The Effects Of JAK/STAT Pathway On The Toxicity Mechanism Of Deoxynivalenol (DON) And T-2 Toxin In RAW 264.7 Cells

The trichothecene mycotoxins are secondary metabolites mainly produced by several fugal species, such as Fusarium. They can result in a worldwide food and feed contamination, which probably makes threat against the human and livestock health. T-2 toxin and deoxynivalenol (DON) which respectively represented as type A and type B trichothecenes, can seriously damage the immune system. Low dose trichothecene exposure can initiate rapid and transient up-regulation of many immune related proinflammatory genes expression with concurrent immune stimulation. In contrast, high dose trichothecene exposure severely injures lymph nodes, spleen, and thymus, with the induction of leukocytes apoptosis, and in the result of immune repression. Trichothecenes can inhibit protein synthesis by binding to the ribosomal peptidytransferase. On the other hand, trichothecenes can trigger a ribotoxic stress response, which actives mitogen activated protein kinase (MAPK) signaling cascade and regulates down-stream mRNA stability and proinflammatory gene expressions. It has been proved that proinflammatory gene upregulations play a crucial role in trichothecenes-induced toxic effects. The over-expressions of proinflammatory genes may contribute to IgA nephropathy (IgAN) leukocytes apoptosis and dysfunction of immune system. JAK/STAT has been found to be associated with inflammation process. However, it's still not clear that whether JAK/STAT play a central role in trichothecenes-induced toxicity and trichothecenes mediated inflammatory gene expressions. We hypothesize that JAK/STAT play a crucial role in trichothecenes mediated IL-6, IL-1βand TNF-a gene expressions. RAW 264.7 murine macrophages were used to test our hypothesis with or without inhibitor AG490 or Stattic. We studied the the effects of JAK/STAT pathway on the toxicity mechanism of DON and T-2 toxin in RAW 264.7 cells, and preliminarily clarified the potential mechanism between JAK/STAT and DON & T-2 toxin induced cell apoptosis and cell cycle arrest.The effect of DON (0.25～16μM) and T-2 toxin (5～320 nM) on the proliferation of RAW 264.4 cells were tested by using MTT method. We found that DON and T-2 toxin treatment evoked a significant dose and time-dependent cell damages in RAW 264.7 cells. When RAW 264.7 cells incubated with DON (1～4μM) or T-2 toxin (7-28 nM) for 12 h, and with or without specific inhibitors AG490 (10μM and 5μM were the pretreated concentrations when co-incubated with DON or T-2 toxin, respectively) and Stattic (10μM and 5μM were the pretreated concentrations when co-incubated with DON or T-2 toxin respectively) pretreated, the mRNA expressions of IL-6, IL-1β, TNF-α, the critical signal molecules in JAK/STAT including JAK1～3, STAT1～3 and SOCSs were detected by qRT-PCR. The results showed that both DON and T-2 toxin could dose-dependently up-regulate the gene expressions of IL-6, IL-1β, TNF-α, JAK1-2, STAT1-3 and SOCSs. The activation of JAK3 induced by DON and T-2 toxin was not significant. With the intervention of AG490 and Stattic, the gene expressions of JAK2 and STAT1-3 all blocked.The protein expressions and activations of STAT1, p-STAT1, STAT3 and p-STAT3 were assessed by western blotting when RAW 264.7 cells treated with DON (2μM) or T-2 toxin (14 nM) for 12 h. The results showed that the phosphorylation of p-STAT1 and p-STAT3 all increased when cells exposed to DON or T-2 toxin. The interference of AG490 and Stattic blocked the activation of STAT1 and STAT3 which was correlated with their mRNA expressions. We also found that the gene expressions of IL-6, IL-1βand TNF-a induced by DON and T-2 toxin were extremely blocked in the presence of AG490 or Stattic, indicating that JAK/STAT pathway could be activated by DON and T-2 toxin in mRNA and protein levels, and it might play a crucial role in proinflammatory gene expressions in response to trichothecenes. The up-regulation of SOCS indicated that it might moderately regulated the induction of proinflammatory induced by DON and T-2 toxin, which avoided extremely response of cells to some extent, and showed an important physiological significance.In the time-effect research, gene expressions of STAT1, STAT3 and p38 were detected by qRT-PCR when RAW 264.7 cells exposed to DON (2μM) or T-2 toxin (14 nM) for 0.25-24 h. In the cross-talk research, with or without inhibitor SB253080 (20μM) or PD98059 (20μM) pretreated, the same method was used to test the STAT1 and STAT3 gene expressions after RAW 264.7 cells exposed to DON (2μM) or T-2 toxin (14 nM) for 12 h. Results showed that p38 could be typically activated by DON and T-2 toxin in a rapid fashion (0.5-1 h). However, the STAT1 and STAT3 caused a delayed activation (12 h). STAT3 gene expression induced by DON could be significantly inhibited by SB253080. But PD98059 did not exhibit inhibited effects on STAT1 and STAT3 gene expressions induced by DON. Both SB253080 and PD98059 inhibited STAT1 gene expression induced by T-2 toxin. While STAT3 induced by T-2 toxin could be suppressed by PD98059, but SB253080 showed no significant effect on it. These results suggested that the activation of STAT1 induced by DON might be p38 & ERK independent, while STAT3 showed p38 dependent and ERK independent. The activation of STAT1 induced by T-2 might be p38 & ERK dependent, while STAT3 showed p38 independent and ERK dependent. We speculated that JAK/STAT might be a downstream molecular target mediated by DON and T-2 toxin.DON and T-2 toxin induced cell cycle arrest and apoptosis were assessed by Flow CytoMeter (FCM). Related cell cycle and apoptosis genes including Bax, Bcl-2, Bcl-xl, Cyclin D1 and p21 were detected by qRT-PCR in the presence of inhibitor intervention. The results showed that the number of apoptosis cells induced by DON and T-2 were increased apparently, which followed by mitochondrial membrane potential (MMP) decreased. In the presence of AG490 or Stattic, the ratio of Bcl-xl/Bax and Bcl-2/Bax significantly decreased, indicating that JAK/STAT might mediate apoptosis-induced by DON and T-2 toxin through the different regulations of Bax, Bcl-2 and Bcl-xl mRNA expressions. The pro-apoptotic effect mediated by STAT1 might play a critical role in the progress of apoptosis induced by DON and T-2 toxin.DON could cause cell cycle arrest in G2/M phase. However, T-2 toxin exhibited G0/G1 cell cycle arrest in RAW264.7 cells. At the same time, we found the cell cycle related gene expressions of p21 and Cyclin D1 which are also the downstream target genes of STAT1 and STAT3, exhibited dose-dependent up-regulation when cells treated with DON for 12 h. The mRNA expression of p21 showed up-regulation when cells treated with T-2 toxin, while the Cyclin D1 was significant suppressed. The results indicated that the different cell cycle arrest induced by DON and T-2 toxin might result from different transcription activity of p21 and Cyclin D1. Together, we firstly reported that JAK/STAT could be activated by DON and T-2 toxin and identified that this pathway might play a crucial role in the regulation of IL-6, IL-1βand TNF-a gene expressions in RAW264.7 cells in response to trichothecenes. The activation of JAK/STAT and MAPK showed different time course. JAK/STAT might be a downstream molecular target in the toxicity effect of DON and T-2 toxin. It played a critical role in cell cycle arrest and apoptosis induced by DON and T-2 toxin.