The Protective Effect Of Sixteen-amino-acid Polypeptide Fragments On CCl₄ Induced Liver Fibrosis And Its Mechanism.

Objective:A variety of factors can lead to liver fibrosis,including alcoholic,chronic hepatitis B,hepatitis C,obesity,autoimmune hepatitis and metabolism disease.Almost all sorts of chronic liver disease are involved with fibrosis,which can easily progress to cirrhosis,liver failure and even carcinoma.Progressive liver fibrosis can be reversed,while cirrhosis is barely reversible.The animal model of carbon tetrachloride induced chronic liver disease in mice has become mature and has been widely used to mimic common pathological process of various chronic liver diseases in human--liver fibrosis.Long terms of carbon tetrachloride stimulating results in liver cell necrosis,inflammation and proliferation of fibrous tissue,with the elevating of serum aspartate aminotransferase and alanine aminotransaminase.Moreover,a large number of inflammatory cell infiltration and extracellular matrix collagen deposition can be observed.In order to seek for the biomarker of early clinical diagnosis of hepatitis,our preliminary study utilizes matrix assisted laser desorption ionization time of flight mass spectrometry?MALDI TOF MS?to detect a differential expression of polypeptide in the serum of chronic hepatitis patients,i.e.,sixteen-amino-acid polypeptide fragments.Also,the in vitro experiments are used to confirm that the polypeptide actually contributes to the proliferation of hepatocytes.There has not been any studies on physiological,pathological and mechanism of the polypeptide in animal models of hepatic fibrosis so far.Referring to the results of previous study of this lab,it has been found out that the polypeptide has protective effect on liver disease including immune hepatitis and fatty liver.Hence,it is reasonable to assume that the polypeptidemay play the similar protective role on chronic liver disease.Hence,in this study,we apply the liver fibrosis model in mice induced by carbon tetrachloride so as to analyze the therapeutic effect of sixteen amino acid polypeptide fragments on liver fibrosis and preliminarily explore its mechanism for the purpose of providing experimental basis for the further clinical treatment.Methods:1 The effect of sixteen-amino-acid polypeptide on liver fibrosis in mice.Fifty male balb/c mice between 6-8 weeks were divided into 5 groups randomly,with 10 mice in each group.In addition to the intraperitoneal injection of normal saline?1ml/kg?to the normal control group at the corresponding points,the other four groups were given 25% CCl₄?1.5?l/g?by intraperitoneal injection once at the interval of 3 days,continuously for 10 times.Meanwhile,after the third injection,apart from the normal control group and model group that are administered with normal saline via intravenous injection at the appropriate time points,the rest groups were given400?g/kg or 800?g/kg body weight sixteen-amino-acid polypeptide via intravenous injection once every three days lasting for 7 times in duration or by intragastric administration of colchicine?200?g/kg?5 days a week continuously for 3 weeks,respectively.Once the treatment has been done,the eyeballs of mice were removed to get blood and then separate the serum,the serum ALT,AST and ALP levels are measured by automatic biochemistry analyzer;ELISA kit for determination of serum HA and ?-C levels.The effect of sixteen-amino-acid polypeptide on CCl₄ induces liver injury was observed by serological markers.2 Histopathological detection of liver tissue.After removing the eyeballs for blood,mice are then killed.Apart the liver and then get two pieces of liver tissues of appropriate size from the similar location of the left lobe and the right lobe respectively?thickness is no more than 0.5 cm in general?and then make them into paraffin blocks.Then stain with hematoxylin and eosin?HE?staining,masson staining after slicethem up serially with thickness of 5um.The morphology and collagen deposition of liver tissues are observed by light microscope and capture images to save.3 The expression levels of liver-fibrosis-related genes in miceThe gene sequences of Col1a1,Col3a1,TIMP-1,MMP-2,CTGF