The Critical Role Of Caveolin-1 In Hepatotoxicity Induced By Aflatoxin B1

Background and AimAflatoxin(AFT)is the most common metabolite of fungi in foods.It is a derivative of dihydrofuran coumarin and is classified as a class I carcinogen by The International Agency for Research on Cancer(IARC).Among the derivatives of aflatoxin,aflatoxin B1(AFB1)is the most toxic and most common in human and animal bodies.The liver is the main target organ of AFB1.After entering the body,AFB1 would be transformed into a highly active intermediate product AFB1-8,9-epoxide(AFBO)through the transformation of cytochrome P450 enzyme system.AFB1 toxicity maily results from AFBO,which can bind to DNA,RNA,protein and other macromolecules,and then block gene transcription,translation and affect gene expression.In this process,AFBO metabolism can lead to accumulation of reactive oxygen species(ROS),lipid peroxidation,DNA and protein damage,gene mutation and cell apoptosis due to the deletion of glutathione(GSH).It can be inferred that oxidative stress caused by AFB1 is the direct cause of hepatocyte damage.At present,providing antioxidants is mainstream measure for the treatment of AFB1 poisoning.However,the mechanism of oxidative stress induced by AFB1 and the key molecules remains unknown.Therefore,exploring the key molecules in oxidative stress of hepatocytes induced by AFB1 may provide a new target for clinical treatment of AFB1.Caveolin-1(Cav-1),a member of the Caveolin family,is located in the membrane and endosome membrane.It gets involved in cholesterol transport and macromolecule endocytosis mainly through autophosphorylation.Cav-1 can regulate multiple signal transduction pathways in the cells,such as receptor tyrosine kinase,Src family tyrosine kinase(Src,Fyn,etc.),endothelial nitric oxide synthase and the related downstream signal molecules H-Ras,MEK,ERK,PI3K/AKT et al.Cav-1 is not only a tumor suppressing protein,but also a oncoprotein.This dual effect is also a hot issue in recent years,depending on the cell type and the different stages of cancer development.Cav-1 is also found to be associated with a variety of stresses,including oxidative stress,ultraviolet radiation,radiotherapy,and stress-related signals.Recent evidence has shown that Cav-1 is an oxidative stress-related protein,while oxidative stress can affect the membrane transport of Cav-1.Under oxidative stress,Cav-1 phosphorylation is closely related to cell apoptosis and cell adhesion.The dual effect of Cav-1 is also reflected in oxidative stress.On the one hand,it has been testified that the increased expression of p-Cav-1 protein has anti-apoptotic effect and can promote the survival of cells after oxidative stress.While on the other hand,it has also been found that decreased or absent expression of Cav-1 can significantly inhibit neutrophil chemotaxis,adhesion and apoptosis of epithelial/ endothelial cells in lung tissue,alleviate diffuse alveolar injury,pulmonary vascular endothelial barrier injury and pulmonary edema.Our previous studies had shown that AFB1 could lead to increased expression of Cav-1 and changes the location of Cav-1 molecules.This suggests that Cav-1 may play an important role in AFB1-induced hepatotoxicity.On this basis,we analyzed the role of Cav-1 in AFB1-induced liver injury by down-regulating or over-expressing it,and we also analyzed its mechanism through oxidative stress pathway.Part ?: The role of Cav-1 in aflatoxin-induced hepatotoxicityMaterials and methods To study the role of Cav-1 in hepatotoxicity induced by AFB1,we detected hepatotoxicity induced by AFB1 by cell proliferation assay,flow cytometry,immunofluorescence western blot et al.on a cell model of human normal hepatocytes(L-02).We first down-regulation or overexpression of Cav-1 expression in hepatocytes through transfection of plasmids or siRNA,and then detected the effect of over-expression or down-regulation of Cav-1 on liver injury induced by AFB1.In addition,the effects of Cav-1 on cell activity and apoptosis were analyzed by down-regulating the expression of Cav-1 to evaluate whether Cav-1 plays a similar role in different liver cells Huh7 and HepG2.Results AFB1 exhibited toxicity to hepatocytes,as evidenced by the decrease in cell viability,the increase in cell apoptosis and the increased level of oxidative stress in the cell(an increase in the level of active oxygen and an increase in MDA content).It is further found that AFB1 could increase cav-1 level and change the cellular localization of Cav-1.Basised On this,we detected the cytotoxicity of hepatocytes induced by AFB1 after down-regulating the expression of Cav-1 in hepatocytes.The results revealed that Cav-1 down-regulation could markedly decrease the viability of hepatocytes loss and the apoptosis of hepatocytes and the level