| Ochratoxin A(OTA)is one of the secondary metabolites produced by several species belonging to the genera Penicillium and Aspergillus.OTA is a pentaketide derived from a dihydroiso-coumarin group amide-linked to L-phenylalanine and its chemical name is L-phenylalanine-N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1-H-2-benzopyrane-7-y1)carbonyl]-(R)-isocoumarin.It is well known that the primary target organ of OTA is kidney,but OTA is also a potential immunotoxin fungal compound.Previous study found that the immune toxicity induced by OTA may become a major reason for the failure of immunization and increasing of susceptibility to diseases in pig farms.However,at home and abroad,the toxicity study of OTA is still focused on its nephrotoxicity and carcinogenicity until now.The toxicity study of OTA on porcine immune cells is rarely reported and the mechanism and pathway remain largely unclear.Porcine circovirus type 2(PCV2)is a non-enveloped,single-stranded,circular DNA virus which is known as one of the smallest animal viruses.PCV2 is the primary causative agent of several syndromes collectively known as porcine circovirus-associated disease(PCVAD).This cluster of diseases includes postweaning multisystemic wasting syndrome(PMWS),which is a newly emerging worldwide swine disease first reported in Canada in 1991;porcine dermatitis and nephropathy syndrome(PDNS);porcine respiratory disease complex(PRDC);proliferative necrotising pneumonia(PNP);and reproductive failure.Monocytes/macrophages(including alveolar macrophages)are considered to be the primary target cells of PCV2.Therefore,the immune system is most severely damaged after the invasion of PCV2.In the domestic pig farms,both OTA exposure levels and PCV2 infection rates are high.Thus,a significant percentage of pig farms may occurred OTA exposure and PCV2 infection at the same time.But so far,the interaction of the immune toxicity induced by OTA and PCV2 and its underlying mechanism has never been studied.The aim of this research was to determine the toxic effects of OTA on procine immune cells in vitro and to explore the effect and the underlying mechanism of PCV2 infection on OTA-induced immune toxicity when OTA co-exist with PCV2,so as to provide a theoretical basis for the combined control against OTA poisoning and PCV2 infection in production practice.Experiment 1:Immune toxicity of OTA on porcine alveolar macrophages and its mechanismThe immune toxicity of OTA was evaluated by using a porcine alveolar macrophage(PAM)cell line 3D4/21 as an in vitro cell culture model.The cells were treated with OTA at different concentrations of 0,0.1,0.5,1.0,1.5,2.0,3.0,4.0 μg/mL.The results showed that,compared with the control group,1.5-4.0 μg/mL of OTA significantly reduced cell viability and 1.0-4.0 μg/mL OTA significantly increased the activity of lactate dehydrogenase(LDH),both in a dose-dependent manner.The alteration of apoptosis index was relatively more sensitive,OTA at 0.5 μg/mL significantly increased Annexin V-binding(25.8%)and reduced Bcl-2/Bax mRNA ratio(16.9%)in a dose dependent manner.In addition,0.5 μg/mL of OTA significantly increased the mRNA levels of interleukin-1α(IL-1α)(29.1%)and 1.0 μg/mL OTA significantly increased the mRNA levels of tumor necrosis factor-α(TNF-α)(68.3%),but not in a dose-dependent manner.The expression of IL-1α and TNF-α was slightly decreased when the concentration of OTAreached 2.0 μg/mL.Meanwhile,0.5 μg/mL of OTA significantly increased intracellular reactive oxygen species(ROS)and malondialdehyde(MDA)levels(increased 43.7%and 20.5%,respectively),and 1.0 μg/mL of OTA significantly reduced the activity of total-superoxide dismutase(T-SOD)(19.8%).N-acetylcysteine(NAC)blocked OTA-induced immune toxicity through increasing cell viability,decreasing LDH activity,and alleviating apoptosis-and oxidative stress-related index,respectively.These results indicated that OTA induced immune toxicity in 3D4/21 and it is mediated by oxidative stress.Experiment 2:Effects of PCV2 infection on OTA-induced immune toxicity on porcine alveolar macrophages and its mechanismThe immune toxicity of OTA was evaluated by using a porcine alveolar macrophage(PAM)cell line 3D4/21 as an in vitro cell culture model.The cells were cultured with or without 0.5 MOI of PCV2 for 24 h and then treated with OTA at different concentrations of 0,0.1,0.5,1.0,1.5,2.0 μg/mL.The results showed that PCV2 infection aggravated OTA-induced decrease of cell viability and increase of LDH activity at OTA concentrations of 1.0-2.0 μg/mL,and decreased the minimum OTA concentration that produced cytotoxicity.In addition,PCV2 infection aggravated OTA-induced apoptosis at 1.0-2.0 μg/mL of OTA;PCV2 infection promoted OTA-induced rise of IL-la and TNF-a mRNA levels and production of ROS over all the OTA concentrations used.Meanwhile,at OTA concentrations of 1.5μg/mL,PCV2 infection aggravated OTA-induced increase of the mRNA(increased 101.9%and 22.6%,respectively)and protein(increased 51.4%and 79.2%,respectively)levels of TLR4 and MyD88,and also aggravated OTA-induced phosphorylation of downstream signaling proteins such as ERK1/2,p38,and NF-κB p65(increased 58.1%,72.0%,and 97.3%,respectively).Further studies have shown that TLR4 knockdown with TLR4-specific siRNA significantly alleviated the promoting effect of PCV2 on OTA-induced immune toxicity,as demonstrated by increasing cell viability(17.6%),and decreasing LDH activity(15.7%),apoptosis ratio(23.2%),and IL-la(26.4%)and TNF-a mRNA levels(23.8%).These results suggest that PCV2 infection aggravated OTA-induced immune toxicity in 3D4/21 and the activation of TLR4/MyD88 signaling pathway should be one of the main mechanisms. |
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