Dietary Flavonoid Hyperoside Induces Apoptosis of Activated Human LX-2 Hepatic Stellate Cell by Suppressing Canonical NF-κB Signaling

Abstract

Hyperoside, an active compound found in plants of the genera Hypericum and Crataegus, is reported to exhibit antioxidant, anticancer, and anti-inflammatory activities. Induction of hepatic stellate cell (HSC) apoptosis is recognized as a promising strategy for attenuation of hepatic fibrosis. In this study, we investigated whether hyperoside treatment can exert antifibrotic effects in human LX-2 hepatic stellate cells. We found that hyperoside induced apoptosis in LX-2 cells and decreased levels of α-smooth muscle actin (α-SMA), type I collagen, and intracellular reactive oxygen species (ROS). Remarkably, hyperoside also inhibited the DNA-binding activity of the transcription factor NF-κB and altered expression levels of NF-κB-regulated genes related to apoptosis, including proapoptotic genes Bcl-Xs, DR4, Fas, and FasL and anti-apoptotic genes A20, c-IAP1, Bcl-X L , and RIP1. Our results suggest that hyperoside may have potential as a therapeutic agent for the treatment of liver fibrosis.

Figure 1

Structure of hyperoside and its inhibitory proliferation effect on LX-2 cells. (a) Chemical structure of hyperoside. (b) Effect of hyperoside on the growth of LX-2 cell lines for 24 h. (c) Effect of hyperoside on the growth of LX-2 cell lines for 48 h. Cell proliferation was analyzed using MTT assay. Cells were treated with different concentration of hyperoside (0, 0.125, 0.25, 0.5, 1.0, and 2.0 mM/L). Results represent the mean ± SEM from three independent experiments (^∗∗means compared with the control group, P < 0.001).

Figure 2

Hyperoside induced proapoptosis effect and its statistical representation of data. The apoptosis rate of Lx-2 cells was analyzed by flow cytometry (Annexin V-FITC/PI). Cells were treated with different dose of hyperoside (0, 0.5, 1.0, and 2.0 mM/L) for 24 h and 48 h, respectively. Results represent the mean ± SEM from three independent experiments (^∗∗means compared with the control group, P < 0.001, n = 3).

Figure 3

Hyperoside attenuated LX-2 activation. (a) Western blotting analysis of α-SMA and collagen I. The relative expression of proteins was calculated according to the reference band of β-tubulin. (b) mRNA levels were quantitated by real-time PCR. The expression was analyzed by 2^-ΔΔCT method. 18s mRNA was used as a housekeeping gene (^∗means compared with the control group, P < 0.05; ^∗∗means compared with the control group, P < 0.001, n = 3).

Figure 4

(a) LX-2 cells were treated in the absence or presence of hyperoside with different concentration for 24 h. The cells were then stained with DCF-DA and subjected to fluorescence microscopy. (b) Intracellular ROS was detected with flow cytometry.

Figure 5

Hyperoside induces HSC apoptosis by blocking NF-κB activation and mediating NF-κB-dependent genes. (a) Hyperoside blocks NF-κB activation in Lx-2 cells. The effect of hyperoside on NF-κB DNA-binding activity in LX-2 cells was evaluated by ELISA. LX-2 cells were incubated for 48 hours in the absence or presence of TNF-α (20 ng/mL) and hyperoside (1 mM/L). Nuclear protein was extracted and subjected to ELISA for measurement of NF-κB DNA-binding activity. (b) Relative expression of NF-κB mediating proapoptotic target genes. (c) Relative expression of NF-κB mediating antiapoptotic target genes (^∗means compared with the control group, P < 0.05; ^∗∗means compared with the control group, P < 0.001, n = 3).