Abstract
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.
Highlights
As global warming worsens, high ambient temperatures, strenuous activity, or their combination can lead to heat stress (HS) that negatively affects animal performance, health, and livestock value [1]
3, 5, and 7 mmol l-arginine markedly increased cell proliferation after heat exposure (P < 0.05), and 5 mmol had the optimal effect on IEC-6 cell proliferation. l-arginine at 10 mmol worsened the damage of heat stress in cells and further suppressed cell proliferation (P < 0.05)
In the present study involving both an intestinal epithelial cell line (IEC-6) and rat intestine model, we showed that l-arginine is critical for intestinal epithelial barrier integrity, under heat stress conditions, because it enhances the expression and production of Tight junctions (TJs) proteins by regulating the AMPK signaling pathway
Summary
High ambient temperatures, strenuous activity, or their combination can lead to heat stress (HS) that negatively affects animal performance, health, and livestock value [1]. HSPs are important for the quality of synthesized proteins by preventing the misfolding of proteins and degrading damaged proteins [8]. They are involved in multiple physiological processes including cell proliferation, apoptosis, and stress responses [9]. According to their molecular size, HSPs are classified into main six families, namely small HSPs, HSP40, HSP60, HSP70, HSP90, and HSP100 [10]. Found that heat stress increases HSP70 expression in the small intestine [12]. Uerlings et al demonstrated that gene expression of HSF1, HSP70, and HSP90 is elevated under HS [13]
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