Fructose Diet Induced Ietm Role In Ms And Its Related Diseases: Glycine Protection Mechanism

The part IThe protective effects of glycine on MS and intestinal mucosal barrier induced by a high-fructose diet in ratsAim:To investigate the role of intestinal endotoxemia (IETM) in metabolic syndrome (MS) induced by a high-fructose diet, and the protective effects of glycine on MS and intestinal mucosal barrier.Methods:In this study, model group rats (high-fructose diet) received 8% fructose, group of glycine intervention (high fructose and glycine diet) received 8% fructose and 1% glycine. The body weight and systolic blood pressure were measured monthly. The animals were sacrificed under deep anesthesia in 2nd month,4th month,6th month and 8th month, and blood was collected by abdominal aorta, and plasma was separated. Upper small intestine was rapidly removed and part of them was put in the 10% neutral formalin, and the remaining samples were stored at-80℃until processed for estimations.1. Evaluation the changes of plasma lipid Plasma triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and free fatty acid (FFA) levels were measured by enzyme method.2. Measurement plasma LPS, insulin, pro-inflammatory cytokines and IR The levels of plasma LPS were assay by LAL chromogenic. and plasma insulin, TNF-a and IL-10 levels were measured with ELISA kits. IR assessed using HOMA-IR, HOMA-IR= plasma glucose (mmol/L)×fasting insulin (EU/mL)/22.5.3. Histopathological detection Small intestine was stained with H&E, and pathological changes were observed by microscopic. The expression of intestinal alkaline phosphatase (AKP) was detected by Gomori's method.Results:During the experiment, all rats were in good condition. The weight gain and systolic blood pressure were significantly higher than control from 3ed month to 6th month and 3ed month respectively (P＜0.05, P＜0.01). Glycine signilicantly reduced the weight gain at 4th month and 6th month, and reduced the systolic blood pressure in 4th month to 6th month (P＜0.05,P＜0.01).1. The changes of blood lipids The levels plasma TG, TC and FFA were increased significantly (P＜0.01, P＜0.05), but HDL-C was decreased significantly (P＜0.01, P＜0.05). Glycine significantly reduced the levels of plasma TG all the time, significantly decreased the levels of plasma FFA in model group at 6th month and 8th month (P＜0.01, P＜0.05).2. The changes of plasma LPS, insulin, pro-inflammatory cytokines and IR The levels of plasma LPS, insulin and HOMA-IR were increased significantly (P＜0.01) after 2nd month, and plasma TNF-αand IL-6 levels were increased significantly at 4th month and 8th month (P＜0.01). Glycine significantly decreased the plasma LPS, insulin, HOMA-IR and pro-inflammatory cytokine levels of model group (P＜0.05, P＜0.01).3. Correlation of plasma LPS and HOMA-IR, FFA Plasma LPS was positively correlated with HOMA-IR (rs=0.76, P=0.0164) and FFA(rs=0.65, P=0.0001).4. Histopathology resultsMucosal structure of model group rats was damaged, and AKP-positive reactions were weakened, and the changes were time-dependent. Intervention with glycine significantly ameliorated the impairment of the intestinal wall and the expression of AKP in model group.Conclusion:1. High-fructose diet induces model group rats MS, which shows obesity, glucose intolerance, dyslipidemia, hypertension. The mechanism may be closely related to IR (especially IR in liver) induced by accompanying IETM. Glycine could improve IR, inflammation by reducing endotoxemia of model rats, and play a role in the protective effects of MS.2. High-fructose diet induces model rats IETM, which shows destruction of the intestinal wall structure, increase of permeability and decrease of expression of APK in small intestine. Glycine could improve IETM by protecting the intestinal wall structure and increase of the intestine APK expression. The partⅡThe roles of IETM in MS-related diseases induced by a high-fructose diet and the protective effects of glycineAim:To investigate the roles of IETM in metabolic syndrome-related diseases (NAFLD, T2DM and metabolic hypertension) induced by a high-fructose diet, and the protective effects of glycine on them.Methods:In this study, model group rats (high-fructose diet) received 8% fructose, group of glycine intervention (high fructose and glycine diet) received 8% fructose and 1% glycine. The systolic blood pressure was measured monthly. The animals were sacrificed under deep anesthesia in 4th month and 8th month, and