| BackgroundAlcohol is habitually consumed in many cultures nowadays.Alcohol abuse,whose detrimental effects have been well realized,however is fairly popular in the modern world.The chronic alcoholism is the most significant symptom after alcohol consumption,besides,there are more than 60 kinds of alcohol-related disease,making it a very dangerous factor not only affecting human's heath,but also burdening the social health care system.The most common hepatic change caused by alcohol consumption is steatosis,or fatty liver.In fact,steatosis helps the body to store lipid from blood and therefore can be seen as a protective mechanism.The steotosis caused by alcohol is partially due to the alcoholic metabolism.As the important multifunctional organ performing detoxification,liver plays an important role in the alcohol metabolism.It is also the major target organ of alcohol-induced injury,which includes steatosis?fatty liver?,steatohepatitis,and fibrosis and/or cirrhosis in severe cases.The pathogenesis of alcoholic hepatotoxicity has been studied for several decades.Basically,the increased production of reactive species?ROS/RNS?due to oxidative stress induced by alcohol is one of the key mechanisms of alcohol-induced liver injury.After alcohol consumption,liver generates abundant reactive species such as O2-?H2O2?OH-?NO and activates phospholipase and lipid peroxidation reaction,even affects the structure and function of DNA and proteins.The mitochondrial respiratory depression effect of NO has fatal influence on the liver metabolism,and the ONOO-produced by the reaction of NO and O2-is a strong oxidant and cytotoxic substance directly inducing the cell injury and apoptosis.The majority of studies on alcoholic liver injury focus on the effects of chronic alcohol consumption.However,clinical evidence highlights the detrimental effects of acute alcohol consumption,and drives more researchers to investigate the binge drinking and some other acute experimental models.Cav-1,a 21-24 KD structural protein component of caveolae membranes,acts as a scaffolding protein to functionally regulate cell growth,apoptosis and cholesterol trafficking.It can modulate all three types of NOS via interaction with NOS-Cav-1 binding motif.Reports indicated Cav-1 knock-out mice also showed an increases of eNOS activity and NO production in endothelial cells and had endothelial hyper-permeability.Besides,an up-regulation of eNOS/iNOS protein levels has been observed in hearts of Cav-1 null mice.Our research team previously has reported the negative association between Cav-1 and NO in brain.However,little has been reported on the pathophysiological roles of Cav-1 in different drinking habits,although increased Cav-1 expression has been documented under a variety of hepatic disease conditions.In this study,we investigated the role of Cav-1 in hepatocytes in the development of alcoholic induced liver injury in mice.These results indicate that Cav-1 induction serves as a protective response to binge and chronic drinking in the development of alcoholic liver injury.Materials and Methods1.Animals study.Experimental protocol was approved by the University of Hong Kong Institutional Animal Care and Ethical Committee.Male Cav-1 knockout mice(Cav-1-/-mice,strain Cav-1tmlMls/J,Jackson Laboratory,Bar harbor,ME,USA),Cav-1 heterozygous littermates(Cav-1+/-)and wild-type littermates?WT,Cav-1+/+?at the age between 8-12 weeks were genotyped and used in the experiments.1.1.Acute binge drinking mice modelAfter 6 hours of fasting,mice were given 50%?vol/vol?ethanol at an accumulative dosage of 5 g/kg body weight by intragastric administration.Vehicle treated mice received the same volume of water.In parallel studies,additional mice were treated with the iNOS inhibitor N-?3-?Aminomethyl?benzyl?acetamidine?1400W;Calbiochem,EMD Millipore;5 mg/kg,ip.?at 30 minutes before ethanol feeding.1.2.Chronic alcohol-induced liver injury mice modelAll male mice?6-8 weeks old?were fed a control Lieber-deCarli liquid diet?TROPHIC Animal Feed,China?for the first 3 days to acclimatize them to the liquid diet.Mice were randomly divided into either ethanol-fed or pair-fed groups.The ethanol-fed group was given the diet with increasing concentrations of ethanol,as follows:1%?vol/vol?and 2%each for 2 days,then 3%for 3 