Impact of Lycium barbarum polysaccharide on apoptosis in Mycoplasma-infected splenic lymphocytes

Purpose: To evaluate the effect of Lycium barbarum polysaccharide (LBP) on apoptosis in Mycoplasma-infected splenic lymphocytes (SLs), and the underlying mechanisms. Methods: SLs isolated from C57BL/6J mice were infected with Mycoplasma. The infected SLs were administered at different concentrations of LBP for 4 h, and the proportions of apoptotic cells and levels of relative reactive oxygen species (ROS) were determined by flow cytometry. The expressions of proapoptotic genes and endogenous antioxidant enzymes were investigated by real-time polymerase chain reaction (RT-PCR) and Western blotting. Results: LBP treatment produced dose-dependent reductions in apoptotic ratio and intracellular ROS levels of SLs (p < 0.05). In addition, the expressions of pro-apoptotic genes were decreased by LBP treatment with respect to mRNA and protein levels (p < 0.05). In contrast, mRNA and protein levels of anti-apoptotic factor Bcl-2 were significantly increased in a dose-dependent manner (p < 0.05). Furthermore, RT-PCR and Western blot results demonstrated that the expression levels of mRNA and proteins in Nrf2, HO-1 and NQO1 were up-regulated by Mycoplasma infection (p < 0.01), and further increased by LBP treatment (p < 0.05). Conclusion: LBP exerts a hyperactive antioxidant response encoded by Nrf2 to protect SLs from apoptosis induced by ROS-related oxidative damage after Mycoplasma infection. These results suggest that LBP may serve as a beneficial and dietary anti-Mycoplasma and anti-apoptotic agent.


INTRODUCTION
Mycoplasma has significant impacts on many human diseases [1]. Immune responses are believed to play roles in protection from Mycoplasma diseases [1]. In Chinese traditional medicine, the use of Lycium barbarum as a functional food for promoting immunity and longevity were documented as far back as 2,800 BC [2]. Studies have shown that Lycium barbarum polysaccharide (LBP), a major active component of Lycium barbarum, attenuates cytotoxicity, lowers blood glucose, enhances immunity, and also possesses anti-cancer properties [3][4][5]. Apoptosis is closely associated with lymphocyte development and homeostasis [6]. Enhanced or inhibited apoptosis in lymphocyte can lead to immunodeficiency or autoimmunity, or lymphoma [6]. LBP plays an important role in lymphocyte proliferation and cytokine production [7,8]. A recent study reported that LBP has beneficial effects on spermatogenesis through regulation of apoptosis and antioxidant activity in diabetic male mice [9]. However, little is known about the effect of LBP on apoptosis in lymphocytes.
Reactive oxygen species (ROS) have been considered as messengers of phagocytes involved in defense against pathogen invasion [10]. They are involved in lymphocytes activation, hypo-responsiveness, and apoptosis [10]. Recent studies indicate that LBP has antioxidant properties [9,11]. Thus, this study was aimed at investigating the effects of LBP on apoptotic signaling induced by ROS-related oxidative damage in splenic lymphocytes (SLs) after Mycoplasma infection.

Mycoplasma species
Mycoplasma (MP) species (ATCC 15488, Ziker Biotech, China) was cultured in modified Friis broth containing 2.5 % CO 2, at 37 °C. The culture medium was centrifuged at 18,000 g for 20 min at 4 °C, to harvest the Mycoplasma. The cells were washed thrice in PBS (pH 7.4), and suspended in RPMI 1640 medium. It was previously reported that at 540 nm, an absorbance of 0.2 was equivalent to 10 8 colonyforming units per ml (CFU/mL) of Mycoplasma [12]. Thus, the concentration of Mycoplasma was determined spectrophotometrically at 540 nm.

LBP isolation
Water extraction and ethanol precipitation were employed to extract LBP from Lycium barbarum fruit. Dried fruits of Lycium barbarum were ground to fine powder and extracted in water at 4 °C overnight. The Lycium barbarum residue was then extracted three times in boiling water. Then, the aqueous extract was concentrated and precipitated using 5 volumes of 100% ethanol. The precipitate was dried to obtain the crude LBP, which was then purified using DEAEcellulose ion exchange chromatography. Prior to administration, the LBP was dissolved in PBS and filtered through a 0.22 μm filter.

Flow cytometry analysis
SLs were harvested and washed twice with PBS. Then, the cell pellet was re-suspended in washing buffer. For the apoptosis analysis, the cells were stained with PI dye at 4 °C for 30 min, followed by examination using a FACSCanto II cytometer (BD Biosciences, Germany). The percentage of apoptotic cells was calculated.
In order to determine the level of ROS, SLs were incubated with fluorescent probe DCFH-DA (10 uM) at 37 °C for 30 min. Then, after washing, intracellular ROS were quantified by FACSCanto II cytometer (BD Biosciences, Germany).

Quantitative real-time PCR
SLs were washed with PBS and RNA was extracted using RNeasy Plus Micro Kit (QIAGEN, USA), followed by cDNA synthesis using SuperScript® IV First-Strand Synthesis System (Invitrogen, USA). PCR primers (with sequences) of the target genes were as follows: β-Actin

Western blot analysis
Cells were lysed in an ice-cold hypotonic lysis buffer (containing proteinase inhibitor cocktail and PMSF) and centrifuged at 10,000 rpm at 4 °C for 3 min. The protein concentration was measured using BCA Protein Assay reagent kit (Thermo Fisher Scientific, USA). The cell lysates (20 g of protein) were subjected to 10 % SDS-PAGE. Then, the gel was electro-transferred onto a PVDF membrane. After blocking with 5 % skimmed milk, the membrane was successively incubated with anti Caspase-3, Bax, Bcl-2, Nrf2, HO-1, and NQO1 primary antibodies (1:1000; Abcam, USA) and goat anti-rabbit IgG-HRP secondary antibody (1:2000; Abcam, USA). Bands were detected by ECL kit (Thermo Fisher Scientific, USA). Target gene expression levels were normalized with an internal control (βactin).

