Deoxynivalenol induces inflammation by activating NF-κB and inflammasome pathway

다국어 초록 (Multilingual Abstract)

The tricothecene toxin deoxynivalenol (DON) is one of the most prevalent and hazardous fungal secondary metabolites which contaminates serial based foods and causing toxicity to human. Many previous studies reported that the amounts of DON only existed in foods; however, whether DON influences on deleterious effects such as the pro-inflammatory responses in the cellular levels has not been understood. Therefore, we evaluated whether DON gives any effects on the pro-inflammatory responses in vitro in murine macrophage RAW264.7 cell. In the current study, we observed that low concentrations of DON (below 1000 nM) exhibited no substantial influence on cell viability and cellular morphological modification. Nevertheless, over at 400 nM DON slightly increased sub-G1 populations (4.65 ± 0.26% at 400 nM, 7.37 ± 0.22% at 800 nM and 7.61 ± 0.61% at 1000 nM DON, respectively) compared to the untreated control (1.66 ± 0.14%); however, the populations were lower than those of LPS-treated group (10.61 ± 0.08%). Then, we found that DON upregulated inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) at the transcription and translation levels, which consequently increased nitric oxide (NO) and prostaglandin E2 production. In addition, DON dose-dependently enhanced the expression of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-12 and tumor necrosis factor-α (TNF-α). DON also remarkably upregulated the nuclear translocation of NF-κB in a concentration-dependent way. Therefore, we suggest that DON could causes pro-inflammatory conditions in macrophages and thereby may lead to inflammatory disorders in human.

다국어 초록 (Multilingual Abstract)

Deoxynivalenol (DON) is one of the most common fungal toxins which contaminate food grains and cereal-derived products. However, whether DON induces inflammasome-mediated inflammation is still unknown. In the current study, we examined whether DON stimulates IL-1β secretion in BV2 microglia cells by activating NLRP3/ACS-mediated inflammasome. Treatment with high doses of DON (over 800 nM) decreased cell proliferation; however, no significant apoptotic sub-G1 was observed, which indicates that DON induces BV2 cells at a stagnant stage. We also found that DON significantly upregulated IL-1β expression in a dose-dependent manner from 0.5 h to 6 h as much as comparing to treatment with LPS and ATP, which was regulated by nuclear factor-κB (NF-κB) activation. In addition, IL-1β was remarkably secreted in the presence of DON at 24 h and a caspase-I inhibitor significantly prohibited DON-mediated IL-1β secretion, which suggest that caspase-1, which is an effector molecule of inflammasome, is an important upregulator of IL-1β. Thus, components of inflammasome such as ASC and NLRP3 substantially increased by DON treatment and transition knockdown of ASC and NLRP3 significantly downregulated DON-mediated IL-1β expression and secretion, which means that DON stimulates NLRP3/ASC-mediated inflammasome, which consequently promotes IL-1β expression and secretion in BV2 microglia cells. Taken together, these data suggest that DON induces IL-1β expression in BV2 microglial cells by activating the NF-κB signaling pathway and subsequently upregulated NLRP3/ASC-mediated inflammasome-mediated IL-1β secretion. Therefore, DON could induce inflammatory diseases or disorders by activating inflammasome-mediated IL- 1β.

목차 (Table of Contents)

• Acknowledgmentsii
• List of Figures vi
• PART 01 vii
• Abstract viii
• 1. Introduction 1
• 2. Material and methods 3
• 2.1. Reagents and antibodies 3
• 2.2. Cell culture and MTT assay 3
• 2.3. Reverse transcriptase polymerase chain reactions (RTPCR). 3
• 2.4. Western blotting assay 4
• 2.5. Flow cytometric analysis 5
• 2.6. ELISA 5
• 2.7. NO assay 6
• 2.8. Statistical analysis 6
• 3. RESULTS 7
• 3.1. Low concentrations of DON exhibit no influence on cell viability of RAW264.7 macrophages 7
• 3.2. DON increases iNOS and COX-2 expression, leading to NO and PGE2 production 9
• 3.3. Stimulation of RAW264.7 cells with low concentrations of DON results the upregulation of inflammatory cytokines such as IL-6, IL-12 and TNF-α ... 12
• 3.4. DON stimulates the NF-kB pathway 15
• 4. Discussion 16
• 5. References 19
• PART 02 23
• Abstract i
• 1. Introduction 22
• 2. Material and Methods 25
• 2.1. Reagents and antibodies 25
• 2.2. Cell culture and viability 25
• 2.3. Reverse transcriptase polymerase chain reactions (RT-PCR). 25
• 2.4. Western blot assay 26
• 2.5. Flow Cytometric Analysis 27
• 2.6. ELISA 27
• 2.7. siRNA transfection28
• 2.8. Statistical analysis 28
• 3. Results 29
• 3.1. DON exhibits no cytotoxic effect on BV2 microglia cells at low concentrations 29
• 3.2. DON induces the expression of IL-1β gene in BV2 microglia cells by activating the NF-κB signaling pathway 32
• 3.3. DON increases the secretion of active IL-1β protein in BV2 microglia cells 35
• 3.4. DON enhances IL-1β secretion by activating caspase-1 37
• 3.5. DON upregulates NLRP3/ASC-mediated inflammasome, which stimulates IL-1β secretion by activating caspase-1 40
• 4. Discussion 44
• 5. Reference 48