blood was collected by abdominal aorta and plasma was separated. Liver, pancreas, thoracic aorta and abdominal adipose tissue were rapidly removed, and parts of them were put in the 10% neutral formalin, and the remaining samples were stored at-80℃until processed for estimations.2. Evaluation of the plasma parametersPlasma glucose, FFA and NO was measured by enzyme method. Plasma insulin, TNF-α, IL-6, ET-1 and ANGⅡwere detected by ELISA kits. Insulin resistance index (HOMA-IR) was calculate, HOMA-IR=fasting glucose (mmol/L)×fasting insulin (EU/mL)/22.5, HOMA-β=20×fasting insulin (EU/mL)/[fasting glucose (mmol/L)-3.5]. Plasma LPS was assay by LAL chromogenic, and the correlation of LPS and HOMA-IR, FFA were analyzed.2. Histopathological detectionThe tissue of liver, pancreas and thoracic aorta were stained with H&E, and pathological changes were observed by microscopic.3. Immunohistochemical detectionThe expression of liver CD68 and vascular eNOS were detected by immunohistochemistry with LP method and two-step method respectively. Taking five sections each slices, the average number of positive cells were analyze with IPP6.0 software.4. Detection ofβ-cell apoptosis The apoptosis of pancreatic cells were detected using in situ hybridization by two-step method. Taking five slices each group, cell apoptosis was analysis using IPP6.0 software.5. RT-PCR detectionThe expression of vascular ET-AR mRNA and adipose angiotensinogen mRNA were analyzed by RT-PCR, obtaining the standard curve, melting curve and the relative quantification of samples according to-2ΔΔCt method.Results:During the experiment, all rats were in good condition. The systolic blood pressure was significantly higher than control from 3ed month to 8th month (P＜0.05, P＜0.01). Glycine significantly reduced the elevated systolic blood pressure in 4th month to 6th month (P＜0.05, P＜0.01)1. Changes of the plasma parametersCompared with control group, the levels of plasma insulin, TNF-α, IL-6, ET-1, HOMA-IR and ANG II were significantly increased (P＜0.01, P＜0.05), the levels of plasma NO were significantly decreased (P＜0.01). But the changes were no significant difference in different stage. Glycine significantly decreased the plasma insulin, TNF-αand IL-6, ET-1, HOMA-IR, LPS levels of model group in 4th month and 8th month (P＜0.05, P＜0.01), and significantly decreased the levels of plasma ANGⅡof model group in 6th month and 8th month (P＜0.05, P＜0.01), and significantly increased plasma NO levels in 4th month (P＜0.01). Plasma LPS was positively correlated with HOMA-IR (rs=0.76, P=0.0164) and FFA(rs=0.65, P=0.0001).2. Histopathological resultsThe results showed that fatty liver was occurred in 2nd month, which was developed into NASH in 8th month. With the time extension of high-fructose diet, the pancreatic islet was hypertrophy and hyperplasia (in 8th month). Intervention with glycine at different times significantly improved the impairment of pathological tissue in model group. Compared with the control group, there was a mild proliferation of vascular smooth muscle in model group and the intervention group.3. Immunohistochemical resultsThe expression of hepatic CD68-positive cells of model group were significantly higher then control group at 4th month and 8th month (P＜0.05, P＜0.01), and the number of positive cells were increased with time prolonged. Compared with control group, the expression of positive eNOS was significantly decreased (P＜0.01), and the changes were more obvious in 8th month. Glycine significantly reduced the CD68 expression of model group in 4th month and 8th month (P＜0.05, P＜0.01), and significantly increaced the eNOS expression of model group in 4th month (P＜0.05). 4. The results of RT-PCRPCR results showed that ETA-R mRNA expression of model group was significantly increased in 4th month and 8th month (P＜0.01). and the angiotensinogen mRNA expression was significantly increased in 4th month (P＜0.01). Glycine significantly reduced the expression of ETA-R mRNA and angiotensinogen mRNA in model group (P＜0.01).Conclusion:1. High-fructose diet induced rats MS-related diseases (NAFLD, T2DM and metabolic hypertension). IR and chronic low-grade inflammation induced by IETM are common pathophysiological of these diseases.2. High-fructose diet induced MS-related NAFLD, T2DM and metabolic hypertension, which accompanies with the IETM, inflammation and IR/hyperinsulinemia. IETM involved in IR by initiating inflammatory mechanisms.3. Glycine could play a role