AUTHOR=Wang Xiaohui , Zhang Tong , Li Wenli , Wang Heliang , Yan Lei , Zhang Xiaowen , Zhao Lianwen , Wang Nianxue , Zhang Beibei 

TITLE=Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway

JOURNAL=Frontiers in Immunology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1357072

DOI=10.3389/fimmu.2024.1357072

ISSN=1664-3224

ABSTRACT=<sec><title>Introduction</title><p><italic>Clostridium perfringens</italic> α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear.</p></sec><sec><title>Methods</title><p><italic>In vivo</italic>, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). <italic>In vitro</italic>, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms.</p></sec><sec><title>Results and discussion</title><p>The results showed that <italic>in vivo</italic>, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin <italic>(IL)-1β</italic>, <italic>IL-6</italic> and <italic>IL-17</italic> mRNA expression. <italic>Clostridium perfringens</italic> α toxin significantly decreased jejunal mechanistic target of rapamycin <italic>(mTOR)</italic> and solute carrier family 38 member 9 (<italic>SLC38A9)</italic> mRNA expression, while arginine supplementation significantly increased <italic>mTOR</italic> and <italic>SLC38A9</italic> mRNA expression. <italic>In vitro</italic>, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines <italic>IL-6</italic>, C-X-C motif chemokine ligand (<italic>CXCL</italic>)<italic>10</italic>, <italic>CXCL11</italic> and transforming growth factor-β (<italic>TGF-β</italic>), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (<italic>Bax</italic>), B-cell lymphoma-2 (<italic>Bcl-2</italic>), B-cell lymphoma-extra large (<italic>Bcl-XL</italic>) and cysteinyl aspartate specific proteinase 3 (<italic>Caspase-3</italic>) and the ratio of <italic>Bax</italic> to <italic>Bcl-2</italic>. Arginine pretreatment significantly increased the α toxin-induced decrease in <italic>mTOR</italic>, <italic>SLC38A9</italic>, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (<italic>4EBP1</italic>) and ribosomal protein S6 kinase (<italic>S6K</italic>) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased <italic>mTOR</italic> mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury <italic>in vivo</italic> and <italic>in vitro</italic>, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.</p></sec>