The Effects Of Azithromycin On Cytokines And Nitric Oxide In Rats With Alcoholic Liver Disease

PrefaceDue to long - term and large quantity of alcohol drinking , alcohol and its metabolic , ie. acetaldehyde, damage hepatocyte by direct or immune mechanisms which show as hepatocyte steatosis, alcoholic hepatitis, fibrosis and cirrhosis. Alcoholic hepatic fibrosis is chronic and can be reversed which is attributed to the loss of equilibrium of secreation and degradation of extracellular matrix ( ECM). It is not only the outcome of alcoholic fatty liver and hepatitis but also the basis of liver carcinoma and hepatic cirrhosis and so it is the focus of the study on the development and retroconversion of alcoholic liver disease. At present it has been founded that many cytokines play an important role in alcoholic hepatic fibrosis, but the studies on the joint action of CTGF ,TNF-α and serum NO is few. In this study, we established a rat model of alcoholic liver disease by direct intragastric inoculation of ethanol, and explored the pathological changes of hepatic tissue and expression of CTGF,TNF -α and serum NO in different stages. At the same time we studied the influence of Azithromycin on their expression which may be helpful to the study of the pathogenesis , prevention and treatment of alcoholic liver disease.Materials1. Animals96 male Wistar rats, 25 rats in normal group and 38 rats in alcoholic group and 33 rats in treatment group.2. reagent and drug(l)Anhydrous ethanol(2)Rabbit - anti - rat antibody : CTGF ( 1:100) , TNF - a ( 1:100 ) , Strept - Avidin -Biotin(SABC)test kit and diaminobenzidine(DAB)color reagent. (3)Nitric Oxide test kit (4)Azithromycin 3. Instrument (l)Paraffin microtome(2)Iight microscope and photography apparatus (3)Micro(fluorescence) -picture analyze systemMethods1. Animal model96 rats were divided into three groups randomly. There were 25 rats in normal group and 38 rats in alcoholic group and 33 rats in treatment group. The alcohol and treatment rats were given alcohol at a dose of40% ,8g ? kg' ? day" , by intragastric perfusion during the first 4 weeks,50% alcohol 9g kg"1 ? day*1 during the second 4 weeks and 50% alcohol 10 g ? kg" day" ? during the third 4 weeks. The normal group was treated with physiological saline in the same way to the alcoholic group. The treatment rats was given with Azithromycin (10 mg kg"1 ? day "^before intragastric perfusion in 4N8A12 weeks. 12^16^10 rats in alcoholic group and 10N14A9 rats in treatment group and 9N8A8 rats in normal group were killed respectively at the end of 4^8^12 weeks.2. Pathologic examinationThe central part of right lobe of hepatic tissue was cut into two fragment which were fixed by formalin, then made into paraffin blocks and cut into 4^m of section. The tissue were stained with HE routinely and the pathologic changes were observed under the light microscopy. The degrees of hepatocyte necrosis and inflammatory cell infiltration were analyzed semi - quantitively. Each slice selected five fields, including the center and circumference.3. Detection of CTGF ^TNF - a by ABC immunohistochemistryThe paraffin section were dewaxed to water, then the endogenetic enzymewas inactived. Repair the antigen,add normal rabbit serum,then add the first antibody CTGF^TNF -a(l :100) ,stay overnight at 4X, and add the second antibody 37X. for thirty minutes. ABC compound was added for thirty minutes. All steps were completely washed by 0. 01MPBS. DAB showed color. The sections were dyed again slightly by hematoxylin and observed under the light microscope. The cells containing light brown particularte in cell plasm were postive cells. Utilize the micro fluorescene picture analysis system to analyze the integrated intensity of immuneohistochemistry of CTGF^TNF - a in selective field of vision.4. Measurement of NO in serum2 ml of blood were collected in apex of heart and were kept for 30 minutes at the room temperature. It was centrifugated for 10 minutes at 2000rpm/min and the serum were collected. Then it was preserved at - 201. Nitrate reductase technique was used to measure NO.5. Statistical analysisAll data were conducted using SPSS11. 5 statistical package. Quantitative variables are expressed as means standard deviation. Comparison between groups was used for the test of the quantitative variables and ANOVA test for qualitative variables.Results1. General conditionThe general condition of rats in alcohol group comparing with that in normal group was bad. The fur of rats in alcohol group was lackluster. They were irritable and body weigh were lost(P <0. 