Figures and data
Leucine ameliorates LPS-induced inflammation. (A) Kaplan-Meier curve showing survival of the mice (n=12). (B) Average daily weight gain of mice (n=8). (C-D) Measurement of IL-6, IFN-γ, and TNF-α secretion in mouse serum and liver by ELISA after treatment with LPS for 6 h. (E-F) mRNA expression of IL-6, IL-1β, NLRP3, MCP1, and iNOS, measured by RT-PCR in the liver and spleen. (G) mRNA expression of IL-6, IL-1β, MCP1, Arg1, Mgl1, and Mgl2, measured by RT-PCR in the bone marrow. Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, and ***P < 0.001, and ****P < 0.0001.
Leucine inhibits M1 polarization and promotes M2 polarization in mice. (A) White blood cell composition and proportion in mice (n=8). (B) Gating strategy for macrophage flow cytometry in the bone marrow. (C) Percentages of CD45+, CD86+, CD206+, and CD86+/CD206+, detected by flow cytometry in the bone marrow (n=8). (D) Percentages of CD45+, CD86+, CD206+, and CD86+/CD206+, detected by flow cytometry in the spleen (n = 8). (E) White blood cell composition and proportion in mice (n=5-6). (F) Measurement of IL-6, IFN-γ and TNF-α secretion in mouse serum by ELISA after treatment with LPS for 6 h (n=6). Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Leucine promotes M2 polarization in BMDMs. (A) Schematic diagram of macrophage polarization. (B) Measurement of IL-6 and TNF-α secretion in cell culture supernatants by ELISA (n=6). (C) mRNA expression of IL-1β, TNF-α, IL-6, and NLRP3, measured by RT-PCR in BMDMs (n=6). (D-E) Detection of arginase-1 activity in the medium and BMDMs (n =5-6). (F) BMDMs isolated from mice were stimulated with leucine, IL-4, or both, and the protein expression of Arg1 was determined. (G) mRNA expression of Arg1, Ym1, Fizz1, and Mgl2, measured by RT-PCR in BMDMs (n=3-4). Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
mTORC1 signaling is necessary for M2 polarization. (A) Protein levels of ARG1, p-STAT6, STAT6, p-mTOR, and mTOR, determined by western blotting. (B-C) Detection of arginase-1 activity in the medium and BMDMs (n=4). (D) mRNA expression of Arg1, Fizz1, Mgl1, and Mgl2, measured by RT-PCR in BMDMs (n=3-5). Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Leucine promotes M2 polarization via mTORC1/LXRα signaling. (A) Protein levels of LXRα, ARG1, p-STAT6 and STAT6, determined by western blotting. (B) The nuclear proteins of BMDMs were extracted, and the protein levels of histones and LXRα were determined by western blotting. (C-D) Detection of arginase-1 activity in the medium and BMDMs (n=4). (E) mRNA expression of Arg1, Fizz1, Mgl1, and Mgl2, measured by RT-PCR in BMDMs (n=5-6). Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Mechanism of leucine alleviating LPS-induced CSS by modulating mTORC1/LXRα signaling. In macrophages, LPS promotes M1 polarization to promote the secretion of inflammatory factors leading to inflammation, and IL-4 promotes M2 polarization to alleviate inflammation. Leucine further promotes IL-4-induced M2 polarization by activating mTORC1/LXRα to alleviate inflammation and repair damaged tissues, while leucine also inhibits LPS-mediated M1 polarization and reduces the expression and secretion of inflammatory factors in the organism.
(A) AST and ALT levels in serum and liver (n=6). Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
(A) Cell viability of 2 mM and 10 mM leucine treatments detected by CCK8 (n=5). Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
(A) mRNA expression of Arg1, Fizz1, Mgl1 and Mgl2, measured by RT-PCR in BMDMs (n=4). (B) mRNA expression of Arg1, Fizz1, Mgl1 and Mgl2, measured by RT-PCR in BMDMs (n=4-6) (C) Protein levels of p-AKT (T308) and p-AKT (S473), determined by western blotting. Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
(A) Measurement of IFN-γ and TNF-α secretion in mouse serum and peritoneal fluid by ELISA after treatment with LPS for 6 h (n=6). Student’s t-test was used to determine statistical significance, defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
qPCR primer sequences.
