| BACKGROUNDType 2 diabetes is a kind of the metabolic syndrome with complex polygenic diseases formed by the interaction between genetic and environmental factors. In recent years, the incidence rises quickly, seriously endangers human health and dramatically increase the social cost of health care. The systemic (body-wide) chronic inflammation plays an important role in insulin resistance under the present research.Lots of researches prove the gram negative bacteria cell wall component lipopolysaccharide (LPS) may be plays key roles in metabolic low degree inflammation. Metabolic endotoxemia is mainly refers to the increasing bacterial lipopolysaccharide breaks into the intestinal mucosal barrier and enter the systemic circulation, resulting the circulation LPS level increased 2-3 times than the normal value.The influence of unhealthy lifestyles such as high fat diet, make the excessive lipid accumulation in the intestinal epithelial,induce the destruction of intestinal tight junction structure, also changes intestinal microenvironment such as intestinal flora,its will trigger intestinal inflammation, damage the intestinal barrier function, intestinal permeability increased, resulting in increased LPS in blood. That the function of intestinal mucosal barrier damage is an important mechanism correlation of metabolic endotoxemia.In many factors to maintain normal mucosal barrier function, tight junction (TJ) plays an important role, it is not only the most important structure of intestinal epithelial barrier, is a rate limiting step regulating paracellular transport material. Tight junction (TJ) is mainly composed of a transmembrane protein claudin, occludin and zonula occludens protein membrane (ZO) etc., the main function is to maintain the intestinal epithelial cell polarity and regulate intestinal permeability, reduce intestinal macromolecules and microorganisms through the intestinal wall into the body inner environment.It has been proved that, regulate the intestinal flora and improve the intestinal inflammation by means such as prebiotics and probiotics can significantly reduce the high fat diet induced intestinal tight junction damage, reduce intestinal LPS enters into the internal environment, so as to ease the systemic inflammation and insulin resistance. Therefore, by reducing the intestinal permeability, reduce intestinal bacterial metabolites such as lipopolysaccharide into blood circulation is a new therapeutic strategy to improve the high fat diet induced obesity, type 2 diabetes and other metabolic diseases.Glucagon like peptide -2 (glucagon-likepeptide-2, GLP-2) is a polypeptide substance secreted by L cells in the gut, and as an intestinal specific growth factor, promote the growth of intestinal mucosa significantly stronger than other intestinal epithelial growth factor, has a strong repair function to damage the intestinal epithelium, can improve the intestinal mucosa a significant barrier. Similar to GLP-1, GLP-2 can also be enzymatic degradation of DPP-4 inactivation.Some other study has point out that the extremely important function in mediating the metabolic inflammation and insulin resistance of protease activated receptor 2 (protease activated 2 receptors, PAR2), also play a decisive role in the promotion of intestinal inflammation, inducing intestinal mucosal injury. Intestinal activated PAR2 can not only induce degranulation and activation of nuclear factor kappa NF- B mast cells, promote cell migration and release of inflammatory cytokines and inflammatory infiltration, intestinal mucosal injury, can also activate the cytosolic ERK1/2, prompted the re distribution of intestinal tight junction proteins and the surrounding actin, increase intestinal permeability.Recent studies have found that DPP-4 may act as the PAR2 agonists involved in the pathophysiology of PAR2, speculated that inhibition of DPP-4 may reduce the activation of intestinal PAR2, mediate the alleviation of damage of intestinal mucosa.The activation of DPP-4 inhibitors inhibit the intestinal hydrolysis of GLP-2 and PAR2, may protect the barrier function of intestinal mucosa in rats with type 2 diabetes, thereby reducing intestinal LPS into the blood, reduce the metabolic endotoxemia.To detect the changes of intestinal tight junction protein ZO-1, occludin expression of type 2 diabetic rats and portal venous plasma LPS concentration and it related inflammatory factors, to investigate the protective effect of DPP-4 inhibitor linagliptin on the intestinal mucosal barrier and metabolic endotoxemia and its possible mechanism, for mining the effects beyond hypoglycemic of DPP-4 enzyme inhibitor in clinical application extensions and brings the new strategyof treatment of obesity and insulin resistance.Materials and methods30 male SD SPF rats, weighing 180-200g, were randomly divided into normal control group (group NC, n=10) and model group (group DM, n=20), normal control group was fed with common diet, diabetes group were fed with high fat diet for eighth weeks, then the rats were fasted for 12 h, diabetes group rats were take the intraperitoneal injection of STZ twice (35 mg/kg weekly) to build model, normal diet group were intraperitoneally injected with equal volume of citric acid sodium citrate buffer solution. After 72 h, blood glucose (FPG), fasting insulin (FINs), insulin resistance and fasting blood glucose is greater than or equal to 11.1 mmol/L as the T2DM diagnostic criteria.The successful model rats were then divided into the model control group and the linagliptin group, the intervention