The supernatant was centrifuged at 13000gfor 25 min at 4C. part of SAC is the induction of the heme oxygenase-1 (HO-1) gene in an NF-E2-related element-2 (Nrf-2) and Akt dependent manner. Specifically SAC was found to induce the phosphorylation of Akt and enhance the nuclear localization of Nrf-2 in cells. PJS Our results were further confirmed by specific HO-1 gene knockdown studies which clearly shown that HO-1 induction indeed played a key part in SAC mediated inhibition of apoptosis and ROS production in HepG2 cells, therefore suggesting a hepatoprotective part of SAC in combating oxidative stress mediated liver diseases. 1. Intro Oxidative stress in liver hepatocytes underlies numerous liver diseases [1]. Hydrogen peroxide (H2O2) takes on a major part in inducing liver oxidative stress, by disrupting the cellular redox circuitry that depends on the redox state of various signaling molecules behaving as redox sensitive molecular switches, or by directly damaging cellular macromolecules including DNA, proteins, and lipids. This alters many fundamental cellular functions including proliferation, differentiation, migration and adhesion [1] alpha-Cyperone and eventually results in sustained alpha-Cyperone hepatocyte apoptosis, a pathological condition regularly associated with the progression of several liver diseases such as hepatic ischemia-reperfusion (I/R) injury, alcoholic liver disease, nonalcoholic fatty liver disease, and hepatitis [2, 3]. H2O2 alpha-Cyperone levels that induce oxidative stress have been shown to downregulate heme oxygenase-1 (HO-1), a phase II anti-oxidant enzyme, involved in the rate limiting step of heme rate of metabolism that catalyzes the conversion of heme into carbon monoxide and biliverdin. Several studies possess depicted that, induction of HO-1 manifestation interferes with the progression of a number of hepatic pathophysiological conditions including ischemia/ reperfusion (I/R) injury, liver swelling, hepatic fibrosis and hepatitis [4]. It has also been shown that HO-1 plays a role in cellular defense mechanism against oxidative stress induced apoptotic cell death [5C7]. S-allyl cysteine (SAC), a potential antioxidant found in the aged garlic extract (AGE) [8], has been reported to possess cytoprotective effects [9]. SAC offers many advantages over additional garlic compounds owing to the facts that SAC is definitely odourless and less harmful, pharmacokinetic studies show that it offers 98 percent bioavailability [10], it is the only reliable marker utilized for studies involving oral garlic intake because it is definitely detectable and raises quantitatively in the blood and it is the only constituent of garlic that does not induce P450 isozymes in the body suggesting that SAC will not cause P450-induced contraindications with medicines [10]. Severalin vivostudies have suggested SAC to protect from oxidative stress induced liver injury. SAC has shown effectiveness in protecting from carbon tetrachloride induced liver cirrhosis [11] and liver injury [9]. SAC improved nonalcoholic fatty liver disease in rats with type 2 diabetes via rules of hepatic lipogenesis and glucose rate of metabolism [12]. SAC alleviated chromium (VI)-induced hepatotoxicity in rats by inhibiting inflammatory markers [13]. However the detailed mechanism behind the antioxidative and antiapoptotic effects of SAC has not been elucidated. The present study has been designed to investigate the mechanism behind the anti-oxidative and anti-apoptotic potential of SAC in hydrogen peroxide stimulated HepG2 cells, a widely usedin vitromodel for the study of oxidative injury in liver. For the first time we demonstrate in our study that SAC alleviates hydrogen peroxide induced oxidative injury and apoptosis through upregulation of Akt/Nrf-2/HO-1 signaling pathway in HepG2 cells. 2. Materials and Methods 2.1. Materials S-allyl cysteine was purchased from Abcam. Trypan blue, 2,7-dichlorodihydrofluorescein diacetate (DCFH2-DA),5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-imidacarbocyanine iodide (JC-1), and Wortmannin were purchased from Sigma-Aldrich, USA. Dulbecco’s revised eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA solutions were purchased from HiMedia, India. INTERFERin siRNA transfection reagent was purchased from Polyplus, USA. Taq polymerase and dNTPs were purchased from Thermo Fisher Scientific, USA. Random hexamer primer and RiboLock RNase inhibitor, and RevertAid reverse transcriptase were purchased from Thermo Scientific, USA. Anti-gfor 10 min at 4C. Then equal volume of TBA remedy (0.375 % TBA, 15 % trichloroacetic acid, and 0.25 N HCl) was added to the supernatants and heated for 15 min inside a boiling water bath followed by centrifugation at 10,000gfor 5 min. Finally absorbance of the supernatant was measured at 535 nm. The ideals are displayed as fold switch over control. 2.9. RNA Extraction, Reverse Transcription and Semiquantitative PCR Cells were seeded in 6-well plates and after treatment; total RNA was isolated using TRIzol reagent (Invitrogen). Equal amount (1.5 alpha-Cyperone gfor 15 min at alpha-Cyperone 4C. The supernatant was used as whole cell lysate. Cytosolic and nuclear components were prepared using a previously.