days and 5%for a further 7 weeks.The pair-fed group was treated with the isocalorical control liquid diet over the entire feeding period.Naringin treatment:after the first 3 weeks of chronic alcohol-induced liver injury mice modeling,mice were fed with the alcoholic liquid daily diet added with a dose of 200mg/kg/d naringin for 4 weeks.1.3.Animal sample preparationAfter the mice model establish,mice was anesthetized by 200mg/kg pentobarbital,then collected blood from heart.The serum was separated from blood by centrifuging under 3000rpm for 10 minutes after holding in 4? overnight,then kept in-80?.The serum was used to detect the serum level of Cav-1,ALT/AST,nitric oxide,nitric oxide synthase?total complex compound enzyme activity and induction of enzyme activity nitric oxide?,superoxide dismutase?Superoxidedismutase,SOD?activity,catalase activity,GSH/GSSG activity and expression,free fatty acids?Free fatty acids,FFA?and triglyceride levels.After the heart perfusion by PBS,the liver of mice was collected,firstly,observe the general morphology,then cut some fresh liver tissue from the left hepatic lobe and fixed it by 4%PFA?frozen and paraffin sections of the middle?for the further HE staining and immunohistochemistry analysis,and detection of the expression liver Cav-1 and related proteins,and TUNEL experiment;at the same time,another liver tissue was homogenated and used for immnoblotting test?Western Blot analysis?to detect the expression of Cav-1 and related proteins,and detections of liver nitric oxide composite enzyme levels?total enzyme activity and composite enzyme activity induced by nitric oxide?,alcohol dehydrogenase?alcohol dehydrogenase ADH?,catalase activity,GSH/GSSG activity and expression levels and superoxide dismutase?Superoxidedismutase,SOD?activity;finally,extract the total RNA of mouse liver for reverse transcription polymerase chain reaction and Real-time PCR assay for detection the mRNA levels of Cav-1 and inflammation factors.1.4.Peroxynitrite Fluorescent Probe Assay.A fluorescent probe,COMPOUND 14,has been developed based on a specific reaction with ONOO"-.This probe is highly sensitive and selective for the detection of peroxynitrite not only in abiotic but also in biological systems.After 3h treated with ethanol,mice were anesthetized and the live in situ reperfusion with COMPOUND 14?20mM/mice liver;Perfusion rate:2ml/min,total 25 ml?.Fresh liver sample sectioned into 15?M cryosection slices.After washed with PBS for 5 min and then incubated with DAPI for 10 min,the sections were monitored by epifiuorescence microscopy.All the operations were controlled in 2 hours.In vitro study,after different dose ethanol treatment 2h,the L02 cells were incubated with COMPOUND 14?20uM?for 30min,then washed three times with PBS buffer and subjected to detect at an excitation wavelength of 543 nm and emission wavelength of 567nm.2.In vitro experimentImmortalized human hepatocyte cell line?L02?was acquired from Laboratory Animal Center of SUN YAT-SEN University?Guangzhou,China?.L02 cells were cultured in Dulbecco's modified Eagle's medium?DMEM?containing 10%FBS?Gibco BRL,Grand Island,NY?and maintained in a humidified incubator at 37?with 5%CO2.After attachment?12h?,cells were stimulated with 100 mM ethanol,200 mM ethanol or vehicle for an additional 24h period.Then the cells were harvested for the other tests.ERK1/2 inhibitor?PD98059,Cell Signal Technology?or JAK2 Inhibitor ?,SD-1029?CAS 118372-34-2,Santa cruz?and Cav-1 small interfering RNA?siRNA?transfection were used in this study follow the previous protocols;using ONOO-fluorescent probe for the detection of liver cells in alcohol stimulating ONOO' production and release.3.Human binge drinking projectStudy populationThe study included a within-subjects,double-blind and placebo-controlled human laboratory paradigm.Participants?n=65?without long-term drinking or alcohol dependence in the binge drinking project were young male adult aged 21-35 years.They are nonalcoholic social drinkers who meet criteria for either binge drinkers?n=40,consumed 5+ drinks in 2 hours?or sham drinkers?n=25,consumed same volume water in 2 hours?.A 'binge' was operationally defined as consuming 5 or more drinks per occasion for men,4 or more for women,according to National Institute on Alcohol Abuse and Alcoholism?NIAAA?.The