Forsythiaside A Regulates Activation of Hepatic Stellate Cells by Inhibiting NOX4-Dependent ROS

Abstract

Hepatic stellate cells (HSCs) activation is an important step in the process of hepatic fibrosis. NOX4 and reactive oxygen species expressed in HSCs play an important role in liver fibrosis. Forsythiaside A (FA), a phenylethanoid glycoside extracted and isolated from Forsythiae Fructus, has significant antioxidant activities. However, it is not clear whether FA can play a role in inhibiting the HSCs activation through regulating NOX4/ROS pathway. Therefore, our purpose is to explore the effect and mechanism of FA on HSCs activation to alleviate liver fibrosis. LX2 cells were activated by TGF-β1 in vitro. MTT assay and Wound Healing assay were used to investigate the effect of FA on TGF-β1-induced LX2 cell proliferation and migration. Elisa kit was used to measure the expression of MMP-1 and TIMP-1. Western blot and RT-qPCR were used to investigate the expression of fibrosis-related COLI, α-SMA, MMP-1 and TIMP-1, and inflammation-related TNF-α, IL-6 and IL-1β. The hydroxyproline content was characterized using a biochemical kit. The mechanism of FA to inhibit HSCs activation and apoptosis was detected by DCF-DA probe, RT-qPCR, western blot and flow cytometry. NOX4 siRNA was used to futher verify the effect of FA on NOX4/ROS pathway. The results showed that FA inhibited the proliferation and migration of LX2 cells and adjusted the expression of MMP-1, TIMP-1, COLI, α-SMA, TNF-α, IL-6 and IL-1β as well as promoted collagen metabolism to show potential in anti-hepatic fibrosis. Mechanically, FA down-regulated NOX4/ROS signaling pathway to improve oxidation imbalances, and subsequently inhibited PI3K/Akt pathway to suppress proliferation. FA also promoted the apoptosis of LX2 cells by Bax/Bcl2 pathway. Furthermore, the effects of FA on TGF-β1-induced increased ROS levels and α-SMA and COLI expression were weaken by silencing NOX4. In conclusion, FA had potential in anti-hepatic fibrosis at least in part by remolding of extracellular matrix and improving oxidation imbalances to inhibit the activation of HSCs and promote HSCs apoptosis.

Mitochondrial dysfunction: A promising therapeutic target for liver diseases

“…Forsythiaside A down-regulates the NADPH oxidase 4/ROS signaling pathway to improve oxidation imbalance, decreases collagen-1 and α -SMA expression, and increases the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. 97 …”

Bazi Bushen alleviates skin senescence by orchestrating skin homeostasis in SAMP6 mice

“…According to the manufacturer's instruction, 27 Hyp content in skin tissue was measured using commercial assay kits (Nanjing JianCheng Bioengineering Institute, Nanjing, China).…”

Small-molecule natural products for reversing liver fibrosis based on modulation of HSCs activation: an update

“…Mechanistically, FA downregulates the NOX4/ROS signal pathway to take a favorable turn to oxidative imbalance as well as inhibits the PI3K/Akt pathway to withhold propagation soon afterwards. Additionally, by silencing NOX4, the effect of FA on TGF-β1-seduced increase in ROS levels, expression of α-SMA as well as COLI was attenuated [186] . Overall, FA has anti-fibrotic properties that quell HSCs activation as well as boost HSCs apoptosis, at any rate partly via mending oxidative imbalance as well as remodeling the extracellular matrix.…”