N-n-Butyl haloperidol iodide ameliorates liver fibrosis and hepatic stellate cell activation in mice.

N-n-Butyl haloperidol iodide (F2) is a novel compound that has antiproliferative and antifibrogenic activities. In this study we investigated the therapeutic potential of F2 against liver fibrosis in mice and the underlying mechanisms. Two widely used mouse models of fibrosis was established in mice by injection of either carbon tetrachloride (CCl4) or thioacetamide (TAA). The mice received F2 (0.75, 1.5 or 3 mg·kg-1·d-1, ip) for 4 weeks of fibrosis induction. We showed that F2 administration dose-dependently ameliorated CCl4- or TAA-induced liver fibrosis, evidenced by significant decreases in collagen deposition and c-Jun, TGF-ß receptor II (TGFBR2), α-smooth muscle actin (α-SMA), and collagen I expression in the liver. In transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 cells (a human hepatic stellate cell line) and primary mouse hepatic stellate cells, treatment with F2 (0.1, 1, 10 µM) concentration-dependently inhibited the expression of α-SMA, and collagen I. In LX-2 cells, F2 inhibited TGF-ß/Smad signaling through reducing the levels of TGFBR2; pretreatment with LY2109761 (TGF-ß signaling inhibitor) or SP600125 (c-Jun signaling inhibitor) markedly inhibited TGF-ß1-induced induction of α-SMA and collagen I. Knockdown of c-Jun decreased TGF-ß signaling genes, including TGFBR2 levels. We revealed that c-Jun was bound to the TGFBR2 promoter, whereas F2 suppressed the binding of c-Jun to the TGFBR2 promoter to restrain TGF-ß signaling and inhibit α-SMA and collagen I upregulation. In conclusion, the therapeutic benefit of F2 against liver fibrosis results from inhibition of c-Jun expression to reduce TGFBR2 and concomitant reduction of the responsiveness of hepatic stellate cells to TGF-ß1. F2 may thus be a potentially new effective pharmacotherapy for human liver fibrosis.

Effects of ghrelin on the intracellular calcium concentration in rat aorta vascular smooth muscle cells.

BACKGROUND/AIMS: Ghrelin has been regarded as a cardioprotective factor with complicated mechanisms. Whether ghrelin is vasodilative or vasoconstrictive in nature is controversial, and the effects of ghrelin on intracellular calcium concentration are still unclear. To explore the mechanisms involved in the vasoactive regulation of ghrelin at the cellular level, we investigated the effects of ghrelin on calcium concentrations in rat aorta vascular smooth muscle cells (VSMCs). METHODS: We obtained VSMCs via cell culture and stained the cells with Furo-2 AM. Western blotting was used to verify growth hormone secretagogue receptor (GHS-R1a) expression in VSMCs. The intracellular calcium variations affected by ghrelin and the interactions of ghrelin with angiotensin II (AngII), Sq22536, and potassium chloride (KCl) were observed using a calcium imaging and analysis system. RESULTS: Western blotting revealed good GHS-R1a expression in VSMCs. The most prominent finding in the present study was that ghrelin inhibited the AngII-induced increase in the calcium concentration. This inhibition was reversed by the adenylate cyclase inhibitor Sq22536 and the GHS-R1a antagonist (D-Lys(3))- GHRP-6. This finding revealed the potential vasodilative effects of ghrelin at the cellular level. We did not observe any effects of ghrelin on intracellular calcium concentrations in resting VSMCs or the increase of calcium concentration induced by KCl. CONCLUSION: Ghrelin inhibited the increase in the intracellular calcium concentration of rat aorta VSMCs induced by AngII, which may depend on the activation of the cAMP/PKA pathway.