study protocol conformed to the ethical guidelines of the 1975 Declaration of Hwlsinki and was approved by the appropriate institutional review committee with written informed consent from all participants.The study was fully approved by the Ethics Committee of Integrated Traditional Chinese and Western medicine of Southern Medical University and registered at Chinese Clinical Trial Registry?ChiCTR-TRC-13003344?3.2 ParticipantsTo strictly control the experiment process in parallel under the premise of eating and drinking water,57volunteers finally?binge drinking group 32,control group 25?completed the trial,blood samples were collected and record the vital signs and breath alcohol concentration at each time point after drinking alcohol?2h,6h,12h and 24h?.The expression level of,blood alcohol concentration,liver function,Cav-1,nitric oxide and inflammatory factors were be detected in the blood samples.Statistical analysisAnalysis using SPSS 20.0 and Graph prism 5.0 statistical software,and results were expressed as meansąSEM.Statistical analysis were carried out by one-way analysis of variance?LSD or Dunnett's T3?,Repeated Measures ANOVA and unpaired student's t-test dependent experimental designs.Correlations were analyzed using Spearman's rank correlation test.P<0.05 was taken as the minimum level of significance.ResultsThe binge drinking project1.1.Binge-drinking induced up-regulation of Cav-1 expression level in normal mice serum and liverBinge-drinking induced up-regulation of the expressions ofCav-1 in serum and liverWe determined the level of serum Cav-1 and observed a up-regulation of serum Cav-1 after binge drinking mice model.The Cav-1 level at 12h,and has a significant difference when compared with the control group?p=0.0136?.After binge drinking,liver Cav-1 expression showed a significant increase in 12h?p=0.0003?.This trend is the same as serum Cav-1 level.1.2.The expression levels of serum Cav-1 and liver Cav-1 have significant positive correlationSpearman rank correlation analysis results revealed that serum Cav-1 level has a significant positive correlation with liver Cav-1 level,rs=0.808,p=0.000.1.3.The increase of liver Cav-1 mainly occurs in endothelial cells and hepatocytes endothelial cellsImmunofluorescent results revealed that liver Cav-1 increased significantly after binge drinking.Immunohistochemical analysis revealed that liver Cav-1 mainly locates in hepatocytes endothelial cells around central veins,and was increased after ethanol treatment.2.1 Cav-1 deficiency leads to increased susceptibility to binge drinking-induced hepatic injury in mice Establishment of three genotypes and identificationMale Cav-1 knockout mice(JAXCav-1-/-mice,strain Cav-1tmlMls/J,Jackson Laboratory,Bar harbor,ME,USA),Cav-1 heterozygous littermates(Cav-1+/-)and wild-type littermates?WT,Cav-1+/+?were genotyped to show a gradient deficiency of Cav-1 expression in mice.2.2 Comparison among the three genotyped mouseThere were no significant differences in body weight between WT and knockout littermates(Cav-1-/-mice and Cav-1+/-mice)at the age of 8-12 weeks?p=0.753?;therefore we choose these mice for further experiments.2.3 Binge-drinking elevated Cav-1 mRNA level of normal mice,whereas showed no significant influence on the level of Cav-1+/-miceAt 12h after binge,Cav-1 was significantly up regulated on mRNA level in the WT livers?t=3.675,p=0.0104?.Comparatively,Cav-1+/-mice as the heterozygous type displayed a relative lower liver Cav-1 expression and no significant difference in both normal and ethanol-treated conditions?p=0.9807?.2.4 Cav-1-/-mice demonstrated higher ALT&AST levels after binge drinkingWith ALT&AST levels as markers of liver function,we observed that a more severe liver injury in Cav-1-/-mice than in WT mice after binge drinking?p=0.039?,indicating that Cav-1 deficiency may lead the liver more vulnerable to ethanol injury.2.5 Cav-1-/-mice demonstrated enhanced hepatocyte apoptosis after bingeRevealed by TUNEL and Cleaved caspase3 staining,a more severe ethanol-induced hepatocyte apoptosis was observed in Cav-1-/-livers than WT livers.Western blot also support that the expression of Bcl-2 was down regulated?p=0.041?while Cleaved caspase-3 was significantly up-regulated?p=0.035?.2.6 Cav-1-/-mice demonstrated no significant changes