The Mechanism Of FNDC3B Regulating The Progression Of Alcoholic Liver Disease Via AMPK Pathway

ObjectiveThe function of the liver is vital,and damage to it can greatly affect the quality of life.In recent years,the incidence of liver diseases has been increasing,placing a significant burden on healthcare systems worldwide.The causes of liver diseases include viral infections,excessive alcohol consumption and high-fat diets et al.Caused by excessive alcohol intake,alcoholic liver disease has attracted more and more attention due to young patients and the prevalence of drinking culture.Excessive alcohol consumption is currently defined as more than 40 grams of alcohol intake per day,which seriously damages the normal function of liver cells,leading to a series of pathological changes in the healthy liver such as steatosis,fibrosis,cirrhosis and even cancer.However,effective treatments and therapeutic targets are still lacking.It is of great guiding significance to clarify the pathogenesis mechanism of alcoholic liver disease and develop novel and effective therapeutic strategy.Steatosis is the most common pathological change as well as the initial stage of alcoholic liver disease,characterized by abnormal accumulation of lipids in hepatocytes,including triglycerides,phospholipids and cholesterol esters.Hepatic lipid metabolism could be disrupted in several aspects,including increased synthesis,decreased oxidation and reduced outward transport of free fatty acids.The study indicated that alcohol can directly destroy the oxidative balance of cells and increase the rate of fatty acids esterification,thus regulate lipid synthesis and metabolism.At the same time,alcohol can also regulate the expression of key transcription factors and signaling pathways,resulting in the imbalance of lipid homeostasis in the liver by disrupting lipid synthesis,oxidation and transport.However.the specific mechanism of lipid metabolism regulation in alcoholic liver disease has not been elucidated.It has been reported that improving steatosis can effectively inhibit the progression of alcoholic liver disease and improve the prognosis of patients.Therefore,a better understanding of the molecular mechanism of alcohol-induced lipid disorders is of great significance for the development of novel therapeutic targets for alcoholic liver disease.Alcohol metabolism in the liver results in lots of reactive oxygen species,contributing to cellular oxidative damage.Alcohol dehydrogenase(ADH)and Cytochrome P450 2E1(CYP2E1)are the key enzymes for the classical and complementary pathways of alcohol metabolism,respectively.The common metabolite is acetaldehyde,which is strongly cytotoxic.In patients with long-term chronic alcohol consumption,the expression of CYP2E1 was upregulated and the proportion of supplement pathway was increased,thus significantly increasing the intracellular ROS level.ROS can destroy many components of cells,such as proteins,DNA and lipids.Among them,ROS attacks and destroys intracellular lipids,resulting in lipid peroxidation.The accumulation of lipid peroxidation products will damage the normal structure and function of proteins and DNA,destroying many normal biochemical processes.At present,the specific mechanism of alcohol-induced lipid peroxidation remains unclear.Alleviating lipid accumulation and lipid peroxidation is significant for alcoholic liver disease.Therefore,in order to develop novel therapeutic strategies to delay the progression of alcoholic liver disease,it is necessary to deeply understand the specific mechanism of alcohol-induced lipid peroxidation.Fibronectin type-Ⅲ domain-containing protein 3B(FNDC3B),one of members in FNDC family,consists of 9 fibronectin type Ⅲ domains,1 transmembrane domain and 1 proline-rich domain.It has been reported that FNDC3B plays an important role in several physiological and pathological processes,such as signal transduction.tumor progression and metabolic regulation.In addition,studies have shown that FNDC3B can affect the expression of sterol regulatory element binding proteins(SREBPs),key transcription factors for lipid synthesis,suggesting that FNDC3B is involved in the regulation of lipid metabolism.However,whether FNDC3B plays a regulatory role in alcoholic liver disease remains unclear.Therefore,this study is divided into three parts.In the first part,the expression of FNDC3B in alcoholic liver disease and its relationship with alcohol-induced lipid deposition were clarified by combining