Current knowledge on epidemiology and evolution of novel porcine circovirus 4.

Porcine circovirus type 4 (PCV4) is a newly emerging virus, with both PCV4 genomic DNA and specific antibodies detected in swine herds in several provinces in China and South Korea. Although the virus was first identified in 2019 in Hunan, China, retrospective research suggests that serum samples collected as early as 2008 were positive for PCV4 antibody. Infections with only PCV4 or co-infections with other pathogens have been associated with several clinical manifestations, but its pathogenesis remains to be determined. The purpose of this review was the following: (1) to characterize PCV4 epidemiology by assessing evolutionary dynamics and genetic diversity of PCV4 strains circulating in swine herds; (2) to reconstruct a computerized 3D model to analyze PCV4 Cap properties; (3) and to summarize the current evidence of PCV4-associated clinical-pathological manifestations. The origin of PCV4 is apparently distinct from other PCV, based on analysis of phylogenetic trees. Of note, PCV4 shares an ancient common ancestor with mink circoviruses. Furthermore, the amino acid residue at position 27 of the PCV4 Cap is a key benchmark to distinguish PCV4a (27S) from PCV4b (27 N), based on PCV4 strains currently available, and variation of this residue may alter Cap antigenicity. In addition, the capsid surface of PCV4 has characteristics of increased polar residues, compared to PCV2, which raises the possibility that PCV4 may target negatively charged host receptors to promote virus infection. Further studies are required, including virus isolation and culture, and more detailed characterization of molecular epidemiology and genetic diversity of PCV4 in swine herds.

The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation.

The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitroIMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus.

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/µL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

Epidemic and genetic characterization of porcine epidemic diarrhea virus strains circulating in the regions around Hunan, China, during 2017-2018.

Outbreaks of porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) infection have caused high mortality of piglets and significant economic losses to the Chinese swine industry. In the current study, 184 specimens from pigs with or without signs of diarrhea were collected from 39 farms across eight provinces, mainly around Hunan, People's Republic of China, in 2017 to 2018 in order to obtain epidemiological information on PEDV infections in these regions. The results indicated an average PEDV-positive rate of 38.04% (70/184) and more-pronounced disease severity in diarrheic pigs (48.76%; 59/121) than in non-diarrheic pigs (17.46%; 11/63). Phylogenetic and sequence analysis demonstrated that 14 representative PEDV strains from 14 swine farms belonged to the G2 group (G2-a and G2-b subgroups) and displayed a high degree of genetic variation. In particular, two out of the 14 PEDV strains were found to have unique indels in the S1 gene. The strain HN-SY-2017-Oct had a 9-nucleotide (T1152GAAGCCAAT1160T) insertion, and the strain ZJ-2018-May had a 3-nucleotide (AAA) deletion at position 1126 in the S1 gene. A three-dimensional structural prediction revealed that these unique insertions might lengthen the loop on the surface or increase the likelihood of the surface protein being phosphorylated at 388Y, thereby affecting the virulence or pathogenicity of PEDV. Collectively, the data show that PED remains a severe threat to the pig industry and that variant PEDV stains are circulating in China. The updated PEDV epidemiological data will facilitate the design of PEDV vaccines and the application of effective measures for PED prevention.

Development and application of a baculovirus-expressed capsid protein-based indirect ELISA for detection of porcine circovirus 3 IgG antibodies.

BACKGROUND: Porcine circovirus type 3 (PCV3), recently widely isolated from pigs with various clinical conditions, is likely globally epidemic. However, development of serological diagnosis for PCV3 in pigs is ongoing. Our objectives were to: 1) establish an indirect ELISA, using PCV3 capsid protein (Cap) prepared by Baculovirus Expression Vector System (BEVS) as a high-quality coating antigen for detection of PCV3-associated antibodies in serum samples; and 2) use this ELISA to conduct a serological survey for PCV3 in various regions of Hunan province, China. RESULTS: The PCV3 positive rate to the ELISA assay (total of 190 serum samples) was higher in sows with reproductive failure compared to healthy sows (34/85, 40.0% versus 30/105, 28.6%), with similar results using qPCR assays. Further, in an additional 1038 serum samples collected from January 2016 to May 2018 in various regions of Hunan province and tested with this established ELISA, 20 to 84% were positive for PCV3 (according to region of sera collection), with high PCV3 seroprevalence (> 50%) in herds in Changde, Hengyang and Yueyang. Moreover, among serum samples from herds in Shaoyang and Changde, PCV3 seroprevalence was higher in sows than in other classes of pigs (i.e., suckling piglets, nursery pigs, gilts, growing-finishing pigs and boars). CONCLUSIONS: We developed a full-length PCV3 Cap-based ELISA using a eukaryotic expression system with excellent potential to elucidate PCV3 epidemiology. Based on this assay, PCV3 has been circulating in Hunan province. PCV3 prevalence was lower in healthy sows than in those with reproductive failure. Further studies are warranted to identify the PCV3 responsible for high seroprevalence in sows and determine pathogenesis of PCV3 in sows with reproductive failure.

