The Studies About The Role Of Metabolic Endotoxemia In Fructose Induced NAFLD Metabolic Diseases

Objective:1 To establish the animal model of NAFLD and other metabolic diseases together with metabolic endotoxemia induced by fructose diet in rats.2 Through LPS simulation of metabolic endotoxemia. to investigate the mechanism role of metabolic endotoxemia in metabolic diseases.Methods:Normal Control group(Normal Control, NC group) gives rats normal diet. In High Fructose Diet group(High Fructose Diet, HFD group), rats are feeding with 8% fructose water. In Lipopolysaccharide group(LPS group), rats are given normal diet(300 μg?kg-1?d-1 subcutaneous injection). The body of weight were measured every weekly. After eight weeks and 12 hours of fasting, when the glucose tolerance test ends, after ether anesthesia, take abdominal aortic blood and 3500r/min centrifugation for 10 minutes, then take the upper plasma and fast subpackage them in-40℃ for backup. Isolate liver tissue rapidly, then place part of them put in 4% paraformaldehyde solution and fix, paraffin embedded, fix, then cut sections, staining with hematoxylin eosin(HE staining); Part of OCT are embedded in-80℃ for preservation, and then make frozen sections(oil red O staining); The remaining samples are placed in liquid nitrogen for quick-frozen, then place them under-80℃ cryopreservation, available for other experiment use.1.The detection of related indexes: blood lipid series, liver enzyme and plasmaUse enzymic method to detect the plasma level change of TG, TC, FFA and HDL in every group; Use enzymic method to detect quantitatively the liver tissue TG. Enzymicmethod to detect fasting plasma glucose(FPG, Fasting plasma glucose); ELISA method for the detection of serum fasting insulin(FINS, Fasting insulin) level, and calculate insulin resistance index, the formula is: HOMA-IR= fasting plasma glucose(mmol/L)* fasting insulin(EU/ml)/22.5.2.The detection of plasma endotoxin and proinflammatory factorsUse limulus reagent chromogenic method to detect plasma endotoxin(LPS) level change; ELISA method for the detection of TNF-α and IL-6 level change in serum, and analyze the correlation between LPS and HOMA-IR.3.Pathological examination of liver tissueTake liver tissue out from 4% paraformaldehyde solution, routine paraffin-embedded sections, 5μm in thickness, stained with H&E, under light microscopic to observe pathological changes of liver tissue; take out OCT embedded tissue in-80℃and place in the temperature of-25℃freezing microtome to rewarming 15 minutes, transecting tissue 8μm, with oil red O staining, observe red lipid of liver tissue in each group under light microscope.4. Insulin receptor substrate 1 and Toll receptor 4 protein expression detectionUse western blot to detect the key protein IRS1 and its phosphorylated protein in liver tissue’s insulin signal transduction, and at the same time to detect LPS receptor TLR4 protein level.Results:During the experiment, the rats in each group are in good condition, and there are a few set off phenomenon. The body weight change of rats in HFD group and LPS group is significantly higher than that of NC group(P<0.01, P<0.05) in two to eight weeks. At the end of 8 weeks’ experiment, rats are fasted for 12 hours and given a gavage of 50% glucose solution(1.5g/kg), respectively collect blood samples from tail vein before gavage(0 min) and after gavag(30 min, 60 min and 120 min), then use the fast blood glucose meter(ROCHE ACCU-CHEK Active) to detect blood glucose levels. Comparedwith NC group, the blood glucose of HFD group and LPS group at each time point are significantly increased(P< 0.01, P< 0.05). The result shows that rats of HFD group and LPS group have abnormal glucose tolerance.1.Test results of blood lipids and liver enzymeThe TG(Triglyceride, plasma and liver quantitative), TC(Total cholesterol), ALT(Alanine aminotransferase) and FFA(Free fatty acid) levels of HFD group and LPS group are significantly higher than those in NC group(P<0.01, P<0.05), while HDL level is significantly decreased(P<0.05). Compared with LPS group, the change of HFD group has no statistical significance.2.The detection of plasma endotoxin, insulin and proinflammatory factorThe plasma LPS(Lipopolysaccharides), FINS(Fasting insulin), FPG(Fasting plasm glucose)and HOMA-IR(homeostasis model assessment of IR) in HFD group and LPS group are significantly higher than those of NC group(P<0.05, P<0.01). The levels of TNF-α and IL-6( tumor necrosis factor–α,TNF-α; interleukin-6,IL-6) in HFD group and LPS group are significantly higher than those of NC group(P<0.05). The difference of HFD group and LPS group has no statistical significance. LPS levels of each group are positively correlated with HOMA-IR(rs=0.85, P<0.01).3.The pathology result of liver tissueThe liver in NC group looks dark red and has no greasy feeling. It is stained with H&E, and the liver cells are centered around the central vein and radiate out to the surrounding. The liver in HFD group and LPS group looks white in appearance, and the section looks obvious greasy. The tissue portal area occurs lipid droplets vacuoles, accompanied with ballooning degeneration. At the same time, hepatic lobule and portal area of LPS group show inflammatory cell infiltration, part of them are accompanied with spotty necrosis. Oil red O staining shows that HFD group and LPS group have a large number of red lipid.4.The results of western blotThe liver tissue TLR4 expression in HFD group and LPS group has increased significantly(P<0.01), and the ratio of IRS1 and p-IRS1Tyr632 /IRS1 has significantlydecreased(P<0.05), and there is no statistical significance between HFD group and LPS group.Conclusion:1. High fructose diet can induce rat to infect with various metabolic diseases, such as NAFLD. These diseases have the following symptoms: weight increase(abdominal obesity), abnormal blood lipid and liver enzyme, glucose tolerance is damaged, IR, fatty degeneration of liver tissue, pro-inflammatory cytokines are released, insulin signaling pathway is impaired(insulin receptor substrate expression decreases), and exists metabolic endotoxemia. The pathogenesis characteristics are very similar to the human disease.2. High fructose diet induces LPS in metabolic diseases to combine with TLR4, triggering the inflammatory factors expression increases, liver and peripheral occur insulin resistance, and promote the occurrence and development of NAFLD and other metabolic diseases.