Protective effects of oxymatrine against arsenic trioxide-induced liver injury

Oxymatrine, a quinolizidine natural drug extracted from Sophora japonica, has been reported to have neuroprotective effect and cardioprotective effect. However, the protective effect of oxymatrine on arsenic trioxide (As2O3)-induced liver injury has not been reported. In the present study, we investigated the protective effects of oxymatrine on As2O3-induced liver injury in rats. Male Wistar rats were administrated 3mg/kg As2O3 intravenous injection on alternate days for 4 days. Oxymatrine was given 1 h before As2O3 treatment. The results showed that oxymatrine inhibited As2O3-induced hepatic pathological damage, liver ROS level and MDA level in a dose-dependent manner. As2O3 decreased the antioxidant enzymes SOD, GPX, and CAT activity and the decrease was inhibited by treatment of oxymatrine. Furthermore, oxymatrine attenuated the retention of arsenic in liver tissues and improved the expression of Nrf2 and HO-1. In conclusion, our results suggested that oxymatrine protected against As2O3-induced oxidative damage by activating Nrf2/HO-1 signaling pathway.


INTRODUCTION
Arsenic is a hazardous substance of global concern that is present in soil, drinking water, and food [1]. Arsenic often causes severe health hazards such as dermatosis, diabetes, and cancers, despite of its beneficial role in the treatment of acute promyelocytic leukemia (APL) [2,3]. Studies showed that arsenic affects all organ systems of human and liver is one of the most important target organs for arsenic [4]. Previous reports demonstrated that arsenicinduced liver injury was closely associated with oxidative stress [5]. Arsenic exposure has been reported to produce reactive oxygen species and depress the antioxidant defense system which leading to the oxidative damage of liver tissues [6]. Recently, studies showed that antioxidants had therapeutic effects against arsenic-induced liver injury [7,8]. Nrf2 is a redox-sensitive transcription factor that plays a critical role in cellular antioxidant defense [9]. Once stimulation, Nrf2 translocates to the nucleus to initiate transcription of cytoprotective genes such as HO-1 [10]. Studies showed that activation of Nrf2 could protect against arsenic trioxide-induced injury.
Oxymatrine, the major quinolizidine alkaloid isolated from the root of Sophora flavescens Ait (kushen), has been reported to have anti-inflammatory, anti-tumor, and antioxidant effects [11,12]. Previous reports showed that oxymatrine exerted a protective effect on ischemia or ischemia/reperfusion damage in liver, intestine and heart [13,14]. Oxymatrine has been reported to protect rat brains against permanent focal ischemia [15]. Oxymatrine also protected against experimental hepatic fibrosis [16]. In addition, oxymatrine was found to prevent adriamycininduced cardiac injury in rabbits, which was associated with its antioxidant and anti-apoptotic activities [17]. However, the protective effect of oxymatrine against arsenic trioxide-induced liver injury has not been reported. Thus, in the present study, we aimed to investigate the protective effects of oxymatrine against arsenic trioxideinduced liver injury.

Oxymatrine reduced As 2 O 3 -induced liver histopathologic changes
The effects of oxymatrine on As 2 O 3 -induced liver histopathologic changes were detected in this study. The results showed that liver tissues of control and oxymatrinetreated groups showed normal lobular architecture and cellular structure. Liver sections of As 2 O 3 -treated group showed significant pathologic changes, such as extensive areas of portal inflammation, inflammatory cell infiltration and cellular necrosis. However, the pathological changes of liver sections were attenuated in As 2 O 3 + oxymatrine (12.5, 25, 50mg/kg) treated group ( Figure 1).

Effects of oxymatrine on As 2 O 3 -induced antioxidant enzymes SOD, GPX, and CAT activity
The effects of oxymatrine on As 2 O 3 -induced antioxidant enzymes SOD, GPX, and CAT activity was shown in Figure 2. The results showed that compared to the control group and oxymatrine-treated group, the levels of SOD, GPX, and CAT decreased significantly in As 2 O 3 -treated group. However, the inhibition of SOD, GPX, and CAT levels by As 2 O 3 -was revised by treatment of oxymatrine (12.5, 25, 50mg/kg).

