Diallyl Trisulfide Suppresses Oxidative Stress-Induced Activation of Hepatic Stellate Cells through Production of Hydrogen Sulfide

Accumulating data reveal that garlic has beneficial effects against chronic liver disease. We previously reported that diallyl trisulfide (DATS), the primary organosulfur compound in garlic, reduced fibrosis and attenuated oxidative stress in rat fibrotic liver. The present study was aimed at elucidating the underlying mechanisms. The primary rat hepatic stellate cells (HSCs) were cultured and stimulated with hydrogen peroxide (H2O2) for inducing HSC activation under oxidative stress. We examined the effects of DATS on the profibrogenic properties and oxidative stress in H2O2-treated HSCs. The results showed that DATS suppressed and reduced fibrotic marker expression in HSCs. DATS arrested cell cycle at G2/M checkpoint associated with downregulating cyclin B1 and cyclin-dependent kinase 1, induced caspase-dependent apoptosis, and reduced migration in HSCs. Moreover, intracellular levels of reactive oxygen species and lipid peroxide were decreased by DATS, but intracellular levels of glutathione were increased in HSCs. Furthermore, DATS significantly elevated hydrogen sulfide (H2S) levels within HSCs, but iodoacetamide (IAM) reduced H2S levels and significantly abrogated DATS production of H2S within HSCs. IAM also abolished all the inhibitory effects of DATS on the profibrogenic properties and oxidative stress in HSCs. Altogether, we demonstrated an H2S-associated mechanism underlying DATS inhibition of profibrogenic properties and alleviation of oxidative stress in HSCs. Modulation of H2S production may represent a therapeutic remedy for liver fibrosis.


Introduction
Hepatic fibrosis represents an overactive wound healing process secondary to a variety of chronic liver injuries. During fibrogenesis, excessive connective tissue accumulates in the liver and results in the distortion of hepatic architecture [1]. Chronic liver injury activates and transforms quiescent hepatic stellate cells (HSCs) from vitamin Astoring pericytes to myofibroblast-like cells. Once activated, HSCs become profibrogenic through acquiring proliferative, contractile, and proinflammatory properties as the primary cellular source of extracellular matrix (ECM) components in the liver [2]. Clinical and basic data suggest that oxidative stress critically participates in the progression of fibrosis and acts as mediators of molecular and cellular events implicated in liver fibrosis. Generation of reactive oxygen species (ROS) plays an important role in HSC activation and initiation of hepatic fibrogenesis [3]. Culture of HSCs treated with oxidative stress-related molecules can mimic the HSC activation caused by oxidative stress in liver fibrosis [4].

Oxidative Medicine and Cellular Longevity
This cellular model is commonly established for investigating the mechanisms of HSC activation and evaluating the effects of antifibrotic candidates. However, currently there are few breakthroughs in the therapeutic intervention of hepatic fibrosis. Identification of antifibrogenic agents that are innocuous is urgently needed.
Garlic has been recognized for prevention and treatment of various diseases by many different cultures throughout history. Recent studies support the effects of garlic and its functional ingredients in a wide range of applications, including anticancer, antithrombotic, antiatherosclerotic, antidiabetic, and antioxidant properties [5]. It is characterized that diallyl trisulfide (DATS), a major organosulfur compound in garlic, is responsible for the pharmacological efficacy of garlic [6]. We previously demonstrated that DATS reduced carbon tetrachloride (CCl 4 )-caused liver injury and fibrogenesis in rats, which was associated with inhibition of HSC activation and attenuation of hepatic oxidative stress [7]. However, the molecular mechanisms underlying DATS's antifibrotic activity are not fully understood. Interestingly, emerging data indicate that drugs that target hydrogen sulfide (H 2 S) or generate safe levels of H 2 S in vivo may be therapeutic options for chronic liver diseases [8]. It is given that DATS is an exogenous donor of H 2 S and the liver has a high hepatic capacity for H 2 S metabolism [9]. We hypothesized that DATS exerted its antifibrotic effects associated with generation of H 2 S. Experiments were performed to verify this hypothesis.

Measurement of Glutathione (GSH).
HSCs were seeded in 6-well plates and cultured for 24 h. HSCs were then treated with various reagents at indicated concentrations for 24 h and were lysated with RIPA buffer. Intracellular GSH levels were determined using kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the protocol. Experiments were performed in triplicate.