database clinical information,in vitro alcoholic model and animal alcoholic model.Also,the microRNAs regulatory mechanism of FNDC3B was explored in depth.In the second part,the correlation between FNDC3B and lipid peroxidation in alcoholic liver disease was clarified by using the mouse alcoholic model with hepatocytes-specific FNDC3B depletion and the cell alcoholic model with stable FNDC3B depletion.Meanwhile,the relationship between FNDC3B and ferroptosis was discussed by using RNA highthroughput sequencing and ferroptosis inhibitor.In the third part,we explored the specific regulation of FNDC3B on steatosis and lipid peroxidation in alcoholic liver disease,and clarified that AMPK signaling pathway is the key pathway.In this study,liver localization magnetic resonance imaging,BODIPY 493/503 fluorescence staining and oil red staining were used to comprehensively evaluate lipid deposition,and BODIPY C11 fluorescence staining,ROS fluorescence staining and 4-HNE histochemical staining were used to comprehensively evaluate lipid peroxidation.This study sheds lights on the regulation of FNDC3B on the progression of alcoholic liver disease and identified the potential molecular mechanism,providing new clues for therapeutic targets of alcoholic liver disease.Part Ⅰ Effect of miR-192-5p on lipid deposition in alcoholic liver disease by regulating FNDC3BMaterials and Methods1.Investigation of FNDC3B expression in alcoholic liver diseaseIn dataset GSE3632 of Gene Expression Omnibus(GEO)database,R software was used to analyze the gene expression information of liver tissues of patients with alcoholic liver disease and normal people.The expression level of FNDC3B in the liver tissues of patients with alcoholic liver disease was detected.Mouse model of alcoholic liver disease was established in 10 weeks old wild-type C57BL/6 mice.The mRNA level of FNDC3B in the liver tissues of alcohol mice and control mice were detected by real-time PCR,and the protein expression of FNDC3B was detected by Western blot.The mouse primary hepatocytes were extracted according to two-step method,and the in vitro alcoholic model was constructed by stimulating the cells with 100 mM alcohol for 48 h.The mRNA level of FNDC3B in cells was detected by qPCR,and the protein expression level of FNDC3B in the in vitro alcoholic model was verified by Western blot,and statistical analysis was conducted.2.Investigation of the effect of FNDC3B on lipid deposition in alcoholic liver disease2.1 Effect of FNDC3B deletion on alcohol-induced lipid deposition in vitroTo generate VL-17A cells,ADH1 and CYP2E1 were stably overexpressed in HepG2 cells.Lentivirus carrying FNDC3B shRNA sequence was transfected into VL-17A cells to construct FNDC3B knockdown cell model according to the protocol.The successful knockdown of FNDC3B was validated by Western blot.The triglyceride detection kit and BODIPY 493/503 fluorescence staining were used to detect the lipid deposition in cells stimulated with 100 mM alcohol for 48 h.2.2 Effect of FNDC3B knockdown on hepatic steatosis of alcoholic mouse modelMice were injected with AAV8-FNDC3B-shRNA through the tail vein,and control group was injected with control virus.After 4 weeks of ordinary diet feeding,mice were divided into two groups respectively,and were fed with alcoholic liquid diet or equal-calorie control liquid diet for 4 weeks.On the second day after feeding,alcohol(5 g/kg)and equal-calorie maltodextrin were given once gavage.7 h after intragastric administration,liver located magnetic resonance scanning was performed to collect the water signal,fat signal and MRS spectrum of mice.The data was analyzed by RadiAnt DICOM Viewer and Topspin software.9 h after intragastric administration,mice were sacrificed and liver tissues were collected.The successful knockdown of FNDC3B was validated by Western blot and immunofluorescence staining.HE staining and oil red staining were used to detect the liver steatosis.The levels of ALT,AST,triglyceride and total cholesterol in blood were determined by biochemical analysis.The content of triglyceride and total cholesterol in liver tissue were also evaluated.3.Researches on regulation of microRNA on FNDC3B resulting to steatosis3.1 Screening of microRNAs bound to FNDC3BUsing bioinformatics analysis tool TargetScan and miRDB,combined with GEO dataset GSE59492 to predict possible microRNAs that bound to FNDC3B.Candidate microRNAs were further screened by qPCR assay.The binding between miR-192-5p and FNDC3B