Effect Of Pterostilbene To Relieve The Injure Of Bovine Mammary Epithelial Cells Induced By Deoxynivalenol

Deoxynivalenol(DON),the most famous feature is that it causes vomiting in animals,also known as vomiting toxin.It is a toxin produced by Fusarium oxysporum that can widely pollute grains and feed,and has a serious toxic effect on humans and animals.In this experiment,bovine mammary epithelial cells(MAC-T)were used as the research object to explore its in vitro cytotoxicity to MAC-T cells and its potential molecular mechanism.It is also of great significance to effectively improve the toxic effects of DON in MAC-T cells.In recent years,natural plant-derived substances have gradually been used by researchers due to their highly effective anti-inflammatory,antioxidant and cell regeneration effects.Pterostilbene(PTE)is a natural substance found and extracted from rosewood and blueberries.Studies in recent years have also shown that PTE has certain positive effects on animal health.MAC-T cells were treated with different concentrations of DON(0.1,0.25,0.3,0.5,0.8,1,2 ?g/mL)for 24 h,and then the cell viability was detected by CCK8 to screen the appropriate concentration DON effect used in this test.The results showed that DON-treated cells at a concentration greater than or equal to 0.25 ?g/mL significantly reduced cell viability.Similarly,After 24 h of DON treated cells at a concentration of 0.25 ?g / mL,intracellular LDH was released,and the integrity of the cell membrane was damaged.DON caused significant GSH depletion,a decrease in T-SOD and T-AOC decreased,MDA content increased.After DON-treated cells at a concentration of 0.25 ?g/mL for different time periods(9 h,15 h,and 24 h),the fluorescence intensity of MAC-T cells was observed by DON in a fluorescence inverted microscope significantly increased,ROS content increased.DON induces inflammatory responses in MAC-T cells by increasing the expression of genes NF-?B P65,NF-?B P50,COX-2,MyD88,TNF-?,IL-1?,IL-6 and IL-8.In addition,the expressions of TNF-?,IL-6 and IL-8 were significantly correlated with time;the expressions of NF-?B P50,COX-2 and MyD88 were significantly correlated with time;NF-?B P65 and IL-1? There was no significant time-related change in the expression.Cells treated with DON at a concentration of 0.25 ?g/mL was used to detect cell cycle changes by flow cytometry after 24 h.The results showed that DON reduced the G0/G1 phase arrest of S-phase of MAC-T cells and affected the cell cycle progression to prevent normal proliferation.In addition,the results of measuring the apoptosis rate using Annexin V-FITC showed an increase in the apoptosis rate of MAC-T cells.DON induces MAC-T cell apoptosis by increasing Bax mRNA expression level,Bcl-2 non-expression and increased Bax/ Bcl-2 expression ratio.The cell viability and fluorescence quantitative PCR gene expression results in the first test continued to use the DON concentration(0.25 ?g/mL)and action time(9 h).First,MAC-T cells were treated with different concentrations of PTE(2,4,8,16 ?M)for 9 h.Cell viability was measured by CCK8 to analyze the non-cytotoxicity of PTE in this concentration range.After that,MAC-T cells were treated with different combinations of PTE and DON(0.25 ?g/mL)for 9 h,and then a suitable protective dose of PTE(8 ?M)was selected.The test was divided into four groups: control group(CON),0.25 ?g/mL DON treatment group(DON),8 ?M concentration PTE treatment group(PTE),PTE(8 ?M)and DON(0.25 ?g/mL)combined treatment In group(P + D),MAC-T cells were incubated for 9 hours.The results of cell proliferation assay by crystal violet method showed that PTE can effectively improve the inhibitory effect of DON on the proliferation of MAC-T cells.PTE can effectively improve the antioxidant capacity(T-AOC)of MAC-T cells to resist the effect of DON on the antioxidant capacity of cells,and reduce DON on reactive oxygen species ROS and MDA improves DON-induced GSH depletion in MAC-T cells.PTE inhibits DON-induced iNOS and NO production in MAC-T cells.PTE can effectively regulate the levels of Nrf2,Keap1,SOD1 and SOD2 mRNA to resist the effects of DON.PTE can significantly inhibit the levels of DON-induced MAC-T activation key transcription factors NF-?B P65,NF-?B P50 and downstream inflammatory mediators inflammatory cytokines COX-2,IL-1?,IL-6 and MCP-1 mRNA levels.ELISA results showed that PTE inhibited the protein expression of TNF-? and IL-6 produced by DON-induced cells.This study proved that DON caused toxic effects on MAC-T cells,damaging MAC-T cells to varying degrees and affecting normal cell function.PTE alleviates the toxic effect of DON on MAC-T cells,improves the oxidative damage and inflammatory response of DON to cells,improves the antioxidant capacity and antioxidant enzyme levels,and reduces nitric oxide,reactive oxygen levels and inflammatory cytokine expression to achieve antioxidant and anti-inflammatory effects.