Department of Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, No. 2 Yuan Ming Yuan Western Road, Hai Dian District, Beijing, 100193, China.
Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Mol Biol Rep. 2019 Dec;46(6):6435-6451. doi: 10.1007/s11033-019-05090-1. Epub 2019 Oct 1.
Heat stress (HS) and secondary restricted blood flow to the intestines cause dysfunction of the intestinal epithelial barrier. Tight junctions (TJs) are essential to maintain intestinal integrity. L-Arginine has beneficial effects on gut functions. However, the underlying mechanisms remain largely unknown. This study tested the hypothesis that L-arginine regulates the TJ network by activating AMP-activated protein kinase (AMPK) signaling, which in turn improves intestinal barrier functions under HS. IEC-6 cells and rat small intestines were used as experiment models of heat stress. AICAR and dorsomorphin were used to activate and inhibit the AMPK pathway, respectively. Cell proliferation, apoptosis, differential gene expression and KEGG pathway analysis, intestinal paracellular permeability, intestinal morphology, and expression of HSP and TJ proteins, and p-AMPK were determined. L-Arginine promoted cell proliferation and reduced apoptosis after heat exposure at an optimal concentration of 5 mmol. Transcriptome sequencing analysis revealed that differentially expressed genes associated with the HSP family and TJs were elevated by L-arginine. According to KEGG pathway analysis, L-arginine activated the AMPK signaling pathway. In vivo, intestinal damage resulted in obvious morphological changes as well as apoptosis with TUNEL and caspase-3 staining under HS and dorsomorphin treatments. Furthermore, HS and dorsomorphin increased the serum D-lactate concentration, diamine oxidase activity, and mRNA expression level of MLCK (P < 0.05). In contrast, L-arginine and AICAR treatments reduced intestinal injury, maintained intestinal permeability, and increased the villus/crypt ratio under hyperthermia. L-Arginine had the same effect as AICAR both in vitro and in vivo, namely increasing p-AMPK protein expression. L-Arginine and AICAR also upregulated the mRNA expression level of HSP70 and HSP90, and downregulated mRNA expression of MLCK (P < 0.05). The protein expression levels of TJ proteins ZO-1 and claudin-1 were suppressed by heat stroke and dorsomorphin, but enhanced by L-arginine and AICAR. Our findings indicate that activation of AMPK signaling by L-arginine is associated with improved intestinal mucosal barrier functions by enhancing the expression of TJs in rat small intestines and IEC-6 cells during HS.
热应激（HS）和肠道二次血流受限导致肠道上皮屏障功能障碍。紧密连接（TJ）对于维持肠道完整性至关重要。L-精氨酸对肠道功能有有益的影响。然而，其潜在机制在很大程度上仍不清楚。本研究通过激活 AMP 激活蛋白激酶（AMPK）信号来检验 L-精氨酸通过调节 TJ 网络来改善热应激下肠道屏障功能的假说，该信号反过来又改善了肠道屏障功能。IEC-6 细胞和大鼠小肠被用作热应激的实验模型。AICAR 和 dorsomorphin 分别用于激活和抑制 AMPK 途径。测定细胞增殖、凋亡、差异基因表达和 KEGG 通路分析、肠道旁细胞通透性、肠道形态以及 HSP 和 TJ 蛋白的表达和 p-AMPK。在最佳浓度 5mmol 下，L-精氨酸促进热暴露后细胞的增殖并减少凋亡。转录组测序分析显示，L-精氨酸上调了与 HSP 家族和 TJ 相关的差异表达基因。根据 KEGG 通路分析，L-精氨酸激活了 AMPK 信号通路。在体内，热应激和 dorsomorphin 处理导致肠道损伤，TUNEL 和 caspase-3 染色显示明显的形态变化和细胞凋亡。此外，HS 和 dorsomorphin 增加了血清 D-乳酸浓度、二胺氧化酶活性和 MLCK 的 mRNA 表达水平（P < 0.05）。相反，L-精氨酸和 AICAR 处理减少了高温下的肠道损伤，维持了肠道通透性，并增加了绒毛/隐窝比。L-精氨酸在体外和体内均具有与 AICAR 相同的作用，即增加 p-AMPK 蛋白表达。L-精氨酸和 AICAR 还上调了 HSP70 和 HSP90 的 mRNA 表达水平，并下调了 MLCK 的 mRNA 表达（P < 0.05）。TJ 蛋白 ZO-1 和 claudin-1 的蛋白表达水平被热射病和 dorsomorphin 抑制，但被 L-精氨酸和 AICAR 增强。我们的研究结果表明，L-精氨酸通过激活 AMPK 信号通路与提高 HSP70 和 HSP90 的 mRNA 表达水平有关，在热应激大鼠小肠和 IEC-6 细胞中增强 TJ 的表达，从而改善肠道黏膜屏障功能。
