Therapeutic effects of different doses of polyethylene glycosylated porcine glucagon-like peptide-2 on ulcerative colitis in male rats

Qi et al. BMC Gastroenterology (2017) 17:34 DOI 10.1186/s12876-017-0593-x RESEARCH ARTICLE Open Access Therapeutic effects of different doses of polyethylene glycosylated porcine glucagon-like peptide-2 on ulcerative colitis in male rats Ke-ke Qi, Jia-jia Lv, Jie Wu and Zi-wei Xu* Abstract Background: Polyethylene glycosylated (PEGylated) porcine glucagon-like peptide-2 (pGLP-2) considerably increases half-life and stability compared with the native pGLP-2, but the effective dose for intestinal damage is still unclear. This study aims to evaluate the available dose of polyethylene glycosylated porcine glucagon-like peptide-2 (PEG-pGLP-2), a modified, long-acting form of pGLP-2 in an experimental rat model of ulcerative colitis. Methods: Thirty-five male rats were randomly assigned into five groups: control, dextran sodium sulphate (DSS), DSS + PEG–pGLP-2(L), DSS + PEG–pGLP-2(M) and DSS + PEG–pGLP-2(H). Rats in control group received only water; other rats were fed with 5% (w/v) DSS and intraperitoneally administered with 12.5, 25 and 100 nmol/kg PEG–pGLP-2 daily for 6 days. Results: Compared with the control treatment, DSS treatment significantly (p < 0.05) decreased body weight change, colonic length, duodenal villus height and expression of zonula occludens-1, whereas significantly (p < 0.05) increased colonic damage score and expression of claudin-1, interleukin (IL)-1, IL-7, IL-10, interferon-γ and tumour necrosis factor (TNF)-α in colon. However, the three doses of PEG–pGLP-2 all reduced these effects; these treatments significantly (p < 0.05) increased body weight change and duodenal villus height, whereas significantly (p < 0.05) decreased colonic damage score and expression of IL-1, IL-7 and TNF-α in colon. Specifically, low-dose (12.5 nmol/kg/d) PEG–pGLP-2 was effective. Conclusions: These results indicated that PEG–pGLP-2 is a novel and potentially effective therapy for intestinal healing in a relatively low dose. Keywords: Porcine glucagon-like peptide-2, Ulcerative colitis, Tight junction, Inflammation cytokines Background Frequent injection of porcine glucagon-like peptide-2 (pGLP-2) because of the rapid degradation of pGLP-2 by the enzyme dipeptidyl peptidase-IV (DPP-IV) is the main impediment of pGLP-2 as a potential therapeutic agent for intestinal dysfunction and damage in weaning piglets [1]. For the first time, our team designed and purified polyethylene glycosylated (PEGylated) pGLP-2 (PEG-pGLP-2), whose half-life (t1/2) is 16-fold longer than that of pGLP-2 in DPP-IV in vitro [2]. PEG-pGLP* Correspondence: Institute of Animal Science, Zhejiang Academy of Agricultural Sciences, 198 Shiqiao Road, Jianggan, Hangzhou 310021, China 2 infusion could alleviate the severity of intestinal injury in weaning piglets [3, 4]. The t1/2 of teduglutide, a GLP2 analogue, which is a candidate for the treatment of short bowel syndrome (SBS) in two phase III clinical studies [5], is 2.99 h in male patient [6]. Recently, our team proved that the t1/2 of PEG-pGLP-2 is 3.38 h in male rats [7]. These findings provide a new perspective to the potential therapeutic use of PEG-pGLP-2 for weaning and diarrhoeal diseases in young pigs. However, no reports about the therapeutic effect of PEG-pGLP-2, except that of our team, are available. Little information is also known about the dose and frequency of administration with PEG-pGLP-2. Thymann © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Qi et al. BMC Gastroenterology (2017) 17:34 et al. [8] found that injection of acylated GLP-2 analogue 25 μg/(kg BW · 12 h) for 5 days increases intestinal weight and activity of brush border enzymes in weaned pigs when only using long-acting analogues or under diarrhoeal conditions. Kaji et al. [9] demonstrated high doses (240 μg/kg/day for 5 days) of continuous infusion result in the largest increases in microscopic and gross intestinal morphologic parameters in parenterally maintained rats. Similarly, Nakame et al. [10] found that high dose of GLP-2 (800 μg/kg BW every 6 h before stress) administration improves the incidence and survival rate of necrotising enterocolitis (NEC) in rats. Nevertheless, Sigalet et al. [11] found the effects of subcutaneous injection of weaning pigs with GLP-2 at a dose of 40 μg/ kg/day for 42 days are limited to the gastrointestinal tract; GLP-2 could increase crypt cell proliferation rates and decrease in apoptosis; no effects were observed on activity, appetite, behaviour or food intake. Deng et al. [12] found that both low and high doses of exogenous GLP-2 (2 or 10 nmol/kg BW per day for 7 consecutive days) improve the growth of weaned piglets and protect them against LPS-induced intestinal damage. The various results of therapeutic methods of GLP-2 may be caused by the different agents, experimental animals and test environments. However, the substantially long t1/2 of PEG-pGLP-2 provides us confidence to utilise it for post-weaning diarrhoea syndrome. The present study was designed to examine the effect of different doses of long-acting pGLP-2, that is, PEGp[Gly2]GLP-2, on growth performance, intestinal morphology and expression levels of tight junction (TJ) proteins and inflammatory cytokines in an experimental rat model of ulcerative colitis. Results of this study may provide the dose reference for application of PEG-p[Gly2]GLP-2 in post-weaning diarrhoea syndrome. Methods Materials Porcine [Gly2] GLP-2 (HGDGSFSDEMNTVLDNLATRDFINWLLHTKITDSL, > 98%) was synthesised by Chinese Peptide Company (Hangzhou, China). Monomethoxy PEG–succinimidyl propionate (mPEG–SPA; molecular weight (MW) = 5 kDa) was purchased from Beijing Kaizheng Biotech Development Co., Ltd. (Beijing, China). The ion-exchange chromatography (IEC) resin and column used were CM Sepharose Fast Flow (GE Healthcare Bio-Science AB, Uppsala, Sweden). Ultrafiltration membranes with a MW cutoff of 3000 were purchased from Millipore Corporation (Billerica, MA, USA). Dextran sodium sulphate (DSS) (MW = 36 000–50 000) was purchased from MP Biomedicals China (Shanghai, China). RNAiso Plus, PrimeScript®RT-reagent with gDNA Eraser Kit and SYBR® Premix Ex TaqTM were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). RNA locker Page 2 of 8 was purchased from TIANDZ (Beijing, China). Unless otherwise specified, all other chemicals and reagents were of analytical grade from Sigma–Aldrich and Fluka (Milan, Italy). Preparation of PEG-p (...truncated)