Effect Of Ferulic Acid On Hepatic Injury Induced By AFB1 In Rats Research

Aflatoxin B1(AFB1)is the most harmful mycotoxin at present.After metabolic activation by cytochrome P450(CYP450)enzyme,a highly toxic metabolite AFB1-8,9-epoxide(AFBO)is generated.The latter can covalently bind DNA,proteins and other biological macromolecules,causing DNA damage,gene mutation,cell metabolic disorder,and even cell death.The liver is the main organ producing CYP450 enzyme,so it becomes the target organ of AFB1.AFB1 not only threatens human health and life,but also causes huge economic losses to the farming industry.Currently,the commonly used AFB1 attenuating methods still have some defects,such as weak effect,high toxicity,and no effect on AFB1 entering animals.Ferulic acid(FA)is an important effective component of ferulic acid,Angelica sinensis,jujube kernel and other Chinese medicinal materials.It has been reported that FA has antioxidant,anti-apoptosis,inhibition of CYP450 and other activities,and can play a protective role in liver injury induced by drugs,high-fat diet and other factors.However,the application of FA in the intervention of AFB1 induced liver injury has not been reported.This study will rats as experimental animals AFB1 induced liver injury model was established and given different doses of FA to intervene,application of pathology technique and Western Blot,q PCR,such as molecular biology experimental technique,by detecting the blood biochemical indexes,AFB1 toxic metabolites(AFB1-DNA and AFB1-alb)content,liver tissue pathological changes,liver cell oxidative stress related indicators,evaluation of FA intervention effect of AFB1 induced liver injury,By measuring the key CYP450 enzyme(this CYP1A2,CYP2A6,CYP2E1 and CYP3A4)expression changes and some ways mitochondrial apoptotic pathways in the expression of key genes change,explore the FA intervention mechanism of action of AFB1 induced rat liver cell damage,eventually to clarify whether FA regulation CYP450 enzyme and affect the mitochondrial apoptosis by giving play to the role of intervention AFB1 cause liver cell damage.Forty-nine healthy male Wistar rats with SPF grade were randomly divided into 7 groups with 7 rats in each group.The blank control group(Group C): 0.3 ml of normal saline was administered daily.AFB1 infected group(AFB1 group): 300 mg/kg AFB1 standard daily;Low dose FA intervention group(L group): 300 mg/kg AFB1+60 mg/kg FA per day;Medium dose FA intervention group(M group): 300 mg/kg AFB1+120 mg/kg FA per day;High dose FA intervention group(group H): 300 mg/kg AFB1+240 mg/kg FA per day;Ferulic acid control group: 120 mg/kg FA per day;Solvent group(DMSO group): 0.3 m L DMSO was administered daily.After continuous gavage for 28 days,the rats in each group were anesthetized,weighed,collected blood,and euthanized.The serum was prepared for blood biochemical indicators and other experiments,and the liver was quickly removed.Some rats were fixed in formalin solution for histopathological detection,and some were frozen in liquid nitrogen for subsequent experiments.The experimental results showed that :(1)compared with group C,AFB1 significantly reduced the body weight of rats(p<0.01),caused hepatocyte apoptosis and hepatocyte necrosis,and significantly increased serum ALT,AST,AKP,GGT and TBA indexes(p<0.01).The weight of the rats in group L,Group M and group H showed an increasing trend(p<0.01),and the serum indexes were significantly reduced(p<0.01),among which the changes in AST,AKP and GGT levels were dose-dependent.Histopathological examination showed necrosis and apoptosis of hepatocytes in rats in AFB1 group,disordered structure of hepatocyte cords,small amount of inflammatory cell infiltration,granular degeneration of hepatocytes in L group,inflammatory cell infiltration in L group,intact structure of hepatocytes in M group and clear cords in H group.Compared with group C,FA control ratsThe body weight was slightly reduced,but there was no statistical significance.The liver cell structure was intact,the staining was uniform,the serum AKP was significantly reduced(p<0.01),and there was no significant difference in other indicators.(2)In the determination of AFB1-DNA and AFB1-ALB adducts,compared with AFB1 group,L group,M group,H group significantly decreased(p<0.05 or p<0.01),and presented a dose-dependent.(3)Compared with group C,ROS and MDA levels of oxidative stress markers in liver cells of AFB1 group were significantly increased(p<0.01),while SOD and GST activities of antioxidant enzymes were decreased,among which GST was significantly decreased(p<0.01).Compared with AFB1 group,ROS and MDA levels in L group,M group and H group decreased in a dose-dependent manner(p<0.01),while SOD and GST activities in liver cells in M group and H group increased(p<0.01).Compared with group C,ROS and MDA in FA group showed a decreasing trend,and MDA showed a very significant decrease(p<0.01),while GST and SOD in FA group showed no significant changes.(4)Compared with group C,AFB1 significantly upregulated pro-apoptotic genes bax,caspase-3,caspase-9,cyt-c,and p53(p<0.01),and down-regulated anti-apoptotic genes bcl-2(p<0.01).Compared with AFB1 group,the relative expression levels of bax,caspase-3,caspase-9,cyt-c and p53 in FA intervention group were significantly decreased(p<0.01),while the expression levels of bcl-2 were significantly increased(p<0.01).Compared with group C,bcl-2 gene in FA group was significantly up-regulated(p<0.01),while other apoptosis-related genes showed no significant difference.(5)The apoptotic cell staining results showed that compared with the control group,the number of apoptosis of hepatocytes in AFB1 group was significantly increased(p<0.01).The number of apoptotic cells in the FA intervention group was significantly lower than that in the AFB1 group.(6)Compared with group C,AFB1 significantly upregulated the expressions of CYP1A2,CYP2A6,CYP2E1 and CYP3A4(p<0.01).Compared with AFB1 group,the relative expression levels of CYP1A2,CYP2A6,CYP2E1 and CYP3A4 in the FA intervention group were significantly reduced(p<0.01).Compared with group C,the expression of CYP1A2,CYP2A6,CYP2E1,CYP3A4 and other enzymes in FA group showed no significant difference(p>0.05).In conclusion,this study result shows that the FA on the one hand can inhibit this CYP1A2,CYP2A6,CYP2E1 and the expression of CYP3A4 inhibitory AFB1 AFBO toxic metabolite production,reduce the liver cell oxidative damage induced by AFB1,on the other hand can by inhibiting mitochondrial apoptosis signaling pathways intervention AFB1 induced rat liver cell apoptosis,play a role of intervention of AFB1 cause liver injury in rats.