Study On The Protective Effect Of Luteolin And Folic Acid On Aflatoxin B₁ Toxicity And The Anti-cancer Effect Of Luteolin

Objective?1?The human esophageal epithelial cells were cultured with aflatoxin B1?AFB1?,luteolin?Lut?and folic acid?FA?,to investigate theintervention of Lut and FA on AFB1-induced toxicity in vitro experiments,observing the human esophageal epithelial cell proliferation,cell cycle and apoptosis,MTHFR,FOLR1 gene expression and MTHFR gene promoter region methylation status,to provide a certain experimental basis and theoretical basis for prevention of AFB,toxicity.?2?The human esophageal cancer Eca109 cells were treated with Lutin vitro experiments to explore their effects on apoptosis and the mechanismsso as to providebasic data for future usage.Methods?1?Effects of AFBi on human esophageal epithelial cell physiology:Cell Counting Kit-8 kit was used toresearch the influence of AFB1 on human esophagus normal cell proliferation;flow cytometry?FCM?is used to detect cell cycle changes and apoptosis.?2?The interventions of Lut and FA on human normal esophageal epithelial cells treated with AFBi:AFBi-treated human normal esophageal epithelial cells were cultured with Lut and FA,CCK8 was used to research the proliferation influence of AFBi on thehuman esophagus cell proliferation;FCMwas used to detect cell cycle changes and apoptosis.?3?The effects of Lut and FA on MTHFR and FOLR1 genes in normal esophageal epithelial cells exposed to AFBi:Real-time PCR was used to detect the expressions of MTHFR and FOLR1 mRNA.The protein expressions of MTHFR and FOLR1 were detected by Western blot.MTHFR gene promoter methylation level was detected by MassArray EpiTYPER platform.?4?The effectsand mechanisms of Luton Eca109 cells:MTT is used to research the proliferation influences of Lut on Eca109 cells.Cell cycle changes and apoptosis were detected by FCM.The mRNA expressions of cell CDK2,cyclinA,caspase9 and caspase3 were detected by Real-time PCR.The related protein levels were determined by Western blot.Results1.Effects of AFB1 on human esophageal epithelial cell physiology:?1?The proliferation rate of human normal esophageal epithelial cells decreased when cells were treated with different concentrations of 13?M,25?M,5?M,100?M,200?M AFB1 for 24h,48h and 72h respectively;?2?The FCM results showed thatcells treated with 50?M,100?M and 200?M AFBi for 24h were arrested in G0/G1 phase?P<0.05?;?3?The percentages of apoptotic cells in 50?M,200?M AFB1 groups were significantly higher than those in blank control groups?p<0.05?.2.The interventions of Lut and FA on human normal esophageal epithelial cells treated with AFB1:?1?The inhibition of human normal esophageal epithelial cell proliferationin Lut,Lut combined with FA groups were significantly lower than the control group?p<0.05?,the effect in FA group was not significant?P>0.05?;?2?The proportions of cells in G0/G1 phase in Lut,Lut combined with FA group were significantly lowerthan that in the exposure group?P<0.05?,and the percentages of S phase cells in Lut and Lut combined with FA groups were significantly higher than the percentage in exposure group.The effect of FA was not significant?P<0.05?;?3?The apoptosis rates of the cells treated with Lut,Lut combined with FA were significantly lower than thatin the exposure group?P<0.05?.3.The effects of Lut and FA on MTHFR and FOLR1 genes in normal esophageal epithelial cells exposed to AFB1:?1?AFB1 could up-regulate the expressions of MTHFR?FOLR1 mRNA?P<0.05?.After Lut intervention,the expressions of MTHFR and FOLR1 mRNA significantly decreased?P<0.05?.20?M and 200?M FA do not affect the expression of MTHFR mRNA significantly?P>0.05?.The expression of FOLR1 mRNA in 200?M FA group decreased significantly?P<0.05?.Lut combined with FA could significantly attenuated the effect of AFB1 on the up-regulation of MTHFR mRNA expression.Lut combined with different concentrations of FA perform different regulatory effects on FOLR1 mRNA?P<0.05?;?2?AFB1 up-regulate the expression of MTHFR protein?P<0.05?,down-regulate the expression of FOLR1 protein?P<0.05?in human normal esophageal epithelial cells.Intervention with Lut,the expression of MTHFR protein decreased,and the expression of FOLR1 protein increased?P<0.05?.In 20?M,200?M FA groups,the expression of MTHFR protein was close to exposure group?P>0.05?,but the expression of FOLR1 protein was close to control group?P>0.05?.Lut combined with FA can significantly attenuate the effect of AFB1 on MTHFR and FOLR1 protein expressions.?3?In exposure group,the level of methylation was significantly higher than that of the control groupon CpG₅?CpG₆.7?CpG₁6?CpG₃5?CpG₄2?P<0.05?.Analyzed CpG₅ and CpG₃5,the levels of methylation in Lut and Lut combined with200?M FA were lower than exposure group?P<0.05?;CpG₆.7,the level of methylation in exposure group was higher than control group?P<0.05?,and the level of methylation in 200?M FA group was lower than exposure group?P<0.05?;the methylationlevels of CpG₁6?CpG 42were affected obviously except 20?M FA group?P<0.05?.4.The effectsand mechanisms of Luton Eca109 cells:?1?The results showed that the proliferation rates of Eca109 cells decreased when cells were treated with different concentrations of 40?M,80?M,120?M,160?M,200?M and 240?M Lut for 24h,48h and 72h respectively?P<0.05?,meanwhile,the shape was inverted U-type,with the Lut concentration increase;?2?40?M,160?M,240?M for the late experimental intervention concentrations:Lut could arrest cell cycle at the Sphase when cells were treated with 160?M?240?M Lut separately,and induce apoptosis in 160?M Lut?p<0.05?;?2?Lut could down-regulate the expression of CDK2 mRNA?P<0.05?,and the effect of Lut on the expressions of CyclinA,Caspase9 and Caspase3 mRNA increased at first,followed with decrease;?3?Lut could down-regulate the expression of CDK2 protein?P<0.05?.The effect of Lut on the CyclinA protein expression was promotion firstly,then becoming inhibition,and the CyclinA protein expressions treated with 160?M and 240?M Lut were inhibited significantly?P<0.05?.The effect of Lut on the expression of Caspase9 and Ccaspase3 proteinswas promotion firstly,then becoming inhibition,the expression of Caspase9 and Caspase3 protein streated with the 240?M Lut group were inhibited significantly?P<0.05?.Conclusion1.AFB1 can inhibit the proliferation of human normal esophageal epithelial cells by arresting the cell cycle and promoting the apoptosis.The interventions of Lut and FA can attenuate the AFB1-induced toxicity on cell proliferation,cell cycle,apoptosis,which plays a protective role.2.Lut and FA can reduce the effect of AFB1 on the expressions of MTHFR,FOLRl mRNA and protein in human normal esophageal epithelial cells,which can improve the metabolism of FA in the cells,then protect the normal esophageal epithelial cells.The interventions of lutand FA could reduce the methylation level of MTHFR gene promoter region increased by AFBi.Lutand FA have different effects on different CpG sites.Lutand FA could attenuate the toxic effects of AFB1 on human normal esophageal epithelial cells and protect human normal esophageal epithelial cells.3.Lut could inhibit the proliferation of esophageal cancer Eca109 cells,arrest cell cycle in the S phase by down-regulating CDK2 and Cyclin A,and induce apoptosis.Its mechanism of inducing Eca109 cell apoptosis involves the expression of Caspase9,Caspase3,but the specific mechanism still need to be researched in future.