The Intestinal Endotoxemia In The Pathogenesis Of NAFLD, T2DM And Its Molecular Mechanisms

In recent years, with the change of people's eating and living habits, fat,sugar and other high calorie foods gradually takes an increasingly important role in their diet. The number of people suffering from overly-fat, fatty liver, diabetes, high blood pressure and cardiovascular disease is also rising year after year. In this circumstance, NAFLD as a symptom of Metabolic Syndrome in liver, is no doubt attracting more and more attention. Our experiments find that the rat injected with small dose of LPS will suffer from IR and NASH in the9th week. Combined with the Cani application in high fat diet can cause metabolic endotoxemia, and can lead to obesity and insulin resistance, This finding raises a possibility that endotoxemia may be a cause of chronic low-grade inflammation and IR and therefore serve as a trigger of NAFLD and T2DM.The research is composed of three sections:Section one The animal model of Metabolic syndrome and its related diseasesSection two The effect of IETM in the development of MS and related diseaseSection there The basic mechanisms of IETM in starting MS related diseaseSection one The animal model of Metabolic syndrome and its related diseasesThere are five experiments in this section.Experiment One The changes of body weight and visceral fat weight in rat.64rats were randomly divided into eight groups. which were normal groups of3months(3C),6months(6C),9months(9C) and12months(12C), and high-sucrose and high-fat groups of3months(3H),6months(6H),9months(9H) and12months(12H) respectively. The rats of high-sucrose and high-fat groups was fed with high sugar and high fat food (70%normal fodder plus20%Lard plus10%table sugar plus1%Cholesterol plus0.25％Cholic acid). Animals in all the above groups are fed before the dusk and water is provides all the time. These animals are weighed monthly. Rats under pentobarbital anesthesia, in the corresponding normal and high-sucrose and high-fat groups at the end of the3th,6th,9th and12th month respectively to get body weight and body length; and calculate the Lee's index. And in sterile conditions,we pick liver, visceral adipose tissue (epididymal and perirenal) weighing, calculations of the liver, visceral fat and rat weight ratio. Results:Compared with normal groups, the level of weighting, Lee's index, liver index, Visceral fat percentage of body of high-sucrose and high-fat groups in rats of every phases were higer. By high-sucrose and high-fat diet feeding, it suggests us that rats occurrence of overweight and obesity.Experiment TWO The changes of plasma LPS level in rats.Rats in the corresponding normal and HSHF groups are killed at the end of the3th,6th,9th and12th month respectively to collect their blood in the aorta and test the plasma LPS level in rats.Result:Compared with normal groups, the level of LPS of high-sucrose and high-fat groups of every phases were higer. It suggests us that by high glucose and high fat feeding rats has been the formation of intestinal endotoxemia at third months and continued to increase to the twelfth months.Experiment There The changes of serum lipid and liver enzyme in rats.Rats in the corresponding normal and HSHF groups are killed at the end of the3th,6th,9th and12th month respectively to collect their blood in the aorta and test the changes of plasmaTG, Tch, HDL-c,LDL-c and ALT level in rats. Result:After one year's feeding with high sugar and fat foods, rats in the HSHF group has begun to suffer from hypertriglyceridemia, hypercholesterolemia and abnormal lipoproteins by the end of the3rd month, and the activity of their ALT increases apparently.Experiment Four The changes of FPG, FIN, HOMA-IR, HOMA-beta and glucose tolerance in ratsAt the end of the2th month, rats experienced Intraperitoneal glucose tolerance. Rats in the corresponding normal and HSHF groups are killed at the end of the3rd,6th,9th and12th month respectively to collect their blood in the aorta and test the changes of FPG,FINS,HOMA-IR and HOMA-β level in rats. Result:Compared with normal groups, the level of FPG, FINS and HOMA-IR of high-sucrose and high-fat groups of every phases were higer, but the level of HOMA-beta of6H group rats first showed the compensatory increase, then progressive decrease in sequence.Experiment Five The changes of Liver, pancreas and adipose tissue pathological and pancreatic beta cell apoptosisRats under pentobarbital anesthesia, in the corresponding normal and HSHF groups at the end of the3th,6th,9th and12th month respectively to get liver,pancreatic and visceral adipose tissue. Fixed in10% neutral formalin solution, Paraffin embedded sections, HE staining. Pathological changes were observed under light microscope. Application of TUNEL in pancreatic islet beta cell apoptosis and liver cell apoptosis. Result:Histopathology revealed serious fatty degeneration of liver, fatty liver and the onset of hepatitis, fibrosis and cirrhosis. Along with the time progress, in islet gradually atrophic and accompanied by inflammatory infiltration. Fat cells gradually increased and with inflammatory infiltration. Compared with normal groups, TUNEL results show that the number of islets cell apoptosis and liver cells of high-sucrose and high-fat groups gradual increasing from march to december.Integrated the first part of the experiment results are summarized as follows:①High glucose and high fat feeding rats appeared to main components of metabolic syndrome, such as obesity and visceral fat increase, dyslipoproteinemia, impaired glucose tolerance and elevated fasting glucose.②After one year's feeding with high sugar and fat foods, rat's liver experienced serious hepatic steatosis, fatty liver and the onset of hepatitis, fibrosis and cirrhosis, consistenting with the whole process of NAFLD.