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells.

BACKGROUND: Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G710 to A710) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively. RESULTS: Though all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID50), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal. CONCLUSIONS: These findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

Background-Free Imaging of a Viral Capsid Proteins Coated Anisotropic Nanoparticle on a Living Cell Membrane with Dark-Field Optical Microscopy.

Exploring the diffusion dynamics of a viral capsid proteins (VCP)-functionalized nanocarrier on a living cell membrane could provide much kinetic information for the better understanding of their biological functionality. Gold nanoparticles are an excellent core material of nanocarriers because of the good biocompatibility as well as versatile surface chemistry. However, due to the strong scattering background from subcellular organelles, it is a grand challenge to selectively image an individual nanocarrier on a living cell membrane. In this work, we demonstrated a convenient strategy to effectively screen the scattering background from living cells for single-particle imaging with a polarization-resolved dual-channel imaging module. By taking advantage of the polarization of anisotropic gold nanoparticles (gold nanorods, GNRs), the signals from cell components could be counteracted after subtracting the sequential images one by one, while those transiently rotating GNRs on the cell membrane still exist in the processed image. In contrast to the previously reported methods, this method does not require a complicated optical setup alignment and sophisticated digital image analysis process. According to the single-particle imaging results, the majority of VCP-GNRs were anchoring on the cell membrane with confined diffusion. Interestingly, on further inspection of the diffusion trajectories, the particles displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. Non-Gaussian step distribution was noted, indicating heterogeneous binding and desorption processes on the cell membrane. As a consequence of the robust background screening capability, this approach would find broad applications for single-particle imaging under a noisy environment, e.g., living cells.

Molecular detection and sequence analysis of porcine circovirus type 3 in sow sera from farms with prolonged histories of reproductive problems in Hunan, China.

A newly emerging porcine circovirus, designated PCV3, has been reported in various countries (USA, Poland, South Korea and China) since 2017. Its presence may be associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystem inflammation. In this study, we report identification of PCV3 in cases of reproductive failure in various regions in Hunan, China. From January 2015 to December 2016, sera were collected from 190 sows from seven farms with reproductive problems. Specifically, 85 samples were from sows with a history of reproductive failure, whereas the remaining 105 were from healthy sows. The PCV3-positive rate was significantly higher in sows with reproductive failure (45.9%) than in healthy sows (21.9%), based on quantitative PCR (qPCR) assays. Although phylogenetic analysis based on the cap gene suggested that these PCV3 isolates belonged to the clade PCV3a, amino acid sequence variations in the Cap protein still occurred among these isolates, and these might have contributed to antigenic alterations of the Cap protein, based on the Jameson-Wolf antigenic index. Finally, we concluded that PCV3 was circulating in sows in Hunan province, China. However, the association of PCV3 with reproductive failure in sows and its potential for vertical transmission need to be studied further.

First molecular detection of porcine circovirus type 3 in dogs in China.

Porcine circovirus type 3 (PCV3) has recently been isolated from diseased pigs within the USA. The objective was to detect the presence of PCV3 in dogs. Nested polymerase chain reactions (PCR) with PCV3-specific primers for the capsid gene were used to detect PCV3 genomic DNA in serum samples from dogs (n = 44) in China. There was PCV3 DNA detected in 4 of 44 dogs [all were negative for PCV2 and canine circovirus (CanineCV)]. Based on sequence analysis, positive sequences were grouped into PCV3 genotypes. However, these isolates had close evolutionary relationships with FoxCV (KP941114) and CanineCV (JQ821392). Further investigations of the epidemiology, evolutionary biology, and pathobiology of PCV3 to dogs are warranted.