Effects of oxymatrine on As 2 O 3 -induced ROS and MDA levels
The effects of oxymatrine on As 2 O 3 -induced ROS and MDA levels were shown in Figure 3. The results showed that ROS and MDA levels increased significantly in As 2 O 3 -treated group in comparison with control group. However, the levels of ROS and MDA in As 2 O 3 + oxymatrine (12.5, 25, 50mg/kg) treated group decreased significantly in comparison with As 2 O 3 -treated group.

Effects of oxymatrine on As 2 O 3 -induced ALT and AST levels in serum
The effects of oxymatrine on As 2 O 3 -induced ALT and AST levels were shown in Figure 4. As shown in Figure 4, the levels of ALT and AST of As 2 O 3 -treated group increased significantly than that of control group. However, the levels of ALT and AST in As 2 O 3 + oxymatrine (12.5, 25, 50mg/kg) treated group decreased significantly in comparison with As 2 O 3 -treated group.

Effects of oxymatrine on As accumulation in liver tissues
The results showed that compared to the control group, As 2 O 3 resulted in a significant increase in the arsenic concentration of liver tissues. However, treatment   are the mean ± SEM (n=12 in each group). p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. As 2 O 3 group. www.impactjournals.com/oncotarget of oxymatrine (12.5, 25, 50mg/kg) significantly attenuated As 2 O 3 -induced accumulation of arsenic in liver tissues ( Figure 5).

Effects of oxymatrine on Nrf2 and HO-1 expression
The effects of oxymatrine on Nrf2 and HO-1 expression were detected by Western blot analysis. As shown in Figure 6, As 2 O 3 treatment increased the expression of Nrf2 and HO-1. However, oxymatrine (12.5, 25, 50mg/kg) up-regulated the expression of Nrf2 and HO-1 induced by As 2 O 3 .

DISCUSSION
Oxymatrine, a quinolizidine natural drug extracted from Sophora japonica, has been reported to have antioxidant effect [12]. In the present study, we investigate the protective effects of oxymatrine on As 2 O 3 -induced toxicity in liver and oxidative stress in rats. The results showed that oxymatrine exhibited protective effects on As 2 O 3 -induced liver injury. Oxymatrine protected against As 2 O 3 -induced liver injury through activating Nrf2 signaling pathway.
Serum AST and ALT were used as biochemical indicator of liver injury [18,19]. Our results showed that the levels of AST and ALT increased significantly in rats exposure to As 2 O 3. Elevated levels of serum AST and ALT in As 2 O 3 -treated rats were associated with the extensive alterations in the pathological changes of liver tissues. Treatment of oxymatrine remarkably inhibited As 2 O 3 -induced AST and ALT production which suggesting oxymatrine could attenuate liver damage. Meanwhile, histological analysis demonstrated that oxymatrine attenuated liver pathologic changes, including inflammatory cell infiltration and cellular necrosis. These results indicated oxymatrine had protective effects against As 2 O 3 -induced liver injury.
Previous studies showed that As 2 O 3 -induced liver injury is associated with increased oxidative stress in liver tissues [20]. Arsenic exposure could induce   the production of ROS which played critical roles in arsenic-induced toxicity [21,22]. Lipid peroxidation generates a variety of relatively stable decomposition end-products, such as MDA, which can be measured as indirect indicators of oxidative stress [23]. In this study, we found that the levels of ROS and MDA in liver tissues increased after As 2 O 3 exposure. Treatment of oxymatrine significantly inhibited As 2 O 3 -induced ROS and MDA production. Arsenic-induced damage in the antioxidant system involves several mechanisms such as altered SOD, CAT and GPX expression [24]. In this study, our results showed that the inhibition of SOD, GPX, and CAT levels by As 2 O 3 -was abolished by treatment of oxymatrine. These results indicated that oxymatrine exhibited antioxidant effects in As 2 O 3 -induced liver injury. Nrf-2 has been reported to play important roles in the induction of antioxidant enzymes against oxidative stress [25,26]. HO-1 expression is controlled by the transcription factor Nrf-2 [27]. Recent studies showed that Nrf-2 signaling pathway played a protective role in As 2 O 3 -induced liver injury [28]. Our results showed that the increases in Nrf2 and HO-1 expression were augmented by oxymatrine.
In conclusion, the present study demonstrated that oxymatrine attenuated As 2 O 3 -induced oxidative damage in liver tissues by activating Nrf2/HO-1 signaling pathway. Oxymatrine may be an agent for preventing and treating As 2 O 3 -induced liver injury.

