Dehydroepiandrosterone Prevents H2O2-Induced BRL-3A Cell Oxidative Damage through Activation of PI3K/Akt Pathways rather than MAPK Pathways

Dehydroepiandrosterone (DHEA) is a popular dietary supplement that has well-known benefits in animals and humans, but there is not enough information about the mechanisms underlying its effects. The present study aimed at investigating these mechanisms through in vitro experiments on the effects of DHEA on rat liver BRL-3A cells exposed to oxidative stress through H2O2. The findings showed that DHEA increased the antioxidant enzyme activity, decreased ROS generation, and inhibited apoptosis in H2O2-treated cells. These effects of DHEA were not observed when the cells were pretreated with known antagonists of sex hormones (Trilostane, Flutamide, or Fulvestrant). Furthermore, treatment with estradiol and testosterone did not have the same protective effects as DHEA. Thus, the beneficial effects of DHEA were associated with mechanisms that were independent of steroid hormone pathways. With regard to the mechanism underlying the antiapoptotic effect of DHEA, pretreatment with DHEA was found to induce a significant decrease in the protein expression of Bax and caspase-3 and a significant increase in the protein expression of PI3K and p-Akt in H2O2-treated BRL-3A cells. These effects of DHEA were abolished when the cells were pretreated with the PI3K inhibitor LY294002. No changes were observed on the p-ERK1/2, p-p38, and p-JNK protein levels in H2O2-induced BRL-3A cells pretreated with DHEA. In conclusion, our data demonstrate that DHEA protects BRL-3A cells against H2O2-induced oxidative stress and apoptosis through mechanisms that do not involve its biotransformation into steroid hormones or the activation of sex hormone receptors. Importantly, the protective effect of DHEA on BRL-3A cells was mainly associated with PI3K/Akt signaling pathways, rather than MAPK signaling pathways.


Introduction
Oxidative stress, which is caused by an increase in the production of reactive oxygen species (ROS), plays an important role in the development of liver diseases like fatty liver, alcohol liver, and liver injury [1]. Oxidative stress affects cell functioning by damaging lipids, proteins, and enzymes, and this subsequently leads to the apoptosis of the affected cells [2]. Hydrogen peroxide (H 2 O 2 ), which is a prominent ROS, is closely involved in the induction of liver oxidative stress [3]. High H 2 O 2 levels are responsible for lipid peroxidation and DNA damage, which eventually lead to the apoptosis of hepatic cells [4,5]. The H 2 O 2 -induced apoptosis of hepatocytes involves the inhibition of antioxidative mechanisms and apoptosis-associated regulatory proteins like Bcl-2 family proteins and caspases. Thus, inhibition of proapoptotic pathways might be a feasible way of preventing or stalling liver damage caused by excess H 2 O 2 production. Furthermore, since oxidative stress has been implicated in the majority of liver injuries [6,7], another treatment strategy for liver injury might be the use of active antioxidant molecules that ameliorate liver oxidative stress.
The incidence of and susceptibility to liver diseases is known to increase with age [8]. There is some speculation that the aging-related degenerative changes observed in humans is associated with an aging-related marked decline in the levels of dehydroepiandrosterone (DHEA). In fact, the decrease in circulating DHEA levels is associated with multiple metabolic consequences including autoimmune diseases, aberrations in lipid metabolism, type 2 diabetes, and oxidative stress-related diseases [9]. Recently, DHEA was reported to exhibit antioxidative effects under conditions of acute as well as chronic oxidative stress [10][11][12], and these antioxidant effects have been confirmed through in vivo [13,14] and in vitro [15] experiments, including our recent study in which DHEA treatment was found to protect various types of cells against oxidative damage [16,17]. Although these beneficial effects of DHEA are known, there is not enough information about the mechanisms through which it exerts these effects.
DHEA is essentially a precursor protein that has the potential to transform into estrogens or androgens in organs such as the kidney, brain, gonads, and liver [18,19], and it can exert various physiological effects by binding receptors, such as the estrogen receptor, androgen receptor, and other highly specific receptors, in the target tissues. Some studies have reported that DHEA executes its effects mainly through conversion into sex steroids and activation of androgen or estrogen receptors [20][21][22]. For example, our recent study found that DHEA reduced lipid droplet accumulation in primary hepatocytes from the chicken through its biotransformation into steroid hormones [23], and Mills et al. showed that DHEA promotes the healing of cutaneous injuries by activating estrogen receptors [20]. In contrast, there is also some evidence that the positive effects of DHEA are independent of the activation of sex steroid receptors [24][25][26]. This means that DHEA may exert its physiological effects as a neurosteroid by directly binding to neurotransmitter receptors. However, the mechanisms underlying its effects and its efficacy remain unclear in the absence of sufficient supporting data. The present study sought to fill in this gap in the literature.
Based on the findings of the literature so far, the aims of the present study were to determine whether DHEA protects H 2 O 2 -exposed BRL-3A cells from oxidative stress and apoptosis and to identify the signaling pathways and mechanisms that may be involved in the effects of DHEA. We believe that these findings will shed light on the antioxidative mechanisms of DHEA, which may have potential for the treatment of oxidative stress-induced conditions in humans.

