| BackgroundDiabetes is popular on the world wide in present, which bring serious consequence to human health and life in developed and developing coutries, especially when the chronic complication of diabetes developed. The incidence of diabetes is increasing gradually along with the improvement in the quality of life and the changes in life style, as a result, the incidence of diabetic vascular complication is gradually increasing, which bring economic and social burden to the patients with diabetes mellitus. The diabetic vascular diseases contain diabetic macrovascular diseases, which including cerebrovascular disease, coronary heart disease and peripheral vascular disease, and diabetic microvascular diseases that including diabetic nephropathy, diabetic retinopathy and diabetic peripheral neuropathy. Diabetic macrovascular diseases remain to be the leading cause of death in patients with type2diabetes mellitus, and its main pathological feature is atherosclerosis. Atherosclerosis, as one of the vascular diseases, is a common chronic inflammatory reaction in vascular, mainly involving the intima of artery, characterized by lipid deposition, monocyte aggregation, foam cell formation, smooth muscle cells proliferation and vascular endothelial dysfunction, which leading to the formation of atherosclerosis. In consideration of the serious consequences of the diabetic vascular disease, it has become an important issue about how to prevent the incidence of diabetic vascular disease. However, the pathogenesis of diabetic vascular disease is still unknown, which bring difficulties to the treatment of diabetic vascular disease.The dipeptidyl peptidase-4inhibitor, as a novel oral hypoglycemic medicine, not only decrease the level of HbA1C, fasting glucose and postprandial glycemia, but also improve the dysfunction of pancreatic cells by increasing the secretion of insulin and inhibiting the secretion of glucagon, without increasing the body weight and the incidence of hyperglycemia. DPP-4inhibitor take the aboved effets maily through selective inhibiting the activation of DPP-4enzyme and preventing the degradation of glucagon-like peptide-1(GLP-1), thereby increasing the activation and duration of GLP-1. GLP-1is a peptide hormone that encoded by human glucagon gene, secreted by the intestical L cells and released into blood in the stimulation of food. GLP-1take an anti-diabetic effect mainly through promoting the transcription of insulin gene, increasing the biosynthesis and secretion of insulin, stimulating the proliferation and differentiation of pancreatic β cells, inhibiting the apoptosis of pancreatic P cells and inhibiting the secretion of glucagon by pancreatic a cells. GLP-1not only can decrease the level of HbA1C, fasting hyperglycemia and postprandial hyperglycemia but also can inhibit the appetite and food intake, which resulting in body weight loss. However, the endogenous GLP-1which possessing a short half-life can be degraded by an enzyme called DPP-4. DPP-4, which also known as T-cell surface antigen CD26, is a serine protein kinase existed in cell surface and partially existed in the circulation in soluble form. Its main effect is to remove the first two amono acid of the amino terminus, whereas the second amino acid of endogenous GLP-1can be removed by DPP-4, which leading to the inactivation of GLP-1rapidly and inreversibly. Recently lots of researches have proved that the GLP-1receptor agonists can attenuate the development of atherosclerosis possibly through activating the cAMP/PKA signaling pathway after interacted with GLP-1receptor. And sitagliptin, as a competitive DPP-4inhibitor, play a role in anti-diabetic effect through inhibiting the activation of DPP-4enzyme by interaction with the glutamate residues and also take a protective role in cardiovascular diseases reported by recently researches. It has been also reported that sitagliptin possibly can inhibite the progression of atherosclerosis, but its definite mechanisms are unclear, maybe through activating the cAMP/PKA signaling pathway or improving the vascular endothelial dysfunction by increasing the synthesis of NO independent of cAMP/PKA and PI3K/AKT signaling pathway. It has been confirmed recently that both of the AMPK and MAPK signaling pathway may take an important part in the anti-atherosclerotic progress. AMPK as one of the important molecules that regulating the biological energy metabolism, express in various organs related with metabolism. It has been found that its activation process