Western Blot Analyses.
HSCs were treated with various reagents at indicated concentrations for 24 h. Whole cell protein preparation, detection, and band visualization were performed as we previously described [7]. -Actin was used as an invariant control for equal loading of total proteins. Representative blots were from three independent experiments.
2.14. Statistical Analyses. Data were presented as mean ± SD and analyzed using SPSS 16.0 software. Histograms were created using GraphPad Prism 5 software (San Diego, CA, USA). The significance of difference was determined by oneway ANOVA with the post hoc Dunnett's test. Values of < 0.05 were considered to be statistically significant.

DATS Inhibits Viability and Reduces Fibrotic Protein Expression Associated with H 2 S in HSCs.
We initially evaluated potential cytotoxicity of DATS on HSCs using LDH assay, showing that DATS at 20 M or higher concentrations had significant cytotoxic effects on HSCs (Figure 1(a)). We thus investigated DATS effects at nontoxic doses subsequently. We herein used H 2 O 2 to stimulate HSC activation mimicking oxidative stress, because H 2 O 2 is the most stable ROS and diffuses readily in and out of cells [10]. DATS at doses of 0.1-10 M inhibited cell viability concentrationdependently in H 2 O 2 -stimulated HSCs (Figure 1(b)). Moreover, DATS reduced the expression of fibrotic marker proteins -smooth muscle actin ( -SMA), 1(I) procollagen, and fibronectin and downregulated the type I and II receptors for transforming growth factor-(TGF-RI and TGF-RII), platelet-derived growth factor-receptor (PDGF-R), and epidermal growth factor receptor (EGF-R) in H 2 O 2stimulated HSCs (Figure 1(c)). We hypothesized that DATS exerted its effects associated with production of H 2 S. The compound IAM has been demonstrated to block both the exofacial and intracellular thiols resulting in elimination of garlic-induced H 2 S production [11]. Here, IAM at concentration range within 100 M did not cause morphological alterations in HSCs, and accordingly we selected 100 M IAM for subsequent experiments (Figure 1(d)). We found that DATS concentration-dependently increased the H 2 S content in HSCs and that IAM at 100 M significantly abrogated DATS elevation of H 2 S production and reduced the base level of H 2 S in HSCs (Figure 1(e)), highlighting IAM as a potent blocker of DATS generation of H 2 S. Furthermore, IAM effectively rescued DATS downregulation of fibrotic marker molecules in H 2 O 2 -stimulated HSCs (Figure 1(f)). Taken together, these data suggested that H 2 S was involved in DATS suppression of the expression of fibrotic proteins in activated HSCs.

DATS Induces Cell Cycle Arrest Associated with H 2 S in HSCs.
We next examined DATS effects on cell fate of HSCs. DATS arrested cell cycle at G2/M checkpoint in H 2 O 2stimulated HSCs (Figure 2(a)). Addition of IAM abrogated DATS-induced G2/M arrest (Figure 2(b)). Cyclin A, cyclin B1, cyclin-dependent kinase (CDK) 1, and CDK2 are four critical regulators at G2/M checkpoint responsible for driving cells into the mitosis process [12]. DATS decreased the expression of cyclin B1 and CDK1, while cyclin A and CDK2 were not apparently affected by DATS (Figure 2(c)). Moreover, DATS inhibition of cyclin B1 and CDK1 was abolished by IAM (Figure 2(d)). These data collectively indicated that H 2 S was involved in DATS modulation of cell cycle regulators and G2/M checkpoint arrest in HSCs.

DATS Stimulates Apoptosis Associated with H 2 S in
HSCs. We subsequently examined DATS effects on HSC apoptosis. HSCs treated with DATS exhibited significant DNA condensation and fragmentation with brilliant blue staining (Figure 3(a)), and DATS increased apoptotic rates concentration-dependently in H 2 O 2 -treated HSCs (Figure 3(b)). However, the increase in apoptotic rate by DATS was abolished by IAM (Figure 3(c)). Furthermore, the antiapoptotic protein Bcl-2 was diminished by DATS; the proapoptotic molecule Bax was increased by DATS (Figure 3(d)). DATS also activated the caspase cascade in H 2 O 2 -stimulated HSCs, because the cleaved forms of caspase-9, caspase-8, caspase-7, and caspase-3, and PARP-1 were all increased by DATS (Figure 3(d)). However, DATS effects on these molecules were abrogated by IAM (Figure 3(e)). Altogether, these findings indicated that H 2 S was involved in DATS induction of caspase-mediated apoptosis in HSCs.

