| BackgroundDiabetes mellitus, the incidence of which increasing year by year, has been listed as the third mighty disease following cardiovascular disease and tumor in the Developed Countries. A research result shows that the times of clinical service and hospitalization in diabetic patients increased obviously, and the use of medical services of diabetic patients was two to four times that of non-diabetic patients, the medical costs of diabetic populations were nine times that of non-diabetic populations with the same age and sex. Another research indicates that for diabetic patients in china less than20%of the diabetic medical expenses were spent in blood-glucose control, and more than80%of that spent in the treatment of diabetic complications. The serious harm of diabetes mellitus to human health is the chronic diabetic complications developed from arteriosclerosis and microangiopathy. The incidence of angiopathy in diabetic patients is three to four times that of non-diabetics patients. It has been suggested that the fundamental pathological characteristics of diabetic angiopathy were atherosclerosis and the anomaly thickening, fibrosis and calcification of tunica media and tunica adventitia. The UKPDS shows conclusively that the incidence of diabetic microangiopathy could be obviously reduced by good glycemia control. However, the three important trials in intensive glucose lowering (ACCORD, ADVANCE and VADT) published in2008show that intensive blood-glucose control could reduce the rate of diabetic microangiopathy, but did not reduce the rate of major CVD events in patients with type2diabetes. So it is desiderated to further explore the mechanisms of the high rate of cardiovascular diseases in patients with type2diabetes, in order to find the theoretical basis of upstream treatment strategies for cardiovascular complications in patients with type2diabetes. Insulin resistance runs through the whole course of type2diabetes. The UKPDS shows that insulin resistance could cause macroangiopathy before the onset of hyperglycemia. Insulin resistance is the primitive cause and pathogenic basis of metabolic abnormality and cardiovascular complications in type2diabetes. Selective insulin resistance could enhance the proliferation of vascular smooth muscle cell and fibroblasts. Studies on selective insulin resistance indicate that in insulin-resistant states, the metabolic Akt pathway of insulin effect is impaired obviously, while the proliferative MAPK pathway is enhanced. TRIB3, served as the common regulatory protein of the Akt and MAPK pathway, is closely related to selective insulin resistance. So we hypothesize that TRIB3not only participates in pathogenesis of type2diabetes by inducing insulin resistance through the metabolic Akt pathway, but also participates in the initiation and progression of diabetic macroangiopathy through the proliferative MAPK pathway. So in this study, we aim to establish the type2diabetic model, determine the effects of TRIB3on the initiation and progression of diabetic macroangiopathy through inducing in vivo TRIB3gene silencing by RNAi technique, and investigate the morph metric and structural changes of great vessels after TRIB3gene silencing by various pathological and histological staining techniques. We aim to explore the effects of TRIB3on diabetic macroangiopathy and its possible mechanisms. The results will provide potential new targets for the early treatment of cardiovascular complications in type2diabetes, and have important academic theoretical significance and potential clinical application perspective.Objectives1.To confirm the feasibility of establishing diabetic macroangiopathy rat model induced by a high fat and sugar diet combined with a small dose of STZ;2.To investigate the improvement of diabetic macroangiopathy by transfection of pAdxsi-TRIB3-shRNA into type2diabetic rat;3. To explore the mechanism by which TRIB3silencing would ameliorate diabetic macroangiopathy in molecular, histological and functional levels.Methods1. Sixty5-week-old male SD (Sprague-Dawley) rats were randomized into Four groups after intraperitoneal glucose tolerance test(IPGTT) and intraperitoneal insulin tolerance test (IPITT):Control, chow+streptozotocin (Chow+STZ), high-fat and sugar diet (HF) and diabetes mellitus (DM).Control and Chow+STZ groups rats were fed on normal chow (20%crude protein,3%crude fat,3%crude fiber, others74%,including carbohydrates, microelements, etc). HF and DM groups