Establishment Of Mouse Model Of Alcoholic Liver Fibrosis And Clinical Analysis Of Alcoholic Liver Disease

Alcoholic liver disease (ALD) is the result of long-term heavy drinking.According to the 2006 ALD guideline of liver diseases branch of the Chinese MedicalAssociation, pathology diagnostic criteria of ALD is divided into 5 types, mildalcoholic disease of the liver(AMI), alcoholic fatty liver(AFL), alcoholic hepatic(AH),alcoholic hepatic fibrosis(AHF), alcoholic liver cirrhosis(AC). ALD is the mostimportant cause of liver cirrhosis disease in the Western country, but also the 10thcommon cause of death. With changes in life style of our country in recent years, theincidence rate of alcohol-induced liver disease upgrades, ALD becomes the secondlargest cause of liver damage only behind virus hepatitis. Liver fibrosis is thepathological procedure of paraplasmic proliferation of the liver fibrous connectivetissue, when the liver cells necrosis or have inflammatory stimulation, Deteriorationof liver fibrosis can cause the reconstruction of hepatic lobules and formation of falsehepatic lobules, namely, liver cirrhosis. The domestic and foreign scholars have noobjection to the invertibility of liver fibrosis. Therefore, ALD plays a decisive role inthe prognosis in the prognosis in the prognosis of alcoholic liver cirrhosis. The evolution and the reversal time of ALD has been paid more attention in recent years.For an in-depth study of the pathogenesis of ALD, establishment of animal model ofALD, which is similar to alcohol-induced liver disease of human, is necessary.[Objective]The purpose of my first part research is to establish a ideal mouse model of ALD,which can provide a model to study the liver stem cell derived from bone marrow.The second part of this study is to analysis the clinical data of 105 patients of ALDadmitted to our hospital in the recent 5 years, to explore the characteristics andclinical outcome of ALD, to raise the awareness and importance of ALD, and toachieve the goals of the early detection, early diagnosis and treatment.[Materials and Results]1. Establish mouse model of alcoholic liver fibrosisThere were 100 Balb/c female mouse (the SPF level, 5～6 week old, weight15～18g), which were randomly divided into two groups (92 of mold group, 8 ofcontrol one). Mice of mold group were intragastriced with 54 degree white liquortwice every day within totall 16 weeks, and the 10% (v/v) white liquor was the onlybeverage. From the first weekend to the third one, mice of mold group were onceintragastriced with 8 mL/kg, once with 10 mL/kg from the 4th to the 7th weekend,and once with 12 mL/kg from the 8th to the 16th weekend. Mice of the control groupwere given the equal volume of distilled water. During the experiment, all mice wereraised with all-nutrition diet. In the end of the 4th, 8th, 12th weekend of model group,six mice were randomly selected, fasting for 12 hours before death, executed by themean of cervical dislocation, get eye blood, and remove liver immediately. Specimensof liver were observed by light microscopy, after weighed in 10% formaldehyde, paraffin-embedded, dehydrated and stained by hematoxylin-eosin (HE) or picricacid-acid fuchsin (VG). Blood samples were detected for the biochemical markers.Mice of control group were killed in the 16th weekend, compared with model group.In the mice of experiment some indicators were observed such as mental stateactivities, superficial gloss, loss of appetite and etc. And weight and size of specimens,shape, color, texture and the plane situation were observed per week. Serumbiochemical indicators, including alanine aminotransferase (ALT), aspartateaminotransferase (AST) and triglyceride (TG), total protein (TP), albumin (ALB).liver pathology were observed. Specimen of liver were paraffin-embedded andstained with hematoxylin-eosin and VG. Pathological phenomenon (liver celldegeneration and necrosis, inflammatory cell infiltration, the proliferation of collagenfibers, etc)were observed, the results are as follows.1.1 quantitative analysis : There were 23 mice died often become of acute orsubacute death. The main reasons were asphyxia, aspiration pneumonia,gastrointestinal perforation, acute gastric dilation, alcoholism, and etc. Mice of thecontrol group were all survived.1.2 Changes in the spirit : The model mice appeared to languish, lethargy, slowresponse, loss of appetite. These symptoms reduced after the 4th week of theexperiment, but superficial gloss was still poor and weight grew slowly. By contrary,the control mice were superficially sheen, lively, and had normal appetite withoutdrowsiness.1.3 The appearance of liver specimens : there are smooth surface, red color,sharp edge, and