Effects Of Ochratoxin A On PCV2 Repication Promotion,Blocking Effects Of Selenium And Their Mechanisms

Porcine circovirus type 2(PCV2)is the primary causative agent of porcine circovirus disease(PCVD).However,not all pigs infected with PCV2 will develop PCVD.PCVD includes postweaning multisystemic wasting syndrome,porcine dermatitis and nephropathy syndrome,porcine respiratory disease complex,and so on.Postweaning multisystemic wasting syndrome is the most serious disease of PCVD.PCVD is a worldwide swine disease.The morbidity and severity of PCVD varies significantly in different area and different pig farms.It has been reported that PCVD development has been linked to animal management,presence of concurrent viral infections,environment stress,and nutrition,but its pathogenic mechanism is not clear until now.Ochratoxin A(OTA)a worldwide mycotoxin that naturally occurs in food and feeds,is a secondary product of several species of Aspergillus and Penicillium metabolism.The contamination level of OTA in feed and blood varies between different farms and countries.OTA is a potent nephrotoxin in humans and animals,but the mechanisms of its toxicity have not been clearly determined.Previous studies have shown that the toxicity of OTA is associated with the ability to interference antioxidant enzyme synthesis and to produce reactive oxygen species.Selenium(Se),an antioxidant trace element for humans and animals,has many important biological functions,including enhancing the antioxidant function and immunity,anti-toxic elements,anti-stress response,anti-viral infections,and so on.It has been reported that Se exerts its biological functions through selenoproteins.Previous studies indicated that organic Se is less toxic than inorganic Se,and the bioavailability of organic Se is relatively high.The present study was conducted to investigate the toxicity of OTA,the effects of OTA on PCV2 replication and their signal pathway mechanisms,the protective effects of Se and mechanisms,which partly explains why the morbidity and severity of PCVD varies significantly in different area and different pig farms,and provides a theoretical basis for the control of PCVD and the application of organic Se in swine industry.Experiment 1,effects of ochratoxin A on PCV2 replicationThis research was conducted to evaluate the effects of OTA on PCV2 replication in vitro and vivo.PK15 cells and PCV2-infected pigs were used in the experiment as the model.In vitro,PK15 cells were infected with PCV2 for 24 h,then incubated with OTA at 0,0.01,0.05,0.1 and 0.5 ?g/ml.In vivo,twenty-seven crossbred weanling piglets randomly allocated to three groups were fed for 42 days ad libitum a basal diet without(Con)and with supplementation of 75?g/kg OTA(OTA group 1)and 150 ?g/kg OTA(OTA group 2).From each group,three piglets were randomly selected for blood collection on Days 0,14,28 and 42 and tissue collection on Day 42.The results in vitro showed that low doses of OTA significantly increased PCV2 DNA copies and the number of infected cells.Maximum effects were observed at 0.05 ?g/ml OTA.The results in vivo showed that PCV2 replication was significantly increased in serum and tissues of pigs fed 75 ?g/kg OTA compared with the control group and pigs fed 150 ?g/kg OTA.The results showed that serum,liver,kidney,spleen,ILN,and BLN tissues had very low OTA concentrations close to the detection limit,and there was no detectable OTA in the lung in pigs fed the basal diet.Levels in serum rose to 0.27-0.47 ?g/ml in the 75 ?g/kg OTA group,and 0.40-0.69 ?g/ml in the 150 ?g/kg OTA group.Levels in tissues increased from 0.05 to 0.11 ?g/g in the 75 ?g/kg group,and from 0.08 to 0.16 ?g/g in the 150 ?g/kg OTA group.A significant increase of PCV2 DNA copies was observed in the 75 ?g/kg OTA group on Day 42 compared with the control group,whereas a slight,but not statistically significant,increase of PCV2 DNA copies was observed on Days 14 and 28.Significant increases in PCV2 DNA copies were observed in kidney,spleen,and lung of pigs treated with 75 ?g/kg OTA,and in