Statistical analysis
All data are presented as mean ± standard deviation (SD). For multiple group comparisons, one-way analysis of variance (ANOVA) and appropriate post-hoc analysis were performed using a statistical software package (Prism5, USA). P < 0.05 and < 0.01 were considered statistically significant.

LBP prevented SLs apoptosis
The LBP-treated groups exhibited dosedependent reductions in the apoptotic ratio of SLs when compared to the MP-infected group (Figure 1). When the LBP concentration reached 100 μg/mL, the apoptotic ratio of SLs was significantly decreased relative to the MPinfected group (p < 0.05). The apoptotic ratio in the 400 μg/mL LBP treated group was decreased to a level similar to the control group. These data indicate that LBP is an effective anti-apoptotic agent in SLs.

LBP prevented accumulation of intracellular ROS
Flow cytometry analysis showed a dosedependent decrease in intracellular levels of ROS in SLs. When the LBP concentration reached 400 μg/mL, ROS levels were significantly decreased when compared to the MP group (p < 0.05, Figure 2). These results indicate that ROS clearance may be a mechanism involved in the protective effect of LBP on the apoptosis of mycoplasma-infected SLs.

LBP regulated the expressions of proapoptotic genes
The expressions of pro-apoptotic genes, caspase-3 and Bax, were decreased by LBP treatment at both mRNA and protein levels. In the 200 and 400 μg/mL groups, the relative expressions of these genes decreased significantly when compared with the MP group (p < 0.01, Figure 3). In contrast, both mRNA and protein levels of the anti-apoptotic factor Bcl-2 were significantly increased in a dose-dependent manner when compared with the 200 and 400 μg/mL MP groups (p < 0.01).  Results of qPCR and Western blot show that the mRNA and protein levels of Nrf2 were significantly up-regulated by Mycoplasma infection (p < 0.01 when compared with the control group. Figure 4). LBP treatment for 4 h further increased the Nrf2 expressions, when compared with the MP group (for mRNA levels: p < 0.01 in the 50 to 400 μg/mL groups; for protein levels: p < 0.01 in the 400 μg/mL group), HO-1 (for mRNA levels: p < 0.01 in the 200 to 400 μg/mL groups; for protein levels: p < 0.01 in the 50 to 400 μg/mL groups), and NQO1 (for mRNA levels: p < 0.01 in the 50 to 400 μg/mL groups; for protein levels: p < 0.01 in the 200 to 400 μg/mL groups; Figure 4). These results support the anti-apoptotic role and ROS-clearing effects of LBP.

DISCUSSION
In this study, Mycoplasma-infected SLs were used to investigate the role of LBP in antioxidant defense against immunologic injury. It was found that Mycoplasma infection increased apoptosis and ROS accumulation in SLs. However, LBP treatment reduced the ROS levels, increased the activation of Nrf2 pathway, and inhibited apoptotic signals. A growing body of evidence indicate that LBP and Lycium barbarum have a variety of immuno-modulatory functions. Some researchers reported that LBP can induce phenotypic and functional maturation of dendritic cells via NF κB signaling pathways [13,14]. It has been shown that LBP can act as an adjuvant for the generation of Tfh cells to enhance T celldependent antibody responses [15]. It has also been demonstrated that LBP can promote cytokine secretion in macrophages, Th1, and Th2 [16]. Researchers have consistently shown that LBP significantly induces proliferation of T lymphocytes [4,17]. However, not much was hitherto know about the anti-apoptotic and antioxidant effects of LBP in lymphocytes. To the best of our knowledge, the present study is the first to demonstrate the anti-apoptotic property of LBP in SLs through the Nrf2 pathway, thereby providing a deeper understanding of its beneficial properties in immunocytes. Numerous studies have reported that the Nrf2 signaling pathway plays a critical role in cellular defense against oxidative stress. Some researchers used Keap1-deficient mice and found that T-cell-specific augmentation of Nrf2 increased antioxidant response and reduced intracellular cytokine production by T-cells [18]. Nrf2-KO mice developed inflammatory lesions and autoimmune syndromes, and decreases in expressions of HO-1 and NQO1 [19]. HO-1 and NQO1 are two well-recognized downstream factors of the Nrf2 signaling pathway. HO-1 is as a potent radical scavenger known to protect cells against oxidative stress [20]. Studies with HO-1knockout mice which exhibit increased susceptibility to oxidative insults proved that HO-1 is involved in the endogenous defense against oxidative stress [20]. NQO1 is a flavoprotein which prevents the participation of quinones in redox cycling, thereby sequestering ROS [21]. However, there is lack of data on the role of Nrf2 signaling pathway in SLs infected with Mycoplasma. Thus, the effect of Mycoplasma on activation of Nrf2 and its downstream antioxidant enzymes (HO-1 and NQO1) was investigated in the present study. The results obtained suggest that the Nrf2 pathway is involved in the survival of Mycoplasma-infected SLs, and that LBP treatment protects the infected SLs through the clearance of ROS.

CONCLUSION
The results of this study indicate that LBP can protect Mycoplasma-infected SLs from ROSinduced apoptosis through a mechanism involving a hyperactive antioxidant response encoded by Nrf2. This finding provides evidence to show that LBP may serve as a beneficial and dietary anti-Mycoplasma and anti-apoptotic agent.

Open Access
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