medium texture in the control group. By contrast, colors of liver werebleak and substance were congested in the model group.1.4 Histological changes : specimens were stained by hematoxylin-eosin in thecontrol group, Liver cells were arranged radially to the central vein, hepatic lobules was clear and outline of liver cable was neat. After the 4th week, there were slightlyfatty degeneration in the liver cell. In the 8th week there were kytoplasm swelling,cell cables disordered, osteoporosis cytoplasmic swelling, and more extensivevacuolar degenerated in the liver cells compared to one of the 4th week. In the 12thweek, there were necrosis points and spots in liver cells. In the 16th week, there wereinfiltration of inflammatory cells, fibroblasts and some necrosis in the exchangedistrict, fibrosis was more evident, and the marked red duct hyperplasia collagenfibers were to extend around the lambs.1.5 Serum biochemical indicators of liver fibrosis: The serum content of ALBwas significant different at each week of the model group compared with the controlone (F=6.490, P=0.001), but there were no significant differences at each week ofthe model group (F=0.620, P=0.610). Compared with the control group in the 12th,14th, 16th week, there were no significant differences with TP (F=1.217, P=0.327) ,there were no significant differences at each week of the model group (F=0.965, P=0.429) .Compared with the control group at each different time points,except there were no significant differences with AST/ALT, TG in the 4th (P=0.083;P=0.318), there were significant differences with AST, ALT, TG, AST/ALT (F=10.281, P＜0.001; F=8.610, P＜0.001; F=3.605, P=0.018; F=4.490, P=0.007).2. Clinical analyses of 105 cases of alcoholic liver diseaseThere were 105 clinical cases of ALD admitted to the hospital in the recent fiveyears. According to 2006 guideline of ALD diagnostic criteria amended by liverdisease branch of the Chinese Medical Associations, the 105 cases were divided into3 groups, nonalcoholic fatty liver (AFL), alcoholic hepatitis (AH), alcoholic livercirrhosis (AC). The data of drinking history, clinical manifestation, liver function andimaging examination was analyzed and discussed respectively. The results were asfollows. 2.1 ALD had similar clinical manifestations like the other liver disease, such asnon-specific symptoms of malaise, anorexia, fever, jaundice, abdominal distension,hematemesis and melena.2.2 The patients of ALD were mainly the men drinking 45～600g wine per day,the average life 21 years. The number of patients among 31-60 years age is the mostin the data(89 cases). There were 67 cases of alcoholic liver cirrhosis, 63.8% of total.The median alcohol intake of AC group was significant differences compared withAFL group and AH group(P=0.001, P=0.013). The median drinking time of ACgroup was (24.13±7.84), there were significant differences (P=0.001, P=0.001)compared with the AFL group (16.21±12.43), AH group (16.00±10.09).2.3 The patients of ALD had varying degrees of abnormal serum enzymelevels. The median serum ALT, AST, GGT of AH group was significantdifference compared with the AFL group(P＜0.001). Compared with AC group, thelevels of AST, ALT, GGT were significant difference (P＜0.001; P＜0.001; P=0.047),but with AST/ALT the difference no significant(F＝0.801, P=0.451). The ALB levelof AC group was obviously low, there were significantly different(P=0.003;P＜0.001)compared with AH and AFL group. The level of TBIL was significantlyincreased in the AC group, and the difference was significantin the three groups(P＜0.001). The leading cause of death in the patients of ALD is the late complicationsof liver cirrhosis.[Conclusion]1 Wine gavage method can be successfully applied to established the model ofalcoholic liver fibrosis, within the 16 weeks, which could emergence variousmanifestations, such as fatty degeneration, inflammatory change, and the proliferationof collagen fibers. The degree of liver damage will be aggravated with the increasing extension of alcohol.2 This method of manufacturing model is simple, low-cost, close to drinkinghabits of our people, and can provide an ideal animal model for studying the stem cellderived from bone marrow in the liver how to repair the alcohol-induced liver injury.3 The high incidence age of ALD was 30- 60 year old in the data. With theincidence of ALD, there was intimate relation between alcoholic liver disease andalcohol intake ,drinking year.4 With alcoholic liver disease there were no-specific clinical manifestations.For early diagnosis and treatment and better prognosis, poeple with drinking habitshould periodically monitor biochemical indicators of AST, GGT, TBIL, combinedwith imaging examination.