Fig. 6
Targeting GPR65 alleviates hepatic macrophage inflammation and fibrosis by suppressing the JNK/NF-κB pathways. a Western blotting was used to determine the level of p-IKK, p-IĸBα, p-NF-κB p65, IKK, IĸBα, NF-κB p65, p-MLK3, p-MKK4, p-MKK7, p-JNK, JNK, p-p44/42, p44/42, p-p38, p38, p-GSK-3β, p-Akt, Akt, p-PDK1, p-PTEN and p–c-Raf in liver tissues from WT, WT + BDL, GPR65-KO and GPR65-KO + BDL mice. b Western blotting was used to determine the level of p-IKK, IKK, p-IĸBα, IĸBα, p-p65, p65, p-MLK3, p-MKK7, p-JNK and JNK in GPR65-KO, GPR65-overexpressed, various pH-treated and GPR65 agonist/inhibitor-treated HMs. The specific inhibitors of JNK, SP600125 (10 μmol/L) and JNK-IN-8 (5 μmol/L), as well as the specific inhibitors of NF-κB, BAY 11–7082 (5 μmol/L) and APDC (20 μmol/L), were used to treat GPR65-overexpressed HMs for 24 h, and qRT-PCR was used to assess the expression of Tnfα, Il6 and Tgfβ3 (n = 3) (c); TGF-β1 and IL-6 level in the supernatant were detected by ELISA (n = 3) (d); the expression and location of TNF-α was assessed by confocal microscopy (e). Scale bar = 20 μm. *P < 0.05 vs. DMSO + pcDNA3.1; #P < 0.05 vs. DMSO + pcDNA3.1-GPR65. CCl4 carbon tetrachloride, BDL bile duct ligation, ELISA enzyme-linked immunosorbent assay, HM hepatic macrophage, KO knockout, qRT-PCR quantitative real-time reverse transcription-polymerase chain reaction, IKK inhibitor of ĸB kinase, IĸBα inhibitor of ĸB α, NF-κB p65 nuclear factor κB p65 subunit, MLK3 mixed lineage kinase 3, MKK4 mitogen-activated protein kinase kinase 4, MKK7 mitogen-activated protein kinase kinase 7, JNK c-Jun N-terminal kinase, GSK-3β glycogen synthase kinase 3β, Akt protein kinase B, PDK1 pyruvate dehydrogenase kinase 1, PTEN phosphatase and tensin homolog, c-Raf c-rapidly accelerated fibrosarcoma, TGF-β1 transforming growth factor-β1, IL-6 interleukin-6, TNF-α tumor necrosis factor-α