The Protective Effect Of 36 Amino Acid Polypeptide Fragment To CCl₄-induced Liver Fibrosis And Its Mechanism

Objective:Liver fibrosis is a chronic and progressive disease,and its character is the formation and accumulation of extracellular matrix which may change the structure of liver.Liver fibrosis is the common result of numbers of chronic liver diseases.The risk of developing liver cirrhosis is highly increased when fibrosis is at an irreversible stage.So it is important for patients with liver fibrosis to diagnose and treat early.However,there is still short of standard treatment and specific drugs.Thirty-six amino acid polypeptide fragment was found in the serum of patients with chronic hepatitis when detecting its biomarkers,and the level of 36 amino acid polypeptide fragment was increased.The previous study showed that 36 amino acid polypeptide fragment could make the cycle of liver cells changed and inhibit apoptosis.Up to now,there is no study investigating the pathological and physiological functions of 36 amino acid polypeptide fragment in liver fibrosis mice.Early we found that 36 amino acid polypeptide fragment had the protective effect on both acute liver injury and non alcohol fatty liver disease.In this study,we are going to explore whether 36 amino acid polypeptide fragment has the similar protective effect on liver fibrosis mice or not.The mice were treated with CCl₄ by intraperitoneal?i.p.?injection to investigate the mechanism involved in.Methods:1 The protective effect of 36 amino acid polypeptide fragment to liver fibrosis miceFifty Balb/c mice were randomly divided into five groups,they are control,model,reference drug,36 amino acid polypeptide fragment low dose?150?g/kg?and high dose?250?g/kg?group respectively.The mice in control group were injected intravenously with saline.Mice in model group were injected intraperitoneally,once every three days,with 25% CCl₄,ten times in total.The other three groups were treated with different drugs?colchicine,36 amino acid polypeptide fragment 150?g/kg and 250?g/kg?after CCl₄ injected.Then the mice were sacrificed after last injection,and tested serum alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,hyaluronidase?HA?and collagen ???-C?levels.2 Pathology test of liver tissue in each groupThe liver tissue in each group was embedded with paraffin and detected with HE and Masson staining in order to observe pathological morphological changes and collagen deposition.3 The expression level of liver fibrosis related genesTotal RNA was extracted from liver tissues.Expression of Col1A1,Col3A1,MMP-2,TIMP-1 and CTGF was analyzed by quantitative RT-PCR.4 The level of reactive oxygen species in the liver cellsReactive oxygen species?ROS?content was tested by flow cytometry to evaluate the level of oxidative stress.Results:1 The result of serum ALT and AST in each groupCompared to control group?31.05±2.91?,mice in model group?45.07±5.32?had significantly increased ALT level?P<0.01?.ALT serum levels for colchicine?34.65±5.57?,36 amino acid polypeptide fragment low?36.08±2.07?and high dose groups?40.08±2.84?were lower than model group?P<0.05?.Similarly,the level of AST in model group?85.48±3.44?was higher than control group?62.05±10.93?.The serum levels of AST in colchicine?73.64±11.76?and 36 amino acid polypeptide fragment low dose group?71.89±11.02?were decreased?P <0.05?.2 The result of serum HA and ?-C in miceOur data showed that serum HA in model mice?291.98±39.16?was higher than in the controls?P<0.05?.The 36 amino acid polypeptide fragment low dose group?240.23±25.75?had a lower HA level than the models?P <0.05?.?-C in model mice?180.95±40.73?was higher than in the controls?P<0.05?,too.And two groups of 36 amino acid polypeptide fragment?104.55±31.77,117.46±26.92 respectively?were in decreased ?-C level?P <0.01?.3 Histopathology of liver tissueHE staining showed that the structure of the liver lobules and portal in control group was fully complete.And no necrosis area was observed.On the contrary,model mice after CCl₄ injection developed apoptosis and necrosis with inflammation,and the hepatic structure was destroyed seriously.Obvious differences existed between drug groups?colchicine group and two 36 amino acid polypeptide fragment groups?and model group that the area of necrosis and inflammation was decreased significantly.There was almost no area of apoptosis and necrosis in low dose group.The result of Masson staining showed that there was no collagen deposition in liver tissue from control group.CCl₄ treatment in the model group remarkably increased the fibrotic area.Bridging fibrosis was showed up developing typical pseudo-lobule.Colchicine and 36 amino acid polypeptide fragment decreased the area of fibrosis among CCl₄-treated mice.4 Expression of fibrosis related genes in each groupThe relative expression of Col1A1 was 1.00,20.67,9.60,11.10,10.47?P<0.05?;the relative expression of Col3A1 was 1.00,12.61,5.58,6.16?6.57?P<0.05?;the relative expression of MMP-2 was 1.00,11.93,5.93,5.26?5.67?P<0.05?;the relative expression of TIMP-1 was 1.00,8.25,3.97,3.36,4.19?P<0.05?;and the relative expression of CTGF was 1.00,3.97,1.91,1.74,2.22?P<0.05?.Compared with the control group,fibrosis related genes,such as Col1A1,Col3A1,MMP-2,TIMP-1 and CTGF,were in a elevated relative expression level?P<0.05?.The mRNA expression level of Col1A1,Col3A1 and MMP-2 was altered by colchicine and two 36 amino acid polypeptide fragment dose group?P <0.05?.The colchicine and 36 amino acid polypeptide fragment low dose group had a lower level of TIMP-1 and CTGF mRNA than model group?P <0.05?.5 The change of ROS level in hepatocyteROS level in the model group(?859.63±337.81?was higher than the control group?220.33±62.69?,the difference was statistically significant?P<0.01?;ROS level in two 36 amino acid polypeptide fragment groups?259.60±118.10 and 327.20±123.63?was lower than that in model group?P<0.01?.Conclusions:1 Thirty six amino acid polypeptide fragment can attenuate CCl₄-induced mice liver injury and fibrosis promoting the damaged liver cells recovery and reversing liver fibrosis.2 The protective effect of 36 amino acid polypeptide fragment to liver fibrosis mice may be associated with inhibiting oxidative stress and down-regulating the expression of MMP-2,TIMP-1 and CTGF.