Gang Qian

Selenium Alleviates Aflatoxin B1-Induced Immune Toxicity through Improving Glutathione Peroxidase 1 and Selenoprotein S Expression in Primary Porcine Splenocytes

Selenium (Se) is generally known as an essential micronutrient and antioxidant for humans and animals. Aflatoxin B1 (AFB1) is a frequent contaminant of food and feed, causing immune toxicity and hepatotoxicity. Little has been done about the mechanisms of how Se protects against AFB1-induced immune toxicity. The aim of this present study is to investigate the protective effects of Se against AFB1 and the underlying mechanisms. The primary splenocytes isolated from healthy pigs were stimulated by anti-pig-CD3 monoclonal antibodies and treated by various concentrations of different Se forms and AFB1. The results showed that Se supplementation alleviated the immune toxicity of AFB1 in a dose-dependent manner, as demonstrated by increasing T-cell proliferation and interleukin-2 production. Addition of buthionine sulfoximine abrogated the protective effects of SeMet against AFB1. SeMet enhanced mRNA and protein expression of glutathione peroxidase 1 (GPx1), selenoprotein S (SelS), and thioredoxin reductase 1 without and with AFB1 treatments. Furthermore, knockdown of GPx1 and SelS by GPx1-specific siRNA and SelS-specific siRNA diminished the protective effects of SeMet against AFB1-induced immune toxicity. It is concluded that SeMet diminishes AFB1-induced immune toxicity through increasing antioxidant ability and improving GPx1 and SelS expression in splenocytes. This study suggests that organic selenium may become a promising supplementation to protect humans and animals against the decline in immunity caused by AFB1.

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells.

Amelioration of CCl4-induced liver injury in rats by selenizing Astragalus polysaccharides: Role of proinflammatory cytokines, oxidative stress and hepatic stellate cells

Selenizing Astragalus polysaccharides (sAPS) were prepared by nitric acid-sodium selenite method. Effect of sAPS on carbon tetrachloride (CCl4)-induced liver injury and the underlying mechanisms were investigated in the rat. Forty male Wistar rats were divided into five equal groups as follows: control group; CCl4 group; CCl4+Astragalus polysaccharides group; CCl4+sodium selenite group and CCl4+selenizing Astragalus polysaccharides group. The results showed that sAPS significantly decreased the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase in the serum, malondialdehyde and hydroxyproline content in liver (P<0.01), and increased the levels of total protein, total antioxidant capacity, glutathione peroxidase, and superoxide dismutase in liver of rats induced by CCl4. In addition, expression levels of antioxidant-related genes (GPX1, SOD1, and Nrf2) were significantly increased following supplementation of the sAPS (P<0.01). Furthermore, sAPS effectively ameliorated CCl4 induced hepatic necrosis and inflammation, and it also reduced the expression levels of proinflammatory cytokines including TNF-α, IL-6, COX-2 and NFκB (P<0.01). Moreover, sAPS significantly decreased the expression levels of α-smooth muscle actin, collagen 1, TGF-β1, but increased the Bcl-2/Bax mRNA ratio in rats administered CCl4 (P<0.01). Taken together, it could be concluded that sAPS could increase the activities of Astragalus polysaccharides and sodium selenite to protect the liver from damage by attenuating hepatic inflammation, oxidative stress, fibrogenesis, and induces apoptosis and cell cycle arrest in hepatic stellate cells.

Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress.

Aflatoxin B1 Suppressed T-Cell Response to Anti-pig-CD3 Monoclonal Antibody Stimulation in Primary Porcine Splenocytes: A Role for the Extracellular Regulated Protein Kinase (ERK1/2) MAPK Signaling Pathway.

