The Protective Effect Of 36 Amino Acid Polypeptide Fragments To High Fat Diet Accompany With CCl₄ Induced Non Alcohol Fatty Liver Disease And Its Mechanism

Objective:Changes in modern diets is contributing to an increase in the prevalence of fatty liver disease, also known as “disease of the wealthy”. The disease(non-alcoholic fatty liver disease NAFLD) which is the second-most prevalent liver disease after viral hepatitis, is a serious health risk to humans. There are numerous causes of fat liver disease that result from pathological changes mainly concentrated in the hepatic lobule. NAFLD is one of the most common diffuse liver disease, sampled with hepatic cellular fat vacuoles as the main pathological characteristics, which can even lead to liver fibrosis, liver cirrhosis or failure. Consequently, it is important for early diagnosis and treatment of the disease. Nonalcoholic fatty liver disease is usually treated without drug therapy or drug treatment. The former apply to those who experience mild symptoms and therapeutic procedures include, physical exercise, weight loss, diet, etc. However, moderate and severe fatty liver disease patients cannot effectively eliminate liver fat without drug treatment. Most lipid-lowering drugs have limited fat elimination ability and they can also lead to different degrees of liver cell damage. Therefore, the status of lipid-lowering drugs in the treatment of fatty liver disease is currently under scrutiny.In a previous study, we found that 36 amino acid polypeptide fragments is a higher content polypeptide detected in the serum from chronic hepatitis patients. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer was used for early clinical diagnosis of hepatitis biomarkers(MALDI-TOF MS). The preliminary study found that high expression of polypeptide fragments could promote proliferation of cultured hepatic cells.To date, no study has been conducted on the pathological and physiological functions of polypeptides in chronic fatty liver disease in an animal model. The purpose of this experiment is to investigate the role of polypeptides in the progression of chronic fatty liver disease. In this experiment we use high fat diet accompanied with CCl4 to induce chronic liver injury model in rats in order to study the function of 36 amino acid fragment and also to explore the action mechanism occuring at the same time. Lastly, to provide experimental basis for further clinical treatment of related liver diseases.Methods:1 The study is divided into seven groups as listed: model group, normal control group, western medicine, Chinese traditional medicine group, polypeptide low dose, medium dose and high dose group. After making a successful fatty liver model, the rats were treated with synthetic 36 amino acid peptide fragments. Then we detect each group with serum alanine aminotransferase(ALT), aspartate aminotransferase(AST) in serum total bilirubin(TBIL), serum total cholesterol(CHOL) and triglyceride(TG). Serological indexes were used to observe the effectiveness of peptides on high-fat diet carbon and accompanying tetrachloride induced NAFLD.2 Each liver tissue was embedded with paraffin and detected with HE staining. By semi quantitative observation in pathology, we try to find out the impaction on the treatment to the NAFLD rats in different groups.3 Liver tissue was stained specifically. The 36 amino acid fragment treatment group were put into model group, the clinical traditional Chinese medicine and western medicine treatment group and compared. Furthermore, we determined the peptides’ fragment protective effects on the NAFLD rats.4 Immunohistochemical method was used to detect each rat liver tissue with their protein expression in MCP-1. The peptide fragments were discussed about the peptides’ fragment protective effects to the NAFLD rats from protein levels, from mechanism relatively.5 Analyze the relevance between fatty degeneration degree with the protein expression conditions of MCP-1, we could draw a quantitatively conclusion which can further confirm that the 36 amino acid peptide fragments play an important role of in the protective mechanism.Results:1 The result about different experimental groups in serum levelsALT serum levels in model groups were significantly higher(P<0.05) than that in low, medium, high, western medicine, Chinese traditional medicine and normal dose groups. AST serum levels for low, medium and normal peptide dose groups were significantly lower than that of the model group(P<0.05). There was an obvious difference between medium peptide dose groups and western medicine peptide dose groups using serum AST level. Using TBIL level, low, medium and high peptide dose groups were significantly lower than that of the model group(P<0.05). Using CHOL levels, medium dose groups were significantly lower than that of the model group(P<0.01). Finally, low, medium, high, western medicine, Chinese traditional medicine, and normal peptide dose groups,(using TG level), were significantly lower than that of the model group(P < 0.01).2 The results showed that liver tissue with HE stainingBased on the pathological section and according to the universal standard to evaluate the degree of liver steatosis level, the results showed that: in normal group of rat liver tissue, the hepatic lobule structured clearly, liver cells surrounded the central veins and were orderly arranged in the portal area, fatty degeneration degree level was 0. In model group of rat liver tissue big fat vacuoles were observed with changes >75% of liver cells cavity. The degree of fat degeneration was 4 and accompanied with lymphocyte infiltration. In Low, high and the Chinese traditional medicine peptide dose groups; 50%~75% of liver cells cavity changed, the degree of fatty degeneration was 3. In medium and western medicine peptide dose groups showed that 25%~50% of liver cells cavity changed, the degree of fatty degeneration was 2.3 The results showed that liver tissue with specific stainingIn the normal control group, liver cell membranes stained using Sudan dye appeared light grey, central vein and portal area scattered black dot particles(free fat particles); in the model group, the central vein and portal area location stained flake black. In the Chinese traditional medicine, low and high dose groups, the central vein area and liver parenchyma were stained as bulk distribution fat particles, compared with model group decreased; middle dose peptides group and the western medicine group show the results that central vein area had a significant reduction in the liver parenchyma parts compared with model group.4 MCP-1protein expression conditions in different liver tissueChinese traditional medicine treatment group, western medicine treatment group and 36 amino acid peptide fragments of three dose groups expressed lower immunohistochemical total scores than that in model group and normal control group,which were statistical significantly(P<0.05); Among Chinese traditional medicine treatment group, western medicine treatment group and 36 amino acid peptide fragments of different dose groups expressed different immunohistochemical scores, but there were no statistical difference between these groups(P>0.05).5 The correlation between hepatocyte fatty degeneration degree and MCP-1protein expression conditionsThree doses of peptide fragments group, western medicine treatment group, Chinese traditional medicine group and normal control group in liver tissue of MCP-1 protein expressed significantly lower than that in model group(P<0.05), the correlation coefficient between HE stained sample fat vacuoles with MCP-1 protein expression is 0.474, P<0.05, statistically significant and the relevance is better. Hepatocyte steatosis positive cells number had decreased to some degrees, and the more the liver tissue of MCP-1 protein positive cells exist, the more the hepatocyte steatosis positive cells appears, with correlation coefficient r = 0.589, P<0.05, statistically significant.Conclusions:1 Exogenous given 36 amino acid peptide fragments can effectively treat the fatty liver rats induced by high-fat diet accompanied with carbon tetrachloride by down-regulating the ALT,AST, TBIL, CHOL and TG serum levels and reducing the fat degeneration.2 Exogenous given 36 amino acid peptide fragments can mitigate the syndrome of rats with NAFLD, which might be associated with the restraining of MCP-1 production.