DATS Inhibits Migration Associated with H 2 S in HSCs.
We next examined DATS effects on migration in HSCs. DATS inhibited the lateral migration of H 2 O 2 -activated HSCs (Figure 4(a)). However, IAM abrogated DATS's inhibitory effects on HSC lateral migration (Figure 4(b)). To confirm the results, we performed transwell migration assay to evaluate the vertical migration of HSCs, showing that H 2 O 2 -stimulated vertical migration was suppressed by DATS ( Figure 4(c)). Similarly, DATS effects were also significantly compensated by IAM (Figure 4(d)). Collectively, these data indicated that DATS inhibition of cell migration was associated with H 2 S in HSCs.

DATS Alleviates Oxidative Stress Associated with H 2 S in HSCs.
We finally determined DATS effects on oxidative stress in HSCs. Fluorescence microscope analyses and flow cytometry assessments consistently demonstrated that H 2 O 2 -treated HSCs had significantly high intracellular levels of ROS, but DATS decreased ROS production (Figures 5(a) and 5(b)). However, IAM significantly rescued DATScaused reduction in ROS levels in H 2 O 2 -stimulated HSCs ( Figure 5(c)). Furthermore, DATS decreased the intracellular levels of LPO and increased the intracellular levels of GSH in H 2 O 2 -stimulated HSCs ( Figure 5(d)). However, DATS inhibition of LPO and elevation of GSH were significantly compensated by IAM ( Figure 5(e)). Altogether, these results suggested that DATS attenuated oxidative stress in HSCs, which was relevant to H 2 S.

