Effects Of Grape Seed Proanthocyanidin Extracts On Aorta In Diabetic Rats And Their Mechanisms

Part OneEffects of Grape Seed Proanthocyanidin Extracts on Aortic Arteriosclerosis in the Streptozotocin Induced Diabetic RatsBackgroundThe incidence of diabetes mellitus (DM) has increased in developed countries; the main reasons are the modern life styles, dietary imbalances and irregular eating hours. All forms of diabetes are characterized by chronic hyperglycaemia and the development of diabetes-specific vascular complications. Since insulin is applied in the diabetic patients in the clinic, the chronic vascular complications are the major cause of morbidity and mortality for the diabetic patients.Grape seed proanthocyanidin extracts (GSPE) derive from grape seeds, have been reported to possess a variety of potent properties including anti-oxidant, anti-inflammation, and anti-tumor. It was reported that GSPE had an antiatherosclerotic effect in protecting aortic wall of cardiovascular diseases. Our previous experiments showed that GSPE displayed anti-nonenzymatic glycation, reducing receptor of advanced glycation end products (RAGE) protein expression, subsequently leading to decreased expression of high level vascular cell adhesion molecule 1 induced by advanced glycation end products (AGEs), and reduced the content of AGEs in the kidneys of diabetic rats. The purpose of this study was to examine the effects of AGEs on the diabetic aorta and to evaluate the efficacy of aortic protection by GSPE. We took streptozocin (STZ) induced diabetic rats as an animal model, determined the aortic pulse wave velocity (PWV), and collected aorta of the control, GSPE treated control rats, untreated, and GSPE treated diabetic rats, and applied them to transmission electron microscope, staining of hematoxylin-eosin (HE) and periodic acid-schiff(PAS).Objective1. To study the effects of GSPE on the function of aorta in the diabetic rats. Aortic PWV, central and peripheral blood pressure was determined by a pressure recording system after 24 weeks.2. To study the effects of GSPE on the morphology of aorta in the diabetic rats. Aortic morphology was observed by light and electron microscopy.MethodsMale Wistar rats (n=80, 180-220g) were purchased from Laboratory Animal Center of Shandong University. Thirty randomly selected rats were divided into two groups: control group (C1) and control group treated with GSPE (C2, administrated with GSPE with a dosage of 250mg/kg), which received a single tail vein injection of 0.1mol/L citrate buffer only. The other fifty rats received a single dose of STZ (55 mg/kg, injected into tail veins) freshly dissolved in 0.1 mol/L sodium citrate buffer (pH 4.5) after a 12 hours' overnight fasting. Only rats with blood glucose higher than 16.7 mmol/L after five days were considered as being diabetic in the fasting state, by using a One Touch II Glucose Analyzer. Ten rats with blood glucose levels of less than 16.7 mmol/L were excluded from the study. The diabetic rats were divided into 2 groups: an untreated diabetic group (DM1, n=20) and another diabetic group treated by GSPE with a dosage of 250mg/kg (DM2, n=20). GSPE was given in normal saline solution by intragastric administration for 24 weeks. Aortic blood pressure and pulse wave velocity were determined in anesthetized rats. Prior to sacrifice after 24 weeks of diabetes, animals were weighted. At the end of the hemodynamic measurements, blood was collected and centrifuged at 1500 g for 20 min for biochemical, fasting blood glucose (FBG), glycated hemoglobin (HbA_1c) and AGEs determination. The thoracic aorta ultrastructure was observed by electronic microscope. The pathologic changes of rat thoracic aorta tissue were studied by HE and PAS staining.Moreover, medial thickness/internal diameter ratio, elastic modulus (EM) and wall stress (WS) were calculated according to the Moens-Korteweg and Lame equation. Immunohistochemistry was performed on the paraffin-embedded tissue sections using a standard avidin-biotin complex peroxidase method using 3, 3'- diaminobenzidine as chromogen.Results1. General dataIn the course of experiment, rats of DM1 group showed hyperdiuresis, polydipsia, polyphagia, emaciation, filthy furs and slowly growth. Above-mentioned symptoms decreased in DM2 group. The rats in C1 and C2 group had no diabetic symptoms, their growth and nurtures were well.2. Effects of GSPE on Body Weight, FBG, HbA_1c and AGEsThe body weight of diabetic rats was significantly smaller than that of control rats at 24 weeks (P<0.01). GSPE improved the body weight of diabetic rats at 24 weeks (P<0.01). The FBG, HbA_1c and AGEs of diabetic rats were higher than those of the control rats (P<0.01). GSPE significantly reduced the AGEs of diabetic rats (P<0.05). But GSPE could not decrease the FBG and HbA_1c of diabetic rats.3. Effects of GSPE on Aortic Blood Pressures, PWV, WS and EMThe central and peripheral SBP, MAP, PP, central DBP and PWV of diabetic rats were higher than those of control rats (P<0.05), while there were no differences in their peripheral DBP and heart rate (P>0.05). After treated