the anti-atherosclerotic effect, mainly through upregulating the Akt/eNOS signaling pathway and then inhibiting the activationg of p38-mediated NF-κB, which leading to the reduction of inflammation. MAPK as one of the intracellular serine/threonine protein kinases, contains p38MAPK, extracellular signal-regulated protein kinase (Erk), c-jun amino-terminal kinase (JNK) and stress-activated protein kinase (SAPK) signaling pathway. It has been proved that MAPK may play an anti-atherosclerotic effect through inhibiting the expression of adhesion molecules, reducing the inflammation and increasing the stability of atheroxclerotic plaques.Although the pathogenesis of diabetic vascular complications remains unclear, it has been considered to be associated with activation of protein kinase C, production of advanced glycationend products, oxidative stress, hyperactivity of polyol pathway and abnormalities of hexosamine pathway. And activation of PKC in vascular tissues induced by chronic hyperglycemia seems to be one of the most important mechanisms. PKC kinase is one of an important serine/threonine kinases, widely presenting in various human tissues and cells. It is a protein family containing different subtypes which phosphorylating proteins in intracellular and regulating the process of growth, proliferation, senescene, apoptosis and so on by acting on different signaling transductive pathways. It has been found that PKC contains11kinds of subtypes and3categories, which including classic PKC, novel PKC and atypical PKC. Classic PKC, containing PKCα, β (Ⅰ Ⅱ) and y, can be activated by calcium, diacylglycerol and phorbol esters; novel PKC, including PKCδ, ε,η and θ, can be activated by diacylglycerol and phorbol esters; and atypical PKC, including PKCζandλ, generally can be activated by phospholipid substances other than calcium, diacylglycerol or phorbol esters. PKC expresses in different tissues and PKCα、PKCβ、PKCδ、PKCε、and PKCζ can be detected in vascular tissues especially the PKCβ isoform. PKC can be activated by different factors and hyperglycemia is the most important reason through increasing the de novo synthesis of diacylglycerol instimulated by its intermediate metabolic productions such as advanced glycationend products, reactive oxygen species, cytokines, growth factors and so on. Previous researches suggest that the activation of PKC maybe accelerate the development of atherosclerosis, but its mechanisms are unknown. And now there is no reports about the relationship between sitagliptin and PKCβ activation, therefore we choose to explore it.According to the unsolved problem aboved, we designed this comprehensive study to investigate the cardioprotective effect of sitagliptin and its mechanisms independent of decreasing hyperglycemia in ApoE-/-mice. In addition, we eatablished the vascular endothelial thansgenic PKCβ mice with ApoE-/- background, using the vascular endothelial PKCβ transgenic mice crossed with ApoE-/-mice, to investigate the effect of activation of PKCβ on atherosclerosis. And then we gave intervenetion to confirm whether sitagliptin can attenuate the progression of atherosclerosis induced by activation of PKCβ and explored its underlying mechanisms. Since diabetic vascular complications are serious and unresolved, it is important to study and provide more clinical theoretical basis for sitagliptin in the prevention of diabetic vascular complications.Part1The DPP-4inhibitor sitagliptin attenuates the progression of atherosclerosis in apolipoprotein-E knockoutmice via AMPK-and MAPK-dependent mechanismsObjective:To investigate the anti-atherosclerotic effect of DPP4inhibitor sitagliptin and explore its relevant mechanisms whether associated with the activation of AMPK signaling pathway and the inhibition of MAPK signaling pathway that caused by sitagliptin.Methods:Twenty four male apolipoprotein-E knockout mice aged8weeks were randomly divided into the control group (HFD, n=12) fed with high-fat diet (HFD) only and sitagliptin treated group (sitagliptin, n=12) fed with HFD plus sitagliptin at a concentration of0.3%both for16weeks.Body weight and food intake of all mice were recorded weekly and intraperitoneal glucose tolerance test (IPGTT), after injected with20%glucose at a dose of2g/kg, was administered with tail vein blood at0,15,30,60and120min after fasting for16h at week14. At the end of the study, the blood samples were collected from orbital sinus of mice that fasting for8hours, after injected with1.5%pentobarbital sodium. And the serum