Journal: Hepatology Communications

Article Title: Arrb2 in hepatocytes promotes M2 macrophage polarization, ameliorates hepatic ischemia–reperfusion injury through upregulating metabolite 6-ketoLCA

doi: 10.1097/HC9.0000000000000916

Figure Lengend Snippet: 6-ketoLCA upregulated by Arrb2 in hepatocytes promotes M2 macrophage polarization, alleviating hepatic IRI. (A–E) Liver and serum samples from liver-specific Arrb2-CKO and WT mice with or without 6-ketoLCA treatment after I/R (n=6). (A) HE and TUNEL staining were analyzed microscopically. Scale bar: 200 μm; Suzuki score and necrosis area were examined. (B) Quantitative analysis of the bile acid metabolite 6-ketoLCA in liver tissue. (C) Liver function levels (ALT, AST, TBA, GGT) were analyzed between groups. (D) qRT-PCR analysis of mouse liver tissue mRNA expression for inflammatory factors (IL-6, TNF-α, IL-10, TGF-β). (E) iNOS, CD86, CD206, and CD163 expression in PMMs was assessed by WB analysis. (F) The CCK8 assay measured the viability of RAW264.7 cells exposed to varying concentrations and durations of 6-ketoLCA (n=3). (G, I) Co-culture of RAW264.7 and AML12 cells. (G) ELISA for culture medium supernatant inflammatory factors (IL-6, TNF-α, IL-10, TGF-β). (I) BCL2, BAX, and GAPDH expression in AML12 cells was assessed by WB analysis. (H, J) Flow cytometry detected the proportions of M1 (CD86, PE) and M2 (CD206, APC) macrophages treated with IL-4 and 6-ketoLCA. The ratio of M1/M2 macrophages was measured. All results are presented as mean ± SD, and statistical significance was assessed. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CKO, conditional knockout; GGT, gamma-glutamyl transferase; HE, hematoxylin and eosin; I/R, ischemia/reperfusion; IL-6, interleukin 6; IL-10, interleukin 10; IRI, ischemia–reperfusion injury; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PMMs, primary mouse macrophages; TBA, total bile acids; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling; WB, western blotting; WT, wild type.

Article Snippet: RAW264.7 were cultured in 10% FBS RPMI-1640 medium with 6-ketoLCA (100 ng/mL) and IL-4 (20 ng/mL) to induce polarization to M2-like macrophage. shRNA targeting TGR5 (GeneCopoeia, Rockville, MD, USA) was transfected into RAW264.7 cells utilizing Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol to achieve TGR5 knockdown.

Techniques: TUNEL Assay, Staining, Quantitative RT-PCR, Expressing, CCK-8 Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Knock-Out, Reverse Transcription, Polymerase Chain Reaction, End Labeling, Western Blot