with GSPE, the SBP, DBP, MAP, PP and PWV decreased in DM2 group than those of DM1 (P<0.05).At the end of research, the thoracic aorta underwent outward aortic remodeling in the diabetic rats. The medial thickness, medial thickness/internal diameter ratio and EM were increased in diabetic rats relative to controls (P<0.01). WS of diabetic rats was significantly smaller than that of control rats (P<0.05), while there were no differences in their internal diameter (P>0.05). After treated with GSPE, the medial thickness, the medial thickness/internal diameter ratio and EM decreased in DM2 group than those of DM1 (P<0.01).4. Effects of GSPE on Aortic Morphology In the diabetic rats, aortic remodeling was observed in the thoracic aorta, and the nuclei were impaired, and the degeneration was exacerbated in the aortic tissue. A lot of PAS positive materials could be found in the thoracic aorta of DM1 group. Moreover, in the diabetic rats, GSPE suppressed the aortic remodeling, and led to light microscopic findings similar to those of the control rats.5. Effects of GSPE on Aortic UltrastructuresUnder electron microscopy, the impaired endothelial cell and the curmbly structure under endothelial tissue were seen from aortic tissue in the diabetic rats. Moreover, many smooth muscle cells inserted the endothelial tissue in diabetic rats, whereas normal ultrastructure was observed in aortic tissue of the control rats. GSPE tended to improve the preservation of the fine structure of endothelial cell and to decrease the number of inserted smooth muscle cells in the endothelial tissue.6. Effects of GSPE on the Protein Expression of RAGE in the Aorta TissueBy immunochemistry, it is found that the grey value of RAGE in the aortic of STZ induced rats decreased comparing to the non-diabetic controls while the number of RAGE positive cells increased (P<0.01).. After treated with GSPE, the grey value of RAGE in the aortic of STZ induced rats increased comparing to DM1 group, while the number of RAGE positive cells decreased (P<0.01).Conclusion1. GSPE improved the aortic function in the diabetic rats. After treated with GSPE, the SBP, DBP, MAP, PP, PWV and EM decreased in DM2 group than those of DM1.2. GSPE suppressed the aortic remodeling, and led to light microscopic findings similar to those of the control rats.3. GSPE tended to improve the preservation of the fine structure of endothelial cell and to decrease the number of inserted smooth muscle cells in the endothelial tissue. Part TwoProteomics Analysis of the Actions of Grape Seed Proanthocyanidin Extracts on Aortic Arteriosclerosis in the Streptozotocin Induced Diabetic RatsBackgroundWith the accomplishment of human genome project, genomes provide new insights into all kinds of dieases in the field of biomedicine. However, biological processes are driven by proteins and the evidence is mounting against messenger RNA (mRNA) being a reliable indicator of protein levels. Many early studies indicated a poor correlation between mRNA and protein for all but the most abundant proteins. The proteome consists of all proteins present in a cell or organism at a given time, including not only those translated directly from genetic material but also the variety of modified proteins arising from alternative splicing of transcripts, from extensive posttranslational processing, or from some combination of the two, resulting in modifications that have the potential to alter protein structure or function. Therefore, the science of proteomics is one of the most important areas of research in the post-genomic era.GSPE derive from grape seeds; have been reported to possess a variety of potent properties including anti-oxidant, anti-inflammation, and anti-tumor. It was reported that GSPE had an antiatherosclerotic effect in the cardiovascular dieases. Our previous experiments showed that GSPE had a protective effect on aortic tissue in the diabetic rats. But the molecular target is unclear in the macrovascular complications of diabetes.At present, the common shortcomings of drugs research are that we only pay attention to the phenotypic observation. Moreover, it is simple to understand the pharmaco-active of drugs in the molecular level. But we are unclear the underlying drug target. In order to make up the disparity of pharmaco-effect and its understanding in the molecular level, we study the drug mechanism and find the drug target by the technology of proteomics. These methods can improve the velocity and efficiency of drug research, and decrease the cost of new drug research. We took streptozocin (STZ) induced diabetic rats as an animal model, collected aorta of the control, untreated, and GSPE treated diabetic rats, and applied them to 2-D difference gel electrophoresis (2-D DIGE), followed by analysis using an AutoFlex martix-assisted laser desorption/ ionization time-of-flight mass Spectrometry with LIFT technology (MALDI-TOF-TOF MS) or liquid chromatography electrospray ionisation mass spectrometry/mass Spectrometry (LTQ-ESI-MS/MS). We expected to figure out which proteins participated in the progress of diabetic macrovascular