rats received high-fat and sugar diet (34.5%fat,17.5%protein and48%carbohydrates). Four weeks later, IPGTT and IPITT were performed again, rats with insulin resistant in DM group were injected once with low dose of STZ (intraperitoneal at27.5mg/kg), and meanwhile Chow+STZ group rats received same dose of STZ. Control and HF groups rats received same dose of citrate buffer. One week after STZ administration, blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index(ISI, ISI=Ln(FINS×FBG)-1)was calculated. Rats with FBG>11.1mmol/1in two consecutive analyses, lower insulin sensitivity and characterized by polyuria, polydipsia and polyphagia were considered the diabetic rat model.2.Thirty5-week-old male SD (Sprague-Dawley) rats were made into diabetic rat model according to the above method, and then were randomized into two groups: DM-Vector group and DM-siRNA group.12weeks after successful modeling of DM, DM-siRNA group rats were injected via the jugular vein with2.5x1010PFU of pAdxsi-TRIB3-shRNA, while DM-Vector group rats were injected with2.5x1010PFU of pAdxsi.Adenovirus transfection was repeated after two weeks. Four weeks after first adenovirus injection, rats were sacrificed.3. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT):Glucose tolerance was assessed by IPGTT after12-hour diet fasting without water fasting. A bolus of glucose (lg/kg, ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60and120min and tested for glucose. Blood glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The area under the curve of glucose (AUCg) was calculated. To evaluate insulin tolerance, IPITT was performed after4-hour diet fasting without water fasting. A bolus of insulin (1unit/kg, ip) was injected, and blood samples were taken for glucose measurement as described above.4. Serum biochemical parameter analysis:blood glucose and serum insulin were measured to calculate the insulin sensitivity index (ISI). Furthermore, serum total cholesterol (TC), triglycerides(TG) and free fatty acids (FFA) were also measured.5. Measurement of blood pressure:Heart rate (HR) and blood pressure (BP) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan). 6. Ultrasonography of abdominal aorta:Successive observation of the changes of abdominal aorta in the initiation and progression of diabetic macroangiopathy by2-D, pulsed-wave Doppler and ultrasonic integrated backscatter technique.7. Pathological analysis:HE staining, Masson trichrome staining, Sirius red staining and Verhoeff staining were preformed on rat aorta. The aortic media thickness, perivascular collagen area/luminal area (PVCA/LA), aortic collagen percentage per medial area, aortic elastin percentage per medial area and aortic collagen/elastin ratio were determined with a computerized image analysis system. The average aortic wall architecture score was graded on Verhoeff-stained sections.8. Immunohistochemical staining for collagen â… and â…¢:The collagen â… and â…¢ depositions of aorta were viewed by immunohistochemical staining under a light microscope. The aortic Collagen â… and â…¢ content were determined with a computerized image analysis system.9. Hydroxyproline analysis:The collagen content of rat aortic tissues was determined by Hydroxyproline assay. The rat aortic tissues dried to constant weight were weighed, and then hydrolyzed in6mol/L HC1at110℃for16hours. The Hydroxyproline content of the hydrolysates was detected by an ELISA kit. Data were expressed as micrograms collagen per milligram dry weight, with the assumption that collagen contains an average of13.5%Hydroxyproline.10. Real-time quantitative RT-PCR:Total RNA of the fresh rat aortic tissues was extracted by Trizol. The mRNA of TRIB3was quantified by Real-time Fluorescent Quantitative RT-PCR.11. Western blot analysis:The proteins of TRIB3, collagen â… and â…¢, phospho-PI3K/PI3K,phospho-Akt/Akt,phospho-MKK4/MKK4and phospho-JNK/JNK were detected by western blotting.Results1. General characteristics of rats in each group at the end of the experimentThe rats in Chow+STZ group had obviously higher values of water intake, food intake and24h urine volume compared with Control group (P<0.05). The rats in HF group had increased values of water intake, food intake and24h urine volume, and significantly increased body weight compared with Control group (P<0.05). The rats in DM group had markedly increased values of water intake, food intake and24h urine volume compared with Control group (P<0.01). The rats in Chow+STZ group had significantly increased values of water intake and24h urine volume compared with HF group (P<0.05～0.001). The rats in DM group had significantly increased values of water intake, food intake and24h urine volume compared with HF group and Chow+STZ group (P<0.001, P<0.001).2. Results of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)(1) Results of IPGTTAfter a4-week high-fat and sugar diet, the levels of blood glucose in DM group were significantly higher than that at baseline at Omin,30min,60min,120min in IPGTT. The area under the curve of glucose (AUCg) increased significantly at week4than at baseline (P<0.05).Results of IPGTT in each group at12weeks after the onset of diabetes (at week17):The Chow+STZ group had significantly elevated blood glucose levels at30min and60min in IPGTT compared with the Control group (P<0.05); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and Control groups. The DM group had significantly elevated blood glucose levels at15min and60min in IPGTT compared with Control group (P<0.01); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and Chow+STZ groups. The DM group had significantly elevated blood glucose level at15min in IPGTT compared with Chow+STZ group (P<0.05), and also at120min in IPGTT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of DM and Chow+STZ groups in IPGTT increased significantly compared with Control group (P<0.05～0.01).Results of IPGTT in each group at the end of the experiment:The Chow+STZ group had significantly elevated blood glucose levels at60min and120min in IPGTT compared with Control group (P<0.05); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and control group. The DM group had significantly elevated blood glucose levels at15min,60min and120min in IPGTT compared with Control group (P<0.01); The DM group had significantly elevated blood glucose levels at15min and30min in IPGTT compared with Chow+STZ group (P<0.05), and also at60min and120min in IPGTT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of DM group, HF group and Chow+STZ group in IPGTT increased significantly compared with Control group (P<0.05). The area under the curve of glucose (AUCg) of DM group increased significantly compared with HF group (P<0.05). (2) Results of IPITTAfter a4-week high-fat and sugar diet, the levels of blood glucose in DM group were significantly higher than at baseline at Omin,15min,30min,60min,120min in IPITT. The area under the curve of glucose (AUCg) in IPITT increased significantly at week4than at baseline (P<0.01).Results of IPITT in each group at12weeks after the onset of diabetes (at week17):The Chow+STZ group had significantly elevated blood glucose levels at15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The HF group had significantly elevated blood glucose levels at Omin,15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The DM group had significantly elevated blood glucose levels at Omin,15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and Chow+STZ group, and between DM group and Chow+STZ group. The DM group had significantly elevated blood glucose levels at Omin,15min,30min and60min in IPITT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of the DM group, HF group and Chow+STZ group in IPITT increased significantly compared with Control group (P<0.05～P<0.01). The area under the curve of glucose (AUCg) of DM group in IGITT increased significantly compared with HF group (P<0.05).Results of IPITT in each group at the end of the experiment:The Chow+STZ group had significantly elevated blood glucose levels at15min in IPITT compared with Control group (P<0.05); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and Control group; The DM group had significantly elevated blood glucose levels at15min,60min and120min in IPITT compared with Control group (P<0.05); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and DM group, and between DM group and Chow+STZ group. The area under the curve of glucose (AUCg) of DM group, HF group and Chow+STZ group in IPITT increased significantly compared with Control group (P<0.05). The area under the curve of glucose (AUCg) of DM group in IGITT increased significantly compared with HF group (P<0.05).3. Serum biochemical parameters analysis:The serum biochemical parameters were of no statistical difference among each group before the experiment. After4weeks of a high-fat diet, serum TG and FFA levels were significantly higher in the HF and DM groups than control and chow+STZ groups (P<0.05). The ISI was markedly decreased in the HF and DM groups (P<0.05). Insulin resistance appeared at week4in rats fed a HF diet. One week after STZ injection, FBG was remarkably elevated in the DM group and remained elevated until the end of the experiment. ISI consistently decreased in the DM group after the onset of diabetes. Simultaneously, in the DM group, serum TC, TG, and FFA levels were maintained at higher levels than the control (P<0.05) during diabetes. Thus, the diabetic model induced by a HF diet and low-dose STZ was characterized by insulin resistance, moderate hyperglycemia and hyperlipidemia resembling the state of chemical diabetes in humans.3. Blood pressures and heart rate:There were no differences in SBP, DBP and MAP among four groups during the experimental process. At the end of the experiment, HR was higher in DM group compared with control and chow+STZ groups, but the difference was not statistically significant.4. Ultrasonography of abdominal aortaAt the end of the experiment, compared to Control group, the HF group showed no significant differences in ultrasonic measurements; In Chow+STZ group, the Peterson’s elastic modulus (Ep) and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the peak systolic velocity (PSV) and cross-sectional distensibility (CD) were deceased (P<0.05～0.001) compared with Control group; In DM group, systolic diameter (Ds), diastolic diameter (Dd), Ep and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the end diastolic velocity (EDV), peak systolic velocity (PSV) and crosssectional distensibility (CD) were decreased (P<0.05～0.001) compared with Control group; In Chow+STZ group, the stiffness index of abdominal aorta was significantly increased compared with HF group (P<0.01); In DM group, the Dd, Ep and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the EDV and PSV were decreased (P<0.05～0.001) compared with HF group; In DM group, the Dd and Ds of abdominal aorta were significantly increased compared with Chow+STZ group (P<0.05～0.001).At the end of the experiment, in Chow+STZ and DM groups, IBS%of abdominal aorta was significantly increased compared with Control group (P<0.01); In Chow+STZ, HF and DM groups, cyclic variation of integrated backscatter (CVIB) of abdominal aorta was significantly decreased compared with Control group (P<0.001); In Chow+STZ and DM groups, IBS%of abdominal aorta was significantly increased compared with HF group (P<0.05～0.01); Cyclic variation of integrated backscatter (CVIB) of abdominal aorta in DM group was significantly decreased (P<0.01).6. Pathological characteristics of aorta(1) HE staining:Compared with Control group, in DM group, the endothelial cells adhering to inner elastic plates showed arrangement disorder, and the vascular smooth muscle cells (VSMCs) of tunica media arranged irregularly, with irregular nucleus and uneven cytoplasm staining. In Chow+STZ, HF, and DM groups, the media thickness of aorta was significantly increased compared with Control group (72.73.09±2.12vs.58.09±0.97μm, P<0.01;62.69±0.67vs.58.09±0.97μm, P<0.05;72.38±2.30vs.58.09±0.97μm, P<0.001). In Chow+STZ and DM groups, the media thickness of aorta was increased compared with HF group (P<0.05). In DM group, the media thickness of aorta was increased compared with Chow+STZ group, but the difference was not statistically significant.(2) Masson staining:The VSMCs in aorta were stained red, while the collagen fibers were stained bluish green. In control group with mild perivascular fibrosis, collagen fibers were thin, intact and appropriately arranged. While in DM group, the collagen content increased significantly, thick collagen fibers connected into reticular or mass structure arranged irregularly.Sirius red staining:In control group with mild perivascular fibrosis, the collagen fibers in aorta were thin and uniform under conventional microscope, and the tunica media showed no staining, tunica adventitia was mainly composed of red collagen â… and green collagen â…¢ under polarization microscope. While in the DM group, collagen fibers in aorta increased significantly, arranged irregularly and was disrupted markedly under conventional microscope. Under polarization microscope, collagen â… and collagen â…¢ in tunica media and tunica adventitia of DM group both increased significantly. Semi-quantitative analysis showed that compared with Control group, the PVCA/LA in Chow+STZ, HF, and DM groups increased significantly (0.06±0.0029vs.0.03±0.0015, P<0.001;0.04±0.0016vs.0.03±0.0015, P<0.05;0.10±0.007vs.0.03±0.0015,P<0.01), the CVF increased significantly (4.42±0.48vs.1.12±0.14, P<0.001;2.45±0.22vs.1.12±0.14, P<0.01;5.70±0.31vs.1.12±0.14, P<0.001); Compared with HF group, the PVCA/LA and collagen percentage per medial area of Chow+STZ and DM groups increased significantly (P<0.01-0.001); Compared with Chow+STZ group, the PVCA/LA of DM group slightly increased (P<0.05); Compared with Chow+STZ group, the collagen percentage per medial area of DM group slightly increased, but the difference was not statistically significant.