of the expressions of ADH1,ALDH2 and catalase as well as SOD1,SOD2 level in liver after bingeWith liver ADH1&ALDH2 and catalase as markers,we found no significant change of ethanol metabolism between Cav-1/-mice and WT mice,as well as the expressions of SOD1&2,indicating the independence of ethanol metabolism on the Cav-1 deficiency.2.7 Cav-1-/-mice demonstrated no significant changes of the activity of SOD catalase and ADH as well as serum GSH level after bingeWith the activity of SOD,catalase and ADH as well as serum GSH level as oxidative stress markers,Cav-1-/-mice showed no significant difference of oxidative stress levels after binge compared to WT mice?p>0.05?.2.8 Cav-1-/-mice demonstrated a significant elevation of serum NO after bingeAfter binge,serum NO was accumulated dramatically in both WT and Cav-1-/-mice.Comparatively,the production level of serum NO in Cav-1-/-mice was significantly higher?p<0.01?,indicating a potential role of NO in the process of ethanol-induce liver injury enhanced by Cav-1 deficiency.2.9 Cav-1-/-mice demonstrated a significant elevation of liver 3-NT expression after bingeWith 3-NT as a biomarker of ONOO-,we found a significant increase of liver 3-NT expression in Cav-1-/-mice after binge,compared to WT mice.2.10 Cav-1-/-mice demonstrated a significant elevation of liver ONOO-expression after bingeFurther,a novel fluorescence probe was applied to detect the liver expression of ONOO-,with the result of a much higher level in Cav-1-/-mice than WT mice after binge.2.11 Cav-1-/-mice demonstrated a significant elevation of liver iNOS expression after bingeAs indicate by Western blot,eNOS expression was elevated in Cav-1/-mice liver after binge,but has no significant difference with that in WT mice liver.However,iNOS expression was significantly up regulated in Cav-1-/-mice liver,compared to WT mice?p<0.05?.Immunohistochemical observation further validated this result,indicating an involvement of iNOS in the process of the accumulation of NO due to Cav-1 deficiency.3.1 INOS inhibitor 1400W significantly improve the alcoholic liver injury in both WT and Cav-1-/-mice1400W has no influence on the expression of liver Cav-1With the presence of 5 mg/kg of 1400 W,the expression of liver Cav-1 and Hif-la had no significant difference between WT and Cav-1-/-mice after binge,indicating the selective inhibitory effect of 1400W is independent on liver Cav-1.3.2 1400W inhibited the up-regulation of the activity and expression of liver iNOS after bingeWith the presence of 1400W,the elevation of the expression of iNOS in Cav-1-/-mice liver after binge was abolished strongly?p<0.01?,validate by immunofluorescence determination.3.3 1400W protected liver against alcoholic injury in both WT and Cav-1-/-miceWith the ALT&AST levels as liver function markers,we observed that the liver injury was reduced with pretreatment of 1400W in both WT and Cav-1-/-mice after binge?p<0.05?.3.4 1400W alleviated hepatocyte apoptosis in Cav-1-/-mice after bingeTUNEL analysis revealed that the hepatocyte apoptosis was alleviate significantly Cav-1-/-mice after binge with pretreatment of 1400W,the apoptosis in WT mice was also reduced.3.5 1400W reduced the up-regulation of liver 3-NT expression in Cav-1-/-mice after bingePretreated by 1400W,the up-regulation of the expression of liver 3-NT was significantly inhibited in Cav-1-/-mice after binge,and the inhibitory effect was not so strong in WT mice after binge.4.1 Cav-1 protect binge-induced liver injury through regulating EGFR/STAT3 signaling pathwayCav-1-/-mice demonstrated an upregulation of liver P-STAT3 and P-EGFR while an inhibition of P-ERK1/2 after bingeWestern blot revealed that,in WT mice liver,the expression of Cav-1,P-ERK1/2 and P-STAT3 were all significantly increased after binge?p<0.05?,while P-EGFR was not influence obviously?p>0.05?.In comparison,there were significant up-regulations of the expression of P-EGFR and P-STAT3?p<0.05?,whereas relative lower expression of P-ERK1/2-1/2 in Cav-1-/-mice liver after binge drinking?p<0.05?,indicating the inhibitory effect of Cav-1 on P-EGFR and P-STAT3,and promotion effect of P-ERK1/2 expression.4.2 In vitro L02 cell demonstrated a dose-dependent upregulation of Cav-1 expression level and ERK1/2 activity,whereas a downregulatin of STAT3 activity after