complications and the efficiency of GSPE therapy.Objective1. To study the the pathogenetic mechanism of macrovascular complications caused by DM and obtain the differential proteome in the aorta of diabetic rats.2. To identify the valid multifunctional targets and therapeutic strategies against diabetic macrovascular complications by GSPE.MethodsTotal protein of aorta from group C1 (n=3), group DM1 (n=3) and group DM2 (n=3) were extracted. Aortic tissue lysates were labeled with Cy2, Cy3, and Cy5 following the protocols described in the Ettan DIGE User Manual. The protens were separated differently using immobilized PH gradients 2-DE. The Cy2, Cy3, and Cy5-labeled images were acquired on a Typhoon 9400 scanner. DIGE images were analyzed with DeCyder software as described in the Ettan DIGE User Manual.These gels were fixed and stained with colloidal Coomassie brilliant blue. Peptides were mixed with MALDI matrix and were spotted on to stainless steel MALDI target plates. Samples on the MALDI target plates were then analyzed using an AutoFlex MALDI-TOF/TOF mass spectrometer with LIFT technology (Bruker Daltonics, Bremen, Germany). The FlexAnalysis 2.4 software suite was used to generate the peak lists of all acquired MS/MS spectra. MS/MS was performed using the MASCOT search employing BioTools 3.0 software (Bruker Daltonics, Bremen, Germany). The data were sent to the National Center for Biotechnology nonredundant (NCBInr) protein database (NCBInr 20070518; 4927571 sequences, 1702359384 residues). The search was performed taking Rattus as taxonomy, which contained 40233 sequences. Proteins matching more than two peptides and with a MASCOT score higher than 30 were considered significant (P<0.05). Unidentified peptide mixtures by MALDI-TOF/TOF MS were measured using on a LTQ-ESI-MS/MS (ThermoFinnigan, San Jose, CA, U.S.A.), using a surveyor high-performance liquid chromatography (HPLC) system. The system was fitted with a C18 RP column (0.15mm×150mm, Thermo Hypersil-Keystone). Mobile phase A (0.1% formic acid in water) and the mobile phase B (0.1% formic acid in ACN) were selected. The temperature of the heated capillary was set at 170℃. A voltage of 3.0 kV applied to the ESI needle resulted in a distinct signal. For protein identification and statistical validation, the acquired MS/MS spectra were automatically searched against the Internation Protein Index (IPI) RAT version 3.15.1 database using the Turbo SEQUEST program in the BioWorks^TM 3.1 software suite. An accepted SEQUEST result had to have a DelCN score of at least 0.1 (regardless of charge state). Peptides with a +1 charge state were accepted if they were fully tryptic and had a cross correlation (Xcorr) of at least 1.9. Peptides with a +2 charge state were accepted if they had an Xcorr≥2.2. Peptides with a +3 charge state were accepted if they had an Xcorr≥3.75. Identifications were validated when considering at least two peptide sequences per protein.Results1.2-D DIGE ImagesIn our experiments, we got five 2-D DIGE images of group C1, group DM1 and group DM2. Spots detected by cCBB staining were excised and used for identification. In total, up to 1279-1563 different spots were detected on the five gels as determined by the DeCyder Differntial Analysis Software. Moreover, Separate preparative gels were run to obtain sufficient amounts of protein for MS analysis. These gels were fixed and stained with colloidal Coomassie brilliant blue.2. Mass Spectrometry Identify the Differently Expressed ProteinsSix protein spots were successfully identified by MALDI-TOF/TOF MS/MS. Nineteen protein spots were successfully identified by LTQ-ESI-MS/MS. A total of 25 protein spots included 17 up-regulated and 8 down-regulated spots in aortic tissue of diabetic rats, and were successfully identified with these two types of MS. Among them, the expression of 16 proteins was also found either down-regulated or up-regulated after treatment with GSPE.3. Localization Analysis of Differently Expressed ProteinsUsing the proteomics tool of ExPASy, the subcellar localization of all differently expressed proteins consists of cytoplasm, secreted protein, mitochondrion, cytolemma, nucleus and peroxisome. Forty percent subcellar localization is cytoplasm.4. Function Analysis of Differently Expressed ProteinsUsing the dynamically controlled vocabulary Gene Ontology and a lot of data, the function of all differently expressed proteins consists of metabolism, proliferation, motility, regulation, signal transport and response to stress. Thirty six percent proteins are metabolism functions.Conclusion1. We obtain the reliable differently expressed proteins from group C1, group DM1 and group DM2.2. A total of 25 protein spots included 17 up-regulated and 8 down-regulated spots in aortic tissue of diabetic rats, and were successfully identified with these two types of MS. Among them, the expression of 16 proteins was also found either down-regulated or up-regulated after treatment with GSPE.3. Most subcellar localization of differently expressed proteins is cytoplasm.4. Most of differently expressed proteins are metabolism functions.