(3) Verhoeff elastin staining:In Control group, the VSMCs in aorta were stained yellow or Yellowish red, and the elastin fibers stained black blue were intact and uniform, and arranged appropriately. While in DM group, the VSMCs were stained red, the elastin fibers showed arrangement disorder, sparsity, nonuniformity, and had significant disruptions. Semi-quantitative analysis showed that compared with Control group, the elastin volume fraction of abdominal aorta in Chow+STZ, HF, and DM groups decreased significantly(5.19±0.76vs.11.96±0.75, P<0.001;8.83±0.98vs.11.96±0.75, P<0.05;4.70±0.36vs.11.96±0.75, P<0.001); The elastin percentage per medial area in DM group decreased significantly compared with HF group(P<0.05); Compared with Chow+STZ group, the elastin percentage per medial area in DM group slightly decreased, but the difference was not statistically significant; Compared with Control group, the aortic collagen/elastin ratio in Chow+STZ, HF, and DM groups, increased significantly(0.57±0.09vs.0.05±0.007, P<0.01;0.17±0.0024vs.0.05±0.007, P<0.01;0.71±0.075vs.0.05+0.007, P<0.001); Compared with HF group, the aortic collagen/elastin ratio in Chow+STZ and DM groups increased significantly (P<0.05～0.001); Compared with Chow+STZ group, the aortic collagen/elastin ratio of DM group slightly increased, but the difference was not statistically significant; Compared with Control group, the average aortic wall architecture score of Chow+STZ, HF and DM groups increased significantly (p<0.001); Compared with HF group, the average aortic wall architecture score of Chow+STZ and DM groups increased significantly (p<0.01～0.001); Compared with Chow+STZ group, the average aortic wall architecture score of DM group slightly increased (p<0.05).(4) Determination of collagen content in rat aorta by Hydroxyproline assay.Compared with Control group, the collagen content in Chow+STZ, HF and DM groups increased significantly (13.34±0.37vs.7.01±0.21ug/mg dry weight, P<0.001;8.47±0.21vs.7.01±0.21ug/mg dry weight, P<0.01;15.34±0.47vs.7.01±0.21ug/mg dry weight, P<0.001); Compared with HF group, the collagen content in Chow+STZ and DM groups increased significantly (P<0.001); Compared with Chow+STZ group, the collagen content in DM group increased significantly (P<0.001). (5) Results of Immunohistochemical staining:Compared with Control group, the protein expression of collagen â… and â…¢ in Chow+STZ group markedly increased, the protein expression of collagen â… and â…¢ in HF group slightly increased, while the expression of collagen â… and â…¢ in DM group increased significantly. Compared with HF group, the protein expression of collagen â… and â…¢ in Chow+STZ and DM groups both increased markedly. Compared with Chow+STZ group, the protein expression of collagen â… and â…¢ of DM group increased markedly.(6) Western blot results of collagen â… and â…¢:Compared with Control group, the protein expression of collagen â… and â…¢ in Chow+STZ group markedly increased (collagen â… :0.82±0.05vs.0.51±0.05, P<0.001; collagen â…¢:0.62±0.03vs.0.31±0.02, P<0.001), the protein expression of collagen â… and â…¢ in HF group slightly increased (collagen â… :0.60±0.03vs.0.51±0.05, P<0.01; collagen â…¢:0.47±0.03vs.0.31±0.02, P<0.05), while the expression of collagen â… and â…¢ in DM group increased significantly (collagen â… :1.04±0.06vs.0.51±0.05, P<0.001; collagen â…¢:0.95±0.04vs.0.31±0.02, P<0.001). Compared with HF group, the protein expression of collagen â… and â…¢ in Chow+STZ and DM groups both increased markedly (P<0.01-0.001, P<0.001). Compared with Chow+STZ group, the protein expression of collagen â… and â…¢ in DM group increased markedly (P<0.05).