ethanol treatmentWe applie human liver cell line L02 to better illustrate the underlying mechanism,after ethanol treatment at the dose of either 100 mM or 200mM,we found that Cav-1 expression was upregulated in a dose-dependent manner,accompanying a dose-dependent upregulation of P-ERK1/2 and down-regulation of P-STAT3 Tyr705?p<0.05?.4.3 STAT3 inhibitor has no significant effect on the expression of Cav-1 in L02 cellsWe applied the selective STAT3 inhibitor SD-1092 to pretreat L02 cells before ethanol,and found that the expression of P-STAT3 Tyr705 was significantly inhibited?p<0.05?,while Cav-1 has no obvious change compared to cells without presence of SD-1092?p>0.05?,indication that STAT3 may be the downstream of Cav-1.4.4 ERK1/2 inhibitor induced the down-regulation of both P-ERK1/2 and Cav-1 in L02 cellsWe applied different doses of ERK1/2 inhibitor PD98059 to pretreat L02 cells and found a dose-dependent down-regulation of both P-ERK1/2 and Cav-1?p<0.05?,indicating that ERK1/2 may be the upstream of Cav-1.4.5 Pretreatment of Cav-1 siRNA upregulated the liver STAT activity while had no effect on the ERK1/2 activityTo further validate the pathway,we apply Cav-1 siRNA to pretreat L02 to generate a Cav-1 deficiency?p<0.01?.Results revealed that STAT3 Tyr705 activity was enhanced significantly in Cav-1 deficient cells?p<0.01?,while ERK1/2 showed no significant change?p>0.05?,supporting our previous results.5.1 Relationship between Cav-1 level and NO release in clinical binge projectThe outcome of the clinic binge projectFinaly,the participation of 32 volunteers in binge group and 25 in sham group complied with the requiremente,we recorded and analyzed a series of parameters.5.2 Time-dependent liver injury after bingeAt 2h after binge,BAC reached the highest value and then decreased continuously to 12h.Serum AST&ALT elevated to the peak value at 12h,indicating that at this time-point the liver suffered most injury?p<0.05?.At the meantime we can conclude that this clinical trial is within safe range due to the normal range of ALT&AST during the whole course.Moreover,placebo group who receive water instead of ethanol showed no significant change of Cav-1 level?p>0.05?,excluding the influence of diet on this project.5.3 Serum Cav-1 expression and NO release was increased in a time-dependent manner in binge drinkersResults revealed that serum NO of binge drinkers was increased time-dependently and reached the highest level at 12h?p<0.001?,which is the same trend with ALT&AST.The same trend was also found on the serum Cav-1 lever in binge drinkers,with the peak-value time of 12 h?p<0.05?.5.4 Correlation between Cav-1 expression and other parameters in binge drinkersSpearman rank correlation analysis also revealed that,serum Cav-1 level as negatively correlated with ALT at 24h?rs=-0.4153,p<0.01?,while Cav-1 expression and BAC showed a positive correlation at 2h in binge drinkers?rs=-0.436,p<0.01?.5.5 Correlation between NO release and ALT&AST in binge drinkersAt 24h after binge,serum NO release also demonstrated a positive correlation with AST levels?rs=0.4688,p<0.01?,indicating that NO release is associated with liver injury due to binge.The chronic alchohol-induced liver injury project1.Establishment of chronic ethanol-induced liver injury in miceWT and Cav-1-/-mice at the age between 6-8 weeks were chosen to feed Lieber-deCarli liquid diet.Groups were as below:WT Pair-feed group?WT C?;WT Lieber-DeCarli liquid diet group?WT M?;Cav-1-/-Pair-feed group?KO C?;Cav-1-/-Lieber-DeCarli liquid diet group?KO M?.2.Cav-1 deficiency enhanced the liver injury after a long time of ethanol administration2.1.Cav-1-/-mice showed significant lower body weightAfter 7 weeks,the models were successfully established,and the Cav-1-/-mice showed significant lower body weight than control group?p<0.05?.2.2.Cav-1-/-mice showed aggregated steatosis after chronic ethanol administrationHE staining on the pathological sections showed that,WT mice livers suffered steatosis after chronic ethanol administration,while the extent is more severe in Cav-1-/-mice.Red oil O staining further supported this finding.2.3.Cav-1-/-mice showed higher ALT&AST level after chronic ethanol administrationResults revealed that ALT&AST level were elevated in both Cav-1-/-mice and WT mice after chronic