The Molecular Mechanism Of Alpha-Lipoic Acid On Alleviating Aflatoxin B₁-Induced Liver Injury In Northern Snakehead （Channa Argus）

Aflatoxin B₁（AFB₁）is the highest toxicity and carcinogenicity aflatoxin,and the toxic substances produced by the metabolism of which in the liver can cause structural damage and dysfunction of the liver.Aquafeeds and their raw materials are easily contaminated by AFB₁ due to the extensive application of plant protein in aquafeeds,the changeable global climate,and improper feed processing or storage.This is a serious threat to the liver health of aquatic animals.However,the mechanism of AFB₁-induced liver injury in fish is still inconclusive and there is a lack of systematic studies.Alpha-lipoic acid（α-LA）is a conditioned essential nutrient,which plays an important role in anti-oxidation,anti-inflammatory,and hepatoprotective,and can alleviate the toxic effects of mycotoxin on the domestic animals.However,the protective effect ofα-LA on AFB₁-induced liver injury in fish and its molecular mechanism are still unclear.Therefore,the northern snakehead（Channa argus）was used as the experimental animal in this study,and the potential molecular mechanism of liver injury induced by AFB₁was investigated by the RNA-Seq technique.Moreover,to explore the mechanism ofα-LA in alleviating liver injury induced by AFB₁,α-LA was used as a functional feed additive in vivo and in vitro,and the methods of immunology,cell biology,molecular biology,and nutrition were carried out in this study.The main research results and conclusions are as follows:（1）Transcriptome analyses reveal the potential mechanism of liver injury induced by AFB₁in northern snakeheadTo evaluate the toxic effects of AFB₁ on northern snakeheads and the potential mechanism of liver injury induced by AFB₁.AFB₁ was supplemented into the basal diet at five levels:0,50,100,200,and 400 ppb.And the experimental period was 56 days.The results showed that high concentrations of AFB₁（200 and 400 ppb）inhibited the growth performance,feed utilization,and immunity of northern snakeheads（P<0.05）.200 and 400 ppb AFB₁ significantly increased the levels of serum AST,ALT,ALP,LDH,and AFB₁ accumulation（P<0.05）.Moreover,the results of histopathological and ultrastructural inspection indicated that AFB₁（100,200,and 400 ppb）destroyed the liver tissue structure,leading to vacuolation and necrosis of hepatocytes,mitochondrial swelling,vacuolation,and endoplasmic reticulum swelling in hepatocytes.In conclusion,AFB₁ could cause liver injury in northern snakeheads.And 200 ppb AFB₁ was the optimal concentration for establishing liver injury models.And then,the CON and AFB₁（200 ppb） treatment group were chosen for transcriptomic sequencing.The results of the liver transcriptomic analyses showed that a total of 1307 DEGs were identified,and 48 pathways were significantly enriched.Meanwhile,the data also revealed that the metabolism of xenobiotics by cytochrome P450,protein processing in the endoplasmic reticulum,glutathione metabolism,and PI3K-Akt signaling pathway were related to AFB₁-induced liver injury in northern snakeheads.（2）AFB₁-induced liver injury through CYP450s/ROS/ERS pathwayIn this experiment,we further verified the main molecular mechanism of AFB₁-induced liver injury in northern snakeheads based on the results of experiment 1.The results showed that dietary AFB₁ could promote the expression of phase I metabolic enzyme cyp1a,cyp1b,and cyp3a in the liver（P<0.05）,inhibit the enzyme activity and gene expression of phase II metabolic enzyme GST,increase ROS,MDA,and 8-OHd G levels,decrease the enzyme activity and gene expression of GSH-Px,SOD and CAT（P<0.05）.Moreover,AFB₁ significantly up-regulated the expression levels of endoplasmic reticulum stress-related genes（grp78,ire1,jnk,etc.）and inflammatory genes（nf-κb,il-1β,il-8,etc.）（P<0.05）,increased the numbers of TUNEL staining positive cells in the liver and up-regulated the expression of proapoptotic genes（cas-3,cyt-c,and bax）（P<0.05）,while down-regulated the expression of bcl-2 and iκbα（P<0.05）.In conclusion,dietary AFB₁ could promote phase I metabolic response（CYP450s）and inhibit phase II metabolic response in the liver of northern snakeheads,leading to the accumulation of toxic metabolites of AFB₁.Furthermore,ROS,a byproduct of phase I metabolism,could cause oxidative damage in the liver and activate ERS pathways,thereby inducing apoptosis and inflammatory response,and exacerbating liver damage.To sum up,AFB₁ could induce liver injury of northern snakeheads through the CYP450s/ROS/ERS pathway.（3）The effects of dietary supplementation ofα-lipoic acid on the growth performance,antioxidant capacity,and disease resistance of northern snakeheadTo investigate the effects ofα-LA on growth performance,antioxidant capacity,and disease resistance of northern snakeheads.Six diets supplemented withα-LA at doses of 0,300,600,900,1200,and 1500 ppm were fed to northern snakeheads for 8 weeks.The results demonstrated that dietaryα-LA increased the FBW,WG,PER,and SGR and reduced the FCR of the fish（P<0.05）.