Abstract
Purpose: Leptin promotes the fibrogenic effect of hepatic stellate cells (HSCs). Galanin is another endogenous factor involved in regulating bioenergic metabolism conteracting a majority effects of leptin. However, little is known about the effects of galanin on HSCs. The current study is aimed to investigate the biological effects of galanin on HSCs. Methods: HSCs isolated from male Sprague Dawley rats by in situ perfusion were cultured for 0 weeks or 2 weeks. Cells were then harvested and the mRNA expression of galanin and galanin receptors were examined by RT-PCR. Immunocytofluorescence were used to confirm the presence of GalR2 protein on HSCs. Cultured HSCs-T6 were subjected to galanin at the concentration of 1 to 10000 nmol/L for 24 hours. Cell proliferation was examined by MTT assay and a working concentration for the following experiment was determined. Cultured HSCs-T6 were divided to four groups: control, galanin treatment group (galanin 100nmol/L), GalR2 small interfering RNA (siRNA) treatment group (GalR2 siRNA+Galanin) and GalR3 siRNA treatment group (GalR3 siRNA+Galanin). The proliferation of HSCs-T6 was tested by MTT assay. Western blot was used to detect transforming growth factor β1 (TGF-β1), α-smooth muscle akin (α-SMA) and peroxisome proliferator-activated receptor gamma (PPAR-γ) in all groups. Results: Galanin mRNA and GalR3 mRNA were all expressed by both quiescent and activated HSCs. Importantly, GalR2 mRNA was undetectable in quiescent HSCs but was markedly expressed by activated HSCs. GalR1 mRNA was not detected in either quiescent or activated HSCs. The presence of GalR2 protein on activated HSC was further confirmed by immunocytofluorescence. Treatment with galanin of low concentrations(1-100nmol) inhibited HSCs-T6 proliferation in a concentration-depended manner (P<0.05). Galanin (100 nmol/L) significantly inhibited cell proliferation (P<0.05), TGF-β1 and α-SMA expression, but upregulated PPAR-γ expression (P<0.05). When compared to controls, GalR2 knockdown in HSC-T6 showed no effect on cell proliferation, TGF-β1 and α-SMA protein expression, in contract to a increase in PPAR-γ expression (p<0.05). However, GalR3 knockdown resulted in decreases in HSC-T6 proliferation, TGF-β1 and α-SMAprotein expression but a increase in PPAR-γ expression (P<0.05). Conclusion: Galanin receptors are differentially expressed by HSCs of different activation status. Galanin downregulates HSCs activation and suppresses its profibrogenic features. The underlying mechanisms may involve inhibition of HSC proliferation, decrease in TGF-β1 expression and increase in PPARγ expression. These biological functions of galanin are very likely mediated by type 2 receptor GalR2.
Published Version
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