Ox-LDL induces expression of genes relevant to the activation of HSCs, which is abrogated by curcumin in cultured HSCs
It was previously demonstrated that ox-LDL prompted biosynthesis of ECM components in HSCs, implying ox-LDL being a pro-fibrogenic stimulator 36. We further examined the effect of ox-LDL on expression of genes closely relevant to HSC activation. Semi-confluent cultured HSCs were serum-starved for 24 hr before the stimulation with exogenous ox-LDL at various concentrations (0-15 μg/ml) in serum-depleted media for additional 24 hr. Serum starvation rendered cells more sensitive to stimuli, including exogenous ox-LDL. Subsequent culture in serum-depleted media excluded the interference from other factors in FBS 37, 38. Total RNA and whole cell protein extracts were prepared from the cells for real-time PCR and Western blotting analyses, respectively. As shown in Fig. 1A & B, ox-LDL increased gene expression of α(I) collagen and alpha-smooth muscle actin (α-SMA), the markers of activated HSCs, in a dose-dependent manner. In addition, ox-LDL dose-dependently stimulated expression of pro-fibrogenic genes, including connective tissue growth factor (CTGF) and type I and II TGF-β receptors (Tβ-RI & Tβ-RII), as well as pro-mitogenic genes, including receptors for PDGF-β (PDGF-βR) and EGF (EGFR), as well as cyclin D1, a key regulator in cell cycle progression. These data collectively indicated that exogenous ox-LDL markedly induced expression of genes closely relevant to HSC activation, suggesting the role of ox-LDL in the stimulation of HSC activation in vitro.
To evaluate the effect of curcumin on the elimination of the stimulatory role of ox-LDL in the activation of HSCs, serum-starved HSCs were stimulated with ox-LDL (10 μg/ml) in the presence of curcumin at various concentrations (0-30 μM) in serum-depleted media for 24 hr. Total RNA and whole cell extracts were prepared to analyze expression of genes related to HSC activation by real-time PCR and Western blotting analyses. As indicated in Fig. 1C & D, curcumin dose-dependently inhibited expression of the pro-fibrogenic and pro-mitogenic genes induced by ox-LDL in cultured HSCs. Taken together, these results demonstrated that ox-LDL induced expression of genes closely relevant to the activation of HSCs, which was dose-dependently abrogated by curcumin.