Study Of SAD Induced By IETM Which Caused By High Sugar And High Fat Diet

The partⅠThe high sugar and high fat diet-induced IETM in rats occured metabolic syndromeObjective:Explore the role of intestinal endotoxemia(IETM) in metabolic syndrome induced by high sugar and high fat diet in rats.Methods:After one week’s adaptive feeding, 48 male SD rats were divided into two groups randomly(24 rats in each group), one was normal group which fed with normal food and the other was model group which fed with high sugar and high fat food(include 70%normal fodder, 20% Lard, 10% tablesugar, 1% Cholesterol, 0.25% Cholicacid). Both normal and HSHF groups were randomly divided into three subgroups(8 rats in each subgroup): 6 month, 9 month, 12 month, respectively. The animals were pentobarbital anesthesia in 6th month, 9th month and 12 th month to get body weight and body length,then the blood was collected by abdominal aorta and separated. And the visceral adipose tissue from epididymal and perirenal was weighed in sterile condition. Then the brain was obtained and the hippocampus and cortex were separated quickly in ice after being decapitated. One half of the tissue from hippocampus and cortex was put into the 10%neutral formalin to be fixed, and others were stored at-80°C after being frozen in liquid nitrogen.1. Detection of the metabolic syndrome related indicators in plasma Plasma triglyceride(TG), total cholesterol(TCH), diacylglycerol(DAG), high-density lipoprotein(HDL), low-density lipoprotein(LDL), free fatty acid(FFA),fasting plasm glucose(FPG) and fasting plasm insulin(FIN) levels were measured by using enzyme method. The index of IR was calculated by using the formula HOMA-IR=FPG(mmol/L) × FIN(EU/m L) / 22.5.2. Detection of the inflammation cytokines in plasma The levels of plasma LPS were assayed by using chromogenic assay, and the levels of TNF-α, IL-1 and IL-6 in plasma were measured with radiation immunization analysis.Results:During the experiment, all rats in normal and model groups were in good condition.The weight of rats in HSHF group were significantly higher than that in the normal group at the different time stages(P<0.05). The percentage of visceral fat in body weight was also increased significantly and the level of the Lee’s index was higher in the HSHF group(P<0.05, P <0.01), which indicated the rats of HSHF occurred obesity.1. The changes of the metabolic syndrome related indicators The levels TG, TCH, DAG, LDL, FFA, FPG, FIN and HOMA-IR in plasma were higher in the HSHF group than those in the normal group(P<0.05), but HDL was decreased obviously in the HSHF group(P<0.05). It is suggested that the disorder of glucose metabolism and abnormal lipoprotein were induced by high glucose and high fat diet in rats.2. The changes of pro-inflammatory cytokines in plasma In the HSHF group, the level of plasma LPS was increased significantly in 6th month(P<0.001) and which were always in high level in the whole experiment. The levels of plasma TNF-α, IL-1 and IL-6 in HSHF group were higher significantly at different stages than that in normal group(P<0.05).Conclusion:1. High sugar and high fat diet induced obesity, abnormal glucose tolerance and abnormal lipid metabolism in rats, which suggested that the animals were suffered from metabolic syndrome.2. IETM induced by high sugar and high fat diet caused the chronic low grade inflammation, lipid accumulation and IR, those may be the basis of MS.The part ⅡStudy the roles of IETM in the development of MS related diseases ADObjective:Investigate if IETM induced by high sugar high fat could cause AD and its role in the process.Methods:The methods of animal grouping and handling in this part were the same as the methods in the part I.1. Detection of the levels of AD marker proteins in brain tissue The levels of Aβ in hippocampus and cortex were tested by ELISA, and the p-Tau protein in hippocampus was assayed by using western blot method.2. Histopathological detection of brain tissue Brain tissue hippocampus and cortex were stained with H&E, and the pathological changes were detected by microscopic observation.3. Detection of the AD related genes’ expression Detection the m RNA levels of IDE, APOE4 and BACE in brain tissue by RT-PCR.4. Detection of the inflammation related indexes in brain tissues The levels of diacylglycerol(DAG) and free fatty acid(FFA) in brain tissue homogenate were measured by ELISA. TNF-α, IL-1 and IL-6 levels in brain tissue homogenate were measured using radiation immunization analysis. The levels of LPS and MCP-1 in plasma were assayed. And CD68 and OX-42 in brain were detected by immunohistochemistry. CD68 and OX-42 immunoreactive cells were analyzed using IPP6.0 software.5. Detection of insulin resistance related proteins in brain Insulin resistance related proteins JNK, PI-3K, AKT, GLUT, GSK-3β and phosphorylation levels in hippocampus were tested by western blot.6. Detection of cell apoptosis in brain The apoptosis of brain