of mice centragrated form the blood samples with3000rpm for10min were used to measure the level of serum lipid profile and serum soluble adhesion molecules respectively by enzyme-linked immune-sorbent assay and automatic biochemical analyzer.Mice were killed after collecting the blood samples with spine dislocation and then dissected. After removing the adventitial fatty tissue, the aortas of mice were opened longitudinally from the aortic root to the renal artery with injection of0.9%physiological saline in left ventricle and fixed in10%formalin for36h. And then the fixed aortas were stained with Sudan IV for10min, differentiated in70%alcohol for15min, washed in water for20min and imbedded between two slides. The stained aortas were photographed using a digital camera connected to a dissection microscope to quantify area of the atherosclerotic lesion.Some aortas of mice were cut into three pieces, the upper section were fixed in10%formalin for24h and cross-sectioned after paraffin embedded in order to analyze the expression of collagen fiber, macrophage cells and vascular smooth muscle cells in plaques respectively by masson’s trichrome staining and immune-histochemistry medthod. The middle section of aortas were used for measuring the phosphorylated expression of AMPK and MAPK signaling pathway after obtaining the aortic protein, while the lower section for analyzing the expression of DPP-4, GLP-1R, MCP-1and IL-1in aortas after obtaining the RNA.Statistical analysis:All analysis were performed using SPSS version13.0for Windows. All data were expressed as mean±SD. Comparisons of means between the two groups were analyzed by un-paired Student’s t test. A P value<0.05was considered statistically significant.Results:1. The metabolic profile between the two groups:There were no statistical differences between the groups in body weight, food intake or blood glucose leves at Omin,15min,30min,60min and120min of IPGTT. Our data indicated that sitagliptin can significantly increase the level of HDL (52.78±5.25vs97.76±8.56, P<0.001) in ApoE-/-mice, whereas with no effect on TG, TC, LDL or VLDL compared with the control group.2. Effects of sitagliptin on the formation of the atherosclerotic lesion and on the histological composition of the atherosclerotic plaques:Our data show that sitagliptin can inhibit the formation of atherosclerotic areas in entire aorta, aortic root and abdominal aorta of ApoE±/-mice. The sitagliptin group showed fewer atherosclerotic plaques compared with the control group(7.64±1.98%vs12.91±1.15%, P<0.001). And atherosclerotic plaque areas decreased respectively1.92-and2.74-fold in the aortic root and abdominal aorta of mice fed HFD plus sitagliptin when compared with mice fed HFD (P=0.011and P=0.006). Aorta cross-section immunochemistry results showed that there was significantly less collagen fiber in aortic plaques after intervention with sitagliptin in ApoE-/-mice (49.86±6.26um2vs59.83±5.82um2; P<0.05). And the area occupied by vascular smooth muscle cells and macrophages tended to be decreased in the sitagliptin group compared to the control, although with no significant difference in the statistical analysis.3. Effects of sitagliptin on the expression of serum soluble adhesion molecules:Sitagliptin can inhibite the expression of serum soluble adhesion molecules. Compared with the control group, the serum levels of soluble VCAM-1and P-selectin decreased significantly in the sitagliptin group in statistical analysis (VCAM-1:1163.16±159.62ng/ml vs1365.18±170.26ng/ml, P<0.05; P-selectin:232.71±64.29ng/ml vs288±44.46ng/ml, P<0.05).4. Effect of sitagliptin on mRNA expression levels of DPP-4, GLP-1R, and inflammatory cytokines in aortic tissues of ApoE-/-mice:There were no differences on the expression of DPP-4and GLP-1R in the aortas between the two groups (P>0.05). But sitagliptin did significantly reduce the expression of MCP-1and IL-6in aortas (P=0.001and P=0.033).5. Effect of sitagliptin on the phosphorylation of the AMPK and MAPK signaling pathwaysCompared to the control, phosphorylation of AMPK and its downstream signaling molecule Akt increased in the aortic tissues of the sitagliptin group in statistical analysis (P<0.05and P<0.01), while the phosphorylation of MAPK signaling pathway that contained p38and Erkl/2decreased (P<0.05and P<0.01).Conclusions:1. Sitagliptin can inhibite the progression of atherosclerosis in ApoE-/-mice and also reduce the expression of smooth muscle cells, macrophages and collagen fiber in the atherosclerotic