Novel insights on the effect of nicotine in a murine colitis model

Abstract

Studies showed that nicotine has a positive influence on symptoms of ulcerative colitis. In the present study, we explored the effect of nicotine treatment using different routes of administration in the dextran sodium sulfate (DSS) colitis mouse model. We also investigated the effects of cotinine, a major metabolite of nicotine, in the model. C57BL6 adult male mice were given DSS solution freely in the drinking water for seven consecutive days, and tap water was given thereafter. Disease severity, length of the colon, colon tissue histology, and inflammatory markers, including colonic myeloperoxidase activity and colonic tumor necrosis factor-α levels, were evaluated. The effect of nicotine and cotinine treatments via various different routes of administration were examined the DSS model. In addition, we measured the plasma levels of nicotine and cotinine in our treatment protocols. Administration of low, but not high, doses of oral nicotine in DSS-treated mice resulted in a significant decrease in disease severity, histologic damage scores, as well as colonic level of tumor necrosis factor-α. However, the anti-inflammatory effect of nicotine was not seen after chronic s.c. or minipump infusion of the drug. Differences in plasma levels of nicotine and cotinine do not seem to account for this lack of effect. Finally, oral cotinine alone failed to show a significant effect in the DSS model of colitis. These results highlight that dose and route of administration play a critical role in the protective effect of nicotine in the DSS mouse colitis model.

Fig. 1.

Colonic inflammation was aggravated after DSS exposure to mice in a dose-related manner. (A) DAI changes among groups exposed to different doses of DSS. (B) AUC of DAI for mice in (A). (C) Percentage of body weight change for mice in (A). (D) Mean colon length (cm) for mice in (A). C57BL/6 mice were given different doses of DSS (1, 2.5, and 5%) in the drinking water for 7 days. All clinical signs were assessed on a daily basis for each mouse and were averaged per day for each group. Results are expressed as mean ± S.E. (n = 6–8). *P < 0.05. Ctrl, water-treated animals.

Fig. 2.

Aggravation of inflammatory markers after 7 days of exposure to different doses of DSS in mice. (A) Colonic MPO activity (midpoint titer/g wet tissue) in groups exposed to different doses of DSS (0, 1, 2.5, 5%). (B) Colonic TNF-α level (pg/mg protein) for mice in (A). Results are expressed as mean ± S.E. (n = 6–8). *P < 0.05. Ctrl, water-treated group.

Fig. 3.

Histologic scores from colons after 7 days of exposure to water or different doses of DSS. (A) Loss of epithelium. (B) Crypt damage. (C) Depletion of goblet cells. (D) Infiltration of inflammatory cells. (E) Total histologic damage score. Histologic scores were calculated blindly, using H&E-stained colonic tissue sections, after 7 days in the water-treated control group (Ctrl) and DSS-treated animals. Results are expressed as mean ± S.E. (n = 3). *P < 0.05.

Fig. 4.

Histologic analysis of representative colons from mice after 7 days of exposure to water or different doses of DSS. (A) Appearance of colon from water-treated control. (B) Appearance of colon treated with 1% DSS. (C) Appearance of colon treated with 2.5% DSS. (D) Appearance of colon treated with 5% DSS. H&E stain; original magnification, 40×. Scale bar, 20 μm.

Fig. 5.

Oral nicotine treatment suppressed the severity of DSS-induced colitis in mice. Effects of various doses (6–100 μg/ml) of chronic oral nicotine (Nic) treatment on the time course of DAI (A), AUC of DAI (B), percentage of body weight change (C), and mean colon length (cm) (D). Results are expressed as mean ± S.E. (n = 6–8). *P < 0.05, compared with control (vehicle-treated mice).

Fig. 6.

Oral nicotine treatment (25 and 100 μg/ml) affects colonic TNF-α levels, histologic damage score, and appearance in DSS-induced colitis. (A) TNF-α levels (pg/mg protein) in homogenized colonic tissue samples. Results are expressed as mean ± S.E. (n = 6–8). (B) Histologic colonic damage score in DSS-induced colitis. Results are mean ± S.E. (n = 3–5 per group). Representative colon sections at 7 days after induction of colitis in a control mouse (C), DSS-treated mouse (D), 12.5 μg/ml nicotine–treated mouse (E), 25 μg/ml nicotine–treated mouse (F), and 100 μg/ml nicotine–treated mouse (G). H&E stain; original magnification 40×. Scale bar, 20 μm. *P < 0.05, versus DSS group, #P < 0.05, versus water control group. Ctrl, water-treated group; Nic, nicotine.

Fig. 7.

Effect of chronic s.c. nicotine administration on the severity of DSS-induced colitis in mice. Effects of chronic s.c. nicotine treatment (0.1, 0.5, and 2 mg/kg) on the time course of DAI (A), AUC of DAI (B), mean colon length (cm) (C), and histologic colonic damage score (D). (E) Colonic TNF-α level (pg/mg protein) for mice in (A). Results are expressed as mean ± S.E. (n = 6–8). *P < 0.05, versus control (vehicle-treated mice). Nic, nicotine; Veh, vehicle.

Fig. 8.

Influence of nicotine infusion via minipump on DAI, histologic damage, and colon length in DSS-treated mice. Effects of chronic minipump infusion of nicotine (2.5 or 25 mg/kg per day) on the time course of DAI (A), AUC of DAI (B), mean colon length (cm) (C), and histologic colonic damage score (D). (E) Colonic TNF-α level (pg/mg protein) for mice in (A). Results are expressed as mean ± S.E.M. (n = 6–8). *P < 0.05, compared with control (vehicle-treated mice). #P <0.05, compared with water group. MP, minipump; Nic, nicotine; Veh, vehicle.

Fig. 9.

Lack of suppression of colitis severity by oral cotinine. Effects of oral cotinine (25 or 250 µg/ml) on the time course (A) and AUC (B) of DAI, as well as mean colon length (cm) (C), in mice. Results are expressed as mean ± S.E.M. (n = 6–8). *P < 0.05, compared with control (water-treated mice).