Mechanistic Studies On The Anti-liver Fibrosis Of Natural Components Curcumin And Tetramethylpyrazine By Targeting Hepatic Stellate Cells

Hepatic fibrosis is an integral clinicopathological condition of chronic liver disease. Prevention and treatment of hepatic fibrosis remain a tough problem in the scientific community worldwide. It has been well accepted that activation of hepatic stellate cells (HSCs) and overproduction of extracellular matrix (ECM) are the pivotal events during liver fibrogenesis. Inhibition of HSC activation and clearance of activated HSCs may reverse liver fibrosis. Recent basic and clinical investigations demonstrate that liver fibrogenesis is accompanied by pathological angiogenesis and sinusoidal remodeling, which critically determine the pathogenesis and prognosis of liver fibrosis. It is highly possible that targeting intrahepatic angiogenesis and sinusoidal remodeling could be a novel therapeutic strategy for liver fibrosis. Currently, there are no effective chemical antifibrotics in the clinical context. Chinese herbal medicines have been demonstrated to be promising remedy for hepatic fibrosis, especially, the bioactive components of herbal medicines have served as an important source of antifibrotic candidates.Our prior studies focusing on the antifibrotic potential of natural product curcumin, the primary bioactive component of Curcuma longa L, have shown that peroxisome proliferator-activated receptor-y (PPARy) is a switch molecule controlling HSC activation and that curcumin can potently inhibit HSC activation by activating PPARy. By virtue of the anti-angiogenic effects of curcumin in tumor therapy, we herein propose that curcumin could attenuate the pathological angiogenesis in liver fibrosis contributing to its antifibrotic properties.We first evaluated the effects of curcumin on liver fibrosis and pathological angiogenesis in rats with carbon tetrachloride-induced hepatic injury. Treatment with curcumin significantly improved liver pathohistology, reduced collagen accumulation, and alleviated fibrosis. Curcumin also significantly decreased the abundance of endothelial cell markers and pro-angiogenic signal molecules in the fibrotic liver, suggesting a reduction of hepatic vascularization. Additional experiments in vitro showed that curcumin at a wide range of doses did not impair the viability of rat liver sinusoidal endothelial cell (LSECs) and had no toxicity, and tube formation of LSEC on Matrigel was not apparently affected by curcumin. Moreover, aortic ring assay showed that curcumin did not inhibit neovascularization ex vivo. These data consistently indicated that curcumin could effectively ameliorate fibrosis-associated angiogenesis in fibrotic liver, and that this effect could not be due to direct inhibition of LSECs. Liver specific pericytes HSCs could be the target cells for curcumin. Therefore, we subsequently explored the molecular mechanisms underlying HSC angiogenic properties.It has been established that HSCs as liver pericytes can produce a variety of angiogenic molecules, of which vascular endothelial growth factor (VEGF) is a predominate one, and obtain enhanced capacity to migrate and invade during liver fibrosis, thus contributing to hepatic pathological angiogenesis. Platelet-derived growth factor (PDGF) is the most powerful stimulus for pericytes during angiogenesis. The current study found that PDGF stimulated VEGF expression in HSCs by activating extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways. PDGF also activated focal adhesion kinase (FAK)/RhoA signaling leading to enhanced invasion and vascularization in HSCs. Activation of PPARy inhibited the gene and protein expression of PDGF-β receptor (PDGF-βR), which in turn prevented the angiogenic effects of ERK, mTOR, and FAK/Rho A cascades. It therefore could be concluded that transrepression of PDGF-βR by PPARy mediated the interruption of angiogenic signal transduction in HSCs. PPARy could be a pivotal molecule for modulating HSC angiogenic properties.Based on the mechanistic findings, we next investigated curcumin intervention of HSC angiogenic properties and the underlying pathways. Results demonstrated that curcumin interrupted PDGF-βR/ERK and mTOR pathways leading to reduced VEGF expression in HSCs and associated LSEC vascularization in vitro. Curcumin also blocked FAK/RhoA signaling resulting in suppressed HSC invasion and vascularization capacity. Activation of PPARy significantly strengthened the effects of curcumin. However, curcumin effects were remarkably abolished by inhibition or knockdown of PPARy. It therefore could be concluded that activation of PPARy was required for curcumin to disrupt the angiogenic signal transduction and inhibit the angiogenic properties of HSCs. Computational molecular docking studies revealed that curcumin and its primary in vivo metabolites could favorably bind to the active domain of PPARy protein structure. PPARy could highly be the target molecule for curcumin.We also investigated the in vitro antifibrotic properties and underlying mechanisms of tetramethylpyrazine, an alkaloid commonly isolated from many kinds of herbal medicines. Our results demonstrated that tetramethylpyrazine had no apparent cytotoxic effects on hepatocytes, but significantly inhibited HSC proliferation and induced HSC cell cycle arrest at the G0/G1checkpoint. These effects were associated with the regulation of cyclin D1, p21, p27and p53expression by tetramethylpyrazine. Furthermore, tetramethylpyrazine disrupted mitochondrial functions and led to activation of caspase cascades in HSCs. Mechanistic investigations revealed that tetramethylpyrazine selectively blocked the ERK signaling and activated p53, which resulted in caspase-dependent mitochondrial apoptosis. Further experiments with in vitro HSC activation model induced by glucose and insulin (model1) or hydrogen peroxide (model2) demonstrated that tetramethylpyrazine disrupted the ERK signaling leading to reduced ECM expression in the model1, and blocked the ERK and p38pathways resulting in inhibited ECM expression in the model2. Molecualr docking evidence indicated that ERK2could be the target molecule for tetramethylpyrazine. These discoveries preliminarily elucidated the potential mechanisms underlying the antifibrotic properties of tetramethylpyrazine.In summary, the current study identified the molecular pathways and targets regulating the angiogenic properties of HSCs, which could deepen the understanding of the critical role for HSCs in the pathological angiogenesis during liver fibrosis. We then elucidated the molecular mechanisms by which curcumin inhibited HSC angiogenic properties implicated in attenuation of sinusoidal angiogenesis associated with hepatic fibrosis. Furthermore, we also used several cellular models to outline the signal pathways underlying tetramethylpyrazine inhibition of HSC activation and ECM expression. Our results provide novel insights into the bioactive components isolated from herbal medicines as promising agents for prevention and treatment of liver fibrosis.