plaque, independent of decreasing glucose and body weight.2. Sitagliptin can increase the serum HDL level in ApoE-/-mice, while with no effects on the other serum lipid levels.3. Sitagliptin reduce the formation of atherosclerotic lesion possibly by activating the AMPK-Akt signaling pathway and inhibiting the p38and Erk1/2MAPK signaling pathway, which resulting in decreaed expression of inflammatory factors (MCP-1and IL-6) in aortas and serum adhesion molecules (sVCAM-1and sP-selectin). Part2The DPP-4inhibitor sitagliptin attenuate the development of atherosclerosis in VEPKCβ2+/-ApoE-/-mice and its relative mechanismsObjective:1、Investigate the anti-atherosclerotic effect of PKCβ2specifically overexpressed in vascular endothelial cells and explore its relevant mechanisms.2、Demonstrate whether DPP-4inhibitor sitagliptin can attenuate the development of atherosclerosis in the aortas of VEPKCβ2+/-ApoE-/-mice.3、Explore the underlying mechanisms of the anti-atherosclerotic effects of DPP-4inhibitor sitagliptin in VEPKCβ2+/-ApoE-/-mice.Methods:1、The VEPKCβ2+/-ApoE-/-mice model were established:Protein kinase C β2isoform was overexpressed in endothelial cells using a promoter of vascular endothelial cell cadherin. The vascular endothelial cell spevific transgenic PKCβ2(VEPKCβ2+/-) mice provided by Joslin Diatetes Center were crossbred with apolipoprotein E knockout (ApoE-/-) mice for many times and their offsprings were made genetyping to screen the male VEPKCβ2+/-/ApoE-/-mice as the experimental model.2、Twelve male apolipoprotein-E knockout mice aged8weeks were selected as control subjects feeding with high food diet (HFD, Containing21.8%fat and1.25%cholesterol) for16weeks. Then twenty four male VEPKCβ2+/-/ApoE-/-mice aged8 weeks after genetyping were selected as experimental subjects and randomly divided into the PKCβ group (n=12) fed with HFD only and the sitagliptin treated group (n=12) fed with HFD plus sitagliptin at a concentration of0.3%both for16weeks.The details are as follows.3、The experimental groups:Group1:The normal control group, male ApoE-/-mice aged8weeks fed with HFD for16weeks (n=12).Group2:The experimental group, male VEPKCβ2+/-/ApoE-/-mice aged8weeks fed with HFD for16weeks (n=12).Group3:The sitagliptin treated group, male VEPKCβ2+/-/ApoE-/-mice aged8weeks fed with HFD plus sitagliptin for16weeks (n=12).5、Body weight and food intake of all mice were recorded weekly and intraperitoneal glucose tolerance test (IPGTT) was administered at week14with tail vein blood to measure the blood glucose level at0,15,30,60and120min.6、At the end of the study after feeding for16weeks, the blood samples were collected from orbital sinus of mice that fasting for8hours. And the serum of mice centragrated form the blood samples were used to measure the level of serum lipid profile and serum soluble adhesion molecules respectively by automatic biochemical analyzer and enzyme-linked immune-sorbent assay.7、Mice were killed after collecting the blood samples with spine dislocation and then dissected. After removing the adventitial fatty tissue, the aortas of mice were opened longitudinally from the aortic root to the renal artery. And then the fixed aortas were stained with Sudan ⅣV. The stained aortas were photographed using a digital camera connected to a dissection microscope to quantify area of the atherosclerotic lesion.8、Aortas were fixed and cross-sectioned after paraffin embedded to analyze the expression of collagen fiber, macrophages and vascular smooth muscle cells in plaques respectively by masson’s trichrome staining and immune-histochemistry medthod. The aortic RNA were used for analyzing the expression of PKCβ, DPP4and GLP-1R, while the aortic protein were used for measuring the phosphorylated expression of AMPK and MAPK signaling pathway.9、Statistical analysis:All analysis were performed using SPSS version13.0for Windows. All data were expressed as mean±SD. Comparisons of means among groups were analyzed by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. Multiple comparisons were analyzed by Dunnett’s T3method when P value<0.05. A P value<0.05was considered statistically significant.Results:1、 The comparisons of metabolic profile among groups: There were no statistical differences among groups in body weight, food intake or blood glucose leves at Omin,15min,30min,60min and120min of IPGTT. VEPKCβ2had no effect on the blood lipid levels containing TG, TC, HDL, LDL and VLDL in ApoE-/-mice, while sitagliptin can significantly increase the level of HDL (86.69±14.61vs60.81±10.82, P<0.0001) in VEPKCβ2+/-ApoE-/-mice with no effect on TG, TC, LDL or VLDL compared with the VEPKCβ2group.2、Effects of VEPKCβ2and sitagliptin on the formation of the atherosclerotic lesion:PKCβ2overexpressed in the vascular endothelial cells can significantly increase the formation of the atherosclerotic lesion (23.58±5.31%vs12.91±1.15%, P<0.0001). And moreover sitagliptin can inhibit the formation of atherosclerotic areas in VEPKCβ2+/-ApoE-/-mice with statistical difference (16.19±2.07%vs23.58±5.31%, P<0.01). Compared with the control group, the atherosclerotic areas both in aortic root and abdominal aorta tended to increase in VEPKCβ2group (30.64±14.9%vs21.31±7.45%;12.33±4.7%vs11.06±3.93%), but with no difference in statistical analysis. While the atherosclerotic areas in aortic root and abdominal aorta tended to decrease when treated with sitagliptin in VEPKCβ2+/-ApoE-/-mice (20.23±4.38%vs30.64±14.9%;9.98±3.75%vs11.06±3.93%), although with no statistical difference.3、Effects of VEPKCβ2and sitagliptin on the histological composition of the atherosclerotic plaques:Compared with the control group, the expression of collagen fiber, macrophages and vascular smooth muscle cells in atherosclerotic plaques tended to increase in VEPKCβ2group (smooth muscle cells:28.28±6.56vs22.74±5.11um2; macrophages:4.45±1.32vs2.91±2.01um2; collagen fiber:66.60±16.16vs59.83±5.82%), albeit with no statistical difference. And we found that there was decreasing tendencies on the expression of collagen fiber, macrophages and vascular smooth muscle cells in atherosclerotic plaques after treated with sitagliptin in VEPKCβ2+/-ApoE-/-mice (smooth muscle cells:23.28±3.03vs22.74±5.11; macrophages:3.31±1.38vs2.91±2.01; collagen fiber:52.59±8.65vs59.83±5.82), although with no significant difference in the statistical analysis.4、Conparision of the mRNA expression levels of PKCβ, DPP-4and GLP-1R in aortic tissues among the three groups:There were no differences on the expression of DPP-4and GLP-1R in the aortas among the three groups (P>0.05). The mRNA expression of PKCβ in aortas increased respectively5.18-and5.21-fold in the VEPKCβ group and sitagliptin group, compared with the control group (P=0.012and P=0.035). While there was no statistical difference in the expression of PKCβ in aortas between the control group and VEPKCβ group (P>0.05).5、The expression level of serum soluble adhesion molecules (VCAM-1and P-slectin) among the three groups:Compared with the control group, the serum levels of soluble VCAM-1and P-selectin iecreased significantly in the VEPKCβ group in statistical analysis (VCAM-1:2157.12±553.37vs1365.18±170.26, P<0.001; P-selectin:429.01±102.89vs288.00±44.46, P<0.001). Compared with the VEPKCβ group, the serum levels of soluble VCAM-1decreased significantly in the sitagliptin group in statistical analysis (VCAM-1:1838.20±374.86vs2157.12±553.37, P<0.05), and the serum levels of soluble P-selectin tended to decrease, but with no statistical difference (383.30±63.60vs429.01±102.89, P=0.181).6、The phosphorylation of the AMPK and MAPK signaling pathways among the three groups:Compared to the control group, phosphorylation of AMPK protein decreased by4-fold in the aortic tissues of the VEPKCp group (P=0.023), while the phosphorylation of AMPK protein increased by5.56-fold after treated with sitagliptin in VEPKCβ2+/-ApoE-/-mice compared with the VEPKCβ group. On the contrary, the phosphorylation of MAPK protein inecreased by1.66-fold in the aortic tissues of the VEPKCβ group compared to the control group (P=0.033), while the phosphorylation of AMPK protein decreased by3.02-fold after treated with sitagliptin in VEPKCβ2+/-ApoE-/-mice compared with the VEPKCp group.Conclusions:1、PKCβ2specifically overexpressed in the vascular endothelial cells can accelerate the development of the atherosclerosis in ApoE-/-mice, independent of the glucose-decreasing, weight loss and lipid-reducing effects. 2、The mechanism of anti-atherosclerotic effect of PKCβ2specifically overexpressed in the vascular endothelial cells might associated with the increasing interaction of leukocytes to the vascular.3、Sitagliptin can attenuate the progression of atherosclerosis in VEPKCβ2+/-ApoE-/-mice, with no effect on the expression of PKCβ2in aortas.4、Sitagliptin reduce the formation of atherosclerotic lesion possibly by activating the AMPK signaling pathway and inhibiting the MAPK signaling pathway, which resulting in decreaed expression of adhesion molecules. |
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