group treated with linagliptin 3mg/kg/d gavage, diabetic model group and normal control group were given an equal volume of saline, continue to raise to 13 weekend. At the end of the 13 week, after the last administration, all rats were fasting overnight, record the body weight. With 10% chloral hydrate,0.35 ml/100 g intraperitoneal injection of anesthesia, liver portal vein blood was taken to EDTA-K2 anticoagulant tube and pyrogen free tube,4 degrees centigrade centrifugal (3500 r/min) 15 min, preserved at-20 C for further analysis; quickly intercept near the ileocecal valve (4cm) of the proximal colon, carefully remove the intestinal contents by PBS solution, and then frozen in liquid nitrogen, preserved at-20 C for further analysis; automatic biochemical analyzer to detect the fasting blood glucose (FPG), triglyceride (TG), total cholesterol (TChol); ELISA was used to measure plasma insulin (FIN), insulin sensitivity index (ISI), insulin resistance index (HOMA-IR).take the ISI log former into the statistical analysis; the plasma LPS levels were measured by using tachypleus amebocyte lysate; using ELISA to detect the degree of plasma tumor necrosis factor alpha, interleukin-6 and diamine oxidase; Western bloting for the detection of proximal colon tissue ZO-1, occludin, glucagon like peptide -2 and PAR2.Statistical analysisAnalysis of the data by the statistical software SPSS 19, take mapping by GraphPad Prism 6, data was showed by the form of mean ± standard deviation, make normality, homogeneity of variance test, if the data are consistent with the state, the mean diversity compared with the completely random design analysis of variance, the groups were compared using LSD test; the approximate F Welch test were used if the Equal Variance not assumed when multiple samples were compared; and the comparison between groups was made by Tamhane’s T2 method and the difference was statistically significant if P<0.05.Results1.9 weeks high-fat diet combined with twice STZ intraperitoneal injection successfully built the rat model of T2DMAt the end of ninth week, after molding,72 hours later, there are 4 rats died and the fasting blood glucose of the rest 16 rats of model group is all more than 11.1 mmol/L, besides that, the rats of model group shows significant insulin resistance, mention that the rat model built successfully. During the process,the normal group rat shows normal activity and neat hair, but the rats in the model group shows decreased activity, listlessness, polyuria and dull hair messy.2. type 2 diabetic rats shows increasing the plasma endotoxin levels, induce the metabolic endotoxemia successfullyAt the end of 13th week,after the experiment finished, compared with normal group, the fasting blood glucose, fasting plasma insulin, index, plasma triglyceride and total cholesterol of model group were significantly increased (p<0.01,p<0.05), insulin sensitivity index and body weight decreased significantly(p<0.01).the plasma endotoxin level of liver portal vein as well as tumor necrosis factor alpha and interleukin-6 was significantly increased (p<0.01) either.3. Intestinal barrier dysfunction were observed in the type 2 diabetic ratsAt the end of 13th week,after the experiment finished, model group show its decreased expression in rat proximal colon of tight junction protein ZO-1, occludin (p <0.01). Decreased expression of glucagon like peptide-2 (p<0.01); plasma amine oxidase levels and PAR2 were significantly increased (p<0.01, p<0.05);4. Linagliptin intervention were improved the glucose, body weight and insulin resistance of rats compared with the model control group.At the end of 13th week,after the experiment finished, compared with the model control group, intervention group has a lower fasting blood glucose, fasting plasma insulin, insulin resistance index, plasma triglyceride and total cholesterol levels (p <0.01), insulin sensitivity index (p<0.01) increased significantly. But there was no significant difference in body weight between two groups (p>0.05).5. Linagliptin can improve the high fat diet induced metabolic endotoxemia in rats with type 2 diabetes mellitus.At the end of 13th week,after the experiment finished, compared with the model control group, plasma endotoxin level of liver portal vein was significantly decreased (p<0.01), the expression of tumor necrosis factor alpha and interleukin -6 were significantly decreased either (p<0.01);6. Linagliptin can improve the intestinal barrier function of rats with type 2 diabetes mellitus.After the experiment finished, compared with the model group, linagliptin intervention group shows the increased expression of tight junction protein ZO-1, occludin and glucagon like peptide -2 in the proximal colon (p<0.01), plasma diamine oxidase and PAR2 levels were reduced(p ＜0.01).Conclusions1. High fat diet combined with small dose of STZ can induce the pathology of type 2 diabetes mellitus of rats.2. Type 2 diabetic rat model shows the intestinal mucosal injury and tigh junction protein decreased, and the metabolic endotoxemia appears.3. Linagliptin can improve the high fat diet combined with small dose of STZ mediated metabolic endotoxemia in rats with type 2 diabetes, and alleviate the systemic inflammation.4. The mechanism of linagliptin alleviate the model high-fat diet combined with low dose of STZ mediated metabolic endotoxemia in rats with type 2 diabetes mellitus probable related to the function of alleviate intestinal mucosal injury and reduce the intestinal permeability. |
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