Glucagon-like peptide 2 attenuates intestinal mucosal barrier injury through the MLCK/pMLC signaling pathway in a piglet model
Glucagon-like peptide 2 attenuates intestinal mucosal barrier injury through the MLCK/pMLC signaling pathway in a piglet model
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DOI：
10.1002/jcp.30068
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发表时间：
2020-09-22
影响因子：
5.6
通讯作者：
Jia, Gang
中科院分区：
文献类型：
--
作者：
Chang, Yaqi;Deng, Qiuhong;Jia, Gang
胰高血糖素样肽-2(GLP-2)是一种肠道营养激素，因其具有促进肠道发育的潜力而受到世界各国的广泛关注。我们在体内外研究了GLP-2抗内毒素(LPS)诱导的肠炎症和损伤的作用及其机制。将40头体重相近的28日龄断奶健康仔猪分成4组，每组10头：(1)非激发对照组；(2)脂多糖激发对照组；(3)脂多糖+低剂量GLP-2组；(4)脂多糖+高剂量GLP-2组。仔猪皮下注射添加GLP-2的磷酸盐缓冲液，剂量分别为0、0、2和10nmol/kg体重，连续7d。仔猪于第14天经腹腔注射内毒素100 mg/kg，造成肠道损伤。采用定量逆转录聚合酶链式反应、Western印迹和酶联免疫吸附试验检测典型紧密连接蛋白和肌球蛋白轻链(MLCK)/磷酸化肌球蛋白轻链(PMLC)基因和蛋白表达水平，以及促炎细胞因子水平。大剂量的GLP-2通过下调和重新分布紧密连接蛋白(p<；.05)增加肠道通透性，例如透明带occluden-1(ZO-1)和occludin。GLP-2可降低小肠中促炎症细胞因子的转录，包括IL-1β(IL-1β)、IL-6、IL-8和肿瘤坏死因子-α(p<；0.05)。GLP-2呈剂量依赖性地抑制脂多糖诱导的MLCK表达增加和十二指肠、空肠、回肠pMLC水平的升高。为进一步探讨GLP-2对内毒素所致的断奶后肠屏障损伤的保护作用及其可能的机制，用IPEC-J2单层细胞建立了体外肠上皮屏障模型，用100mU g/mlLPS100 mU/mlGLP-2加或不加1×10~(-8)mol/L GLP-2预处理。体外分析包括对照组、内毒素和GLP-2+内毒素处理。GLP-2通过恢复ZO-1和occludin的表达和超微结构，减轻了内毒素对屏障通透性的破坏作用(p<；0.05)。此外，GLP-2还可逆转内毒素诱导的MLCK过度表达和pMLC过度磷酸化(p<；0.05)。综上所述，我们的研究结果揭示了GLP-2通过MLCK/pMLC信号通路减轻脂多糖攻击的断奶仔猪和IPEC-J2细胞的肠屏障损伤和炎症的机制。
Glucagon-like peptide-2 (GLP-2), an intestinotrophic hormone, has drawn considerable attention worldwide due to its potential to promote intestinal development. We investigated the effects and mechanisms of GLP-2 against lipopolysaccharide (LPS)-induced intestinal inflammation and injury both in vitro and in vivo. Forty healthy piglets weaned at the age of 28 days with similar body weight (BW) were assigned to four in vivo treatments with ten piglets each: (i) nonchallenged control; (ii) LPS-challenged control; (iii) LPS + low dose GLP-2; and (iv) LPS + high dose GLP-2. Piglets were subcutaneously injected with phosphate-buffered saline supplemented with GLP-2 at doses of 0, 0, 2, and 10 nmol/kg BW per day for seven consecutive days. The piglets were challenged with an intraperitoneal injection with 100 mu g/kg LPS on day 14 to induce intestinal damage. After that, the gene and protein expression levels of representative tight junction proteins and myosin light-chain kinase (MLCK)/phosphorylated myosin light chain (pMLC), as well as proinflammatory cytokine levels were determined using quantitative reverse transcription polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay methods. A high dose of GLP-2 pretreatment increased intestinal permeability by downregulating and redistributing tight junction proteins (p < .05), for example, zona occluden-1 (ZO-1) and occludin. GLP-2 decreased the transcription of proinflammatory cytokines genes including interleukin-1 beta (IL-1 beta), IL-6, IL-8, and tumor necrosis factor-alpha in small intestines (p < .05). GLP-2 prevented the LPS-induced increase in the expression of MLCK dose-dependently and the increase in pMLC levels in the duodenum, jejunum, and ileum. To assess further the protective effect of GLP-2 on LPS-induced intestinal barrier injury after weaning and its possible mechanism, an in vitro intestinal epithelial barrier model was established with IPEC-J2 monolayers and treated with 100 mu g/ml LPS with or without 1 x 10(-8) mol/L GLP-2 pretreatment. The in vitro analysis included control, LPS, and GLP-2 + LPS treatments. GLP-2 treatment alleviated the destructive effect of LPS on barrier permeability by restoring the expression and ultrastructure of ZO-1 and occludin (p < .05). In addition, GLP-2 reversed the LPS-induced MLCK hyperexpression and pMLC hyperphosphorylation (p < .05). Taken together, our findings revealed a mechanism by which GLP-2 alleviated LPS-challenged intestinal barrier injury and inflammation in weaned piglets and IPEC-J2 cells via the MLCK/pMLC signaling pathway.