Real-life exposure to Fusarium toxins deoxynivalenol and zearalenone triggers apoptosis and activates NLRP3 inflammasome in bovine primary theca cells.

Cattle are deemed less susceptible to mycotoxins due to the limited internal exposure resulting from rumen microbiota activity. However, the significant amounts of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) frequently detected in bovine follicular fluid samples suggest that they could affect ovarian function. Both mycotoxins trigger several patterns of cell death and activate the NLRP3 inflammasome in the intestine. In vitro studies have reported a number of adverse effects on bovine oocytes. However, the biological relevance of such findings with regard to realistic concentrations of DON and ZEN in bovine follicular fluid is still not clear. Hence, it is important to better characterize the effects of dietary exposure to DON and ZEN on the bovine ovary. Using bovine primary theca cells, this study investigated the effects of real-life patterns for bovine ovary exposure to DON and ZEN, but also DON metabolite DOM-1, on cell death and NLRP3 inflammasome activation. Exposure to DON starting from 0.1 µM significantly decreased theca cell viability. The kinetics of phosphatidylserine translocation and loss of membrane integrity showed that ZEN and DON, but not DOM-1, induce an apoptotic phenotype. qPCR analysis of the expression of NLRP3, PYCARD, IL-1ß, IL-18, and GSDMD in primary theca cells at concentrations of mycotoxin previously reported in cow follicular fluid clearly indicated that DON and DOM-1 individually and in mixture, but not ZEN, activate NLRP3 inflammasome. Altogether, these results suggest that real-life dietary exposure of cattle to DON may induce inflammatory disorders in the ovary.

Fusarium Mycotoxins Zearalenone and Deoxynivalenol Reduce Hepatocyte Innate Immune Response after the Listeria monocytogenes Infection by Inhibiting the TLR2/NFκB Signaling Pathway.

Zearalenone (ZEA) and deoxynivalenol (DON) are two common mycotoxins produced by the genus Fusarium and have potential immunotoxic effects that may lead to a weak immune response against bacterial infections. Listeria monocytogenes (L. monocytogenes), a food-borne pathogenic microorganism ubiquitous in the environment, actively multiplies in the liver, where hepatocytes are capable of resistance through mediated innate immune responses. At present, it is not clear if ZEA and DON affect hepatocyte immune responses to L. monocytogenes infection or the mechanisms involved. Therefore, in this study, in vivo and in vitro models were used to investigate the effects of ZEA and DON on the innate immune responses of hepatocytes and related molecules after L. monocytogenes infection. In vivo studies revealed that ZEA and DON inhibited the toll-like receptors 2 (TLR2)/nuclear factor kappa-B (NFκB) pathway in the liver tissue of L. monocytogenes-infected mice, downregulating the expression levels of Nitric oxide (NO), in the liver and repressing the immune response. In addition, ZEA and DON inhibited Lipoteichoic acid (LTA)-induced expression of TLR2 and myeloid differentiation factor 88 (MyD88) in Buffalo Rat Liver (BRL 3A) cells in vitro, downregulating the TLR2/NFκB signaling pathway and resulting in the decreased expression levels of NO, causing immunosuppressive effects. In summary, ZEA and DON can negatively regulate NO levels through TLR2/NFκB, inhibiting the innate immune responses of the liver, and aggravate L. monocytogenes infections in mouse livers.

Role of Nrf2 Nucleus Translocation in Beauvericin-Induced Cell Damage in Rat Hepatocytes.

Beauvericin (BEA), a food-borne mycotoxin metabolite derived from the fungus Beauveria Bassiana, is proven to exhibit high hepatotoxicity. However, the molecular mechanism underlying BEA-induced liver damage is not fully understood. Herein, the effect of Nrf2 nuclear translocation-induced by BEA in hepatocytes was investigated. CCK8 solution was used to determine the appropriate concentrations of BEA (0, 1, 1.5 and 2 µmol/L), and BRL3A cells were then exposed to different concentrations of BEA for 12 h. Our results reveal that BEA exposure is associated with high cytotoxicity, lowered cell viability, damaged cellular morphology, and increased apoptosis rate. BEA could lead to oxidative damage through the overproduction of ROS and unbalanced redox, trigger the activation of Nrf2 signaling pathway and Nrf2 nuclear translocation for transcriptional activation of downstream antioxidative genes. Additionally, BEA treatment upregulated the expression of autophagy-related proteins (LC3, p62, Beclin1, and ATG5) indicating a correlation between Nrf2 activation and autophagy, which warrants further studies. Furthermore, ML385, an Nrf2 inhibitor, partially ameliorated BEA-induced cell injury while CDDO, an Nrf2 activator, aggravated liver damage. The present study emphasizes the role of Nrf2 nuclear translocation in BEA-induced oxidative stress associated with the hepatotoxic nature of BEA.