ethanol administration,and the increase in Cav-1-/-mice was significantly higher?p<0.05?.2.4.Cav-1-/-mice showed higher serum FFA level after chronic ethanol administrationResults also revealed that,serum FFA level was significantly increased in Cav-1-/-model group,compared to both Cav-1-/-control group?p<0.05?and WT model group?p<0.05?,indicating Cav-1 deficiency enhanced the FFA accumulation in mice after chronic ethanol administration.2.5.Cav-1-/-mice showed higher serum triglyceride level after chronic ethanol administrationSerum triglyceride level was significantly increased in Cav-1-/-model group,compared to both Cav-1-/-control group and WT model group,p<0.01,indicating Cav-1 deficiency enhanced the triglyceride accumulation in mice after chronic ethanol administration.3.Mechanism study on the role of Cav-1 in chronic alcoholic liver injuxy3.1.The expression levels of Cav-1 and PPAR-y was reduced after chronic administration of ethanolqPCR,ELISA and Western blot analysis revealed that chronic ethanol administration caused a down-regulated consequence of the liver Cav-1 mRNA level and the expressions of liver and serum Cav-1?P<0.05?,different from the elevation under stress condition in binge mice.Western blot analysis revealed that the reduce of the expression of Cav-1 was accompanied by a significant decrease of the expression level of PPAR-y after chronic administration of ethanol,indicating a potential relationship between PPAR-? and Cav-1.Biological targets for the treatment of traditional Chinese medicine2.Chinese herbal medicines screening and the alchoholic liver injuryAdministration of naringin in chronic alcoholic mice model As previous,after 3 weeks of administration of ethanol,we co-administered naringin with ethanol diet for 4 weeks at the dosage of 200mg/kg/d.3.Naringin restored the down-regulation of Cav-1 and PPAR-? in mice Western blot results showed that,liver Cav-1 and PPAR-y expression was significantly decreased in model group,while this decrease was inhibited significantly in Naringin-treated group.4.Naringin alleviated chronic alcoholic liver injuryIn order to validate the hepatoprotective effect of naringin,we determined serum FFA and TC levels in treated group compared with model group after chronic ethanol administration,and found that the elevation of both levels were inhibited by naringin in a large extent?p<0.01?,supporting our hypothesis that naringin protects liver against chronic alcoholic injury,and providing important experimental evidence that Cav-1 is a reliable target for screening drugs with hepatoprotective effect against chronic alcoholic liver injury.ConclusionIn the present study,we first established a binge drinking mice model to elucidate the protective effect of Cav-1 against the acute alcoholic liver injury.The underlying mechanism involves the inhibition of iNOS activity,increasing of RNS production and regulation of EGFR/STAT3 pathway.Next,we investigated the role of Cav-1 in the chronic alcoholic liver injury in vivo and in vitro.Through comparison,we found that in the process of the acute injury,the expression level of liver Cav-1 demonstrated a time-dependent increasing after binge drinking,however,this level decreased significantly after chronic alcohol consumption,accompanying with the symptom of steotosis.Together with other findings in the experiment,we conclude that the increasing of the liver Cav-1 expression in the acute liver injury caused by alcohol is a stress reaction associated with RNS accumulation,whereas in the chronic process of alcoholic liver injury,the reduction of the Cav-1 expression is an injury consequence associated with the decrease of PPAR-y.The underlying mechanism deserves further investigation.At last,we apply the in vivo chronic alcoholic model to screen out a compound derived from traditional Chinese medicine,naringin,which can reverse the alcohol-induced reduction of liver Cav-1 and PPAR-y level,and its hepato-protective effects has been validated by the reduction of serum TC and FFA level.This application supports the feasibility of Cav-1 as a potential target for screening drugs with therapeutic effect,or even as a therapeutic molecule for clinic treatment,against both acute and chronic alcoholic liver injury. |
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