was verified by double luciferase reporter gene assay.3.2 Effect of miR-192-5p overexpression on the regulation of FNDC3B expression and steatosisTo construct miR-192-5p overexpression cell model,AML 12 cells were transfected with miR-192-5p mimic.The mRNA and protein levels of FNDC3B were detected by qPCR and Western blot assay.Cells were stimulated with 100 mM alcohol for 48 h.The accumulation of lipid was determined by triglyceride detection kit and oil red staining.3.3 Effect of miR-192-5p inhibition on the regulation of FNDC3B expression and steatosisTo construct miR-192-5p inhibition cell model,AML12 cells were transfected with miR192-5p inhibitor.The mRNA and protein levels of FNDC3B were detected by qPCR and Western blot assay.Cells were stimulated with 100 mM alcohol for 48 h.Triglyceride detection kit and oil red staining were used to evaluate the steatosis.Oil red staining was used to evaluate lipid content in AML12 cells which simultaneously knockdown miR-192-5p and FNDC3B.Results1.The expression of FNDC3B was significantly upregulated in alcoholic liver diseaseR software analyzed the gene expression data in the liver tissues of patients with alcoholic liver disease and normal people in GSE3632 of GEO database.The results showed that the expression of FNDC3B in the liver tissues of patients with alcoholic liver disease was significantly increased.The mouse alcoholic model and corresponding control group were constructed to detect the mRNA and protein expression of FNDC3B.The results of qPCR and Western blot assays showed that FNDC3B was significant upregulated in alcoholic group on both mRNA and protein levels.Mouse primary hepatocytes were extracted by two-step method.Cells were stimulated with 100 mM ethanol to construct alcoholic cell model.The results of qPCR and Western blot assays verified that FNDC3B was significantly upregulated after alcohol stimulation on both mRNA and protein levels.2.FNDC3B significantly alleviates lipid accumulation in alcoholic liver disease2.1 FNDC3B knockdown increased lipid deposition of cells under alcohol stimulationHepG2 cells were stably overexpressed with ADH1 and CYP2E1 to construct VL-17A cells.Lentivirus with FNDC3B shRNA sequence was transfected in VL-17A cells to construct FNDC3B knockdown cells.After the cells were stimulated by 100 mM alcohol for 48 h,the triglyceride detection kit and BODIPY 493/503 fluorescence staining assays were performed to evaluate the lipid content.The results showed that FNDC3B knockdown significantly increased alcohol induced lipid deposition.2.2 FNDC3B knockdown aggravated liver steatosis in alcoholic mouse modelHepatocyte-specific FNDC3B knockdown mouse model and control group were constructed.After 4 weeks of normal diet,mice were divided into two groups and treated with alcohol diet and control diet respectively.The inhibition of FNDC3B was verified by Western blot and tissue immunofluorescence staining.Water signal,fat signal and MRS spectrum were collected using liver located magnetic resonance scanning.The data were analyzed by RadiAnt DICOM Viewer and Topspin.The results showed that the lipid deposition shadow in liver was significantly increased in FNDC3B knockdown alcoholic group.The results of HE staining and oil red staining showed that FNDC3B knockdown significantly aggravated liver steatosis.The biochemical test showed that ALT and AST levels were increased significantly in FNDC3B knockdown group.Content of triglyceride in plasma and liver were increased,while levels of total cholesterol did not change significantly.3.miR-192-5p aggravated lipid deposition via down regulating FNDC3B3.1 miR-192-5p bound to FNDC3BTargetScan,miRDB and GEO dataset GSE59492 were used to predict and screen the possible microRNAs that regulate FNDC3B expression.100 mM alcohol stimulated mouse primary hepatocytes for 48 h.qPCR assay showed that the expression of miR-192-5p was significantly downregulated.Dual luciferase reporter assay further confirmed the direct interaction between miR-192-5p and FNDC3B.3.2 Overexpression of miR-192-5p significantly downregulates FNDC3B andaggravates alcohol-induced lipid depositionAML 12 cells were transfected with miR-192-5p mimic to construct miR-192-5p overexpression cell model.qPCR and Western blot assays showed that the overexpression of miR-192-5p significantly inhibited the transcription and protein expression of FNDC3B.Cells were stimulated with 100 mM alcohol for 48 h,and the intracellular lipid content was