2: Attenuated cell damage with complete ulcer healing. A1-F1 (× 400), A2-F2 (× 200).

Figure 3

Effect of naringenin on total glutathione sulphadryls (A), non-protein sulphadryls (B) and thiobarbituric acid reactive substances levels (C) as well as catalase (D) and superoxide dismutase activities (E) in colonic tissue of rats with acetic acid-induced ulcerative colitis (n = 6). Values are expressed as mean ± SE and analyzed using one way analysis of variance followed by Newman-Keuls post hoc test. ^bP < 0.01 control vs AA; ^cP < 0.05, ^dP < 0.01 AA vs NG25 + AA, NG50 + AA, NG100 + AA or MES + AA groups. T-GSH: Total glutathione sulphadryls; NP-SH: Non-protein sulphadryls; TBARS: Thiobarbituric acid reactive substances; CAT: Catalase; SOD: Superoxide dismutase; AA: Acetic acid; UC: Ulcerative colitis; MES: Mesalazine; NG: Naringenin.

Figure 4

Effect of naringenin on tumor necrosis factor-α (A), interleukin-1β (B), interleukin-6 (C), prostaglandin E₂ (D) and nitric oxide (E) levels in colonic tissue of rats with acetic acid-induced ulcerative colitis (n = 6). Values are expressed as mean ± SE and analyzed using one way analysis of variance followed by Newman-Keuls post hoc test. ^bP < 0.01 control vs AA; ^cP < 0.05, ^dP < 0.01 AA vs NG25 + AA, NG50 + AA, NG100 + AA or MES + AA groups. TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β; IL-6: Interleukin-6; PGE₂: Prostaglandin E₂; AA: Acetic acid; NG: Naringenin; MES: Mesalazine.