Role Of Kupffer Cells Activation In The Pathogenesis Of Acute Alcoholic Fatty Liver

ObjectiveEthanol is an important liver toxicant.Excessive drinking can cause a series of progressive liver diseases-alcoholic liver disease(ALD),which includes alcoholic fatty liver(AFL),alcoholic hepatitis,liver fibrosis and liver cirrhosis.Nowadays,effective treatments for ALD especially severe ALD are still missing.Because of the reversibility,AFL is considered to be the optimal stage for ALD prevention and treatment.Exploration of the molecular mechanism of AFL is helpful to reveal the potential therapeutic targets for ALD treatment.Kupffer cells(KCs)are the resident macrophages in liver which play important roles in the onset and development of many liver diseases.Activated KCs can release reactive oxygen species(ROS).and cytokines like TNF-?.Previous studies have demonstrated that KCs play an important role in the pathogenesis of chronic ALD.However,relevant research about the effects of KCs on acute ALD is still missing.In this study,we investigated the roles of KCs inhibitor(GdCl3)and Etanercept against AFL by using binge drinking-induced acute AFL mice model and cultured adipose tissues.We also tried to elucidate the underlying mechanisms by investigating the hepatic oxidative stress,hepatic lipid metabolism,and adipose fat mobilization,etc.Methods1.In vivo study50 Specific Pathogen Free(SPF)male C57BL/6 mice(18-22 g)were randomly divided into 5 groups after acclimation for 1 week,i.e.Control group,Ethanol group,GdCl3/Ethanol group,Etanercept/Ethanol group and NAC/Ethanol group(n = 10).The mice in GdCl3/Ethanol group,Etanercept/Ethanol group and NAC/Ethanol group were treated with 2 doses of GdCl3(10mg/kg b.w.)in 24 h interval,3 doses of Etanercept(1 mg/kg b.w.)in 12 h interval,3 doses of NAC(100 mg/kg b.w.)in 12 h interval by intraperitoneal injection,respectively.The mice in all of three groups and ethanol group were exposed to ethanol(6g/kg b.w.)by garage in 12 h interval for a total of 3 doses.Mice in control group received equal volume of saline and isocaloric/isovolumetric maltose-dextrin solution.All mice were sacrificed at 6 h after the last dose of ethanol,the blood samples were collected and used for serum separation,livers and epididymis fat were quickly dissected into several parts for histopathological examination,biochemical assay,Western Blotting and RT-qPCR,respectively.H&E staining,Oil Red O staining were used for histopathological examination.The levels of alanine transaminase(ALT),aspartate transaminase(AST),malondialdehyde(MDA),triglyceride(TG).and free fatty acid(FFA)were measured by using commercial assay kits.The proteins and mRNA levels of factors involved in fatty acid metabolism such as peroxisome proliferator activator receptor-a(PPAR-a),sterol regulatory element-binding protein-lc(SREBP-1c).acyl-CoA oxidase(ACOX-1),fatty acid synthase(FAS).acyl-CoA oxidase(ACC),and stearoyl-CoA desaturase(SCD)were detected using WB and RT-qPCR methods.2.In vitro studySPF male Kun-Ming mice(25-30 g)were sacrificed by decapitation.The adipose tissue in epididymis was quickly removed to Petri dishes with sterile PBS and cut into 0.5-1mm3 small part After cleaning and purification,the adipose tissue were cultured in incubator(37?.5%CO2.saturated humidity)for 1.5 h.Then these cultured tissues received TNF-?(10 ng/ml)for 1 h,Supernatant was collected to measure fatty acid.The levels of key proteins involved in lipid mobilization(hormone sensitive triglyceride,HSL)and its phosphorylation level were measured using Western Blotting.Results1.The effects of GdCl3 and Etanercept on the fat accumulation in mice liver induced by acute ethanol exposure.Compared with Control group,the content of TG in Ethanol group is significantly increased(P<0.05).Compared with Ethanol group,the TG level in three intervention groups were significantly reduced by 21.69%.20.88%,and 35.57%,respectively(P<0.05).Pathological examination showed that the liver section of Ethnaol group mice were filled with numerous lipid droplets,which was obviously attenuated in three intervention groups.2.The effects of GdCl3 and Etanercept on the activation of Kupffer cells induced by acute ethanol exposure.Compared with Control group,the protein and mRNA levels of liver F4/80 in mice of Ethanol group were increased by 309.0%and 62.32%(P<0.05),respectively.In contrast,Compared with Ethanol group,the protein and mRNA level of F4/80 in GdCl3/Ethanol group were decreased by 64.62%and 52.40%(P<0.05),respectively.No significant changes of F4/80 protein and mRNA levels were found in Etanercept/Ethanol group or NAC/Ethanol group.3.The effects of GdCl3 and Etanercept on liver oxidative stress induced by acute ethanol exposure.Compared with Control group,the concentration of NO and MDA in liver of Ethanol group were increased by 45.35%and 217.57%(P<0.05),respectively.Compared with Ethanol group,the contents of NO and MDA in GdCl3/Ethanol group,Etanercept/Ethanol group and NAC/Ethanol group were decreased by 22.77%,28.86%,and 27.09%(P<0.05).respectively.4.The effects of GdCl3 and Etanercept on SREBP-lc regulated fat synthesis pathway in liver of acute ethanol exposed rats.The protein levels of matured SREBP-1c(mSREBP-1c,68 kD)showed no differences among different groups.Compared with Control group,the protein levels of ACC.FAS,and SCD-1 in Ethanol group mice liver were all significantly decreased(P<0.05).Compared with Ethanol group,the protein levels of ACC and FAS in mice of GdC13/Ethanol group and Etanercept/Ethanol group were not significantly altered(P>0.05),while the protein level of ACC and FAS in NAC/Ethanol group were significantly increased(P<0.05).5.The effects of GdCl3 and Etanercept on PPAR-a regulated fatty acid oxidative metabolism pathway in liver of acute ethanol exposed rats.Compared with Control group,the protein and mRNA level of PPAR-a in liver of Ethanol group mice were decreased significantly(P<0.05).Compared with Ethanol group,no significant changes were found in pretein and mRNA levels of PPAR-a and ACOX in GdCl3/Ethanol and NAC/Ethanol group.However,the protein level of ACOX was increased in Etanercept/Ethanol group compared with ethanol group(P<0.05).6.The effects of GdCl3 and Etanercept on fat mobilization in epididymis fat of acute ethanol exposed rats.Compared with Control group,the protein content of p-HSL(p563,p565,p660)in Ethanol group showed a significant increase(P<0.05).Compared with Ethanol group,HSL protein level in GdCl3 and NAC groups were reduced significantly(P<0.05).p-HSL563,p-HSL565 and p-HSL660 in three intervention groups showed different level of decrease.7.The effects of TNF-? on fat mobilization in epididymis fat cultured in vitro.1 hour after treated with 10 ng/ml TNF-a,the protein levels of HSL and p-HSL in epididymis fat showed a significant increase compared with control group,which was followed by decrease.Furthermore,treatment with different concentration of TNF-a,the FFA content in supernatant of cultured fat tissue was significantly increased.From the TNF-a concentration of 0.1.ng/ml?5 ng/ml,FFA level was increased with the increase of TNF-a.However,FFA concentration have no more increase between 10 ng/ml?20 ng/ml.Conclusions1.GdCl3 and Etanercept could attenuate fatty liver induced by acute ethanol exposure.2.The protection of KCs against acute alcoholic fatty liver might be relative to the fat mobilization induced by TNF-?.