Animals
Seventy-two healthy male Wistar rats (110-130 g) were purchased from the Center of Experimental Animals of Shandong University (Shangdong, China). The rats were housed under standard conditions (temperature, 23±2°C; humidity: 60±5%). The rats were acclimatized to the environment for 6 days prior to the experiments. The rats had free access to water and food. All animal experiments carried out in this study were approved by the NIH Guide for the Care and Use of Laboratory Animals.

Experimental protocol
A total of 72 rats were randomly divided into six groups (n=12 each group): normal control group, oxymatrine (50mg/kg) treatment group, As 2 O 3 exposure group, and As 2 O 3 + oxymatrine (12.5, 25, 50mg/kg) treatment group. In control group, rats were given equal amount of 0.9% normal saline. In oxymatrine treatment group, rats were given by an intraperitoneal injection of oxymatrine (50mg/kg). In the As 2 O 3 exposure group, rats were administrated 3mg/kg As 2 O 3 intravenous injection on alternate days for 4 days. In As 2 O 3 + oxymatrine treatment group, rats were given by an intraperitoneal injection of oxymatrine (12.5, 25, 50mg/kg) 1 h before As 2 O 3 administered. On the 8th day, rats were killed and the blood samples and livers from each group were collected for various biochemical analyses.

Histological analysis
The liver tissues were collected and fixed in 10% formalin. The liver tissues were dehydrated, embedded in paraffin, and sliced at 4μM thickness. Then, the sections were stained with hematoxylin and eosin (H&E) reagent and visualized with a microscope (Olympus, Japan).

Analysis of oxidative stress and antioxidant defense
Liver MDA and ROS level, the antioxidant enzymes SOD, GPX, and CAT activity were detected by using commercial kits purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China).

Blood clinical analyses
Blood samples were collected and centrifuged at 3000 g for 8 min to obtain serum. The ALT and AST levels were measured using test kits purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China).

Determination of total arsenic in the liver
The liver tissue was digested in HNO 3 -HCLO 4 solution for 48 h at 130°C. Then the digested samples were diluted with deionized water. The concentrations of arsenic were detected using atomic fluorescence spectrometry.

Western blot analysis
Proteins of liver tissues were extracted using T-PER Tissue Protein Extraction Reagent Kit according to the manufacturer's instructions (Thermo). The protein concentration was determined through BCA method. 40 μg proteins were separated on 10% SDS-PAGE gel and transferred onto PVDF membranes. After blocking with 5% skim milk for 2 h, the membranes were incubated with the specific primary antibodies Nrf-2 (1: 1000), HO-1 (1: 1000) at 4 °C overnight. After washing three times, the membranes were probed with HRP-conjugated secondary antibody at room temperature for 2h. Blots were then developed with the ECL Plus Western Blotting Detection System (Amersham Life Science, UK).

Statistical analysis
The results are expressed as the mean ± SEM of three independent experiments. For comparison among groups were determined by one-way ANOVA followed by the Tukey post-hoc test. The P< 0.05 was considered statistically significant.