The Protective Effect Of36Amino Acid Polypeptide Fragments To Con A-induced Acute Liver Injury And Its Mechanism

Objective: Liver disease is a common kind of serious disease thatinfluences human health. If long-term liver damage cannot be curedeffectively and timely, it will eventually progress into hepatocellularcarcinoma or liver failure, which will reduce the quality of patients’ lifesignificantly and be a threat to life. Although hepatitis comprises aheterogeneous group of diseases with different etiologies and complexpathogeneses, previous studies have indicated that the immune system plays apivotal role in the majority of these processes. Concanavalin A (ConA)-induced acute liver injury model is a well-established to show T cellmediated the hepatitis in mice, in order to simulate human viral andautoimmune liver disease.In previous study,36amino acid polypeptide fragments is a highercontent polypeptide that is detected in the serum of chronic hepatitis patients,through searching early clinical diagnosis of hepatitis biomarkers by usingmatrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS). The preliminary study found that the high expression ofthe polypeptide fragments could change the proliferation cycle of culturedHepG2cells, promoting cells to enter S phase.So far, there is no study about physiological and pathological functions ofthe polypeptide in hepatitis animal model. The purpose of our experiment is toinvestigate that it may play a role in the progression of hepatitis. In this studywe used Con A to induce acute liver injury model in mice for researching thefunction of36amino acid fragment in immune induced liver injury, at thesame time, to explore the mechanism of action and provide experimental basisfor further clinical treatment of liver diseases. Methods:1Pretreated mice with the synthetic36amino acid polypeptide fragmentsor dexamethasone, inject Con A to induce liver injury in an hour. Afterinjecting Con A12hours, detect the serum levels of alanineaminotransaminase(ALT) and aspartate aminotransferase（AST）, then livertissues were embedded in paraffin and stained by HE, in order to observe theeffect of the peptide preconditioning on liver injury induced by Con A.2Pretreated mice with the synthetic36amino acid polypeptide fragmentsor dexamethasone, an hour later, given a lethal dose of Con A. Recorded andanalyzed the number of dead mice within72hours, then determine theprotective effect of the polypeptide fragment of Con A induced liver injury.3Flow cytometry (FCM) was used to test the liver cell apoptosis of miceand mitosis in each group of mice, in order to explore the mechanism ofpolypeptide fragment in Con A induced hepatic injury.4Lymphocytes (T cells, NK cells and NKT cells) of mice were detectedthe infiltration in liver tissue by FCM, in order to explore the actionmechanism of peptide fragments of Con A in liver injury induced by immuneresponses.5Through the tail vein injection of Con A to induce liver injury,0.5hours later, the mice were injected the36amino acid polypeptide fragments tocure, and then detected serum ALT and AST levels after injecting Con A12hours. Liver tissues were embedded in paraffin and stained by HE, to observethe therapeutic effects of Con A induced hepatitis. In addition, with a lethaldose of Con A treated mice, peptide fragments or dexamethasone to be used tocure, and then observed and analyzed the number of dead mice in72hours, inorder to determine the treatment effect of the peptide fragment of Con Ainduced liver injury.Results:1The effect of Con-A induced liver injury by prophylactic the36aminoacid polypeptide fragments.Compared with the model group, each of polypeptide fragments anddexamethasone preventive medication group were significantly reduced the serum ALT and AST levels (P<0.05). HE staining showed that the liver tissueof model group mice showed typical liver necrosis and lymphocyticinfiltration; peptide fragment of75μg/kg group, necrosis of liver cellsdecreased significantly, other dose groups (with the dose of150μg/kg,300μg/kg and600μg/kg) and dexamethasone group did not appear the necrosisareas of liver cell.2The effect of lethal dose Con-A induced death by prophylactic the36amino acid polypeptide fragments.The group of prophylactic polypeptide fragments and dexamethasonefor the survival rate of mice were70%and80%, both of the median survivaltime was72hours, significantly higher than those in the model group(20%,11hours). It shows that polypeptide fragment can effectively reduce themortality of mice (P=0.004).3The effect of the cell cycle and apoptosis of hepatocytes byprophylactic the36amino acid polypeptide fragments.Calculated the proportion of group, which prophylactic polypeptidefragment and dexamethasone, the liver cell apoptosis was14.73±3.37%,10.01±0.78%,6.41±0.98%,12.86±1.79%,8.4±2.01%, significantly lowerthan Con A group30.24±1.29%, P <0.01; with increasing the polypeptidefragment dose, the percentage of apoptotic cells decreased, when thepolypeptide fragments up to600μg/kg, the percentage of apoptotic cellsincreased. The hepatocytes of the polypeptide fragment of600μg/kg group,the percentage of hepatocytes in the G0/G1phase was markedly higher thanthat of model group, the G2/M and S phase of hepatocytes was significantlylower than that in the model group (P<0.01); the polypeptide fragment of75μg/kg,150μg/kg and300μg/kg group G2/M hepatocytes ratio is lower thanthe model group (P<0.05); G0/G1liver cells was markedly higher than that ofmodel group, the G2/M and S phase of hepatocytes was significantly lowerthan that in the model group (P<0.01).4The effect of hepatic tissue infiltration of lymphocytes number byprophylactic the36amino acid polypeptide fragments. There were no significant differences among the each group mice ofpreventive polypeptide fragment with liver infiltrating T cells, NK cells andNKT cell ratio compared with the model group, but dexamethasone group thatthe proportion of T cells and NKT cells was significantly lower than that inthe model group (P<0.01).5The effect of therapeutic of Con A-induced liver injury by prophylacticthe36amino acid polypeptide fragments.The serum ALT and AST levels of polypeptide fragments treated group anddexamethasone treated group were significantly lower than those in the modelgroup (P<0.01), HE staining showed no obvious hepatocytes necrosis area.After injecting the lethal dose Con A to induce liver injury, given thepolypeptide fragments on the mice, the survival rate and median survivaltime were50%and58hours; after dexamethasone treatment, mice survivalrate and median survival time were60%and72hours. Both of the groups ofsurvival rates and the median survival time were significantly higher thanthose in the model group (20%,13hours, P<0.026, P<0.011). It explainsthattherapeutic administration can improve the survival rate and median survivaltime.Conclusions:1Exogenous injected36amino acid polypeptide fragment can be effective in the prevention and treatment of Con A induced hepatitis.2The mechanism of exogenous injected36amino acid peptide fragmentsalleviated Con A induced liver injury may relate to the inhibition apoptotic ofhepatocytes and mitosis.