The aim of the present study is to investigate whether aflatoxin B1 (AFB1)-induced immunotoxicity is associated with oxidative stress and the expression of extracellular regulated protein kinases (ERK) 1/2. The primary splenocytes isolated from healthy pigs were activated and proliferated by anti-pig-CD3 monoclonal antibodies (mAb) in the present experiment, which is an antigen-specific stimulant. Results indicated that cell proliferation and interleukin-2 (IL-2) production were significantly suppressed by AFB1 from 4 to 8 μg/mL in a dose-dependent manner compared to the control group. Furthermore, AFB1 significantly increased malondialdehyde (MDA) levels, decreased reduced glutathione (GSH) and total superoxide dismutase levels, and up-regulated p-ERK1/2 expression in the activated splenocytes. N-Acetyl-l-cysteine blocked anti-CD3-induced T-cell suppression by AFB1 through increasing intracellular concentrations of GSH levels, decreasing MDA levels, and down-regulated p-ERK1/2 expression, respectively. Inhibition of the ERK1/2 expression by ERK-specific iRNA attenuated the decrease of T-cell proliferation and IL-2 production induced by AFB1. It was concluded that AFB1 inhibits anti-CD3-induced lymphocyte proliferation and IL-2 production by the oxidative stress mediated ERK1/2 MAPK signaling pathway.

Selenizing astragalus polysaccharide attenuates PCV2 replication promotion caused by oxidative stress through autophagy inhibition via PI3K/AKT activation

Our previous studies have shown that oxidative stress could promote the porcine circovirus type 2 (PCV2) replication, and astragalus polysaccharide (APS)/selenium could suppress PCV2 replication. However, whether selenizing astragalus polysaccharide (sAPS) provides protection against oxidative stress-induced PCV2 replication promotion and the mechanism involved remain unclear. The present study aimed to explore the mechanism of the PCV2 replication promotion induced by oxidative stress and a novel pharmacotherapeutic approach involving the regulation of autophagy of sAPS. Our results showed that H2O2 promoted PCV2 replication via enhancing autophagy by using 3-methyladenine (3-MA) and autophagy-related gene 5 (ATG5) knockdown. Sodium selenite, APS, the mixture of sodium selenite and APS, and sAPS significantly inhibited H2O2-induced PCV2 replication promotion, respectively. Among these, sAPS exerted maximal inhibitory effect. sAPS could also significantly inhibit autophagy activated by H2O2 and increase the Akt and mTOR phosphorylation. Moreover, LY294002, the specific phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor, significantly alleviated the effects of sAPS on autophagy and PCV2 replication. Taken together, we conclude that H2O2 promotes PCV2 replication by inducing autophagy and sAPS attenuates the PCV2 replication promotion through autophagy inhibition via PI3K/AKT activation.

Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium. Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. Recently, we reported that low doses of OTA promoted PCV2 replication in vitro and in vivo, but the underlying mechanism needed further investigation. The present studies further confirmed OTA-induced PCV2 replication promotion as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. Our studies also showed that OTA induced autophagy in PK-15 cells, as assessed by the markedly increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II, autophagy-related protein 5 (ATG5), and Beclin-1 and the accumulation of green fluorescent protein (GFP)-LC3 dots. OTA induced complete autophagic flux, which was detected by monitoring p62 degradation and LC3-II turnover using immunoblotting. Inhibition of autophagy by 3-methylademine (3-MA) and chloroquine (CQ) significantly attenuated OTA-induced PCV2 replication promotion. The observed phenomenon was further confirmed by the knock-down of ATG5 or Beclin-1 by specific siRNA. Further studies showed that N-acetyl-L-cysteine (NAC), an ROS scavenger could block autophagy induced by OTA, indicating that ROS may be involved in the regulation of OTA-induced autophagy. Furthermore, we observed significant increases in OTA concentrations in lung, spleen, kidney, liver and inguinal lymph nodes (ILN) and bronchial lymph nodes (BLN) of pigs fed 75 and 150 μg/kg OTA compared with controls in vivo. Administration of 75 μg/kg OTA significantly increased PCV2 replication and autophagy in the lung, spleen, kidney and BLN of pigs. Taken together, it could be concluded that OTA-induced autophagy in vitro and in vivo promotes PCV2 replication.