Discussion
Many studies demonstrated that garlic extracts elicited therapeutic effects against liver fibrosis. Garlic extracts restored liver histology and accelerated ECM degradation, resulting in regression of liver fibrosis in CCl 4 -intoxicated rats [13]. Administration of aqueous garlic extracts alleviated hepatic oxidative injury and reduced collagen content in bile duct ligation-induced fibrosis in rats [14]. However, these investigations did not identify the specific functional ingredients responsible for the antifibrotic efficacy of garlic. Our previous data showed that DATS attenuated collagen deposition, inhibited HSC activation, and ameliorated oxidative stress in rat fibrotic liver, strengthening the therapeutic value of garlic for liver fibrosis [7]. Elucidation of the underlying mechanisms would be essentially important for developing DATS as a promising antifibrotic candidate.
Our current experiments could provide consistent support for the prior in vivo data. We found that DATS inhibited HSC activation in culture, which confirmed that HSCs could be target cells for DATS, because DATS was able to inhibit nearly all aspects of HSC activation. To explore the underlying mechanisms, we concentrated on the potential biotransformation of DATS. A 2007 study firstly described the real-time kinetics of H 2 S production of DATS within cells, showing that DATS with allyl moieties and three sulfur atoms reacted with exofacial membrane thiols and cross the cell membrane to react with GSH to produce H 2 S, which was a thiol-dependent manner for DATS to produce H 2 S [11]. Thereafter, increasing evidence reveals that the beneficial effects of garlic-derived organic polysulfides are mediated by production of H 2 S. Herein, we thus strongly speculated that H 2 S contributed to DATS inhibition of HSC activation. As expected, DATS could significantly increase H 2 S levels concentration-dependently in HSCs in current study. Moreover, we reasoned that IAM could be a proper blocker of DATS production of H 2 S, because their thioldependent mechanisms of action were exactly matching. Consistently, we here observed that IAM not only abolished DATS elevation of H 2 S production, but also reduced basal H 2 S synthesis in HSCs. Accordingly, we used IAM to testify the role of H 2 S in DATS inhibition of HSC activation properties. The obtained results clearly demonstrated that this H 2 S manipulation significantly weakened DATS effects. Cleaved-caspase-9 Cleaved-caspase-3 Pro-caspase-3 Full-length PARP-1 Cleaved-PARP-1 Cleaved-caspase-7 Pro-caspase-8 Cleaved-caspase-8 Pro-caspase-7 -Actin -Actin Cleaved-caspase-9 Cleaved-caspase-3 Pro-caspase-3 Full-length PARP-1 Cleaved-PARP-1 Cleaved-caspase-7 Pro-caspase-8 Cleaved-caspase-8 Pro-caspase-7 -Actin -Actin We identified the H 2 S as a molecular link implicated in DATS's antifibrotic activity. Actually, the role of H 2 S in hepatology has been studied increasingly during recent years. Malfunction of hepatic H 2 S metabolism may be involved in many chronic liver diseases, including hepatic fibrosis and cirrhosis [15]. Exogenous H 2 S could reduce hepatotoxicity, liver cirrhosis, and portal hypertension through multiple functions including antioxidation, anti-inflammation, cytoprotection, and antifibrosis [16]. Our current data were consistent with these observations and provided evidence that DATS could be transformed to be H 2 S that mediated the potent hepatoprotective and antifibrotic effects in HSCs. We demonstrated that H 2 S was involved in DATS effects on many aspects of HSC activation, including fibrotic marker expression, cell cycle, apoptosis, migration, and the status of oxidative stress. Consistent with our data, recent investigations showed that sodium hydrosulfide, a well-known inorganic H 2 S-releasing molecule, suppressed proliferation and induced G1 phase cell cycle arrest in HSCs [17] and attenuated collagen expression in rats with CCl 4 -induced hepatic fibrosis [18]. These findings collectively suggested H 2 S as a signaling molecule regulating the pathophysiology of HSCs. Actually, H 2 S promotes a number of cellular signals that regulate metabolism, proliferation, and apoptosis in various types of cells. For example, exogenous treatment with H 2 S led to growth inhibition with cyclin D1 downregulation in rat smooth muscle cells [19] and stimulated apoptosis by downregulation of Bcl-2 and Bcl-xL in portal vein smooth  muscle cells [20]. These observations were recaptured in HSCs by our present study and potentiated the role of H 2 S as a gaseous signaling molecule with a DNA damaging function. Interestingly, there was evidence that administration of sodium hydrosulfide significantly inhibited hepatocyte apoptosis in hepatic ischemia reperfusion-induced injury in rats, suggesting a hepatoprotective role for H 2 S in vivo [21]. However, it should be noted that this study examined apoptosis molecules in whole liver tissues rather than in single type of cells. This may raise a possibility that the proapoptotic effects of H 2 S on HSCs could be masked due to the overwhelming amount of hepatocytes in the liver. It could also be assumed that the different effects of H 2 S on apoptosis were cell-specific and disease-specific. Furthermore, we found that DATS inhibited HSC migration associated with H 2 S. Consistently, recent studies showed that DATS suppressed migration and invasion in human colon cancer cells [22] and breast cancer cells [23]. Another important issue is that increasing evidence elucidates the fundamental role of HSC in liver immunology, because HSCs represents a versatile source of many soluble immunological active factors and may act as an antigen presenting cell [24]. Understanding the role of HSCs as central regulators of liver immunology may lead to novel therapeutic strategies for chronic liver diseases. Interestingly, H 2 S has multiple and complex cellular signaling roles in relation to immunological modulation under certain pathophysiological conditions [25]. However, studies addressing how H 2 S regulates HSC immunology during hepatic fibrosis are not seen, which could be an attractive field in hepatology.
We herein evaluated DATS effects in an oxidative stressinduced HSC activation model, because signs of oxidative stress are concomitant or precede HSC activation and collagen deposition [3]. We also suggested a role for H 2 S in regulation of oxidative stress in HSCs. H 2 S could increase intracellular GSH concentrations and suppress oxidative stress in mitochondria [26]. H 2 S could also protect murine liver against ischemia reperfusion injury through upregulation of intracellular antioxidant pathways [27]. The antioxidant effects of H 2 S have recently been examined through use of H 2 S-releasing derivative ACS67, which was found to inhibit ROS formation and NADH oxidation in endothelial cells [28]. In current study, H 2 S also contributed to DATS attenuation of oxidative stress in HSCs. However, whether H 2 S exerted its antioxidant effects by virtue of its undoubted reducing activity or by its activation of endogenous defense systems merits further investigation. Furthermore, it is necessary to use an additional HSC line and another blocker of H 2 S generation to make the results more credible. There is also a need to carry out in vivo studies using certain H 2 S generation blocker to validate the H 2 S-dependent mechanism underlying the antifibrotic effects of DATS, which is ongoing in our laboratory.
In summary, DATS inhibited the profibrogenic properties and alleviated oxidative stress in HSCs. These effects were  associated with production of H 2 S within cells. Our data elucidated, at least partially, the mechanisms underlying DATS's antifibrotic activity and indicated a therapeutic role of targeting H 2 S for treatment of liver fibrosis.