| Objective: Liver disease is one of the most crucial global public hygiene problems, the long-term liver injury without prompt and efficient treatment will lead to hepatic failure or liver cancer, and also decrease the living quality of patients or even threaten their lives. With the improvement of the society and human being’s living quality, fatty liver disease has become the second high-incident liver disease, which is nearly as severe as the viral liver disease and has started to threaten human being’s health. The most possible reasons behind such phenomenon may refer to the rapid modern living pace and bad diet habits. Nonalcoholic fatty liver disease model induced by HFD and CCl4 is a relatively ideal rat model with fatty liver disease which is used to imitate the fatty liver disease caused by people’s bad diet habit.In order to seek for the biomarker of early clinical diagnosis of hepatitis, in the first period, this lab applies matrix-assisted laser desorptionion to detect a polypeptide in the serum of chronic hepatitis patients, which is sixteen amino-acid polypeptide fragments. The preliminary study finds out that this polypeptide cultivated in vitro can change the multiplication of Hep G2 cells and promote the cell into S period.Up to now, there has not been any physical and pathological study about this polypeptide’s functioning mechanisms in the chronic fatty animal’s liver model. Referring to the results of previous study of this lab, it has been found out that the polypeptide have protective effect on the liver disease of acute liver injury mice model induced by the Con A and thus presumably this polypeptide may play the similar protective effect on the development process of chronic fatty liver disease. Having applied the nonalcoholic fatty liver disease model in rats induced by HFD and CCl4, the study analyzes the effect of sixteen amino-acid polypeptide fragments in the treatment of nonalcoholic fatty liver disease and initially explores its mechanism for the purpose of providing experimental basis for the further clinical treatment.Methods:1 The effect of sixteen amino-acid polypeptide fragments on chronic fatty liver injury in rats.Firstly, divide 56 male SD rats into 7 groups randomly. In addition to the intraperitoneal injection of physiological saline and the feeding of common feed to the normal group at the appropriate time points, for the other groups, intraperitoneal injection of CCl4 are given for four weeks at 3-day intervals(3ml/kg),along with fatty feed continuously for 4 weeks. As for the 56 rats that have been divided into groups with chronic fatty liver injury, in addition to give physiological saline through tail intravenous injection at the corresponding time points of the normal group, as for the other groups, give compound glycyrrhizin diluents(120mg/kg) via lavage, Jiangzhitongluo capsules diluents(80mg/kg) or give sixteen amino-acid polypeptides whose dosages are 200, 400, 800 μg/kg through tail intravenous injection. The injection period lasts for six weeks at 3-day intervals. Once the treatment has been done, the animals are killed. Then, collect the blood of rats and separate the serum, examine the ALT, AST, TBIL, CHOL and TG level using fully automatic biochemistry analyzer. Lastly, observe the effect of peptide therapeutic drug delivery on the hepatic injury caused by high fat diet and CCl4 through serum index.2 The detection of liver histopathology.Kill the animals after therapeutic drug delivery and split the liver.Cut down the appropriate hepatic tissue and fix them from the same position separately. Then embed them with paraffin, stain HE staining after slicing them into 5μm, observe the pathological form of hepatic tissues under optical microscope and collect pictures.3 Sudan B fatty special staining of hepatic tissuesPay attention to the extraction of tissues while making frozen section, which should not be too big or too thick; the proper size is about 24×24×2mm. Staining the Sudan after slicing it into about 8μm and observe the pathological mode of the tissues, then collecting pictures.4 Measurement of the expression level of MCP-1 in hepatic tissues.Use IHCA method to examine the albumen expression level of hepatic tissue MCP-1.Manage the systemic software through multifunctional true color cell images and catch the organizational pictures collected by microscope. Choose five of the most masculine views to calculate and then draw the number of masculine cells and the color intensity of masculine cells to get the statistic of HIS. Discuss the mechanism of the peptide treatment effect on the hepatic injury induced by high fat diet and CCl4.5 Correlation analysis of the degree of steatosis and the protein expression level of MCP-1.There are some differences of the protein expression level of MCP-1 among the steatosis of hepatic cells with different degrees. We are supposed to explore the protection mechanism of the peptide on liver by analyzing the influence of the steatosis of hepatic cells with different degrees on the protein expression level of MCP-1.Such procedure depends on the correlation analysis between steatosis degree of hepatic cells observed by HE staining and the protein expression level of MCP-1.Results:1 The influence of the therapeutic drug delivery of sixteen amino-acid peptide fragments on chronic hepatic injury induced by high-fat diet and CCl4The serum ALT levels of peptide fragments 200μg/kg group, 800μg/kg group, Chinese medicine group, western medicine group and the normal group are all significantly lower than the model group(P<0.05); what’s more, the serum AST level of peptide fragment 800μg/kg group is obviously lower than the western group(P<0.05); the serum TBIL level of each group has no obvious differences; the serum CHOL levels of peptide fragments 200μg/kg group, 400μg/kg group and 800μg/kg group are all significantly lower than the western medicine group(P<0.01);the serum TG level of peptide fragments 200μg/kg group, western group, Chinese medicine group and the normal group are all significantly lower than the model group(P<0.01), the serum TG levels of peptide fragments 400μg/kg group and 800μg/kg group have no obvious differences compared with the model group.2 The pathological change of HE stainingThe pathological slice shows that: a large number of fatty vacuoles has appeared in the hepatic tissue of rats in model group. The therapeutic drug delivery of western medicine group has improved the fatty vacuole situation of hepatic cells. Also, different dosages of peptide fragments groups and the Chinese medicine groups have decreased the injury of hepatic cells induced by high-fat diet and CCl4 when compared with the model group. Moreover, it has been easily observed that there is a significant reduction in the number of fatty vacuoles of hepatic cells.3 The results of frozen slice Sudan black B stainingThe results show that: there are black dot fat particles around the central vein and portal area in the model group; the black fat particles of peptide fragment 200μg/kg groups, 400μg/kg groups,Chinese medicine groups and western medicine groups decreased compared with the model group. And there is a significant decline in 800μg/kg groups.4 The influence on the expression of albumen of hepatic tissue MCP-1 of 16 amino acid peptide fragments therapeutic drug deliveryThe results of IHC shows that: the total scores of immunohistochemistry of MCP-1 of western medicine group, Chinese medicine group, peptide fragment 200μg/kg group, 400μg/kg group, 800μg/kg group all have statistic significance when compared to the model group and the normal group, respectively.( P<0.05) While there is no statistical significance among various peptide fragments group, western medicine group and Chinese medicine group.( P>0.05)5 Results of the correlative analysis between the degree of liver steatosis and the expression levels of MCP-1 albumen.The degree of liver steatosis is positively correlated with the expression levels of MCP-1 albumen.(r value is 0.643, P<0.01). And the more the liver steatosis positive cell numbers are, the higher the positive cells of MCP-1 protein in liver tissue, the correlation coefficient is 0.566 and there is statistical significance.(r=0.566, P<0.01)Conclusions:1 Exogenous administration of sixteen amino-acid peptide fragments may be effective on nonalcoholic hepatic disease that induced by high-fat diet and CCl4.2 The mechanism of attenuating chronic liver injury which is induced by high-fat diet and CCl4 through exogenously giving sixteen amino-acid peptide may be related to the inhibition of the expression of MCP-1 in the liver tissues. |
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