Inhibition Of Ferulic Acid On Fusarium Graminearum And Intervention On The Toxicity Of Deoxynivalenol

Fusarium graminearum is a pathogen that can infect wheat,corn,barley and other grain crops and cause diseases such as grain crop fusarium wilt,resulting in the reduction of grain yield and nutritional value that cause serious harm to public health.Its secondary metabolite,deoxynivalenol（DON）,is one of the most widely distributed and most polluting mycotoxins in the world.DON intaked through food contact can cause acute poisoning symptoms such as vomiting,fever and diarrhea;long-term exposure to DON can cause adverse reactions such as weight loss,immune disorders and intestinal dysfunction.The toxicity of Fusarium graminearum contamination and its toxin DON has become one of the urgent food safety problems to be solved.Ferulic acid（FA）is an active ingredient in a variety of traditional Chinese medicines such as ferula asafoetida and angelica,and its antibacterial,antioxidant,anti-inflammatory and anti-apoptotic functions have been extensively studied.Based on its efficacy,this paper studies the inhibition of FA on the growth of Fusarium graminearum,the intervention of FA on the cytotoxicity of DON and presents the detailed mechanism towards it.The main research contents and results are as follows:1.FA inhibits the growth of Fusarium graminearum.The EC₅₀ value of FA on the growth inhibition of Fusarium graminearum was about 20μg/m L,and the EC₉₀ value was about 100μg/m L by mycelial dry weight test.The inhibitory activity of FA on Fusarium graminearum was evaluated by colony growth and spore germination experiments.The results showed that different concentrations of FA had obvious inhibitory effects on colony expansion and spore germination in a dose-dependent manner.The results of scanning electron microscopy showed that under the treatment of different concentrations of FA,the hyphal structure of Fusarium graminearum became loose with wrinkles and depressions on the surface;the results of transmission electron microscopy showed that after treatment with high concentration of FA,the density of mycelial cells of Fusarium graminearum decreased,the color became lighter and uneven,and the content was lost.With the increase of FA concentration,the fluorescence intensity of Fusarium graminearum after PI staining increased（p<0.001）,the extracellular relative conductivity increased,and the leakage of proteins and nucleic acids increased.The results of ergosterol synthesis experiments showed that with the increase of FA concentration,the degree of inhibition of ergosterol synthesis in Fusarium graminearum cells gradually increased.2.FA interfered with DON-induced decrease in cellular activity,oxidative stress,inflammatory response and apoptosis in IPEC-J2 cells.Cell viability experiments demonstrated that 60μmol/L FA could maximize cell viability after 12 h of incubation,and 40μmol/L DON was closest to its IC₅₀ for IPEC-J2 cell viability.In addition,compared with the DON group,pretreatment with FA could up-regulate cell activity and ensure its normal physiological role.The results of ROS assay showed that FA effectively scavenged ROS（p<0.0001）and attenuated DON-induced oxidative stress injury in IPEC-J2 cells.After the determination of antioxidant system indicators,it was found that FA up-regulated the levels of CAT,SOD（p<0.01）,GSH-Px,and GSH（p<0.05）,and enhanced the antioxidant level in IPEC-J2 cells,thereby preventing the occurrence of oxidative stress in cells.The decrease in the release of pro-inflammatory factors IL-1β,IL-6（p<0.0001）,IL-8（p<0.05）and IFN-γin cells proved that FA could inhibit DON-mediated cellular inflammatory response.After measuring the apoptosis rate by flow cytometry,it was found that FA could effectively reduce the apoptosis rate and protect IPEC-J2cells from DON-induced damage.3.The mechanism of FA interfering with DON-induced oxidative stress,inflammatory response and apoptosis in IPEC-J2 cells.The effects of FA on the expression and transcription of related node proteins in the DON toxicity pathway were analyzed by Western blot and RT-q PCR techniques.The results showed that FA could activate the Nrf2-Kepa1 pathway and promote the nuclear translocation of Nrf2 to promote the occurrence of downstream antioxidant reactions.At the same time,FA could also inhibit the phosphorylation of MAPKs pathway and the activation of NF-κB pathway in IPEC-J2 cells,thereby preventing the nuclear translocation process of NF-κB,inhibiting the release of pro-inflammatory factors in cells,and protecting IPEC-J2 cells from DON-induced inflammatory damage.In addition,FA down-regulated the expression of pro-apoptotic proteins Bax and caspase-3,up-regulated the expression level of Bcl-2,thereby preventing the occurrence of apoptosis and protecting cells from DON-induced toxic damage.In conclusion,FA could act on the cell membrane of Fusarium graminearum to inhibit its growth activity.Meanwhile,FA could protect IPEC-J2 cells from DON-triggered cytotoxicity by activating the Nrf2-Keap1 pathway,inhibiting the phosphorylation of MAPKs and NF-κB pathways,and regulating the mitochondrial apoptosis pathway.