Evidence of natural co-infection with PCV2b subtypes in vivo.

Porcine circovirus type 2 (PCV2) is the causative pathogen of porcine circovirus-associated diseases (PCVAD). This virus evolves mostly through point mutations and genome recombination between different PCV2 genotypes (e.g. PCV2a and PCV2b), as has been confirmed in swine herds. In the current work, the complete PCV2 genome sequences of 69 clones derived from various tissues (lymph node, spleen and lung,) of an infected individual, were subjected to phylogenetic and alignment analyses. The results not only demonstrate co-infection with distinct PCV2b subtypes (e.g. 1B and 1C) in the same animal, but also highlight another mechanism of evolution - diverse point mutations acquired during immune evasion by this virus.

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome.

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

In silico analyses of antigenicity and surface structure variation of an emerging porcine circovirus genotype 2b mutant, prevalent in southern China from 2013 to 2015.

Porcine circovirus type 2 (PCV2) is the pivotal pathogen causing porcine circovirus-associated diseases. In this study, 62 PCV2 isolates were identified from seven farms in southern China from 2013 to 2015 and phylogenetic trees were reconstructed based on whole-genome sequences or the cap gene. In this investigation, PCV2b was the main genotype in circulation throughout these farms. Furthermore, an emerging mutant (PCV2b-1C), isolated from PCV2-vaccinated farms, was the predominant strain prevalent on these farms. In addition, we isolated a new cluster that may represent evolution of the virus through recombination of PCV2b-1A/1B and PCV2b-1C. Finally, we discuss evidence that antigenicity and surface structure variation of the capsid resulted from mutation of the C-terminal loop (Loop CT) of the PCV2b-1C Cap in silico.

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap).

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis.

Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay.

Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.

6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes.

It is well known that disrupted brain iron homeostasis was involved in Parkinson's disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10 µM 6-OHDA treatment for 24 h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12 h, however, down-regulation at 24 h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1 + IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.

TIPE2 regulates the response of BV2 cells to lipopolysaccharide by the crosstalk between PI3K/AKT signaling and microglia M1/M2 polarization.

Tumor necrosis factor (TNF)-α-induced protein 8-like 2 (TIPE2) is a crucial negative regulator of both adaptive and innate immunity, which helps maintain the dynamic balance of the immune system by negatively regulating the signaling of T-cell receptors (TCR) and Toll-like receptors (TLR). In this study, we aimed to investigate the role and molecular mechanism of TIPE2 using a lipopolysaccharide (LPS)-induced inflammatory injury model in BV2 cells. Specifically, we constructed a BV2 cell line of TIPE2-overexpression or TIPE2-knockdown via lentiviral transfection. Our results demonstrated that overexpression of TIPE2 downregulated the expression of pro-inflammatory cytokines IL-1ß and IL-6, which was reversed by knockdown of TIPE2 in the inflammation model of BV2 cells. In addition, overexpression of TIPE2 resulted in the conversion of BV2 cells to the M2 phenotype, while the knockdown of TIPE2 promoted the transformation of BV2 cells to the M1 phenotype. Notably, our co-culture experiments with neuronal cells SH-SY5Y showed that the overexpression of TIPE2 in inflammation-injured BV2 cells exhibited a protective effect on the neuronal cells. Finally, western blot analysis demonstrated that TIPE2 significantly reduced the expression of p-PI3K, p-AKT, p-p65, and p-IκBα in LPS treated BV2 cells, and inhibited the activation of NF-κB through the dephosphorylation of PI3K/AKT. These results suggest that TIPE2 plays an important role in mediating neuroinflammatory responses and may be involved in neuroprotection by modulating the phenotypic changes of BV2 cells and regulating the pro-inflammatory responses through the PI3K/AKT and NF-κB signaling pathways. In conclusion, our study provides new insights into the crucial role of TIPE2 in regulating neuroinflammatory responses and highlights its potential as a therapeutic target for neuroprotection.

A non-linear relationship between lesion length and risk of recurrent cerebral ischemia after stenting for symptomatic intracranial stenosis with hemodynamic impairment.