Discovery of SHR2415, a Novel Pyrrole-Fused Urea Scaffold ERK1/2 Inhibitor.

ERK1/2 kinase is a key downstream node of the RAS-RAF-MEK-ERK signaling pathway. A highly potent and selective ERK1/2 inhibitor is a promising option for cancer treatment that will provide a potential solution for overcoming drug resistance. Herein we designed and synthesized a novel scaffold featuring a pyrrole-fused urea template. The lead compound, SHR2415, was shown to be a highly potent ERK1/2 inhibitor that exhibited high cell potency based on the Colo205 assay. In addition, SHR2415 displayed favorable PK profiles across species as well as robust in vivo efficacy in a mouse Colo205 xenograft model.

ZEA and DON inhibited inflammation after L. monocytogenes infection and induced ribosomal hyperfunction.

The complex microbial community in food environment is a major problem of human or animal health and safety. Mycotoxins and food-borne bacteria can both induce inflammation in the body and cause a series of changes in biological functions. In this study, mice were gavaged with low doses of ZEA, DON, or ZEA + DON, and then infected with L. monocytogenes. A cytokine microarray, including 40 inflammation-related serum cytokines, and proteomics were used to verify the effects of ZEA, DON, and ZEA + DON on the host inflammation and biological function after L. monocytogenes infection. The results showed that mononucleosis after bacterial infection was inhibited by ZEA, DON, and ZEA + DON, while the balance of macrophage differentiation was shifted toward M2-type. ZEA, DON, and ZEA + DON decreased the levels of serum proinflammatory cytokines IL-1ß and IL-12 after infection. In addition, the signal of the NF-κB pathway was inhibited. Proteomic results showed that ZEA, DON, and ZEA + DON led to biological dysfunction in ribosomal and metabolic cells, primarily leading to abnormal ribosomal hyperfunction. This study showed that ZEA, DON, and ZEA + DON can aggravate disease progression by inhibiting the inflammatory response following foodborne bacterial infection. These metabolites may also disrupt normal biological functions, which may lead to ribosomal hyperfunction, making bacterial clearance more difficult.

Zearalenone and deoxynivalenol reduced Th1-mediated cellular immune response after Listeria monocytogenes infection by inhibiting CD4+ T cell activation and differentiation.

Based on the fact that mycotoxins and the food-borne bacteria coexist in the natural environment and pose a significant health hazard to humans and animals, it is important to investigate the immunosuppressive mechanism of ZEA (zearalenone), DON (deoxynivalenol), and their combination in bacterial infections. In this study, we established a mouse model of mycotoxin low-dose exposure combined with Listeria monocytogenes infection and investigated the effects of ZEA, DON and their combination on Th1-mediated anti-intracellular bacterial infection based on CD4+ T cell activation and differentiation using both in vitro and in vivo analyses. The present study showed that both ZEA and DON aggravated Listeria monocytogenes infection in mice and affected the activation of CD4+ T cells and Th1 differentiation, including the effects on costimulatory molecules CD28 and CD152 and on cross-linking of IL-12 and IL-12R, by inhibiting T cell receptor (TCR) signaling. When compared with ZEA, DON was found to have a greater impact on many related indicators. Surprisingly, the combined effects of ZEA and DON did not appear to enhance toxicity compared to treatment with the individual mycotoxins. Our findings more clearly revealed that exposure to low-dose ZEA and DON caused immunosuppression in the body by mechanisms including inhibition of CD4+ T cells activation and reduction of Th1 cell differentiation, thus exacerbating infection of animals by Listeria monocytogenes.

Zearalenone and deoxynivalenol inhibited IL-4 receptor-mediated Th2 cell differentiation and aggravated bacterial infection in mice.

