Effect of Sophora subprosrate polysaccharide on oxidative stress induced by PCV2 infection in RAW264.7 cells

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Abstract

In this study, an oxidative stress model was first developed in a mouse macrophage cell line (RAW264.7 cells) by infecting the cells with porcine circovirus type 2 (PCV2). The regulatory effect of Sophora subprosrate polysaccharide (SSP) on PCV2-induced oxidative stress was investigated. The results showed that after infection with PCV2, reactive oxygen species (ROS) and nitric oxide (NO) production, myeloperoxidase (MPO) activity, and inducible nitric oxide synthase (iNOS) expression were significantly increased. Meanwhile, the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) and hydroxyl radical prevention capacity were greatly reduced. These data indicate successful creation of an oxidative stress model in RAW264.7 cells. A dramatic decrease in cell viability was observed in the cells exposed to oxidative stress compared to the control. When the cells were treated with SSP in concentrations of 100, 200 or 400 μg/mL post PCV2 infection, an increase in the GSH/GSSG ratio and hydroxyl radical prevention capacity was observed. We also observed decreased ROS and NO production, MPO activity, and iNOS expression in the infected cells. Our results demonstrated that PCV2 infection was able to induce oxidative stress in RAW264.7 cells and that SSP could reduce the negative effects resulting from the PCV2 infection.

Introduction

Oxidative stress is defined as an imbalance between the production and elimination of ROS that mainly consist of hydrogen peroxide (H2O2), hydroxyl radicals (radical dotOH) and superoxide anions (O2radical dot) [1]. This imbalance leads to damage of important biomolecules and cells, which has potential impacts on disease processes in the whole organism [2]. A variety of chronic and acute diseases are related to oxidative stress, including diabetes, cardiovascular diseases, cancer, aging, inflammation and neurological disease [3], [4], [5], [6], [7], [8]. Viral infection is often accompanied by oxidative stress, which is induced by the alteration of the intracellular redox state [9], [10]. For example, HIV-1 infection increased ROS generation in the host cells and decreased the cellular antioxidant capacity, which further enhanced viral replication [11], [12], [13], [14]. It was reported that antioxidants were able to suppress HIV replication in chronically infected cells [15], [16], [17], [18], [19], indicating that antioxidants may play an important role in antivirus infection.

Porcine circovirus type 2 (PCV2) is also an immunosuppressive virus in pigs. It is a small, nonenveloped, single-stranded DNA virus that belongs to the circoviridae family [20]. PCV2 infection usually accompanies lymphocyte or monocyte depletion and thus further results in immune suppression in the disease [21], [22], [23]. The immunosuppressive disease mainly presents as postweaning multisystemic wasting syndrome (PMWS), which caused a great economic loss worldwide [24], [25], [26]. PCV2 induces oxidative stress in porcine kidney cells (PK-15) by generating extra ROS during the infection, which was found to be associated with alterations in NF-κB activity [27]. Furthermore, oxidative stress induced by H2O2 was reported to enhance PCV2 replication in PK-15 cells [27], indicating that oxidative stress plays a crucial role in PCV2 infection. Previous studies found that the T-SOD activity, total antioxidant capacity (TAOC) and GSH level in the spleen and thymus were markedly decreased in the PCV2-infected mice. Treatment with carboxymethylpachymaran (CMP) improved the spleen and thymus index, promoted the proliferative activities of T and B lymphocytes, and increased the production of GSH, SOD capacity, and the total antioxidant capacity in the spleen and thymus in PCV2-infected mice [28]. These data suggest that the polysaccharide treatment was an effective therapy against PCV2 infection. However, the oxidative stress in PCV2-infected cells, especially in cells from the macrophage lineage, has not been evaluated.

Sophora subprosrate polysaccharide (SSP), a polysaccharide extracted from a traditional Chinese medicine herb named S. subprosrate, shows various biological activities, such as antioxidant and immunoregulatory properties. It was reported that SSP induced the production of NO and IL-2 in murine splenic T lymphocytes by increasing the intracellular calcium levels and PKC activity [29]. SSP was able to overcome cyclophosphamide-induced immunosuppression by raising the GSH level, SOD activity, total antioxidant capacity, and the indices of the thymus and spleen in mice [30]. The enhanced proliferation of splenic lymphocytes, elevated levels of IL-6, TNF-α and GSH and activities of lysozyme and GSH-Px have been observed in SSP-treated mice with dextamethasone-induced immunosuppression [31], suggesting that SSP is a potent immunomodulator and antioxidant.

The objective of the present study was to investigate the regulatory effect of SSP on the oxidative stress induced by PCV2 in RAW264.7 cells.

Section snippets

SSP and reagents

SSP was extracted and purified as described in previous studies [30]. Its structure was characterized in a previous study [32]. The purity of SSP used in this study was 99.9%, and the amount of endotoxin was less than 0.005 ng/mg. The white SSP powder was dissolved in a complete medium at a concentration of 400 μg/mL. The solution was then filtered using a 0.22-μm membrane. In this experiment, SSP was further freshly diluted to a final concentration of 100 or 200 μg/mL. All of the other chemicals

PCV2 infection in RAW264.7 cells

The PCV2 antigen in the RAW264.7 cells was detected at 4, 8, 12 or 24 h post infection (Fig. 1). According to the results of the PCR amplification, a strong specific fragment was found in the cells infected with PCV2 for 24 h when a dose of 104.7 TCID50 was used, while slight fragments was observed at 4, 8, 12 or 24 h when a dose of 102.7 TCID50 was used for PCV2 infection. These findings indicated that the cells were positive for the PCV2 antigen. An indirect immunofluorescence assay showed that

Discussion

In the present study, an oxidative stress model was successfully established by infecting RAW264.7 cells with PCV2, and the model was used to investigate the regulatory effect of SSP on oxidative stress in PCV2-infected RAW264.7 cells. Phagocytosis by macrophages is an important process that removes infectious invaders and participates in inflammation, the immune response, and tissue remodeling [38]. RAW264.7 cells, which form a macrophage cell line, have been frequently used to investigate

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 31260619), Project of the Guangxi Scientific Research and Technique Development(GKG0992014-6), the Guangxi Natural Science Foundation (GKQ0991003), the Guangxi Natural Science Foundation (2010GXNSFD013022) and the Guangxi University Science Foundation (No. X-071026).

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