Peptide YY 3-36 attenuates trinitrobenzene sulfonic acid-induced colitis in mice by modulating Th1/Th2 differentiation

Abstract

Peptide YY (PYY) 3-36, the main circulatory form of PYY, plays important roles in gastrointestinal motility, secretion, and absorption. However, it is unknown whether PYY 3-36 has underlying functions in colitis. The Crohn's disease (CD)-like mouse model in which CD is induced by trinitrobenzene sulfonic acid (TNBS) was established and utilized to investigate this potential role for PYY 3-36. The results showed that the expression of colonic mucosal PYY and PYY receptors Y1, Y2, Y4 were significantly increased in mice with TNBS-induced colitis. In vitro, PYY 3-36 remarkably inhibited the production of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from lipopolysaccharide (LPS)-induced macrophages. In vivo, a high concentration of PYY 3-36 robustly decreased the weight loss and death rate and attenuated the pathological colon tissue damage observed in mice with TNBS-induced colitis. Further studies uncovered that PYY 3-36 treatment reduced the levels of colon myeloperoxidase (MPO) and both colonic and systemic TNF-α and IL-6 observed in murine colitis. Furthermore, flow cytometric analysis showed PYY 3-36 altered the proportion of Th1/Th2 splenocytes in the disease model of colitis. Collectively, these results suggest that PYY 3-36 may be a promising candidate for the improvement of colitis, reflected by the attenuation of colon inflammatory responses observed in experimental murine colitis.

Keywords: PYY 3–36; Th1/Th2 cells; colitis; inflammation; trinitrobenzene sulfonic acid.

Graphical abstract

Figure 1.

Expression of PYY and PYY receptors in mice with TNBS-induced colitis. Balb/c mice (n = 6 for each group) were used to establish a model of colitis induced by intracolonic administration of TNBS (2 mg per 20 g of body weight) mixed with 50% ethanol. All mice were sacrificed on day 3 after administration of TNBS. The colon mucosa was collected to extract its RNA. (a) The expression for PYY and its receptors Y1, Y2, Y4, Y5, and Y6 was measured using quantitative PCR. (b) The brightness of the PCR products was scanned using Quality One software (Data are presented as Mean ±SD). (c) The expression of PYY proteins in the colon mucosa was determined by ELISA. Each dot represents one mouse. n ≥ 5 mice per group; *, p < 0.05; **, P < 0.01; ***, P < 0.001 versus control mice.

Figure 2.

PYY 3–36 treatment improves the weight loss and death rate of mice with TNBS-induced colitis. Mice treated with 50% ethanol in PBS were used as the vehicle control. Colitis was induced by TNBS (2 mg per 20 g of body weight) at day 0. After 12 h, mice received intraperitoneal injections of 0.1 nM or 10 nM PYY 3–36. (a) All experimental Balb/c mice were weighed every day and sacrificed on day 10. (b) Weight change normalized to weight data on day 0 and (c) survival rate was calculated to assess the disease progression. Three individual experiments were performed n ≥ 5 mice in each group.

Figure 3.

PYY 3–36 treatment abrogates the development of TNBS-induced colitis. Mice treated with 50% ethanol in PBS were used as the vehicle control. Colitis was induced by TNBS (2 mg per 20 g of body weight) at day 0. After 12 h, mice received intraperitoneal injections of 0.1 nM or 10 nM PYY 3–36. (a) All experimental Balb/c mice were weighed every day and sacrificed on day 3. Clinical evolution and severity were assessed by (b) macroscopic observation, (c) colon length, (f) histologic score of (d) hematoxylin and eosin-stained sections, and (e) myeloperoxidase (MPO) level in colon tissues at day 3 after TNBS administration (Original magnification 20×) *, p < 0.05; **, p < 0.01 versus the mice injected with TNBS.

Figure 4.

PYY 3–36 treatment reduces the level of serum TNF-α and IL-6 in mice with TNBS-induced colitis. Colitis was caused by TNBS via intracolonic administration. Mice treated with 50% ethanol in PBS as the vehicle control. Mice were injected i.p. with 0.1 nM or 10 nM PYY 3–36 12 h after TNBS instillation. Serum was obtained from the collected blood after sacrificing control and experimental mice at peak of disease progression on day 3. TNF-α and IL-6 were determined by ELISA. *, p < 0.05 **, P < 0.01 versus the mice injected with TNBS.

Figure 5.

PYY 3–36 inhibited the production of TNF-α and IL-6 in the colons of mice with colitis. Mice treated with 50% ethanol in PBS were used as the vehicle control. Colitis was induced by TNBS (2 mg per 20 g of body weight) at day 0. After 12 and 36 h, respectively, mice received i.p. injections of 10 nM PYY 3–36. After 3 d, mice were sacrificed and the colon tissues were collected. The tissues were used for the measurement and gene expression of TNF-α and IL-6 by ELISA (a and b) or PCR (c and d). *, p < 0.05 **, P < 0.01 ***, P < 0.001 ****, P < 0.0001 versus TNBS-injected mice.

Figure 6.

Modulations of IFN-γ- or IL-4-producing CD4⁺ T cells induced by PYY 3–36. Splenocytes were isolated from normal mice with or without pretreatment with 10 nM PYY 3–36 for 2 h and from TNBS-injected mice with or without pretreatment with 10 nM PYY 3–36. The splenocytes were ex vivo stimulated with PMA (20 ng/ml), ionomycin (1 μg/ml), and brefeldin A (1 μg/ml). Flow cytometry was performed to quantify CD4⁺ T cells producing IFN-γ (a) and IL-4 (b).