Effect Of Alcohol Consumption On High-fat High-cholesterol Diet Induced Liver Inflammation And Fibrosis

Background and aims:Nonalcoholic fatty liver disease(NAFLD)is a worldwide popular chronic liver disease,with a spectrum of simple fatty liver,hepatitis,liver fibrosis,and cirrhosis.NAFLD is a component of metabolic syndrome and plays a role in the development of type 2 diabetes mellitus,atherosclerosis,and metabolic associated tumors.Alcohol is one of the most important factors that influence the development of NAFLD.It has been shown that chronic and heavy drinking can lead to alcoholic liver disease,while the role of chronic,regular,and moderate alcohol consumption(MAC)on NAFLD is still unclear.Chronic and regular MAC is popular in daily life.Studies have proven that chronic and regular MAC has protective effects on cardiovascular system.As far as liver,the role of chronic and regular MAC is controversial.The conclusions drawn from different populations are different.The incidence of NAFLD in chronic MAC population was lower than in nondrinkers.MAC also could improve the histology changes of NAFLD.Some degree of regular MAC over the span of a lifetime appears to have a protective effect on the histological severity of liver disease comparing with minimal intake.On the other side,some studies supposed long-term and frequent drinking could aggravate the severity of liver fibrosis in patients with NAFLD and elevated liver enzymes.Besides,frequency of drinking is also important in the role of alcohol on NAFLD.Mice administrated high fat diet(HFD)and alcohol at a dose of2.0 g/kg/d twice a week for a total of 12 weeks showed much worse hepatic steatosis,inflammation and fibrosis than those administrated HFD without alcohol.A pattern of daily drinking by gavage could improve liver steatosis and inflammation.The main aim of this study is to explore the role of chronic and daily MAC on NAFLD in murine models.First,we set up a rat model of high-fat high-cholesterol(HFHC)diet induced nonalcoholic steatohepatitis.At the beginning of the fourth week,moderate Er Guo Tou(EGT),a common Chinese spirits,or ethanol was administrated daily in a manner of intra-gastric gavage.At the end of 12th week,all the animals were sacrificed and liver samples and blood samples were collected.The roles of MAC on HFHC induced liver inflammation and fibrosis were analyzed by serology,histology,and gene expression.In order to distinguish the role of chronic and regular MAC from short-time binge,we set up a mouse model of 3-day HFD plus binge and compared the severity of liver steatosis and inflammation among groups.Interleukin 1?(IL-1?)plays important roles in systemic and organic inflammation.In chronic liver injury,activated Kupffer cells produce massive IL-1?and TNF-?and aggravate liver injury.In our experiments,we first set up a cell model to disguss the role of light ethanol on the activation of macrophages and the production of inflammatory factors;then in hepatic stellate cell lines,we explored the role of IL-1?on the activation of hepatic stellate cells,and verified the results in the rat and mice liver tissue via mRNA sequencing and RT-qPCT.Materials and methods:Male Sprague–Dawley rats were fed with standard chow(SC)or HFHC diet in a clean room.One of rat from each group was sacrificed at the end of third week.Determination of serum ALP,ALT,and AST and histological analysis were carried out to confirm the establishment of nonalcoholic steatohepatitis.At the beginning of the fourth week,all the animals were divided into five groups,including SC group,SC plus EGT group,HFHC group,HFHC plus EGT group,and HFHC plus ethanol(EtOH)group.EGT or EtOH(v/v,50%)was administrated at a dose of 4 g/kg body weight per day in a manner of intra-gastric gavage for a total of 9weeks.There were 10-15 rats in each group.All the rats were fed with standard chow or HFHC chow for a total of 12 weeks.At the end of twelfth week,all the animals were sacrificed and serum ALT,AST,and ALP and plasma endotoxin were determined.Hematoxylin and eosin staining,Sirius red staining and immunohistochemistry of liver sections were performed to compare the severity of liver steatosis,inflammation,and fibrosis.The relative mRNA expression of CXCL1,CXCL2,CD68,F4/80,NF-?B,TNF-?,PPAR-?,IL-1?,IL-6,IL-10,IL-1RA,?