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 (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. (D) Gating strategy for macrophage flow cytometry in the spleen. (E) Percentages of CD45+, CD86+, CD206+, and CD86+/CD206+, detected by flow cytometry in the spleen. 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. (C) mRNA expression of IL-1β, TNF-α, IL-6, and NLRP3, measured by RT-PCR in BMDMs. (D-E) Detection of arginase-1 activity in the medium and BMDMs. (E) BMDMs isolated from mice were stimulated with leucine, IL-4, or both, and the protein expression of Arg1 was determined. (F) mRNA expression of Arg1, Ym1, Fizz1, and Mgl2, measured by RT-PCR in BMDMs. 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. (D) mRNA expression of Arg1, Fizz1, Mgl1, and Mgl2, measured by RT-PCR in BMDMs. 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-4EBP1, and 4EBP1, 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. (E) mRNA expression of Arg1, Fizz1, Mgl1, and Mgl2, measured by RT-PCR in BMDMs. 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.

qPCR primer sequences.

Antibody information.