The immunotoxicity of zearalenone (ZEA) and deoxynivalenol (DON), two of the most common environmental mycotoxins, has been well investigated. However, due to the complexity of the immune system, especially during bacterial infection, many types of immune cells are involved in invasion resistance and bacterial clearance. Of these, T helper 2 (Th2) cells, which are members of the helper T cell family, assist B cells to activate and differentiate into antibody-secreting cells, participate in humoral immune response, and, ultimately, eliminate pathogens. Thus, it is important to identify the stage at which these toxins affect the immune function, and to clarity the underlying mechanisms. In this study, mice infected with Listeria monocytogenes (Listeria) were used to study the effects of ZEA, DON, and ZEA + DON on Th2 differentiation, Interleukin-4 Receptor (IL-4R) expression, costimulatory molecules expression and cytokine secretion after Listeria infection. Naive CD4+ T cells, isolated from mice, were used to verify the in vivo effects and the associated mechanisms. In vivo experiments showed that these toxins aggravated spleen damage after Listeria infection and reduced the differentiation of Th2 cells by affecting the synthesis of IL-4R of CD4+ T cells. In addition, the level of the costimulatory molecule CD154 decreased. Consistent with this, in vitro studies showed that these toxins inhibited the differentiation of mouse naive CD4+ T cell into Th2 subtype and decreased IL-4R levels. In addition, the levels of costimulatory molecules CD154, CD278 and the Th2 cells secrete cytokines IL-4, IL-6, and IL-10 decreased. Based on our in vivo and in vitro experiments, we suggest that ZEA, DON, and ZEA + DON inhibit the expression of costimulatory molecules on CD4+ T cell, and inhibit the IL-4R-mediated Th2 cell differentiation. This may indicate that the body cannot normally resist or clear the pathogen after mycotoxin poisoning.

Decrease in immune function and the role of mitogen-activated protein kinase (MAPK) overactivation in apoptosis during T lymphocytes activation induced by zearalenone, deoxynivalenol, and their combinations.

Currently there are few reports on the combined immunotoxicity of zearaleone (ZEA) and deoxynivalenol (DON). Since the two coexist naturally, it is necessary to understand the immunotoxicity caused by the two mycotoxins alone and in combination. To examine T lymphocytes activation and immune effect during activation, we used mouse primary spleen T lymphocytes as the experimental material and concanavalin (Con A) as the stimulator. The effects of ZEA, DON, and their combined exposure on T lymphocytes immune related function and the relationship between the activation of the mitogen-activated protein kinase (MAPK) signaling pathway and mycotoxin induced T lymphocytes apoptosis were studied in vitro. Specifically, T lymphocytes were isolated from primary mouse splenic lymphocytes, activated by Con A and then exposed to different concentrations of ZEA, DON, and their combinations. Our results showed that ZEA and DON alone and their combinations (20:1) can decrease the cell viability of T lymphocytes activated by Con A. The inhibitory effect of the combined groups was greater than that of the single mycotoxins, showing a synergistic effect. In addition, single or combined mycotoxins can lead to intracellular and surface ultrastructure damage of T lymphocytes, inhibit the expression of CD25 and CD278 and inhibit the synthesis of effect molecules poreforming protein (PFP), granzyme A (GZMA), and tumor necrosis factor-α (TNF-α). Meanwhile, the single mycotoxin or combined mycotoxins can promote the apoptosis of T lymphocytes which was accompanied by the overactivation of MAPK. After using the inhibitors of extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase (JNK) in the MAPK pathway, we found that the apoptosis of the cells induced by the ZEA was significantly decreased, and the apoptosis of the cells induced by DON had no significant changes. This suggests that the activation of MAPK induced by ZEA can promote the apoptosis of T lymphocytes, but the activation of MAPK induced by DON is not directly related to T cell apoptosis.

Zearalenone inhibits T cell chemotaxis by inhibiting cell adhesion and migration related proteins.

Zearalenone (ZEA) is a phenolic resorcylic acid lactone mycotoxin produced by several Fusarium species that grow on temperate and tropical crops. The number of reports documenting the immunotoxic effects of ZEA is increasing, but the underlying mechanism is not clear. The purpose of this study was to investigate the effects of ZEA on T cell chemotaxis and evaluate changes in adhesion and migration proteins associated with this process. Specifically, T cells were isolated from BALB/C mouse splenic lymphocytes, activated by concanavalin A (Con A), and then exposed to different concentrations of ZEA. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used observe the ultrastructural changes inside the cell and on the cell surface, respectively. The transwell migration assay was used to evaluate the effect of ZEA on T cell chemotaxis in the presence of CCL19 or CCL21. A confocal 3D laser was used to capture the morphology of perforated cells and western blot was used to detect the expression of proteins associated with cell migration and adhesion. Additionally, we used flow cytometry to examine the expression of chemokine receptors on T cells. Finally, the chemokine (RANTES and MIP-1α) levels secreted by T cells were assessed using cytometric bead array. Overall, our data showed that treatment with ZEA caused ultrastructural damage on the surface as well as inside of T cells. Moreover, ZEA inhibited T cell chemotaxis which was mediated by CCL19 or CCL21 and disrupted the balance of T cell subtypes. The expression of T cell adhesion and migration proteins was also inhibited by ZEA. The expression of T cell chemokine receptor as well as secretion of RANTES and MIP-1α by T cells was suppressed after ZEA treatment. In summary, our results indicate that ZEA reduced the chemotactic effect of T cells mediated by chemokines, which was likely linked to the inhibition of T cell motility and accompanied by decreased expression of adhesion and migration proteins.

