Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, China.
Department of Nephrology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
Biochem Biophys Res Commun. 2024 Nov 19;734:150656. doi: 10.1016/j.bbrc.2024.150656. Epub 2024 Sep 4.
The mesothelial-mesenchymal transition (MMT) of mesothelial cells has been recognized as a critical process during progression of peritoneal fibrosis (PF). Despite its crucial role in amino acid transport and metabolism, the involvement of L-type amino acid transporter 1 (LAT1) and the potential therapeutic role of its inhibitor, JPH203, in fibrotic diseases remain unexplored. Considering the paucity of research on amino acid-mediated mTORC1 activation in PF, our study endeavors to elucidate the protective effects of JPH203 against PF and explore the involvement of amino acid-mediated mTORC1 signaling in this context.
We established the transforming growth factor beta 1 (TGF-β1) induced MMT model in primary human mesothelial cells and the peritoneal dialysis fluid (PDF) induced PF model in mice. The therapeutic effects of JPH203 on PF were then examined on these two models by real-time quantitative polymerase chain reaction, western blotting, immunofluorescence staining, Masson's trichrome staining, H&E staining, picro-sirius red staining, and immunohistochemistry. The involvement of amino acid-mediated mTORC1 signaling was screened by RNA sequencing and further verified by western blotting in vitro.
LAT1 was significantly upregulated and JPH203 markedly attenuated fibrotic phenotype both in vitro and in vivo. RNA-seq unveiled a significant enrichment of mTOR signaling pathway in response to JPH203 treatment. Western blotting results indicated that JPH203 alleviates PF by inhibiting amino acid-mediated mTORC1 signaling, which differs from the direct inhibition observed with rapamycin.
JPH203 alleviates PF by inhibiting amino acid-mediated mTORC1 signaling.
间皮细胞的间皮-间质转化（MMT）已被认为是腹膜纤维化（PF）进展过程中的一个关键过程。尽管 L 型氨基酸转运蛋白 1（LAT1）在氨基酸转运和代谢中起着至关重要的作用，但它在纤维化疾病中的作用及其抑制剂 JPH203 的潜在治疗作用仍未得到探索。考虑到 PF 中氨基酸介导的 mTORC1 激活的研究甚少，我们的研究旨在阐明 JPH203 对 PF 的保护作用，并探讨氨基酸介导的 mTORC1 信号在这方面的作用。
我们在原代人腹膜间皮细胞中建立了转化生长因子-β1（TGF-β1）诱导的 MMT 模型，在小鼠中建立了腹膜透析液（PDF）诱导的 PF 模型。然后通过实时定量聚合酶链反应、western blot、免疫荧光染色、Masson 三色染色、H&E 染色、苦味酸-天狼猩红染色和免疫组织化学，在这两种模型上检测 JPH203 对 PF 的治疗作用。通过 RNA 测序筛选氨基酸介导的 mTORC1 信号通路的参与，并通过体外 western blot 进一步验证。
LAT1 在体外和体内均显著上调，JPH203 显著减弱纤维化表型。RNA-seq 显示 JPH203 处理后 mTOR 信号通路显著富集。Western blot 结果表明，JPH203 通过抑制氨基酸介导的 mTORC1 信号通路缓解 PF，这与雷帕霉素的直接抑制作用不同。
JPH203 通过抑制氨基酸介导的 mTORC1 信号通路缓解 PF。
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