are obtained from NCBI.Design primers and take GAPDH as the reference.The primers are synthetized by Shanghai Biological Engineering Co.,Ltd.Extract total RNAs of liver tissue of mice,then reversely transcript them into cDNA,respectively with a 10 fold dilution of Col1a1,COL3a1,TIMP-1,MMP-2,CTGF and GAPDH cDNA as template to detect by real-time fluorescence quantitative PCR.After reaction,amplification curve and melting curve are confirmed,as well as the data?relative gene expression of RQ?for further study.The aim is to investigate the mechanism of the effect of sixteen-amino-acid polypeptide on liver fibrosis induced by CCl₄.4 The detection of reactive oxygen species?ROS?in liver cells.ROS content are tested by flow cytometry in order to evaluate the level of oxidative stress.Results:1 The effect of sixteen-amino-acid polypeptide on liver fibrosis induced by CCl₄ in mice.The serum ALT and AST levels of sixteen-amino-acid polypeptide low dose group?36.85±3.36,71.03±7.42?,high dose group?35.27±4.34,73.03±8.13?,colchicine group?34.65±5.57,69.49±11.76?and the control group?31.05±2.91,62.02±9.46?are all significantly lower than the model group?45.07±5.32,85.48±3.44??P<0.05?;while the serum ALP levels has no obvious differences?P>0.05?;as compared to the control group?229.15±32.43?,the serum levels of HA in model group?286.95±37.82?are significantly higher?P<0.05?,other groups are lower than the model group,but no significant differences are identified.;the serum ?-C levels of sixteen-amino-acid polypeptide low dose group,high dose group and the control group?129.68±29.87,109.26±30.16,79.10±30.33?are all significantlylower than the model group?180.96±40.73??P<0.05?,though the serum ?-C levels of the colchicine group?157.12±32.26?decreases compared to the model group,but there is no significant difference?P>0.05?,the serum ?-C levels of sixteen-amino-acid polypeptide low dose group,high dose group are all lower than the colchicines group?P<0.05?.2 The pathological change of liver tissue.As had been showed in HE staining,the hepatocytes are messed up in model group,fibrous structure surrounds the central veins and portal area,along with large numbers of inflammatory cells infiltration and massive hepatocyte necrosis regions.The colchicine positive control group efficiently improves the conditions of necrosis.Also inflammatory cells infiltration decreases.No obvious necrosis and inflammatory cell infiltration has been observed in the two doses sixteen-amino-acid polypeptide groups.According to the Masson staining,a great quantity of green collagen fibers are visible around the central vein and portal area.The hepatic lobule is divided into pseudolobuli by different widths of fibrous septum.The grade of hepatic fibrosis decreases in colchicine positive control group and only a small amount of fibrous tissue has been observed.Fibrous tissue of the two doses of sixteen-amino-acid polypeptide treatment group significantly reduces when compared to the model group.3 The expression levels of liver-fibrosis-related genes in miceFluorescence quantitative PCR results show that the expression levels of Col1a1 in sixteen-amino-acid polypeptide low dose group?7.18±1.61,3.44±1.73,4.37±1.56,3.39±3.19,1.45±0.77?,high dose group?5.66±2.07,3.77±2.64,3.43±1.42,3.43±2.92,1.68±1.21?,colchicine group?9.60±0.52,5.58±0.65,3.97±4.01,5.93±4.84,1.91±1.03?and the control group?1.00?are significantly lower than those in the model group?20.67±6.60,12.61±1.35,8.25±1.77,11.93±6.82,3.97±1.90??P<0.05?.As for the Col3a1,TIMP-1,MMP-2 and CTGF,we draw to the same conclusion.4 The change of ROS in liver cells in miceThe ROS levels of sixteen-amino-acid polypeptide low dosegroup?431.83±174.80?,high dose group?404.50±199.43?,colchicine group?523.00±218.61?and the control group?220.33±62.69?are all significantly lower than the model group?859.63±337.81??P<0.05?.Conclusions:1 Exogenous administration of sixteen-amino-acid polypeptide can significantly improve the liver fibrosis induced by CCl₄.2 The mechanism of sixteen-amino-acid polypeptide's protective effect on CCl₄ induced liver fibrosis may in relation to the attenuating of ROS and down-regulating expression of TIMP-1,MMP-2 and CTGF.