Testosterone and Estradiol Measurement by
Radioimmunoassay. BRL-3A cells were grown in 6-well plates (1 × 10 6 cells/well) and treated with DHEA at doses of 0, 1, 10, or 100 μM DHEA for 24 h. The BRL-3A cells were then harvested and ultrasonically disrupted on ice. Next, the cells were centrifuged at 2500 ×g for 10 min at 4°C. The supernatants were extracted, and the estradiol and testosterone contents were measured using radioimmunoassay kits (Beifang Biotechnology Research Institute, Beijing, China).

Cell Apoptosis Analysis.
The cell treatments were the same as described in Section 2.4 for ROS analysis, and cell apoptosis was examined as reported in our studies [16,17]. Briefly, BRL-3A cells were collected and washed with cold PBS, placed in 195 μL Annexin V-FITC binding solution, incubated in 5 μL Annexin V-FITC and 10 μL propidium iodide in the dark for 30 min, and immediately subjected to flow cytometry analysis with FACSCalibur™.
2.6. Measurement of Antioxidant Parameters. The treatments for the different cell groups were the same as described for the ROS analysis. For the measurements, the cells were harvested, disrupted ultrasonically in ice, and centrifuged at 2500 ×g for 10 min at 4°C. The supernatants were collected and stored -20°C for subsequent analysis. The activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were measured using commercial kits following the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the data were normalized to the protein concentration as determined by a BCA protein assay kit.

2.7.
Real-Time Quantitative RT-PCR. BRL-3A cells were grown in 6-well plates (1 × 10 6 cells per well) and treated with 0, 1, 10, or 100 μM DHEA for 24 h, then they were exposed to 150 μM H 2 O 2 for another 2 h. After incubation, the cells were harvested and total RNA was extracted using the TRIZOL reagent kit (Invitrogen, USA) according to the manufacturer's protocols. Total RNA (2 μg) were reverse transcribed into cDNA using the SuperScript II kit (Promega, USA) according to the manufacturer's recommendation. An aliquot of a complementary DNA sample was mixed with 20 μL SYBR Green PCR Master Mix (Roche, Switzerland) in the presence of 10 pmol of each forward and reverse primers for β-actin (used as an internal control), Bcl-2, and Bax (Table 1). All samples were analyzed in duplicate using the iQ5 Sequence Detection System (Bio-Rad, California, USA) and programmed to conduct one cycle (95°C for 3 min) and 40 cycles (95°C for 20 s, 60°C for 30 s, and 72°C for 30 s). The 2 -ΔΔCT method was used to calculate the fold change in mRNA levels. The primers used were designed by Primer Premier 5 (Premier Biosoft International, Palo Alto, USA) and synthesized by Invitrogen Biological Co. (Shanghai, China).
2.9. Data Analysis and Statistics. Data were expressed as means ± standard error (SE). Differences were analyzed using one-way analysis of variance (ANOVA) followed by post hoc tests. Differences were considered significant at P < 0 05. All statistical analyses were performed with SPSS 20.0 for Windows (StatSoft Inc., Tulsa, USA).

Results
3.1. Biotransformation of DHEA in BRL-3A Cells. The testosterone and estradiol content were not detected in the vehicle-treated group, while the content of both hormones was significantly higher in the DHEA-treated BRL-3A cells than in the vehicle-treated cells, and it increased as the dose of DHEA increased from 1 to 100 μM (P < 0 05) (Figures 1(a) and 1(b)). In keeping with these findings, DHEA treatment resulted in a significant increase in the protein expression of 3β-HSD and 17β-HSD in a dosedependent way (P < 0 05) (Figures 1(c)-1(e)). These findings are in keeping with the known biotransformation pathways through which DHEA is converted to sex hormones.