③At the early of one year's feeding with high sugar and fat foods, rats suffer from IR. At the end of one year's feeding with high sugar and fat foods, pathological observation of pancreatic atrophy. TUNEL confirmed the number of islet cell gradually reduce leads to the presence of islet cell secretion function progressive decrease, and failure to the dominant T2DM ultimately.④Nonalcoholic fatty liver disease and type Ⅱ diabetes mellitus belong to the MS related diseases. This group of diseases whose nature is chronic low-grade inflammatory, their common underlying pathogenesis is insulin resistance. After one year's feeding with high sugar and fat foods, rats appear to chronic low-grade inflammation, and insulin resistance index was significantly increased and always maintain a high level.⑤Although the experiment time is too long, but consistent with clinical practice. And the animal model experiences the gradual process of in-depth understanding of the pathological. So it is the study of metabolic syndrome and its related diseases successful animal model.Section Two The effect of IETM in the development of MS and related diseaseExperiment Six IETM induce Chronic low-grade inflammationRats in the corresponding normal and HSHF groups are killed at the end of the3th,6th,9th and12th month respectively to collect their blood in the aorta and test the changes of LPS, TNFα, CRP and MCP-1level in rats. The expression of CD68protein were detected by immunohistochemistry for observing mononuclear cells expression in fat, liver and pancreatic.Result:Compared with normal groups, the level of LPS,TNFα,CRP and MCP-1level of high-sucrose and high-fat groups of every phases were hige, and continued continued to increase to the twelfth months. Immunohistochemistry showed that mononuclear cells expression in fat,liver and pancreatic of high-sucrose and high-fat groups gradual increasing from march to december.Experiment Seven IETM and insulin resistanceDetection of the various stages of rat liver TG content. And analysis the correlation of liver TG and IR, LPS and IR. Results:all stages of liver TG content increased significantly, and the liver TG. LPS showed a positive correlation with IR.Experiment Eight Ectopic lipid depositionDetection of the various stages of rat plasma contents of FFA. And observation of fatty deposition in liver, pancreas by the Sultan IV staining. Results:After a year of high glucose and high fat feeding, at the end of third month, the FFA level of the rats in the model groupt has increased significantly, to a peak in June and continued to rise to twelfth months, liver, pancreas tissue can be visibled varying degree of fat deposition.Integrated the second part of the experiment results are summarized as follows:①High glucose and high fat induced nonalcoholic fatty liver disease in the presence of intestinal endotoxemia, and continue to the emergence of T2DM.②IETM can induce chronic low-grade inflammation reaction.③By lipolysis, IETM can promote ectopic lipid deposition in liver and pancreas, therefore it can affect and determine the occurrence of IR.Section there The basic mechanisms of IETM in starting MS related diseaseExperiment Nine Oxidative stressDetection of various stages of rat plasma SOD, MDA levels and observation the change of ROS in the liver, pancreas. Results:the plasma SOD level was significantly lower in model rats, and MDA levels were significantly higer in model rat. LPS was negatively correlated with SOD, LPS was positively correlated with MDA. At the third months, the content of ROS in Liver and pancreas were elevated. At the sixth months, the content of ROS in Liver reached its peak, and the content of ROS in pancreatic remained persistently elevated to experiment the twelfth months. These results suggest the presence of oxidative stress.Experiment Ten Endoplasmic reticulum stressThe expression JNK1,p-JNKl,NF-KBp65,I-κB and GRP78in liver and pancreatic were analyzed by Western blot. Results:Compared with normal groups, the expression of p-JNK1, NF-KBp65and GRP78in liver were increased from march to december, but the expression of I-κB in liver was reduced; the expression of p-JNK1,NF-KBp65in pancreatic were increased from june to december, but the expression of I-κB in pancreatic reduced. However the expression of GRP78in pancreatic were increased from march to December. These results suggest the occurrence of endoplasmic reticulum stress in the liver and pancreas.Integrated the third part of the experiment results are summarized as follows:①The plasma MDA level of rats in model group were increased and The plasma SOD level of rats in model group were reduced. And LPS was negatively correlated with SOD, LPS was positively correlated with MDA. At the same time, ROS levels in liver and pancreas were increased in different degrees, further supporting that LPS involved in the occurrence and development of. NAFLD, T2DM and other MS related diseases by inducing oxidative stress.②Elevated levels of LPS in the blood was agreed with the levels of P-JNK, NF kappa B and GRP78in liver and pancreatic. LPS may induce endoplasmic reticulum stress through the infiltrating mononuclear phagocytes in the liver and islet. Therefore the GRP78from the transmembrane protein loss into the blood, p-JNK and NF kappa B signal protein expression increased.Sum up, this study reached the following conclusions:1. Fed with high glucose and high fat diet, rats can replicate the animal model of the metabolic syndrome and its related diseases, which is closer to the real situation of human clinical case.2. Nonalcoholic fatty liver disease induced high glucose and high fat diet accompanied with intestinal endotoxemia, and it has continued for over a year.3. IETM can induce chronic low-grade inflammation n insulin resistance and ectopic lipid deposition caused by lipolysis, so the liver cells experienced Lipid accumulation inflammation and fibrosis. so the pancreatic islet beta cell experienced IR, inflammation, reduced insulin secretion and islet cell apoptosis.4. LPS induced oxidative stress and endoplasmic reticulum stress may be the underlying mechanism of strating NAFLD and T2DM.