Alleviating Effect Of Resveratrol On AFB₁-Induced Liver Injury And Its Mechanism

Mildew of feed has great influence on animal husbandry,among which mycotoxin is the number one public enemy.Aflatoxin B1（AFB1）is a highly toxic mycotoxin.It is widely found in feed and raw materials.AFB₁ contaminated feed will lead to poor growth and reproduction performance,immune dysfunction,nutritional metabolism disorders and other problems.Meanwhile,AFB₁ is a toxic substance that causes liver cancer.AFB₁ and its metabolites will accumulate in the liver and cause liver damage.People who eat livestock and poultry exposed by AFB₁ also pose a threat to human health and public health safety.Currently,DNA methylation and histone modification in AFB₁-induced liver injury have been precisely understood.However,the mechanism on AFB₁ and m⁶A RNA methylation modification is not clear.Resveratrol,as a plant polyphenol,plays a significant role in resisting hepatic toxicity,hepatic inflammation,hepatic oxidative damage and regulating hepatic nutritional metabolism.Resveratrol is also an epigenetic factor,however,the relationship between resveratrol and m⁶A modification is still lacking.Therefore,we cultured mice liver organoids in vitro for preliminarily investigating the biological mechanism of AFB₁-induced liver injury.Futhermore,we performed in vivo experiments to study the role of resveratrol on AFB₁-treated mice liver and further explore the effect of resveratrol and AFB₁ on m⁶A RNA methylation and their crosstalk in the regulation of mice liver function,providing reference for the application of resveratrol in animal production.Experiment 1:The effect of AFB₁ on liver organoids in mice and its regulatory mechanismThe fresh liver of 6-week-old male C57BL/6J mice was used to extract liver stem cells immediately for organoid culture.The experiment was divided into two groups:the WT group and the AFB₁ group.The WT group was not treated with AFB₁,and the AFB₁ group was treated with 80μm AFB₁.Three mice in each group were inoculated with three holes.The organoid culture period was 14 days.AFB₁ treatment was performed on the 14th day of organoid culture,and samples were collected at 48 h after AFB₁ treatment.Compared with the WT group,the liver organoid phenotype of AFB₁-treated mice showed obvious disintegrated state,AFB₁ obviously inhibited the transcriptional levels of Lgr5 and Prominin（P<0.01）,significantly increased the ROS content（P<0.01）and the m RNA expression of Nrf2,HO-1,GCLC,NQO1 and GCLM（P<0.05）.Meanwhile,AFB₁ significantly induced apoptosis of liver organoids（P<0.01）.At the transcriptional level,AFB₁ significantly increased the expression level of Bax（P<0.01）,and extremely decreased the expression level of Bcl-2（P<0.05）and the expression ratio of Bcl-2/Bax（P<0.01）.Futhermore,the m RNA expressions of FTO and ALKBH5 in liver organoids of mice in the AFB₁ group were markedly decreased（P<0.05）,the m RNA expressions of m⁶A reader proteins YTHDF1 and YTHDF2 were significantly decreased（P<0.01）,and AFB₁ significantly increased the content of m⁶A（P<0.05）.Experiment 2:Effect of dietary resveratrol supplementation on growth performance and hepatic structure in AFB1-injured miceThirty-two healthy 6-week-old male C57BL/6J mice were selected,total mice were fed in Nanjing agricultural university barrier condition,after two weeks of adaptive feeding,mice were randomly assigned to four groups（n=8）,they are the control group（CON）,the resveratrol control group（RES）,the aflatoxin B₁group（AFB₁）,and the resveratrol treatment group（ARE）.The CON group was fed a basal diet;the RES group was supplemented with 500 mg/kg resveratrol;the AFB₁group was supplemented with 600μg/kg AFB₁;the ARE group was supplemented with500 mg/kg resveratrol and 600μg/kg AFB₁.The CON group was fed basal diet;The RES group was fed with 500 mg/kg resveratrol.The AFB₁ group was fed with 600μg/kg AFB₁.The ARE group was supplemented with 500 mg/kg resveratrol and 600μg/kg AFB₁.The feeding cycle was 28 days.The results showed that the body weight of AFB₁ group was always lower than that of the other three groups.Compared with mice without AFB₁ supplementation,AFB₁ supplementation significantly decreased the average daily gain（ADG）of mice（P<0.01）.At 12 weeks of age,dietary resveratrol supplementation（ARE group）significantly upregulated body weight related to the AFB₁ group（P<0.05）.Compared with the CON group,AFB₁significantly increased the activities of serum AST and ALT（P<0.05）,and liver section results showed that vacuolation and cell edema in hepatocytes were very common.In the ARE group,the activities of serum AST and ALT were obviously below the AFB₁ group（P<0.05）,and the vacuolation and cell edema of hepatocytes were significantly reduced.In addition,no significant changes in AST and ALT activity and hepatic structure were observed between the CON and the RES groups.Experiment 3:Effect of dietary resveratrol supplementation on AFB₁-induced hepatic oxidative stress,apoptosis,and m⁶A RNA methylation in miceThe experimental design is the same as experiment 2.In this study,AFB₁supplementation induced oxidative damage in the liver.Compared with the CON group,AFB₁ supplementation significantly increased ROS content and MDA content,and significantly decreased CAT activity and T-AOC level（P<0.05）in liver.AFB₁significantly reduced the m RNA expression level of Nrf2,HO-1,GPX,and CAT in liver（P<0.05）.AFB₁ also induced apoptosis in the liver.Compared with the CON group,AFB₁ extremely enhanced the transcriptional levels of Bax,Bcl-2 and Caspase-3（P<0.05）;At the translational level,cleaved-caspase-3 protein expression of AFB₁ mice was significantly increased（P<0.05）,and bcl-2/bax expression ratio was significantly decreased.In addition,AFB1 significantly reduced the protein expression of FTO（P<0.05）and obviously enhanced m⁶A abundance（P<0.05）in liver.Compared with the AFB₁ group,dietary resveratrol supplementation in the ARE group could significantly remove hepatic ROS content and MDA content（P<0.05）,and extremely promote CAT activity and T-AOC level（P<0.05）.At the transcriptional level,hepatic expression of Nrf2,HO-1,GPX and CAT in ARE group were significantly increased（P<0.05）.Resveratrol also inhibited AFB₁-induced apoptosis.Specifically,the m RNA expression level of Bax,Bcl-2,Caspase-3 and cleaved caspase-3 protein expression were significantly down-regulated（P<0.05）.In addition,resveratrol could obviously reverse AFB₁-induced up-regulation of m⁶A levels.In addition,three healthy 8-week-old C57BL/6J male mice and three healthy 8-week-old METTL14^alb/cre,f/f homozygous mice were selected as WT group and METTL14^alb/cre,f/f group,respectively,Feeding conditions were the same as experiment2.Compared with wild-type mice,METTL14 knockout obviously increased the transcriptional level of Bax in liver（P<0.05）,and decreased the the transcriptional level of Bcl-2（P<0.05）and the ratio of Bcl-2/Bax（P<0.05）.Furthermore,Me RIP-seq showed that m⁶A modification sites were present on Bax,Bcl-2,and Caspase-3.In conclusion,dietary AFB₁supplementation could increase the content of ROS,up-regulate the m⁶A level,cause oxidative stress and cell apoptosis,and finally lead to liver injury.However,dietary resveratrol supplementation of could remove AFB₁-induced ROS accumulation,reduce the abundance of m⁶A,alleviate oxidative stress and cell apoptosis,and attenuate liver damage.This study provides a significant reference for ensuring feed safety and animal health.