-SMA,TGF-?,MMP2,MMP9,and TIMP1 in the liver was calculated.Male C57B6 mice were fed with high-fat diet(HFD)for three days in a clean room.Gavage of EGT or EtOH(v/v,50%)at a dose of 2.5 g/kg/d or 5.0 g/kg/d was performed on the third night.All the animals were fasted 4 hours after gavage.Serum samples and liver tissues were then collected after a 12-h fast.Serum ALP,ALT and AST,histology(including liver steatosis,inflammation and fibrosis),mRNA expression of IL-1?,Ly6G,F4/80,CXCL1 and CXCL2 were compared among groups.Mouse macrophage cell line,RAW 264.7 cells,were cultured in DMEM medium and stimulated by LPS10 ng/ml and/or EtOH 50 mmol/L,10 mmol/L and 0.25 mmol/L for6 h or 24 h.Cell survival rate was then calculated based on CCK-8 test.The mRNA expression of F4/80,TNF-?,and L-1?in RAW 264.7 cells stimulated by LPS 10ng/ml+EtOH 0.25 mmol/L or LPS 10 ng/ml for 6 h or 24 h was determined by RT-QPCR.The level of TNF-?in the supernate was measured by a method of ELISA.Rat hepatic stellate cell line,HSC-T6 cells,were cultured in DMEM medium and stimulated by LPS10 ng/ml and/or mouse recombinant IL-1?protein for 6 h,10 h,24h,36 h or 72 h.The activition of cells was determined by the mRNA expression of?-SMA,Collagen and TGF-?and the protein expression of?-SMA.The mRNA expression of?-SMA,Collagen and TGF-?was also determined after stimulated by IL-1?siRNA for 24 h.Results:In rat model,average body weight was highest in SC group than in HFHC diet groups,while liver index was much lower in the former group(P<0.000).Differences of liver index among HFHC diet groups were not significant(P>0.05).The level of serum ALP,AST,and ALT was lower in SC group and SC plus EGT group than in HFHC group,HFHC plus EGT group and HFHC plus EtOH group.The level of serum ALP and AST was a little higher in HFHC plus EGT group than in HFHC group without statistical significance(P>0.05).On the other hand,the level of serum ALP and AST was a little higher in HFHC plus EtOH group than in HFHC group with statistical significance(P<0.01).Plasma endotoxin was less than minimum detection level in SC group and SC plus EGT group(<0.04 EU/ml).Differences of plasma endotoxin among HFHC group,HFHC plus EGT group and HFHC plus EtOH group were not statistically significant(P>0.05).In histology,there was no obvious hepatic steatosis,inflammation or fibrosis in the liver tissue from SC group and SC plus EGT group.There was not obvious difference of hepatic steatosis or inflammation in the liver tissue among HFHC group,HFHC plus EGT group and HFHC plus EtOH group.Liver fibrosis was serious in HFHC group that most were at stage 2~stage 3.A tendency of pseudo-lobule formation was also observed in a few slides.Liver fibrosis was at stage 1 in most slides from HFHC plus EGT group and HFHC plus EtOH group.Difference of the area of fibrosis in the liver was statistically significant among HFHC group,HFHC plus EGT group and HFHC plus EtOH group(P<0.000).The mRNA expression of CXCL1,CXCL2,F4/80,TNF-?,IL-1?,IL-6,IL-10,?-SMA,TGF-?,MMP2,MMP9 and TIMP1 was lower in HFHC plus EGT group and HFHC plus EtOH group than in HFHC group.There was a qualified biological repeatability among samples for mRNA sequencing(R~2>0.92).The number of expressed genes was 11808 in HFHC+EGT group and11236 in HFHC group,including 11073 genes expressed in both groups.There were24 up-regulated genes and 35 down-regulated genes in HFHC+EGT group with a threshold of-log10(p adj)=1.3.In interleukin family,the gene expression of IL-1?,IL-33,IL-34 and IL-36?was down-regulated in HFHC+EGT group.The mRNA expression of IL-1?and IL-33 was much lower in HFHC+EGT group as shown by RT-qPCR.In mouse model,there was not significant difference of serum ALT and AST in HFD group and HFD plus low-dose EGT(2.5 g/kg/d)group or HFD plus high-dose EGT(5g/kg/d)group.Serum AST was higher in HFD