Combination of Selenomethionine and N-Acetylcysteine Alleviates the Joint Toxicities of Aflatoxin B1 and Ochratoxin A by ERK MAPK Signal Pathway in Porcine Alveolar Macrophages

Our previous studies showed that aflatoxin B1 (AFB1) and ochratoxin A (OTA) could trigger joint immune toxicity. Little is known about the combined effects of selenomethionine (SeMet) and N-acetylcysteine (NAC) on the joint toxicities of the two toxins. In this study, results showed that SeMet or NAC alone or in combination significantly alleviated the downswing of cell viability, glutathione production, and phagorytosis induced by AFB1 and OTA in porcine alveolar macrophages. The uptrend of lactate dehydrogenase activities, apoptosis, reactive oxygen species levels, and the relative mRNA of inflammatory cytokines triggered by the two toxins was decreased. Combination of them was more effective than single application. Knockdown of p38, c-JUN N-terminal kinase (JNK), or extracellular signal-regulated kinase (ERK) via use of the corresponding specific siRNA could alleviate the joint toxicities of AFB1 and OTA. However, the ERK but not p38 or JNK pathway was involved in the protection of SeMet and NAC against the immunotoxicity. In conclusion, combination of SeMet and NAC might be a new therapeutic orientation for preventing the joint toxicities induced by AFB1 and OTA.

Activation of AMPK-dependent SIRT-1 by astragalus polysaccharide protects against ochratoxin A-induced immune stress in vitro and in vivo

Recent studies have highlighted the immune stress caused by ochratoxin A (OTA), but little attention was paid to its alleviation. In the present study, the protective effects of astragalus polysaccharide (APS) against OTA-induced immune stress in vitro and in vivo and its mechanism/(s) involved were investigated. The in vitro results showed that APS (20 μg/ml) induced a significant decrease in cytotoxicity, apoptosis and pro-inflammatory cytokine expressions elevated by OTA (1.5 μg/ml) in porcine alveolar macrophages (PAMs). In vivo, APS (200 mg/kg b.w.) significantly alleviated OTA-induced (75 μg/kg b.w.) spleen damages and decreased the expressions of OTA-promoted apoptosis-related protein and pro-inflammatory cytokine. Further study indicated that APS caused significant enhancement of AMPK/SIRT-1 and inhibition of NFκB in PAMs and mice. The down-regulation of SIRT-1 by EX527 in vivo or EX527 and SIRT-1 knockdown in vitro abolished the inhibitory effects of APS on OTA-induced cytotoxicity, apoptosis, spleen damages and pro-inflammatory cytokine expressions. Taken together, these findings indicate that APS could attenuate the immune stress induced by OTA in vitro and in vivo via activation of the AMPK/SIRT-1 signaling pathway.

Ochratoxin A induces cytoprotective autophagy via blocking AKT/mTOR signaling pathway in PK-15 cells

Ochratoxin A (OTA) could cause a variety of toxicological effects especially nephrotoxicity in animals and humans. Autophagy is a highly conserved metabolic process that plays an important role in the maintenance of cellular homeostasis under stress. However, the role of autophagy in OTA-induced nephrotoxicity is unknown. In the present study, we demonstrated that OTA treatment at 2.0-8.0 μM could increase cytotoxicity of PK-15 cells by inducing apoptosis as shown by the increased Annexin V/PI staining, increased caspase-3 and PARP cleavage and increased apoptotic nuclei. Meantime, autophagy was triggered when OTA was administrated, as indicated by markedly increased expressions of LC3-II, ATG5 and Beclin-1, accumulation of GFP-LC3 dots and increased double- or single-membrane vesicles. OTA treatment decreased p-AKT and p-mTOR activities, and OTA-induced autophagy was inhibited when insulin was applied. Furthermore, OTA treatments with autophagy inhibitors (3-methyladenine or chloroquine) or knockdown of autophagy-related genes (ATG5 or Beclin-1) resulted in significantly reduced autophagy level and enhanced cytotoxicity. In conclusion, OTA induces cytoprotective autophagy against its cytotoxicity and inactivation of AKT/mTOR axis plays a critical role in autophagy induction.