(7) Western blot results of TRIB3:Compared with Control group, the protein expression of TRIB3in Chow+STZ group increased in1.33folds (P<0.001), the protein expression of TRIB3in HF group had a increase of2.3372%(P<0.001), the protein expression of TRIB3in DM group increased in1.78folds (P<0.001); Compared with HF group, the protein expression of TRIB3in Chow+STZ group had a increase of35%(P<0.001), the protein expression of TRIB3in DM group had a increase of61%(P<0.001); Compared with Chow+STZ group, the protein expression of TRIB3in DM group had a increase of19%(P<0.001).(8) Western blot results of the key signal molecules in Akt and MAPK pathwayâ‘ The phospho-PI3K/PI3K ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.97,0.67,0.75,0.45. Compared with Control group, the phosphorylation level of PI3K in Chow+STZ, HF and DM groups declined significantly (P<0.001for all); Compared with HF group, the phosphorylation level of PI3K in Chow+STZ and DM groups declined significantly (P<0.001); Compared with Chow+STZ group, the phosphorylation level of PI3K in DM group declined markedly (P<0.01).â‘¡The phospho-Akt/Akt ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.90,0.48,0.61,0.29. Compared with Control group, the phosphorylation level of Akt in Chow+STZ, HF and DM groups declined significantly (P<0.01～P<0.001), and among them the DM group had the Lowest phosphorylation level of Akt, which was the32%of Control group (P<0.001); Compared with HF group, the phosphorylation level of Akt in Chow+STZ and DM groups declined significantly, and between the two groups the DM group had Lower phosphorylation level of Akt, which was the47%of Control group (P<0.001); Compared with Chow+STZ group, the phosphorylation level of Akt in DM group declined slightly, but the difference was not statistically significant.â‘¢The phospho-MKK4/MKK4ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.59,0.85,0.64,1.03. Compared with Control group, the phosphorylation level of MKK4in Chow+STZ, HF and DM groups increased significantly (P<0.05～P<0.001), and among them the DM group had the highest phosphorylation level of MKK4, which was1.86times that of Control group (P<0.001); Compared with HF group, the phosphorylation level of MKK4in Chow+STZ and DM groups increased significantly (P<0.001for both); Compared with Chow+STZ group, the phosphorylation level of MKK4in DM group increased markedly (P<0.01).â‘£The phospho-JNK/JNK ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.45,0.78,0.60,0.97. Compared with Control group, the phosphorylation level of JNK in Chow+STZ, HF and DM groups increased significantly (P<0.01～P<0.001), and among them the DM group had the highest phosphorylation level of JNK, which was2.15times that of Control group (P<0.001); Compared with HF group, the phosphorylation level of JNK in DM group increased significantly (P<0.001), while the phosphorylation level of JNK in Chow+STZ group slightly increased, but the difference was not statistically significant; Compared with Chow+STZ group, the phosphorylation level of JNK in DM group increased slightly, but the difference was not statistically significant.7. The mRNA and protein expression of TRIB3after TRIB3gene silencingThe mRNA and protein expression of TRIB3in aorta:The results of RT-PCR showed that Compared with DM-Vector group, the mRNA expression of aortic TRIB3in DM-siRNA group declined by66%(P<0.001). The results of Western blot showed that compared with DM-Vector group, the protein expression of aortic TRIB3in DM-siRNA group declined by67%(P<0.001).8. TRIB3gene silencing ameliorated the general characteristics of type2diabetic ratCompared with DM-Vector group, the DM-siRNA group rats had obviously lower values of water intake (P<0.05) and24h urine volume (P<0.05) after TRIB3gene silencing. The DM-siRNA group rats had lower values of food take compared with DM-Vector group, but the difference was not statistically significant. The body weight, Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) of DM-siRNA group had no statistical difference compared with DM-Vector group after TRIB3gene silencing.9. The effects of TRIB3gene silencing on IPGTT and IPITT(1) Results of IPGTT:Compared with DM-Vector group, the DM-siRNA group had significantly decreased blood glucose levels at30min and120min in IPGTT (P<0.05for both), and significantly decreased area under the curve of glucose (AUCg)(P=0.041) in IPGTT after TRIB3gene silencing.