α-LA decreased the activities of AST and ALT in serum,increased the activities of antioxidant enzymes such as GST,GSH-Px,and SOD,and the expression levels of related genes（gst,gsh-px,and gr）in the head kidney,spleen,and liver（P<0.05）.Moreover,it improved the serum immune indices（C3,C4,Ig M,etc.）（P<0.05）,and survival rate and promoted the expression of anti-inflammatory genes such as il-10 and tgf-β（P<0.05）,while inhibiting the expression of il-1β,il-8,and tnf-α（P<0.05）,and the above indexes have extreme values in the 600 and/or 900 ppm groups,respectively.Our results demonstrated that suitableα-LA supplementation in the diet could effectively improve the growth,immunity,and the capability of antioxidant,anti-inflammatory,and disease resistance of northern snakeheads.And the recommended dose ofα-LA in the diet of northern snakeheads is 600-900 ppm.（4）The protective effects ofα-LA on the liver injury induced by AFB₁ in northern snakeheadBased on the results of chapters 1,2,and 3,this experiment was divided into four groups:the basal diet was served as a control group,and the 200 ppb AFB₁ diet was supplemented withα-LA at 0,600,and 900 ppm,and named as CON,AFB₁,600α-LA,and 900α-LA,respectively.And the experimental period was 56 days.The results indicated that dietaryα-LA could reverse the changes in AFB₁-induced growth performance parameters（FBW,WG,SGR,etc.）,immune parameters（C3,C4,Ig M,etc.）,and the accumulation levels of AFB₁ in the liver,and decrease serum AST,ALT,ALP,and LDH levels,and alleviate the liver tissue structure and hepatocyte damage caused by AFB₁.Furthermore,compared with the AFB₁group,α-LA significantly up-regulated the cyp1a,cyp1b,cyp3a（P<0.05）,and Nrf2 signaling pathway genes（nrf2,ho-1,and gst,etc.）and proteins（Nrf2 and Ho-1）expression levels（P<0.05）,significantly increased the activity of antioxidant enzymes（GST,SOD,CAT,and GSH-Px,etc.）（P<0.05）,significantly decreased the contents of ROS,MDA,and 8-OHd G（P<0.05）;α-LA significantly inhibited the expression levels of endoplasmic reticulum stress（grp78,ire1,perk,etc.）,inflammatory response（nf-κb,il-1β,il-8,etc.）and apoptosis（cas-3,cyt-c,bax,etc.）related genes（P<0.05）,the TUNEL positive cell rate and protein expression levels of Grp78、Nf-κb and Cas-3 were significantly down-regulated（P<0.05）.The results of this experiment suggested thatα-LA had protective effects against AFB₁-induced growth inhibition,immunosuppression,and liver damage in northern snakeheads,which was related to the activation of the Nrf2 signaling pathway.（5）The molecular mechanism ofα-LA alleviating the northern snakehead hepatocyte injury induced by AFB₁A hepatocyte injury model was established to clarify the protective effect ofα-LA on AFB₁-induced hepatocyte injury.By measuring the cell proliferation rate and biochemical indicators,the optimal concentration and culture time of AFB₁ modeling were determined to be 0.12μM and24 h,respectively.In addition,we further selected three concentrations ofα-LA（100,200,and400μM）for intervention.The results showed thatα-LA intervention significantly increased the rate of hepatocyte proliferation and decreased the levels of biochemical parameters,and increased the gene expression levels of cyp1a,cyp1b,and cyp3a and the levels of GST,GSH,and GSH-Px in hepatocytes（P<0.05）,the ROS,MDA,and 8-OHd G levels were significantly decreased（P<0.05）,and the expression levels of grp78,ire1,perk,chop,nf-κb,il-1β,cas-3,cyt-c,and bax genes and the protein expression levels of Grp78（cell）and Cyt-c（cytoplasmic）were significantly decreased（P<0.05）.Moreover,α-LA intervention could significantly up-regulate the expression of the genes related to the Nrf2 signaling pathway,and significantly increase the expression of Nrf2 protein in hepatocytes,cytoplasm,and nucleus,and Ho-1 protein in hepatocytes（P<0.05）.In conclusion,200μMα-LA intervention had the best effect.Subsequently,we inhibited the Nrf2signaling pathway by specific inhibitor ML385 to further explore the molecular mechanism ofα-LA alleviating hepatocyte injury induced by AFB₁.The results showed that the inhibition of Nrf2protein significantly reduced the relief effects ofα-LA on AFB₁-induced hepatocyte oxidative stress,endoplasmic reticulum stress,inflammation,apoptosis,and AFB₁ metabolism inhibition,which suggested thatα-LA alleviated AFB₁-induced hepatocyte injury by regulating the Nrf2signaling pathway.To sum up,in vivo and in vitro experiment results demonstrate thatα-LA nutritional intervention can alleviate AFB₁-induced liver and hepatocyte injury in the northern snakehead by modulating the Nrf2 signaling pathway.The results of this study provide a new perspective and direction for the prevention and treatment of AFB₁-induced liver injury in fish and provide the theoretical basis for solving liver injury of fish in actual production,as well as provide the theoretical basis for promoting the healthy and efficient aquaculture of fish and the effective utilization ofα-LA in the aquaculture industry.