ANGPTL4 induces Kupffer cell M2 polarization to mitigate acute rejection in liver transplantation

Figures

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  Fig. 1. Analyzing AR in rat liver transplant models. (A) Morphological alterations detected 7 days post-liver transplantation (original magnifications: x100 and x400). (B) RAI scoring according to the Banff classification criteria. (C) TUNEL staining at 200x magnification was used to identify apoptotic cells in liver tissues from each group post-transplantation. (D) Serum liver enzyme levels post-transplantation, as measured by microtiter assay. (E) ELISA was employed to quantify serum inflammatory factor levels in rats post-liver transplantation. ns P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; n = 6.

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  Fig. 2. ANGPTL4 expression in the liver following transplantation. (A) RT-qPCR evaluation of ANGPTL4 levels in rat liver tissues. (B) ELISA results of serum ANGPTL4 protein levels. (C) Immunohistochemical staining of ANGPTL4 in liver graft sections (200x magnification). ns P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; n = 6.

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  Fig. 3. ANGPTL4 recombinant protein mitigates AR following liver transplantation. (A, B) Representative HE images of liver grafts taken 7 days post-transplantation at original magnifications of ×100 and ×400, along with RAI scoring of the liver. (C) TUNEL staining to identify apoptotic cells in liver tissues across different groups post-transplantation (200× magnification). (D) Measurement of serum liver enzyme levels after transplantation using a microtiter assay. (E) ELISA evaluation of serum inflammatory factors in rats following liver transplantation. ns P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; n = 6.

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  Fig. 4. ANGPTL4 facilitates the in vivo shift of KCs towards the M2 phenotype. (A) WB analysis of polarization-related protein expression in KCs, with quantitative analysis based on band intensity. (B) Immunohistochemical staining of CD86 and CD163. ns P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; n = 6.

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  Fig. 5. ANGPTL4 recombinant protein promotes M2 polarization of KCs. (A) RT-qPCR and ELISA were used to measure IL-10, IL-1β, and TNF-α expression levels in KCs isolated from rat liver and their corresponding levels in the cell supernatant. (B) WB analysis of polarization-related protein levels in KCs, with quantitative evaluation based on band intensity. (C) Flow cytometry was employed to assess M1-polarized KCs, using FITC-labeled CD68 for KC identification and APC-labeled CD86 to detect M1 polarization. ns P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; n = 3.

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  Fig. 6. Hepatocyte-derived ANGPTL4 is crucial for suppressing inflammatory activation in KCs. (A) Schematic of the experimental procedure: co-culture of HCs with LPS-stimulated KCs. (B) ELISA analysis of ANGPTL4 levels in the KCs culture medium. (C) RT-qPCR and ELISA analysis of IL-10, IL-1β, and TNF-α expression levels. (D) WB analysis of polarization-related proteins in KCs, with quantitative assessment based on band intensity. (E) Flow cytometry analysis of M1-polarized cells, identifying KCs with FITC-labeled CD68 and M1 markers with APC-labeled CD86. (F) Western blot analysis of proteins related to the NF-κB pathway, with quantification based on band intensity. (G) Immunofluorescence staining to localize phosphorylated p65. ns P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; n = 3.

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  Fig. 7. Knockdown of ANGPTL4 expression in HCs within the co-culture system promotes M1 polarization of KCs. (A) Schematic representation of the experimental procedure: siRNA-mediated knockdown of ANGPTL4 in HCs co-cultured with LPS-stimulated KCs. (B) RT-qPCR and ELISA assessments of IL-10, IL-1β, and TNF-α levels. (C) WB analysis of polarization-related proteins in KCs, with quantification of band intensity. (D) Flow cytometry of M1-type polarized cells, with FITC-labeled CD68 and APC-labeled CD86. (E) Western blot assessment of proteins associated with the NF-κB pathway, including band intensity quantification. (F) Immunofluorescence staining to identify the localization of phosphorylated p65. ns P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001; n = 3.