Pathology, research and practice · 2026
BMSC therapy alleviates conA-induced acute immune mediated liver injury via mir-27b-5p-mediated inhibition of the MARCO/NF-κB axis and macrophage M1 polarization.
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Zhang Jing, Hui Ziye, Shen Huanjun, Li Yanpeng, Wang Xining, Tian Shuyu, Kang Bibo, Jia Zhansheng, Ye Chuantao
Аннотация
BACKGROUND: Autoimmune hepatitis (AIH) is an immune-mediated inflammatory liver disease in which dysregulated macrophage polarization contributes to disease progression. Concanavalin A (ConA)-induced acute immune-mediated liver injury is a widely used model of acute AIH. Bone marrow-derived mesenchymal stem cells (BMSCs) possess potent immunoregulatory properties and show therapeutic potential in AIH, but the underlying mechanisms remain unclear. This study investigated the effects and mechanisms of BMSC therapy in ConA-induced acute immune-mediated liver injury.
METHODS: Mouse BMSCs were characterized by morphology, immunophenotypic surface marker expression, and trilineage differentiation potential. ConA-induced acute immune-mediated liver injury was induced in C57BL/6 mice via intravenous injection of ConA. Twelve hours later, mice received intravenous BMSCs. Liver function, cytokine levels, body weight, survival, and histopathology were assessed. Macrophage polarization in liver and spleen was evaluated by flow cytometry and qPCR. MSC biodistribution was tracked using DiR labeling, followed by ex vivo fluorescence imaging. Transcriptomic analysis identified Marco as a top differentially expressed gene associated with macrophage modulation. In vitro, LPS-stimulated RAW264.7 macrophages were co-cultured with BMSCs in Transwell systems to assess paracrine effects. Parallel experiments employed siRNA-mediated Marco knockdown or transfection with mmu-miR-27b-5p mimic to dissect molecular mechanisms. Downstream NF-κB activation was detected by western blot, and hepatocyte apoptosis was analyzed by flow cytometric analysis. Finally, direct targeting of Marco 3'-UTR by miR-27b-5p was confirmed using dual-luciferase reporter assays in RAW cells, including mutational validation of the predicted binding site.
RESULTS: BMSC treatment ameliorated ConA-induced acute immune-mediated liver injury, reducing TNF-α, IFN-γ, IL-6, ALT, AST, ALP, γ-GGT and increasing IL-10, HGF, CHE. Histopathological analysis revealed substantial attenuation of inflammatory infiltration and tissue damage in both liver and spleen. Mechanistically, BMSCs shifted macrophage polarization away from pro-inflammatory M1 phenotype toward anti-inflammatory M2 phenotype, with preferential in vivo homing of administered BMSCs to inflamed hepatic tissues. Transcriptomic analyses demonstrated that Marco, a pattern recognition receptor upregulated during ConA-induced acute immune-mediated liver injury, was significantly downregulated following BMSC treatment. In vitro, BMSC indirect co-culture or Marco knockdown reduced M1 markers, inhibited NF-κB p65 phosphorylation and nuclear translocation, and decreased hepatocyte apoptosis. Furthermore, mechanistic validation identified mmu-miR-27b-5p directly targeted Marco 3'-UTR, suppressing MARCO expression and macrophage inflammatory responses.
CONCLUSIONS: BMSCs mitigate ConA-induced acute immune-mediated liver injury by inhibiting the MARCO/NF-κB axis, reducing M1 polarization, and promoting M2 polarization. BMSC-associated mmu-miR-27b-5p is a key mediator, representing a novel therapeutic strategy for AIH.
METHODS: Mouse BMSCs were characterized by morphology, immunophenotypic surface marker expression, and trilineage differentiation potential. ConA-induced acute immune-mediated liver injury was induced in C57BL/6 mice via intravenous injection of ConA. Twelve hours later, mice received intravenous BMSCs. Liver function, cytokine levels, body weight, survival, and histopathology were assessed. Macrophage polarization in liver and spleen was evaluated by flow cytometry and qPCR. MSC biodistribution was tracked using DiR labeling, followed by ex vivo fluorescence imaging. Transcriptomic analysis identified Marco as a top differentially expressed gene associated with macrophage modulation. In vitro, LPS-stimulated RAW264.7 macrophages were co-cultured with BMSCs in Transwell systems to assess paracrine effects. Parallel experiments employed siRNA-mediated Marco knockdown or transfection with mmu-miR-27b-5p mimic to dissect molecular mechanisms. Downstream NF-κB activation was detected by western blot, and hepatocyte apoptosis was analyzed by flow cytometric analysis. Finally, direct targeting of Marco 3'-UTR by miR-27b-5p was confirmed using dual-luciferase reporter assays in RAW cells, including mutational validation of the predicted binding site.
RESULTS: BMSC treatment ameliorated ConA-induced acute immune-mediated liver injury, reducing TNF-α, IFN-γ, IL-6, ALT, AST, ALP, γ-GGT and increasing IL-10, HGF, CHE. Histopathological analysis revealed substantial attenuation of inflammatory infiltration and tissue damage in both liver and spleen. Mechanistically, BMSCs shifted macrophage polarization away from pro-inflammatory M1 phenotype toward anti-inflammatory M2 phenotype, with preferential in vivo homing of administered BMSCs to inflamed hepatic tissues. Transcriptomic analyses demonstrated that Marco, a pattern recognition receptor upregulated during ConA-induced acute immune-mediated liver injury, was significantly downregulated following BMSC treatment. In vitro, BMSC indirect co-culture or Marco knockdown reduced M1 markers, inhibited NF-κB p65 phosphorylation and nuclear translocation, and decreased hepatocyte apoptosis. Furthermore, mechanistic validation identified mmu-miR-27b-5p directly targeted Marco 3'-UTR, suppressing MARCO expression and macrophage inflammatory responses.
CONCLUSIONS: BMSCs mitigate ConA-induced acute immune-mediated liver injury by inhibiting the MARCO/NF-κB axis, reducing M1 polarization, and promoting M2 polarization. BMSC-associated mmu-miR-27b-5p is a key mediator, representing a novel therapeutic strategy for AIH.