DHEA Increased Antioxidant Enzyme Activity in H 2 O 2 -
Induced BRL-3A Cells. As shown in Figure 2, the SOD, POD, CAT, and GSH-Px activities were significantly lower in the H 2 O 2 -treated BRL-3A cells than in the vehicletreated cells (P < 0 01). Pretreatment with 1-100 μM DHEA before H 2 O 2 treatment caused the SOD, POD, CAT, and GSH-Px activities to be significantly higher than those in the cells that were treated only with H 2 O 2 (P < 0 05). These effects of DHEA improved as its dose was increased.
The effects of 100 μM DHEA on the activity of the antioxidant enzymes were not altered when the cells were pretreated with Trilostane (the 3β-HSD inhibitor), Flutamide (the AR antagonist), and Fulvestrant (the ER antagonist) ( Figure 2). Furthermore, in H 2 O 2 -induced BRL-3A cells that were pretreated with 12.0 nM testosterone and 6.3 nM estradiol, the antioxidant enzyme activities were  (Figure 3).

Discussion
In the present study, our in vitro experiments on rat liver cells show that DHEA exerts antioxidative and antiapoptotic effects through pathways that are independent of steroid hormones and their receptors; instead, the PI3K/p-AKT pathways seem to be closely involved with these protective effects of DHEA on the liver cells. BRL-3A rat liver cells were used as an in vitro model for studying the effects of H 2 O 2 , as this cell type is susceptible to oxidative damage. Furthermore, H 2 O 2 is well known as    an ROS that easily penetrates cells and reacts with metal ions to produce highly reactive hydroxyl radicals that can cause severe cellular injuries. Here, exposure to 150 μM of H 2 O 2 led to the loss of antioxidant activity and generation of ROS in the BRL-3A cells. Similarly, H 2 O 2 has been shown to induce oxidative damage via its inhibitory effects on antioxidant enzyme activity [27,28]. Furthermore, in the present study, treatment with DHEA reversed these effects of H 2 O 2 . This is also in keeping with the findings of previous studies in which DHEA was reported to inhibit in vitro glucoseinduced ROS generation [29] and in vivo ROS generation, as well as to increase the in vivo protein expression of NADPH oxidase [30]. In particular, DHEA was found to exert antioxidant effects by inducing an increase in catalase expression, activating the thioredoxin system, and suppressing superoxide anion production [14,31]. Also in this study, DHEA was found to promote the activity of the antioxidant enzymes such as SOD, CAT, POD, and GSH-Px in the H 2 O 2 -treated cells. Thus, all these findings demonstrate that DHEA protects the cell against oxidative damage by promoting the activity of antioxidant enzymes and inhibiting ROS production. Another target of DHEA is cellular apoptotic pathways, as we found that BRL-3A cells that were pretreated with DHEA were protected against the apoptosis-promoting effects of H 2 O 2 . Among the proteins involved in apoptosis, Bax and Bcl-2, which are members of the Bcl family of proteins, play an important role as regulators of the initial stages of apoptosis [32]. In this study, DHEA inhibited the H 2 O 2 -induced downregulation of Bcl-2 mRNA expression and upregulation of Bax mRNA expression. Caspases are also important players in the apoptosis pathways, and activated caspase-3, in particular, plays a major proapoptotic role [33]. Our results showed that pretreatment with DHEA led to a significant decrease in the protein expression of Bax and caspase-3 in BRL-3A cells that were treated with H 2 O 2 . Therefore, DHEA might protect liver cells from the effects of H 2 O 2 through its regulatory effects on downstream apoptosis-related proteins of the Bcl-2 and caspase family.
As DHEA has been reported to exert its physiological effects via bioconversion into sex hormones and activation of estrogen/androgen receptors, we also investigated whether DHEA exerted its antioxidative effects via such hormonal mechanisms. First, direct measurement of the testosterone and estradiol contents showed that they had increased in the BRL-3A cells after DHEA treatment. Consistent with these changes in the active hormone content, a significant increase was observed in the protein expression of 3β-HSD and 17β-HSD after DHEA treatment. In agreement with our findings, DHEA has been shown to promote the testosterone and estradiol levels in both in vivo and