l-Arginine alleviates heat stress-induced intestinal epithelial barrier damage by promoting expression of tight junction proteins via the AMPK pathway
l-Arginine alleviates heat stress-induced intestinal epithelial barrier damage by promoting expression of tight junction proteins via the AMPK pathway
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DOI：
10.1007/s11033-019-05090-1
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发表时间：
2019-10
影响因子：
2.8
通讯作者：
Z. Xia;Liqing Huang;P. Yin;Fenghua Liu;Yanhan Liu;Zhenbiao Zhang;Jiabao Lin;Wenshu Zou;Chunxiao Li
中科院分区：
文献类型：
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作者：
Z. Xia;Liqing Huang;P. Yin;Fenghua Liu;Yanhan Liu;Zhenbiao Zhang;Jiabao Lin;Wenshu Zou;Chunxiao Li
热应激（HS）和继发性限制肠道血流导致肠上皮屏障功能障碍。紧密连接（TJ）对维持肠道完整性至关重要。l-精氨酸对肠道功能具有有益作用。然而，其潜在机制在很大程度上仍然未知。本研究验证了L-精氨酸通过激活AMP激活蛋白激酶（AMPK）信号调节TJ网络，进而改善HS下肠道屏障功能的假设。采用IEC-6细胞和大鼠小肠作为热应激的实验模型。AICAR和dorsomorphin分别用于激活和抑制AMPK通路。结果表明，热暴露后，L-精氨酸促进细胞增殖，抑制细胞凋亡，并通过KEGG途径分析其表达，肠壁通透性，肠组织形态学，HSP和TJ蛋白的表达，以及p-AMPK的表达。转录组测序分析显示，与HSP家族和TJ相关的差异表达基因被精氨酸升高。根据KEGG通路分析，l-精氨酸激活AMPK信号通路。在体实验中，HS和dorsomorphin处理后，肠损伤引起明显的形态学改变，TUNEL和caspase-3染色显示细胞凋亡。此外，HS和dorsomorphin还增加了血清D-乳酸浓度、二胺氧化酶活性和MLCK mRNA表达水平（P< 0.05）。L-精氨酸和AICAR均能显著增加p-AMPK蛋白表达，上调HSP 70和HSP 90 mRNA表达，下调MLCK mRNA表达（P< 0.05）。热休克和dorsomorphin抑制TJ蛋白ZO-1和claudin-1的蛋白表达，而byl-arginine和AICAR增强TJ蛋白ZO-1和claudin-1的蛋白表达。我们的研究结果表明，AMPK信号的激活与改善肠粘膜屏障功能的精氨酸通过增强TJ的表达在大鼠小肠和IEC-6细胞在HS。
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 thatl-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 byl-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 byl-arginine and AICAR. Our findings indicate that activation of AMPK signaling byl-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.