Zearalenone induces apoptosis of rat Sertoli cells through Fas-Fas ligand and mitochondrial pathway.

Zearalenone (ZEA) is an estrogen-like toxin produced by Fusarium that is widely found in cereals worldwide. In recent years, ZEA has been found to cause reproductive dysfunction in male animals, but the underlying mechanism remains unclear. This study examined the apoptosis of rat Sertoli cells induced by different concentrations (0, 5, 10, and 20 µmol/L) of ZEA via Fas-Fas ligand and mitochondrial signaling pathway in vitro. Apoptosis rate was detected by flow cytometry. The mitochondrial membrane potential was detected by immunofluorescence assay and flow cytometry. Western Blot and qRT-PCR were used to identify the signaling pathway. The results revealed that ZEA induced apoptosis of rat Sertoli cells, significantly reduced the transcription and expression of the anti-apoptotic protein Bcl-2, increased the transcription and expression of pro-apoptotic proteins Bax and tBID, and Fas, FasL, FADD, and caspase-8. ZEA also increased the activation of caspase-8 and caspase-9, and promoted the release of cytochrome C from mitochondria to cytoplasm. Moreover, addition of caspase-8 inhibitor Z-IETD-FMK led to significant decrease in the mitochondrial membrane potential and apoptosis rate of the ZEA + Z-IETD-FMK group as compared to the ZEA treatment group. The release of cytochrome C from mitochondria to cytoplasm and the activation of caspase-9 and caspase-3 were significantly decreased in the ZEA + Z-IETD-FMK group. These results suggested that ZEA can induce apoptosis of rat Sertoli cells, activate the Fas-Fas ligand signaling pathway and participate in the regulation of mitochondrial apoptosis pathway.

Mechanism and effects of Zearalenone on mouse T lymphocytes activation in vitro.

Zearalenone (ZEA) is particularly toxic to the female reproductive system. Nevertheless, the effect of ZEA on the immune system is still not fully understood. The following study investigates the effects and mechanism of ZEA on mouse T cell activation in vitro. Briefly, T lymphocytes were extracted from primary splenic lymphocyte in mice, activated by concanavalin A, and then were exposed to different concentrations of ZEA for a certain period of time. Flow cytometry was used to detect the expression of activating and co-stimulatory molecules, and the secretion of cytokines in T cells at various stages. The expression of initiation regulatory protein in T cell activation, nuclear factor protein and co-stimulatory molecule related PI3K-Akt-mTOR signaling pathway proteins were detected by western blot. Our data showed that ZEA exposure inhibits the activity of T cell, and inhibits the expression of different activation signals in T cell. Additionally, ZEA exposure reduces the expression of initiative regulatory protein, i.e. LAT, Lck, Zap-70 during the activation of T cells. Thus, the results showed that ZEA exposure inhibits the formation and transmission of activated signal in T cells, interferes with signal pathway of T cell activation nuclear factor NFAT and NFκB, and decreases the secretion of cytokines after activation. Moreover, ZEA exposure interferes with co-stimulatory molecule CD28 during T cell activation, and with the activity of the PI3K-Akt-mTOR signaling pathway downstream of CD28. To conclude, our results indicated that ZEA toxin interferes with the activation of mouse T lymphocytes by affecting TCR signal and co-stimulatory signal, thus playing an essential role in immune toxicity.

Multiplex analysis of serum hormone and cytokine in patients with cervical cOPLL: towards understanding the potential pathogenic mechanisms.

