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.
Highlights
Liver fibrosis was the abnormal proliferation of connective tissue and the excessive accumulation of extracellular matrix proteins in the liver caused by various pathogenic factors, which usually occurred in most types of chronic liver disease such as hepatitis B and C, alcohol liver, nonalcoholic liver disease, cholestasis and autoimmune hepatitis [1, 2]
Our results showed that Forsythiaside A (FA) at a safe dose could significantly inhibit the proliferation and migration of LX2 cells induced by TGF-β1, indicating that FA could effectively inhibit the activation of Hepatic stellate cells (HSCs)
Our research demonstrated that FA alleviated TGF-β1induced LX2 cells activation at least in part through remolding of extracellular matrix and improving oxidation imbalances by NOX4/reactive oxygen species (ROS) pathway to inhibit the proliferation, migration and promote apoptosis of HSCs (Figure 11)
Summary
Liver fibrosis was the abnormal proliferation of connective tissue and the excessive accumulation of extracellular matrix proteins in the liver caused by various pathogenic factors, which usually occurred in most types of chronic liver disease such as hepatitis B and C, alcohol liver, nonalcoholic liver disease, cholestasis and autoimmune hepatitis [1, 2]. Multiple factors directly or indirectly induced HSCs activation, such as fibrotic tissue microenvironment, intestinal malnutrition, immune and systemic metabolic abnormalities and Oxidative Medicine and Cellular Longevity hepatitis virus products and so on [5]. Targeted drug molecular intervention to induce HSCs inactivation therapeutically was a more effective and less toxic precise anti-hepatic fibrosis therapy [5]
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