measured by triglyceride detection kit and oil red staining.The results showed that the overexpression of miR-192-5p significantly increased lipid deposition.3.3 Knockdown of miR-192-5p significantly upregulates FNDC3B and alleviates alcohol-induced lipid depositionAML 12 cells were transfected with miR-192-5p inhibitor to construct miR-192-5p knockdown cell model.qPCR and Western blot assays showed that the expression of FNDC3B was significantly upregulated after miR-192-5p knockdown.Cells were stimulated with 100 mM alcohol for 48 h.The results of triglyceride detection and oil red staining showed that miR192-5p knockdown reduced steatosis.In miR-192-5p and FNDC3B bi-knockdown cells,oil red staining showed that lipid droplets were significantly increased in bi-knockdown cells,compared to miR-192-5p knockdown cells.Part Ⅱ Effect of FNDC3B on lipid peroxidation in alcoholic liver disease by regulating ferroptosisMaterials and Methods1.Effect of FNDC3B on alcohol-induced lipid peroxidation1.1 To detect the effect of FNDC3B depletion on lipid peroxidation in alcoholic mouse modelAAV8-FNDC3B-shRNA virus was injected into mice through tail vein to construct hepatocytes-specific FNDC3B knockdown model.Control group was injected with control virus.After 4 weeks of ordinary feeding,mice were divided into two groups,which were fed alcohol liquid diet and equal calorie control diet respectively.On the second day after feeding,the mice were given once gavage with alcohol(5 g/kg)or isocaloric maltodextrin.The mice were sacrificed 9 h later,and liver tissues were collected.Immunofluorescence staining was used to detect the ROS level.The content of lipid peroxidation end product 4-HNE was detected by immunohistochemical staining,and the levels of MDA and GSH in mouse liver tissue were detected by biochemical analysis.1.2 To detect the effect of FNDC3B knockdown on lipid peroxidation in alcoholstimulated cellsAlcohol-sensitive VL-17A cells were constructed by stably overexpressing ADH1 and CYP2E1 in HepG2 cells.Lentivirus with FNDC3B shRNA sequence was transfected into VL17A cells to construct FNDC3B knockdown cell model.Cells were stimulated with 100 mM ethanol for 48 h.ROS fluorescence staining and flow cytometry were performed to detect the intracellular ROS level.Intracellular MDA content was evaluated by biochemical assay.FNDC3B knockdown cells were stimulated with 100 mM alcohol for 48 h.Transcriptome highthroughput sequencing was performed to detect the altered KEGG pathways.2.To explore the regulation of FNDC3B on ferroptosis2.1 The effect of Fer-1 on lipid peroxidation in FNDC3B knockdown cellsFNDC3B knockdown cells were treated with 1 μm Fer-1 and 100 mM alcohol for 48 h simultaneously.ROS fluorescence staining and flow cytometry were used to detect the ROS level.The cells were stained by BODIPY C11 fluorescence and photographed by confocal laser microscopy.2.2 The effect of Fer-1 on lipid peroxidation in FNDC3B knockdown miceAt the last week of alcohol liquid diet,FNDC3B knockdown mice and control group were given intraperitoneal injection of Fer-1(1 mg/kg)for seven consecutive days.On the second day after feeding,once gavage of ethanol(5 g/kg)was performed.Liver located magnetic resonance scanning was performed 7 h later and the mice were sacrificed 9 h after intragastric administration.Blood and liver tissue of mice were collected.ROS fluorescence staining was used to detect ROS level in liver tissue of FNDC3B knockdown mice treated with Fer-1.Immunohistochemical staining was used to detect 4-HNE content,and biochemical detection was performed to determine MDA and GSH content in liver tissue.2.3 The effect of Fer-1 on steatosis in FNDC3B knockdown miceLiver located magnetic resonance scanning was performed to collect water imaging,fat imaging and MRS spectrum of mice.The data are analyzed and processed by software.Liver steatosis was evaluated by oil red staining.Blood and liver tissue were collected to detect the levels of ALT,AST,triglyceride and total cholesterol.Results1.FNDC3B alleviates alcohol-induced lipid peroxidation1.1 FNDC3B knockdown increased lipid peroxidation in alcoholic mouse modelImmunofluorescence and immunohistochemical staining results showed that the content of ROS and 4-HNE were significantly increased in livers of FNDC3B knockdown mice,compared to control group.Moreover,the MDA produced and GSH consumed significantly increased.1.2 FNDC3B knockdown increased lipid peroxidation in alcoholic cell modelFNDC3B