Mechanisms Of Grape Seed Extract On Arsenic Induced Liver Fibrosis

A large number of occupational researches, epidemiological and experimental studies demonstrated that organic arsenic exposure can cause repeated or prolonged chronic liver parenchymal inflammation, necrosis. In the repairing process, the imbalance of liver extracellular matrix (ECM) synthesis and degradation result in ECM deposition and liver fibrosis. Although the association between arsenic toxicity and hepatic fibrosis has been firmly established, the mechanism of liver damage remains unclear. Recent observations suggest that excessive generation of reactive oxygen species (ROS) play a central role in the development and progression of As-induced hepatic damage. ROS stimulate hepatic stellate cells (HSCs) proliferation and transformation into a smooth muscle actin (a-SMA)-positive myofibroblast-like cells. These HSCs are referred to as activated cells and are responsible for the abnormal extracellular matrix (ECM) proteins during hepatic fibrosis. Transforming growth factor-beta (TGF-β) is the most potent profibrogenic cytokine during hepatic fibrosis, accelerating liver fibrosis by activating TGF-β/Smads signaling pathway. Therefore, inhibiting TGF-β/Smad pathway and HSC activation are the main aim of anti-fibrogenic therapies.The potential role of oxidative stress in arsenic exposure suggests that inhibition of ROS/oxidative stress may protect against arsenic-induced hepatotoxicity. Therefore, many scientific researches focused on exploring safe and effective antioxidant compounds. Grape seeds are rich in polyphenols, including catechin, epicatechin, and procyanidin. The outstanding feature of Grape seed extract (GSE) is its antioxidant activity that is greater than vitamins C, E and β carotene; and functioning as antioxidants and anti-inflammatory agent by directly scavenging ROS and decreasing low-grade inflammation in liver tissue. Thus, we hypothesis that GSE might be employed to reduce arsenic induced oxidative damage and provide a protective mechanism against liver fibrosis.In light of the above hypothesis, the initial purpose of the current study was to clarify whether dietary supplementation of GSE could inhibit chronic arsenic-induced hepatic injury and fibrosis. The secondary goal was to explore the underlying molecular mechanisms involved in arsenic-induced liver injury and potentially to provide assistance in clinical intervention.Methods60healthy male Sprague-Dawley rats (aged6week, weight159.8±11.1g) were divided into four groups randomly:Control group (received only drinking water), As group (received arsenic as NaAsO2in drinking water at a concentration of30mg/L), GSE group (received GSE100mg/kg, dissolved in drinking water, every other day by oral gavage), As+GSE group (received arsenic along with GSE). After12months of experimental period, Liver histomorphology changes were observed by HE staining. Liver functions, the levels of pro-inflammatory cytokines, antioxidant defense, ROS production, protein carbonyls, and lipid peroxidation were analysis in every group. Real Time-PCR was performed to detect the mRNA levels of fibrogenic genes. Western blot was performed to analysis the protein levels of pSmad2, pSmad3, and NADPH oxidase subunits, including Nox2, Nox4and P47phox.The effects of different dose of GSE on As induced ROS production and of NADPH oxidase activities in HSC-T6were analysis. DPI, a flavoprotein inhibitor of NADPH oxidase, was used to further address whether As-induced ROS production was derived from NADPH oxidase. Given that DPI is regarded as a non-specific NADPH oxidase inhibitor, we further depletion of Nox4by specific siRNA for Nox4in HSCs. Adenoviral Nox4expressing vector was transfected into HSC-T6. Then the effects of over-expression of Nox4on As-induced ROS production, NADPH oxidase activities, and mRNA levels of fibrogenic genes were analysis. The plasmid p3TP-Lux, a TGF-β-inducible smad response element, was used to measure TGF-β/Smad signaling. Adenoviral Smad3expressing vector was transfected into HSC-T6, Real Time-PCR was performed to gain the effects of over-expression of Smad3on As-induced mRNA levels of fibrogenic genes and the protective mechanisms of GSE. Results1. A significant decrease in body weight was observed in As treated animals when compared to that of control group (P<0.05). Co-treatment of rats with GSE exhibited a significant decrease in the level of AST, ALT, and bilirubin, pro-inflammatory cytokines, fibrogenic genes, and an increase in the level of albumin when compared with rats receiving As only (P?<0.05). Results of HE staining showed that chronic arsenic exposure caused fibrosis changes in rats liver, while GSE inhibited these changes.2. Chronic arsenic exposure resulted in large amounts of ROS production, hepatic content of lipid peroxidation, and endogenous protein oxidation (P<0.05), whereas co-treatment of GSE restored these changes almost near to control (P>0.05). GSE prevented As-induced decreases in antioxidant defense and improved liver antioxidant defense in As-treated rats.3. The results of Western blot showed that the protein expressions of Nox2, p47phox, Nox4, pSmad2, and pSmad3were significantly elevated in As-administration group comparing with controls (P<0.05), and these inductions were significantly inhibited by GSE co-treatment (P>0.05).4. GSE dose-dependently blocked As-induced ROS levels and activity of NADPH oxidase (P<0.05). Results showed that pretreatment of the HSCs with DPI markedly reduced As-induced ROS production in cultured rat HSCs (P<0.05). Further depletion of Nox4by specific siRNA for Nox4in HSCs revealed that siRNA mediated knockdown of Nox4also abrogate As-induced intracellular ROS production (P<0.05). Over-expression of Nox4markedly attenuated the inhibitory effects of GSE on As-induced ROS production and of NADPH oxidase activities, as well as mRNA expression of fibrogenic genes(P<0.05).5. Results demonstrated that chronic As-administration activated TGF-β/smad signaling pathway(P<0.05), but GSE co-treatment corrected these changes(P<0.05). GSE interrupted the TGF-β/smad signaling pathway directly (P<0.05). Forced expression of Smad3effectively prevented the inhibitory effects of GSE on TGF-β1-stimulated levels of Coll-I and a-SMA mRNA(P<0.05). Conclusions1. Chronic arsenic exposure induces hepatic oxidative stress and inflammatory, activates NOX and TGF-β/smad signaling pathway.2. Meanwhile GSE, a powerful antioxidant with diverse beneficial effects such as against reactive oxygen species and oxidative stress as well as being anti-inflammatory, might be a promising agent in protecting hepatic tissue against oxidative damage and in preventing hepatic fibrosis and dysfunction.3. Suppression of NADPH oxidase activities and TGF-β/Smad activation are involved in the therapeutic benefits of GSE.