bronchial lymph nodes(BLN)of pigs treated with 75 ?g/kg and 150 ?g/kg OTA.The results indicates that low doses of OTA are potentially harmful to animals as they enhance virus replication and partly explains why the morbidity and severity of PCVD varies significantly in different pig farms.Experiment 2,research of the mechanisms of ochratoxin A promoting porcine circovirus type 2 replicationOTA promotes PCV2 replication in vitro and vivo.In order to study the mechanism of OTA promoting PCV2 replication,PK15 cells were used in the experiment as the model.We measured the oxidative stress indices,p38 and ERK1/2 phosphorylation and the effects of NAC/p38/ERK1/2 inhibitor and siRNAs on OTA-promoted PCV2 replication.The results showed that low doses of OTA significantly depleted reduced glutathione and mRNA expression of NF-E2-related factor 2 and y-glutamyl-cysteine synthetase,increased reactive oxygen species,oxidants,and malondialdehyde,and induced p38 and ERK1/2 phosphorylation in PK15 cells.Adding N-acetyl-L-cysteine reversed the changes induced by OTA.Knock-down of p38 and ERK1/2 by their respective specific siRNA or inhibition of p38 and ERK1/2 phosphorylation by their respective inhibitor(SB203580 and U0126)eliminated the increase in PCV2 replication induced by OTA.These data indicate that low doses of OTA promoted PCV2 replication via the oxidative stress-mediated p38/ERK1/2 MAPK signaling pathway.Experiment 3,effects and mechanism of selenium blocking the promotion of porcine circovirus type 2 replication induced by ochratoxin AThe study was conducted to evaluate the mechanisms of selenium affecting the promotion of PCV2 replication.induced by ochratoxin A in PK15 cells.We used over-expression and low-expression SelS PK15 cell as the model and measured PCV2 DNA copies and the positive-infected cells,ROS and GSH and p38 phosphorylation levels.The results showed that 0.05 ?g/ml of OTA promotes PCV2 replication,and selenium blocked the promotion of PCV2 replication induced by ochratoxin A.Over-expression of SelS decreased PCV2 DNA copies and the number of infected cells,while low-expression of SelS increased PCV2 DNA copies and the number of infected cells,but not significant.Over-expression of SelS significantly reversed the promotion of PCV2 replication induced by 0.05 }ig/ml of OTA,and low-expression of SelS has the opposite effects.0.05 ?g/ml of OTA induced ROS production,GSH depletion and p38 phosphorylation.Over-expression of SelS significantly decreased the ROS production,GSH depletion and p38 phosphorylation induced by 0.05 ?g/ml of OTA,and low-expression of.SelS has the opposite effects.These results indicated that SelS plays a key role in the effects of selenium blocking the promotion of porcine circovirus type 2 replication induced by ochratoxin A.Experiment 4,protective effects and mechanism of selenium on porcine nephrotoxicity induced by ochratoxin AIn this study,the protective effects of selenomethionine against OTA-induced nephrotoxicity were investigated using the porcine kidney 15(PK15)cells as a model.We measured cell viability,LDH activity,caspase 3 activity,annexin V-binding,ROS and GSH levels,and selenoprotein expression.The results showed that OTA induced nephrotoxicity in a dose-dependent manner.Se at 0.5,1,2 and 4 ?M had significant protective effects against OTA-induced nephrotoxicity.Furthermore,selenomethionine enhanced the activity and mRNA and protein expression of glutathione peroxidase 1(GPxl),mRNA expression of GPx4,and mRNA expression of thioredoxin reductase 1 in the presence and absence of OTA.Among them,promoting effect of selenomethionine on GPxl was maximal.Knock-down of GPx1 by using a GPx1-specific siRNA eliminated the protective effects of selenomethionine against OTA-induced nephrotoxicity.The results suggest that selenomethionine alleviates OTA-induced nephrotoxicity by improving selenoenzyme expression in PK15 cells.Therefore,selenomethionine supplementation may be an attractive strategy for protecting humans and animals from the risk of kidney damage induced by OTA.