knockdown cells were stimulated with 100 mM alcohol for 48 h.ROS fluorescence staining was performed and the results showed that the average fluorescence intensity of ROS increased significantly in FNDC3B knockdown cells.The biochemical detection showed that MDA level increased significantly in FNDC3B knockdown cells.High-throughput sequencing was performed on alcohol-stimulated FNDC3B knockdown cells.KEGG pathway analysis was performed and the results showed that ferroptosis pathway was significantly changed.2.FNDC3B alleviates alcohol-induced lipid peroxidation by inhibiting ferroptosis2.1 Fer-1 significantly reduced lipid peroxidation in FNDC3B knockdown cellsFNDC3B knockdown cells were treated with 1 μm Fer-1 and 100 mM alcohol simultaneously for 48 h.ROS fluorescence staining showed that Fer-1 significantly reduced the ROS level in FNDC3B knockdown cells.BODIPY C11 fluorescence staining showed that Fer-1 significantly alleviated lipid peroxidation.2.2 Fer-1 significantly alleviates lipid peroxidation in FNDC3B knockdown miceFer-1 injection was performed on alcoholic FNDC3B knockdown mice and control mice.The results of ROS fluorescence staining showed that ROS levels of FNDC3B knockdown mice was significantly reduced after Fer-1 treatment.Immunohistochemical staining showed that Fer-1 decreased the content of 4-HNE in liver.The contents of MDA and GSH in mouse liver tissue were determined by biochemical assay.The results showed that Fer-1 alleviated MDA accumulation and reduced GSH consumption in liver of FNDC3B knockdown mice.2.3 Fer-1 significantly alleviates liver steatosis in FNDC3B knockdown miceHepatic located magnetic resonance scanning showed that the liver fat shadow and fat value was significantly decreased in Fer-1 treated groups.Oil red staining showed that Fer-1 treatment significantly reduced liver steatosis in FNDC3B knockdown mice.The results of liver function and lipid accumulation showed that Fer-1 significantly reduced the levels of ALT,AST,triglyceride and total cholesterol in plasma as well as significantly alleviated the content of triglyceride and total cholesterol in liver.Part Ⅲ Effect of FNDC3B on the progression of alcoholic liver disease through AMPK signaling pathwayMaterials and Methods1.The regulation of miR-192-5p/FNDC3B/AMPK signaling axis on alcohol-induced lipid deposition1.1 To detect the binding and regulatory effect between FNDC3B and AMPKPathway Commons was used to predict the potential molecules that interact with FNDC3B.Co-immunoprecipitation(Co-IP)was performed to detect the interaction between FNDC3B and AMPK.FNDC3B knockdown was stably constructed in VL-17A cells.Cells were treated with 100 mM alcohol for 48 h.Western blot assay was used to detect the phosphorylation and protein expression of AMPK,as well as the expression of downstream molecules ACC1 and CPT1.The effect of AMPK activator AICAR on FNDC3B knockdown induced lipid deposition was detected by oil red staining.1.2 To detect the regulatory effect of miR-192-5p/FNDC3B/AMPK signal axis on lipid depositionMiR-192-5p overexpression and inhibition were constructed in AML 12 cells and stimulated with 100 mM alcohol for 48 h.Western blot was performed to detect the phosphorylation and protein expression of AMPK.The effects of FNDC3B knockdown and AICAR treatment on the lipid content of miR-192-5p knockdown cells were detected by oil red staining.2.Effect of FNDC3B on alcohol induced lipid peroxidation via AMPK signaling pathway2.1 To detect the effect of AICAR on lipid peroxidation and iron overload in FNDC3B knockdown cellsHepG2 cells was stably overexpressed ADH1 and CYP2E1 to construct VL-17A cells.Lentivirus with FNDC3B shRNA sequence was transfected into VL-17A cells to construct FNDC3B knockdown cells.The cells were stimulated with 100 μm AICAR and 100 mM alcohol simultaneously for 48 h.BODIPY C11 fluorescence staining was performed to detect the lipid peroxidation levels.The iron content in blood and liver tissue samples of mice was quantitatively assessed and statistically analyzed with iron ion detection kit.The effect of AICAR treatment on iron content in FNDC3B knockdown cells was detected.2.2 To detect the regulatory effect of AICAR on transferrin expressionFNDC3B knockdown cells was stimulated with 100 mM alcohol for 48 h.High-throughput RNA sequencing data of cells were analyzed to explore differentially expressed genes in ferroptosis pathway.qPCR assay were used to detect the mRNA level of transferrin in liver