in vitro settings [34,35]. These findings can be explained through the process of bioconversion wherein 3β-HSD catalyzes the rapid conversion of DHEA into androstenedione in peripheral organs such as the brain, liver, kidney, and gonads, after which androstenedione is converted into testosterone and estradiol by 17β-HSD and aromatase, respectively [36]. However, although there was evidence of the bioconversion of DHEA, we found that pretreating H 2 O 2 -treated BRL-3A cells with Trilostane (a 3β-HSD inhibitor), Flutamide (an AR antagonist), and Fulvestrant (an ER antagonist) did not alter the effects of DHEA on the ROS content, antioxidant enzyme activity, or apoptosis rate. Furthermore, pretreatment with testosterone and estradiol did not have the same effects as pretreatment with DHEA on the ROS content, antioxidant activity, or apoptosis rate of H 2 O 2 -treated BRL-3A    cells. From these findings, it can be speculated that although a small amount of DHEA was converted into active steroid hormones in BRL-3A cells, the antioxidant and antiapoptotic effects of DHEA were independent of steroid hormonal mechanisms. PI3K/Akt and mitogen-activated protein kinases (MAPKs) such as ERK1/2, JNK, and p38 are known to participate in oxidative stress-induced cell apoptosis [37]. Specifically, MAPKs were found to play a proapoptotic role and PI3K/Akt was found to play a prosurvival role in cells exposed to oxidative stress [38]. Based on these findings, we analyzed the expression of these proteins in H 2 O 2 -treated BRL-3A cells that were protected by DHEA pretreatment. There was a significant increase in the protein expression of p-ERK1/2, p-JNK, and p-p38 after H 2 O 2 treatment, but pretreatment with DHEA did not alter these effects of H 2 O 2 . Thus, the antioxidant mechanisms of DHEA may not involve the activation of MAPK signaling pathways. However, previous studies have shown that DHEA induced a decrease in the level of phosphorylated JNK and p38 in H 2 O 2 -treated muscle cells and thereby decreased their apoptosis rate [39]. The differences in the findings might be related to the use of different cell types in the studies.
In contrast to the findings on MAPK signaling, in the current study, DHEA pretreatment resulted in a significant decrease in the protein expression of PI3K and p-Akt in the H 2 O 2 -treated cells. In agreement with our findings, it has been reported that DHEA protects human neuroblastoma SH-SY5Y cells from apoptosis induced by serum deprivation via the PI3K/Akt pathway [40]. Furthermore, in our study, pretreatment with DHEA also repressed the increase in the protein expression of Bax and caspase-3 that was induced by H 2 O 2 . These effects of DHEA on the expression of PI3K, p-Akt, Bax, and caspase-3 were eliminated when the cells were pretreated with the PI3K inhibitor LY294002. This confirms the involvement of the PI3K/Akt pathways in the DHEA-mediated protection of BRL-3A cells against apoptosis induced by H 2 O 2 . Based on all the findings, it can be concluded that DHEA protected BRL-3A cells against H 2 O 2 -induced oxidative damage by activating PI3K/Akt signaling pathways, rather than by activating MAPK signaling pathways.    In conclusion, our data demonstrate that DHEA exerted a protective effect on BRL-3A cells that were exposed to H 2 O 2 by inhibiting the production of ROS, promoting the activity of antioxidant enzymes, and regulating the expression of apoptosis-related proteins. Furthermore, the antiapoptosis mechanisms of DHEA involved the activation of the PI3K/Akt signaling pathways, rather than the MAPK signaling pathways. Importantly, these effects of DHEA are independent of androgen and estrogen receptor pathways (Figure 8). This could mean that DHEA directly interacts with specific receptors to exert these effects, but this needs to be explored through future investigations into its mechanisms. This information not only increases our understanding of the molecular mechanisms of DHEA, but it also highlights the potential applications of DHEA in the treatment of diseases, especially liver diseases, caused by oxidative stress.   (2) reducing caspase-3 protein levels through the activation of PI3K/Akt signaling pathways, rather than MAPK signaling pathways; and (3) activating specific receptors rather than androgen and estrogen receptors.