Fig. 5.

GPR65 promotes HSCs activation indirectly by paracrine secretion of TGF-β from macrophage. The CM from control, GPR65-overexpressed, pH 6.6-treated or GPR65 agonist-treated macrophage cells, with 20 μg/ml TGF-β neutralization antibody 1D11 or mouse IgG, were used to treat primary HSCs for 24 h. qRT-PCR was used to assess the mRNA level of Gpr65, Acta2, Col1α1, Col1α2, Timp1, Mmp2 and Tgfβ1 (n = 3) (a-c); Western blotting was used to determine the protein level of COL1α1, α-SMA, MMP2 and TIMP1 (d); the expression and location of α-SMA and COL1α1 was assessed by confocal microscopy (e). Scale bar = 20 μm. qRT-PCR was used to assess the expression of Tgfβ1 and Tgfβ3 in GPR65-KO (f), Gpr65-overexpressed (g), or GPR65 agonist/inhibitor (h) treated HMs (n = 3). i TGF-β1 level in the supernatant of HMs transfected with pcDNA3.1 or pcDNA3.1-GPR65 was detected by ELISA (n = 4). HMs isolated from WT and GPR65-KO mice were treated with or without GPR65 agonist (j) or pH 6.6 (k) for 24 h, TGF-β1 level in the supernatant was detected by ELISA (n = 3). ^*P < 0.05 vs. pcDNA3.1, pH 7.4, DMSO, WT, or WT + DMSO/pH 7.4; ^#P < 0.05 vs. pcDNA3.1-GPR65, pH 6.6, GPR65 agonist, or WT + GPR65 agonist/pH 6.6. CM conditioned medium, ELISA enzyme-linked immunosorbent assay, HM hepatic macrophage, HSC hepatic stellate cell, KO knockout, qRT-PCR quantitative real-time reverse transcription-polymerase chain reaction, COL1α1 collagen type I alpha 1, α-SMA α-smooth muscle actin, MMP2 matrix metalloproteinase 2, TIMP1 tissue inhibitor of metalloproteinases 1, TGF-β1 transforming growth factor-β1