Cervical ossification of the posterior longitudinal ligament (cOPLL) is one of the major causes of myelopathy. However, the mechanism underlying remains elusive. In the present study, using MILLIPLEX magnetic bead panel, we investigated four serum hormones and six serum cytokines in cOPLL patients and healthy subjects. The results showed that tumor necrosis factore-α (TNF-α) were significantly increased, and DDK-1 was significantly decreased in the serum from male and female cOPLL patients compared with those from healthy controls, respectively. Osteopontin (OPN) and fibroblast growth factor-23 (FGF-23) were significantly increased in male cOPLL patients compared with that in healthy male controls. Further analysis showed that FGF-23 and OPN significantly increased, dickkopf-1 (DKK-1) decreased in the extensive cOPLL group. In addition, a significant positive correlation between the OPN and FGF-23 was observed in male cOPLL patients. The results are useful for understanding the mechanism underlying cOPLL.

Ossification of the posterior ligament is mediated by osterix via inhibition of the ß-catenin signaling pathway.

Ossification of the posterior longitudinal ligament (OPLL) involves ectopic calcification of the spinal ligament preferentially at the cervical spine. OPLL is associated with different diseases and occurs by endochondral ossification, which is associated with the activity of different transcription factors. However, the pathogenesis of OPLL remains unclear. Here, we investigated the role of osterix (Osx), a transcription factor that functions downstream of Runx2 and is an important regulator of osteogenesis, in the process of OPLL in a dexamethasone (Dex)-induced model of spinal ligament ossification. Our results showed that Osx is upregulated in patients with OPLL and during the ossification of ligament cells in parallel with the upregulation of osteogenic markers including osteocalcin (OCN), alkaline phosphatase (ALP) and collagen-1 (Col-1). Dex-induced ossification of ligament cells was associated with the downregulation and inactivation of ß-catenin, and these effects were offset by Osx knockdown. Activation of ß-catenin signaling abolished the effect of Dex on ossification and the upregulation of osteogenic markers. Taken together, our results suggest that OPLL is mediated by Osx via a mechanism involving the Wnt/ß-catenin signaling pathway, providing a basis for further research to identify potential targets for the treatment of OPLL.

[Preparation of multi-walled carbon nanotubes decorated with Fe3O4 nanoparticles for determination of trace pyrethroid pesticides in water and honey samples].

A magnetic carbon nanotube hybrid material was prepared using a chemical co-precipitation method. The Fe3O4 nanoparticles were enclosed onto the surface of the acid multi-walled carbon nanotubes (AMWNTs), and they were identified as Fe3O4/AMWNTs composites. This hybrid materials displayed typical superparamagnetic behavior, good dispersibility, and good adsorption capacity for pyrethroid pesticides. A magnetic solid-phase microextraction (MSPE) procedure based on Fe3O4/AMWNTs composites, combined with gas chromatography, was developed for the quantification of six pyrethroid pesticides in water and honey samples. Several important parameters affecting the extraction efficiency for six pyrethroid pesticides were optimized in sequential order, including ionic strength, extraction time and desorption time. Under the optimized conditions, this method showed wide linearity ranging from 0.5 µg/L to 50 µg/L with correlation coefficients (R2) higher than 0.990. The limits of detection (LODs) ranged from 0.07 µg/L to 0.20 µg/L at a singal-to-noise ratio of 3. The relative standard deviations (RSDs) ranged from 3. 8% to 8. 1%. Satisfactory recoveries (> 78.4%) were obtained for the simultaneous analysis of the six pyrethroid pesticide residues in river water, fish-pond water and two honey samples. This method is sensitive and simple. It can meet the actual requirement for the analysis of trace analytes from environmental water and honey samples.

[Pseudomonas aeruginosa elastase inhibits immune phagocytosis mediated by pulmonary surfactant protein A].

OBJECTIVE: To investigate the relationship between Pseudomonas aeruginosa (P.aeruginosa) elastase and pulmonary surfactant protein A (SP-A) in host infected by P.aeruginosa. METHODS: C3H mice were intranasally infected with P.aeruginosa wild-type PAO1, ΔlasB mutant and genetic complement strain PDO240LasB to determine the difference of virulence between wide type and mutant. The ability to degrade SP-A in vitro by PAO1, ΔlasB and PDO240LasB was observed through co-incubation of equal bacteria and SP-A and detected by Western blotting. The susceptibility of bacteria to phagocytosis was assayed by in vitro experiment that bacteria treated with SP-A was incubated with mouse Raw264.7 macrophages. RESULTS: Compared with wide-type PAO1, the virulence of ΔlasB mutant was attenuated in the mouse model of P.aeruginosa infection because of the knock down of elastase expression. The ΔlasB mutant lost the ability to degrade SP-A when incubated with SP-A in vitro. The in vitro phagocytosis experiments showed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the wild-type PAO1. CONCLUSION: P.aeruginosa elastase provides a protection from phagocytic cells by degrading SP-A.