(2) Results of IPITT:Compared with DM-Vector group, the DM-siRNA group had significantly decreased blood glucose levels at Omin and120min in IPITT (P<0.05for both), and significantly decreased area under the curve of glucose (AUCg)(P=0.030) in IPITT after TRIB3gene silencing.10. The effects of TRIB3gene silencing on serum biochemical parameters4weeks after TRIB3gene silencing, serum TG level of DM-siRNA group was markedly decreased compared with DM-Vector group (4.98±1.62vs2.57±0.62mmol/L, P<0.05). Serum TC level of DM-siRNA group was markedly decreased compared with DM-Vector group (3.96±1.01vs.7.74±2.15mmol/L, P<0.05). Serum FFD level of DM-siRNA group was markedly decreased compared with DM-Vector group (355.85±26.24vs.464.69±31.23μmol/L, P<0.05). The fasting blood-glucose level of DM-siRNA group was remarkably decreased compared with DM-Vector group (15.63±2.04vs.24.58±3.04mmol/L, P<0.05). The ISI of DM-siRNA group was markedly increased compared with DM-Vector group (-5.17±0.14vs.-5.67±0.14, P<0.05). The fasting insulin level of DM-siRNA had no statistical difference compared with DM-Vector group. 11. The effects of TRIB3gene silencing on ultrasonography of abdominal aortaFour weeks after TRIB3gene silencing, The Dd of abdominal aorta in DM-siRNA group was markedly decreased compared with DM-Vector group (P<0.05); The EDV and PSV of DM-siRNA group were increased markedly compared with DM-Vector group (P<0.01); The Peterson’s elastic modulus (Ep) and crosssectional distensibility (CD) of abdominal aorta in DM-siRNA group were increased markedly compared with DM-Vector group (P<0.01); The stiffness index of abdominal aorta in DM-siRNA group was decreased markedly compared with DM-Vector group (P<0.001);4weeks after TRIB3gene silencing, IBS%of abdominal aorta in DM-siRNA group was significantly decreased compared with DM-Vector group (1.76±0.14vs1.42±0.15, P<0.001); The cyclic variation of integrated backscatter (CVIB) of abdominal aorta in DM-siRNA group was significantly increased compared with DM-Vector group (4.17±0.81vs5.21±0.57, P<0.01).12. TRIB3gene silencing ameliorated diabetic arterial remodeling(1) HE staining:In DM-siRNA group there were only some of the endothelial cells of aorta showed disruption and structure abnormity compared with DM-Vector group. The endothelial cells in DM-siRNA group arranged more regularly, and had a looser adhesion to inner elastic plates compared with DM-Vector group. The VSMCs of abdominal aorta in DM-siRNA group with regular nucleus and even cytoplasm staining were fusiform shaped and arranged more irregularly compared with DM-Vector group. Results showed that the media thickness of aorta in DM-siRNA group was significantly decreased compared with DM-Vector group (74.88±1.21μm vs.69.38±1.11μm,P<0.01).(2) Masson staining:The collagen content of aorta in DM-siRNA group was decreased significantly, and the collagen fibers were thinner, arranged more regularly and distributed more evenly around the vascular smooth muscle cells compared with DM-Vector group. Sirius red staining:The collagen fibers of aorta in DM-siRNA group decreased significantly arranged more irregularly and showed less disruption under conventional microscope compared with DM-Vector group. The yellowish red collagen â… and the yellowish green collagen â…¢ in tunica media and tunica adventitia of aorta in DM-siRNA group decreased significantly, and had less disruption under polarization microscope compared with DM-Vector group. Semi-quantitative analysis showed that compared with DM-Vector group, the PVCA/LA of aorta in DM-siRNA group was decreased significantly compared with DM-Vector group (0.10±0.005vs.0.06±0.006,P<0.001). The aortic collagen percentage per medial area in DM-siRNA group was decreased markedly compared with DM-Vector group (3.77±0.36vs.2.48±0.22,P<0.01).(3) Verhoeff elastin staining:The elastin fibers of aorta in DM-siRNA group arranged more regularly and had less disruptions compared with DM-Vector group. Semi-quantitative analysis showed that compared with DM-Vector group, the aortic elastin percentage per medial area in DM-siRNA group was increased significantly(5.29±0.36vs.7.79±0.72, P<0.01); The aortic collagen/elastin ratio of abdominal aorta in DM-siRNA group was decreased significantly compared with DM-Vector group (0.75±0.09vs.0.36±0.07, P<0.01); The average aortic wall architecture score in DM-siRNA group was decreased markedly compared with DM-Vector group (P<0.05).(4) Determination of collagen content in rat aorta by Hydroxyproline assay: Compared with DM-Vector group, the collagen content of aorta in DM-siRNA group was decreased significantly (14.16±0.30vs.10.70±0.19ug/mg dry weight, P<0... |
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