cells was detected by TUNEL. Take 5 slices in each group,and choose 5 fields to view in each slices, IPP6.0 software was used to analyze the cell apoptotic rate.Results:1. The changes of AD marker proteins in brain tissue P-Tau199 was increased at 6 month and continued to 12 month in the HSHF group;P-Tau396 was not significantly increased in 6month, but was increased significantly in9 month and 12month; Expression of P-Tau212 was gradually increased with time pass over in the HSHF group compared with that in the NC group. Aβ levels in rat cortex and hippocampus were significantly increased at all stages in the HSHF group compared with those in the NC group(P< 0.05).2. Histopathology results in the brain tissue The cerebral cortex and hippocampus were infiltrated by a little amount of inflammatory cells with the proliferation of microglia and lymphocytes in the 6month,and it was gradually increased over time, and inflammatory cells infiltration was most obviously in 12 month.3. The changes of AD related genes The expression of IDE m RNA in hippocampus was decreased gradually in the HSHF group with the passage of time, but the expressions of BACE and APOE4 m RNA were significantly higher than those in the NC group(P< 0.05).4. The changes of inflammation related indexes in the brain tissue The levels of DAG, FFA, TNF-α, IL-1 and IL-6 in brain tissue were increased significantly in the HSHF group(P<0.001, P<0.05). The levels of LPS and MCP-1 in plasma were higher in the HSHF group than those in the NC group(P<0.01).Immunohistochemical results showed that the expression of CD68 was increased obviously in each stage. The microglia was activated and the activation degree increased gradually over time.5. The changes of IR related proteins in brain The levels of p-JNK, p-PI-3K, p-AKT and GLUT in hippocampus were significantly decreased compared with those in the NC group at each stage, but GSK-3βwere increased as time goes on.6. Cell apoptosis in brain The apoptosis results showed that with the time extension of high sugar and high fat diet feeding, a large number of cell apoptosis appeared in the HSHF group, the difference was significantly compared with that in the NC group.Conclusion:1. MS that was induced by high sugar and high fat diet in the model group was always accompanied with IETM, and the characteristic change of AD was showed in the brain tissue. The animal model of MS accompanied with AD was successfully constructed.2. IETM can cause inflammation and insulin resistance in peripheral as well as in brain.3. The occurrence of AD is closely related to central inflammation and insulin resistance.The part ⅢThe basic mechanism of IETM initiated MS related disease ADObjective:To discuss the basic mechanism of MS related disease AD that was initiated by IETM.Methods:The methods of animal grouping and handling in this part were the same as the methods in the part I.1. Detection of oxidative stress related factors in plasma.The level of Maleic Dialdehyde(MDA) in plasma was tested by ELISA.2. Detection of the reactive oxygen species in the brain tissue The level of reactive oxygen species in the brain tissue was detected by immunofluorescence and was observed under the fluorescence microscope.3. Detection of the endoplasmic reticulum stress related proteins The levels of GRP78 and CHOP in hippocampus were tested by using western blot.4. Detection of DAG and FFA in plasma.The levels of DAG and FFA in brain were tested by using ELISA.Results:1. The MDA level in plasma in the HSHF group was significantly higher than that in the control group. And ROS in brain tissue increased significantly, the increase of ROS is more obviously with the passage of time.2. The WB results showed that GRP78 was gradually increased in 6 month and 9month, reached the maximum in 9 month, and was decreased in 12 month but still higher in the HSHF group than that in the NC group. There was almost no expression of CHOP in the NC group, but it was gradually increased over time in the HSHF group and reached the maximum value in 12 month. The levels of DAG and FFA in the brain tissue homogenate were increased significantly in the HSHF group than those in NC group.Conclusion:1. Oxidative stress and endoplasmic reticulum stress induced by IETM may be the basic mechanism of all the metabolic diseases.2. The mechanism of AD is consistent with that of MS, and it can be considered that AD also belongs to MS related disease.Sum up, this study reached the following conclusions:1. Rats fed with high sugar and high fat diet can accompanied with the IETM, and the degree of IETM was gradually increase; IETM can lead to the occurrence of MS related diseases.2. Chronic low-grade inflammation, lipid accumulation and IR induced by IETM were the common mechanism of the MS and AD, It can be considered that AD is also a MS related disease.3. Oxidative stress and endoplasmic reticulum stress induced by IETM may be the basic mechanism of all metabolic diseases.