Fig 2: Blockage of ER stress abrogated ARRB1-modulated activation of liver macrophages in vivo and in vitro.Mice were intraperitoneally administered LPS injection (5 mg/kg) 8 h after gavage with GSK414 (150 mg/kg) or vehicle solution. n = 6 in each group. A TUNEL staining of livers in LPS-induced mice with or without GSK414 challenge (scale bar: 100 µm). B–D F4/80 and MCP-1 staining of livers in LPS-induced mice with or without GSK414 challenge (scale bar: 50 µm), and the positive F4/80 and MCP-1 indexes were scored. E, F Serum ALT and AST levels in LPS-induced mice with or without GSK414 challenge. G TNF-a and IL-1ß staining of livers in LPS-induced mice with or without GSK414 challenge (scale bar: 50 µm), and the positive TNF-a and IL-1ß indexes were scored. C–G *P < 0.05 compared with the respective vehicle treatment; #P < 0.05 compared with LPS-induced WT mice by Student’s t test. H–J Raw264.7 and THP-1 cells were challenged with LPS (1 µg/ml) for 6 h with or without GSK414 (1 µM) treatment after knockdown of ARRB1 by siRNA. H Western blotting analysis of the indicated protein expression. I, J TNF-a, IL-1ß, and IL-6 mRNA levels in Raw264.7 and THP-1 cells in the indicated treatments were analyzed by real-time PCR. *P < 0.05 by Student’s t test. Data are presented as the mean ± SD of three separate experiments.

Fig 3: ARRB1 suppressed the activation of hepatic macrophages induced by LPS.A Representative morphology images of vector- and ARRB1-overexpressing Raw264.7 and THP-1 cells with LPS (1 µg/ml) challenge for 6 h by an inverted microscope (scale bar: 50 µm). B Representative morphology images of WT and ARRB1-KO mouse primary hepatic macrophages with LPS (0.5 µg/ml) were challenged for 6 h (scale bar: 50 µm). C–K TNF-a, IL-1ß, and IL-6 mRNA levels in vector- and ARRB1-overexpressing Raw264.7 and THP-1 cells with LPS (1 µg/ml) challenge for 6 h and mouse primary hepatic macrophages with LPS (0.5 µg/ml) challenge for 6 h were analyzed by real-time PCR. Data are presented as the mean ± SD of three separate experiments. *P < 0.05 by Student’s t test.

Fig 4: Summary of the proposed regulatory mechanism of ARRB1 in this study.ARRB1 suppresses LPS-induced release of inflammatory cytokines in response to activated macrophages. ARRB1 regulates the ER stress signaling pathway by inhibiting the phosphorylation of eIF2a and downstream ATF4 and CHOP expression in liver macrophages.

Fig 5: Overexpression of ARRB1 suppressed LPS-induced ER stress in hepatic macrophages.A Double immunofluorescent staining for F4/80 and GRP78 in the indicated mouse liver sections. Nuclei were stained with DAPI in blue. F4/80 was stained green, and GRP78 was visualized red. The merged positive signals of F4/80 and GRP78 are visualized in yellow (scale bar: 50 µm). B, C GRP78 and CHOP mRNA levels in primary hepatic macrophages with LPS (0.5 µg/ml) challenge for 6 h from WT and ARRB1-KO mice were analyzed by real-time PCR. *P < 0.05 compared with the respective vehicle treatment; #P < 0.05 compared with LPS-induced WT mice by Student’s t test. D Protein expression levels of ARRB1, GRP78, eIF2a, p-eIF2a, CHOP, and ATF4 in ARRB1-overexpressing Raw264.7 and THP-1 cells with or without LPS (1 µg/ml) challenge for 6 h. E–H GRP78 and CHOP mRNA levels of ARRB1-overexpressing Raw264.7 and THP-1 cells with LPS (1 µg/ml) were challenged for 6 h and analyzed by real-time PCR. *P < 0.05 compared with the respective vehicle treatment; #P < 0.05 compared with vector cells following LPS treatment by Student’s t test. Data are presented as the mean ± SD of three separate experiments.