in the protective effects of MS-related metabolic diseases (NAFLD, T2DM and metabolic hypertension) by reducing the IETM, inflammation and IR. The partⅢThe roles of IETM in AD-like lesions induced by a high fructose diet and the protective effects of glycineAim:To investigate the roles of IETM in Alzheimer Disease (AD) -lik lesions induced by a high-fructose diet, and the effects of IETM on cognitive impairment, expression of Aβ1-40 and p-tauSer262 and SP in brain, and the protective mechanisms of glycine on them.Methods:In this study, model group rats (high-fructose diet) received 8% fructose, group of glycine intervention (high fructose and glycine diet) received 8% fructose and 1% glycine. The animals were sacrificed under deep anesthesia in 8th month, and blood was collected by abdominal aorta and plasma was separated. Brains were rapidly removed and the cerebral cortex was isolated, and parts of them were put in the 10% neutral formalin, and the remaining samples were stored at -80℃until processed for estimations.1. Evaluation of the plasma parameterThe levels of plasma TG, HDL-C. TC, FFA and glucose were measured by enzyme method. Plasma insulin was detected by ELISA kit, and plasma LPS was detected by LAL chromogenic method. IR assessed using HOMA-IR, HOMA-IR=fasting plasma glucose (mmol/L)×fasting insulin (EU/mL)/22.5.2. Evaluation of pro-inflammatory cytokinesTNF-αand IL-6 of plasma and brain tissue (10% of cerebral cortex homogenate) were detected by ELISA method.3. Measurement of Aβ1-40The levels of amyloid beta-peptide (Aβ1-40) in plasma and brain tissue (10% of cerebral cortex homogenate) were detected using with ELLISA kit.5. Histopathological detectionBrain tissue was stained with H&E, SP was detected by modified Bielschowsky'method.6. Cognitive function testCognitive function test was tested by Morris water maze.7. Western blot Proteins of insulin signal transduction and p-tau protein were analysis by Western blot, which including insulin receptor criticalβsubunit (IRβ), phosphatidylinositol 3-kinase a subunit (P13-ka) and phosphorylated proteins.Results:During the experiment, all rats were in good condition.1. The changes of blood lipids The levels plasma TG, TC and FFA were increased significantly (P＜0.01, P＜0.05), HDL-C were decreased significantly of model group (P＜0.01, P＜0.05). Glycine significantly decreased the levels of plasma TG and FFA of model group (P＜0.01, P＜0.05).2. The changes of pro-inflammatory cytokinesThe levels of TNF-αand IL-6 in plasma and brain tissue were increased significantly of model group (P＜0.01). Glycine was significantly decreased pro-inflammatory cytokine levels in plasma and brain tissue of model group (P＜0.05).3. The changes of Aβ1-40The levels of Aβ1-40 in plasma and brain tissue of model group were significantly higher then control group (P＜0.01). Glycine decreased the Aβ1-40 levels in plasma and brain of model group, but the changes were not statistically significant.4. Histopathology resultsCells degeneration and nuclear concentration were noted in CA4 region of model group. The results of staining by Bielschowsky showed that SP were appeared in deep cerebral in part of the model group rats, but the numbers and size were not rules. After intervention of glycine, there were still some SP in deep cerebral, but the numbers were reduced.4. The results of Morris water mazeThe mean escape latency of the model group was significantly longer than that of the control group (P＜0.01). Glycine significantly shortened the mean escape latency of model group in the 4th and 5th day (P＜0.05). In the probe trial experiments, the time spent in the target quadrant was significantly lower in model group as compared to control group (p＜0.01). Glycine significantly improved the time of model group (P＜0.05).6. The results of Western blotThe ratios of p-IRpTyr1162/1163/IRβand p-PI3-kp85aTyr508/PI3-kp85 were increased, and the expression p-tauSer262 was decreased in cortex of model group (P＜0.01). Glycine significantly increased the ratios of the two proteins (P＜0.01), and significantly decreased the expression p-tauSer262 (P＜0.05).Conclusion:Long-term high fructose diet induced AD-like lesion in rats, manifestations of cognitive impairment, increase expression of Aβ1-40 and p-tauSer262,occurrence SP in the brain tissue, which characterized with IR and inflammation in the central and peripheral tissue. Glycine plays a role in the protective effects of them by reducing the IETM, inflammation and IR.