tissue of FNDC3B knockdown mice.Gradient concentration of AICAR was used to treat FNDC3B knockdown cells for 48 h.Western blot assay was performed to detect the protein level of transferrin and the phosphorylation of AMPK.Results1.Regulation of miR-192-5p/FNDC3B/AMPK signaling axis on alcohol-induced steatosis1.1 FNDC3B alleviate alcohol-induced steatosis by AMPK pathwayPathway Commons predicted that the α2 subunit of AMPK might interact with FNDC3B,and the combination of FNDC3B and AMPK was further confirmed by co-immunoprecipitation experiments.FNDC3B knockdown cells were stimulated with 100 mM alcohol for 48 h.Western blot was used to detect the phosphorylation of AMPK and the expression of downstream molecules ACC1 as well as CPT1.The results showed that FNDC3B knockdown significantly reduced the phosphorylation of AMPK and ACC1 and the expression of CPT1.Oil red staining assay showed that AMPK activator AICAR significantly decreased lipid accumulation caused by FNDC3B knockdown.1.2 miR-192-5p/FNDC3B/AMPK signaling axis regulates alcohol-induced lipid depositionMiR-192-5p overexpression and knockdown cells were constructed respectively to detect pAMPK expression.The results showed that the overexpression of miR-192-5p decreased the phosphorylation of AMPK,while miR-192-5p knockdown significantly increased the phosphorylation of AMPK.Oil red staining results showed that in alcohol-treated cells,deletion of FNDC3B reversed the protective effects against lipid deposition induced by miR-192-5p inhibition,while lipid accumulation was subsequently ameliorated upon administration of AICAR.2.FNDC3B regulates AMPK signaling pathway to alleviate alcohol-induced lipid peroxidation2.1 AICAR significantly alleviates lipid peroxidation and iron overload in FNDC3B knockdown cellsBODIPY C11 fluorescence staining indicated that alcohol stimulated FNDC3B knockdown cells showed remarkable lipid peroxidation.Treatment of AICAR significantly reduced the lipid peroxidation induced by FNDC3B knockdown.The iron content in blood and liver of mice was evaluated with iron ion detection kit.The results showed that FNDC3B depletion resulted in reduced iron content in blood and increased iron accumulation in liver.In alcoholic FNDC3B knockdown cells,AICAR treatment significantly reduced the iron content.2.2 AMPK increases the expression of transferrinFNDC3B knockdown cells was treated with 100 mM alcohol for 48 h,and high-throughput transcriptional sequencing was performed.Data related to ferroptosis pathway were analyzed and the results showed that FNDC3B knockdown significantly downregulated transferrin.qPCR results further confirmed that the mRNA level of transferrin was decreased in FNDC3B knockdown mice.The FNDC3B knockdown cells were treated with gradient concentration of AICAR for 48 h.Western blot assay was performed to detect the protein expression of transferrin.The results showed that the expression of transferrin was significantly upregulated with the increased phosphorylation of AMPK.Conclusions1.Our studies on clinical database,alcoholic mouse models and cell models indicated that the increased expression of FNDC3B in alcoholic liver disease,which alleviated the progression of alcoholic liver disease by reducing lipid accumulation and lipid peroxidation.2.MiR-192-5p targets to and downregulates FNDC3B,aggravating lipid accumulation in alcoholic liver disease.3.FNDC3B directly interacted with AMPK,and exerts an suppressing effect on alcoholinduced steatosis via activating AMPK and downstream molecules ACC1 and CPT1.4.FNDC3B upregulates transferrin via activating AMPK,thus preventing lipid peroxidation in alcoholic liver disease through inhibiting ferroptosis.Innovation and Significance1.The study for the first time confirmed that the expression of FNDC3B in alcoholic liver disease is significantly upregulated.The absence of FNDC3B leads to aggravated steatosis and lipid peroxidation in alcoholic liver disease,suggesting that FNDC3B plays an important protective role in lipid metabolism of alcoholic liver disease.2.FNDC3B alleviates lipid accumulation and lipid peroxidation in alcoholic liver disease by binding to and activating AMPK,which provides molecular theoretical basis for clarifying the pathogenesis of alcoholic liver disease.3.FNDC3B inhibits ferroptosis and alleviates lipid peroxidation in alcoholic liver disease via upregulating transferrin expression,which provides a new direction for clinical strategies of alcoholic liver disease.