plus low-dose EGT group than in HFD group(P<0.05),and serum ALT was higher in HFD plus low-dose EGT group than in SC group(P<0.01).In histology,both EGT and EtOH at low-dose and high-dose aggravate the severity of hepatic steatosis.There was no liver inflammation was observed in HFD group,while inflammation foci were observed in HFD plus high-dose EGT group and HFD plus high-dose EtOH group.There was no liver fibrosis in all the slides.The mRNA expression of Ly6G and CXCL1 was much higher in HFD plus high-dose EGT group and HFD plus high-dose EtOH group.The mRNA expression of F4/80 in HFD plus high-dose EGT group was much lower than in HFD plus low-dose EGT group and HFD plus low-dose EtOH group.In RAW 264.7 cells,we found stimulation with ethanol at different doses with or without LPS did not decrease the cell survival rate of cells.The mRNA expression of TNF-?decreased after stimulated by both LPS and ethanol(0.25 mmol/L)for 6 h(P<0.05).The mRNA expression of IL-1?decreased significantly after 24 h(P<0.01),and the level of TNF-?in the cell supernate decreased significantly at the same point(P<0.01).In HSC-T6 model,the mRNA expression of?-SMA?Collagen and TGF-?did not increase comparing with control groups after stimulated by mouse recombinant IL-1?or LPS respectively for 6 h or 24 h.Different mRNA expression of?-SMA?Collagen and TGF-?was observed in HSC-T6 cells after stimulated by both mouse recombinant IL-1?and LPS for 6 h,10 h,24 h,and 36 h.The mRNA expression of TGF-?and?-SMA did not significantly increase in 6 h and 10 h.The mRNA expression of TGF-?and?-SMA significantly increased in 24 h(P was 0.002 and0.000,respectively).The mRNA expression of TGF-?did not continue to rise in 36 h,while mRNA expression of?-SMA continued to rise in 36 h(P was<0.01 and 0.004,respectively).Significant increase of mRNA expression of Collagen appeared at 36 h in HSC-T6 cells stimulated by both mouse recombinant IL-1?and LPS.Protein expression of?-SMA increased in HSC-T6 cells stimulated by both mouse recombinant IL-1?and LPS at 72 h.The mRNA expression of?-SMA and IL-1?decreased in HSC-T6 cells stimulated by IL-1?siRNA for 24 h(P was<0.001 and0.05,respectively).The mRNA expression of Collagen showed a tendency of decreasing(P>0.05).There is no difference of the mRNA expression of TGF-?between groups.Conclusions:Chronic and regular MAC does not influence liver index,serum liver enzymes and plasma endotoxin on the basis of standard chow.Still,chronic and regular MAC does not lead to liver steatosis,inflammation or fibrosis as shown in histology and genetics.Chronic HFHC diet feeding can cause serious liver injury,showing as significantly increased liver wet weight and liver index,elevated level of serum ALP,AST and ALT and plasma endotoxin.In histology,chronic HFHC diet feeding leads to serious liver steatosis,inflammation and fibrosis.More importantly,chronic HFHC diet feeding causes the continuous activation of Kupffer cells and further the continuous activation of hepatic stellate cells,which are quite crucial in the development of liver inflammation and liver fibrosis.Chronic and regular MAC has beneficial effects on alleviating liver inflammation and liver fibrosis caused by chronic HFHC diet.Chronic and regular MAC does not aggravate liver wet weight and liver index,serum ALP,AST and ALT and plasma endotoxin on the basis of long-term HFHC diet feeding.Besides,chronic and regular MAC could relief the activation of Kupffer cells and hepatic stellate cells.The possible mechanism is that low-dose ethanol down regulates the activation of macrophages and then the expression of IL-1?.Inadequate activation of macrophages cannot activate hepatic stellate cells.IL-1?per se could not activate hepatic stellate cells directly but could promote sustained activation of hepatic stellate cells on the basis of LPS.In 3-day HFD plus binge model,a single low-dose or high-dose ethanol or EGT aggravated liver steatosis and inflammation as shown in histology and genetics.This effect is different from the role of chronic and regular MAC on the liver.