01) .2. Hepatic pathologyic changesHE stain was conducted and hepatic cells were distributed emitly around central vein in normal group. At the end of 4 weeks, there are a little steatosis and vacuolar degeneration of local liver lobe around centeral vein, and dot necrosis of hepatic cell and inflammatory cell infiltration. At the end of 8 weeks, steatosis and vacuolar degeneration are increased appantly and location of necro-sis is increased. At the end of 12 weeks,there are diffused vacuolar degeneration and necrosis in liver small lobe.3. Expression of CTGF^TNF -a in hepatic tissueExpressions of CTGF and TNF - a were increased in hepatic tissue ( P < 0.01) of alcohol and treatment group comparing with that of normal group. There were significant differences among three groups. The expression of CTGF^TNF -a in hepatic tissue of alcohol and treatment group increased gradually (P < 0.05) along with the prolonged and strengthened stimulation of alcohol ( P < 0.01).DiscussionsRecently, the incidence of alcoholic liver disease ( ALD ) has been increasing gradually in our country, and it has become the second liver disease in some areas. At present the study on the mechanism was few and unenligntened in our country because the animal model of ALD was difficult to make and the study in vivo was controlled. The method of drinking freely to make animal model spent more time and was interfered by many conditions. The animal model of ALD was made succeedly by intragastric administration of ethanol on the basis of repeating trials,and the dose and concentration given increased gradually in order to primarily investigate the mechanism and treatment of ALD.Long term and large quantity of alcohol drinking may induce. It has been reported that the rate of high level of endotoxin in people with ALD was about 67. 3%. Endotoxin (ET) is a kind of lipopolysaccharide in G~ cell wall and in normal state a small quantity of LPS enter liver through instines penetration. It was reported that ethanol can change entero - microbial flora and its PH . In addition it can promote instinal penetration to endotoxin and decrease phagocytosis of Kuffer cell, which lead to the development of endotoxemia. High level of LPS activate hepatic stellate cellA endothelial cell and vascular smooth muscle cell and induced Nonase which can compose a large quantity of NO and activate Kuffer' s cell,resulting that the release inflammatory mediator which will injure hepatic cells.The joint action of CTGF and TNF - a could activate hepatic stellate cell (HSC) and induce the cascade reaction of cytokine, specially more active product, for example connective tissue growth factor at downstream of transforming growth factor -13. These cytokines may activate HSC and make it transform into myofibroblast which will synthesize and excrete lots of collage. At mean time degradation of collage was reduced because activity of collagenase decrease. So the balance between synthesis and degradation of extracellular matrix was disrupted which led to the development of fibrosis and even cirrhosis of liver.Inhibition of any links above may inhibit development of ALD. Selective depollution in the intestines by using antibiotic in early stage, which was not absorbed in bowel, may reduce loading of bacterium in the intestines and spontaneous pollution of bacterium which may decreased the level of endotoxin in plasma and the serum level of NO, and disrupt the cascade reaction of cytokine, so that the level of CTGF, TNF - a in circulation decreased. It may get to the aim of preventing liver injury induced by ethanol stimulation and occurrence and development of ALD.The study showed that the serum level of NO increased gradually along with the progression of ALD; The result of immunohistochemistry showed there was small expression of CTGF, TNF - a in tube area of normal group, the expression of CTGF, TNF - a increased gradually in fibrotic area. In addition, the expression of CTGF was more significant than that of TNF - a. The early use of Az-ithromycin can not only significantly relieve tissue injury but also decrease the expression of CTGF, TNF - a- So the early use of antibiotic is very important in order to decrease the level of endotoxin in intestines which play an important role in the prevention and development of ALD.Conclusions1. The hepatic ethanol injury become more and more serous with the time going , resulted from the long term and chronic ethanol stimulation, which showed as infiltration of inflammatory cells, hepatocyte steatosis, vacuolar de-generatton and point necrosis. After some times a large area of necrosis and pro-liferation of fibrous tissue was significant.2. The serum level of NO and the expression of CTGF and TNF - a are paralleled with the injury degree of liver.3. The expression of TNF - a and CTGF increase gradually along with the NO in the serum.4. Azithromycin may decrease the serum level of NO induced by chronic ethanol stimulation, and inhibit the expression of CTGF and TNF - a.