of oxidative stress.On the contrary,the effects of Cav-1 overexpression were opposite.The similar results were obtained on two hepatoma cell lines Huh7 and HepG2.Conclusion AFB1 could produce hepatotoxicity by inducing oxidative stress,resulting in hepatocyte apoptosis.In this process,AFB1 could promote the expression of Cav-1,which could enhance oxidative stress.While down-regulation of Cav-1 expression in hepatocytes could inhibit the occurrence of oxidative stress induced by AFB1.Part ?: The mechanism of Cav-1 in hepatocytotoxicity induced by AFB1Materials and methods In order to explore the mechanism,we started with the Keap1-Nrf2 signaling pathway,which was a key antioxidant pathway.Firstly,we detected the effects of down-regulation or over-expression of Cav-1 on the expression of Nrf2,together with HO-1 and NQO-1,the two important molecules of downstream antioxidant stress in Nrf2 pathway.Then we detected the interaction between Cav-1 and Nrf2 and the effects of Cav-1 on Keap1-Nrf2 signaling pathway through immunoprecipitation et al.In addition,we explored the regulated pathways of Cav-1 on autophagy by western blot and fluorescein.Furthermore,we aplied autophagy atimulator and autophagy inhibitor to study the role of Cav-1 in autophagy.At the same time,the mechanism of Cav-1 on oxidative stress and autophagy were further verified by two cell lines of Huh7 and HepG2.Results Down-regulation of Cav-1 could promote the expression and transcription of Nrf2,HO-1 and NQO-1,and significantly inhibit the ROS and MDA levels in hepatocytes stimulated by AFB1.On the contrary,over-expression of Cav-1 played the opposite role.Furthermore,we found that Cav-1 could regulate the oxidative stress level induced by AFB1 by direct interaction with Nrf2.After overexpression of Cav-1,the transcription of Nrf2 was inhibited and the interaction between Cav-1 and Nrf2 was enhanced,which affected the dissociation of Nrf2 and Keap1,and finally enhanced the oxidative stress of cells.In addition,the increase of the autophagy marker LC3 revealed that the down-regulation of Cav-1 significantly promoted the autophagy level of hepatocytes after AFB1 treatment,and the similar result was obtained in the formation of autophagysome in fluorescent labeled cells.Autophagy stimulators could significantly reduce AFB1-induced cytotoxicity and inhibit apoptosis,while autophagy inhibitors could promote the cytotoxicity of AFB1 and promote cell apoptosis.Cav-1 could regulate autophagy to affect the hepatocyte damage induced by AFB1.Further analysis of the signal pathway of Cav-1 regulation of autophagy showed that Cav-1 did not change the expression of classical autophagy marker molecule Beclin1 and ATG5 under the treatment of AFB1,suggesting that its regulation was not mediated by the classical autophagy pathways.AFB1 improved phosphorylation of epidermal growth factor receptor(EGFR),which promoted phosphorylation of PI3 K downstream of EGFR and activation of autophagy regulator mTOR,thus inhibiting autophagy.After the treatment with EGFR inhibitor afatinib,the autophagy and viability of the cells were obviously promoted,which further confirmed the results.In addition,the results were similar to those of L-02 cells in Huh7 and HepG2 cell lines.Conclusion AFB1 promoted direct interaction between Cav-1 and Nrf2,which was a key molecule of oxidative stress.Cav-1 enhanced oxidative stress by affecting the transcription level of Nrf2 molecule and the Nrf2-Keap1 interaction,and promoted the hepatocyte injury induced by AFB1.In addition,Cav-1 could inhibit autophagy of cells by promoting the activation of the EGFR-PI3K-mTOR signaling pathway,and promoted the liver cell injury induced by AFB1.SummaryAfter AFB1 enters the body,it is mainly detoxified and metabolized in the liver by interacting with enzyme systems or molecules in the liver cells.AFB1 could induce oxidative stress reaction in metabolic process,resulting in hepatocyte injury and even apoptosis.In this study,we found that Cav-1 may serve as a key molecule regarding AFB1-induced hepatocyte damage.AFB1 promoted the upregulation of Cav-1 expression and changed the subcellular localization of Cav-1 molecules.Cav-1 played two important regulatory roles in hepatocyte injury induced by AFB1: on the one hand,the up-regulated Cav-1 molecule could promote oxidative stress and apoptosis,decrease cell viability by direct interaction with Nrf2;on the other hand,Cav-1 molecules inhibited cell autophagy by activating EGFR-PI3K-mTOR,a non-classical autophagy regulatory pathway,thus promoting cell apoptosis and decreasing cell viability.Therefor we had digged out Cav-1 as a key molecule for the understading of AFB1 toxicity,and may provide a new therapeutic target for future prevention and treatment of AFB1-induced liver injuries.