Background: Stent placement can be an effective treatment for patients with symptomatic intracranial stenosis (sICAS) and hemodynamic impairment (HI). However, the association between lesion length and the risk of recurrent cerebral ischemia (RCI) after stenting remains controversial. Exploring this association can help predict patients at higher risk for RCI and develop individualized follow-up schedules. Method: In this study, we provided a post-hoc analysis of a prospective, multicenter registry study on stenting for sICAS with HI in China. Demographics, vascular risk factors, clinical variables, lesions, and procedure-specific variables were recorded. RCI includes ischemic stroke and transient ischemic attack (TIA), from month 1 after stenting to the end of the follow-up period. Smoothing curve fitting and segmented Cox regression analysis were used to analyze the threshold effect between lesion length and RCI in the overall group and subgroups of the stent type. Results: The non-linear relationship between lesion length and RCI was observed in the overall population and subgroups; however, the non-linear relationship differed by subgroup of stent type. In the balloon-expandable stent (BES) subgroup, the risk of RCI increased 2.17-fold and 3.17-fold for each 1-mm increase in the lesion length when the lesion length was <7.70 mm and >9.00 mm, respectively. In the self-expanding stent (SES) subgroup, the risk of RCI increased 1.83-fold for each 1-mm increase in the lesion length when the length was <9.00 mm. Nevertheless, the risk of RCI did not increase with the length when the lesion length was >9.00mm. Conclusion: A non-linear relationship exists between lesion length and RCI after stenting for sICAS with HI. The lesion length increases the overall risk of RCI for BES and for SES when the length was <9.00 mm, while no significant relationship was found when the length was >9.00 mm for SES.

Betulinic acid mitigates zearalenone-induced liver injury by ERS/MAPK/Nrf2 signaling pathways in mice.

Zearalenone (ZEA) is a mycotoxin commonly found in cereals and feedstuffs, which can induce oxidative stress and inflammation to cause liver damage in humans and animals. Betulinic acid (BA) is extracted from pentacyclic triterpenoids of many natural plants and has anti-inflammatory, and anti-oxidation biological activities in many studies. However, the protective effect of BA on liver injury induced by ZEA has not been reported. Therefore, this study aims to explore the protective effect of BA on ZEA-induced liver injury and its possible mechanism. In the mice experiment, ZEA exposure increased the liver index and caused histopathological impairment, oxidative damage, hepatic inflammatory responses, and increased hepatocyte apoptosis. However, when combined with BA, it could inhibit the production of ROS, up-regulate the proteins expression of Nrf2 and HO-1 and down-regulate the expression of Keap1, and alleviate oxidative damage and inflammation in the liver of mice. In addition, BA could alleviate ZEA-induced apoptosis and liver injury in mice by inhibiting the endoplasmic reticulum stress (ERS) and MAPK signaling pathways. In conclusion, this study revealed the protective effect of BA on the hepatotoxicity of ZEA for the first time, providing a new perspective for the development of ZEA antidote and the application of BA.

Citrinin Exposure Induced Testicular Damage and Spermatogenesis Disorder by Triggering Endoplasmic Reticulum Stress.

Damage to the reproductive system is the key factor leading to male infertility. Citrinin (CTN) is produced by Penicillium and Aspergillus in nature, and is definitely found in food and animal feed. Studies have revealed that CTN can cause damage to male reproductive organs and reduce fertility, but the mechanism of toxicity has not been revealed. In the present study, male Kunming mice were given different doses of CTN (0, 1.25, 5 or 20 mg/kg BW) by intragastric administration. The results demonstrated that CTN exposure caused disorder of androgen, a decline in sperm quality, and histopathological damage of testis. The inhibition of the expression of ZO-1, claudin-1 and occludin suggests that the blood-testis barrier (BTB) was damaged. Simultaneously, CTN inhibited the activity of antioxidant enzymes such as CAT and SOD, and promoted the production of MDA and ROS, resulting in oxidative damage of testis. Additionally, apoptotic cells were detected and the ratio of Bax/Bcl-2 was increased. Not only that, CTN activated the expression of endoplasmic reticulum stress (ERS)-related proteins IRE1, ATF6, CHOP, and GRP78. Interestingly, 4-Phenylbutyric Acid (4-PBA, an ERS inhibitor) treatment blocked the adverse effects of CTN exposure on male reproduction. In short, the findings suggested that CTN exposure can cause damage to mouse testis tissue, in which ERS exhibited an important regulatory role.