Study On The Expression Of TSP-1and TGF-β1in Diabetic Complications Of Diabetic Rats

BackgroundDiabetes (Diabetes Mellitus, DM) has become the one of highest incidence and the most serious disease to human health. One important mechanism of type2diabetes was insulin resistance and insulin dysfunction, then leading to islet failure. Disorder of glucose metabolism, that leading to microvascular disease, and toxic effects of high blood sugar on tissue cells, could give rise to a series of complications, including diabetic erectile dysfunction, diabetic nephropathy, diabetic cardiomyopathy, and diabetic dementia, etc. The research data indicated that about half of patients diagnosed with diabetes after10years with a possible merger erectile dysfunction (ED). ED was the first manifestation in about12%of ED patients with diabetes. Hyperglycemia itself has toxic effects on cardiac, epidemiological studies have found that more than70%of patients with diabetes died of cardiovascular diseases. Diabetic cardiomyopathy was a major cardiac complications in patients with diabetes, and closely related to high incidence and high mortality of cardiovascular disease. It is well established, diabetic cardiomyopathy was an independent primary disease, and was independent on hypertension, coronary artery disease and other known heart disease. Diabetes was also closely related to the cognitive impairment and dementia, as in Europe, the incidence rate of dementia in diabetes patients older than65is6.4%. Leibson et al reported1455cases of adult onset diabetes,981patients were observed after one year later,101had dementia, of which77cases of senile dementia. The mechanism of multi-organ damage in diabetes has still unclear. The non-enzymatic formation of advanced glycation end products, protein kinase C signaling pathway, oxidative stress, polyol pathway activation and increased hexosamine pathway activity were the some roles involved in. With further research, we found that the multiple organ injury in diabetes, that had interstitial fibrosis, apoptosis-related or pathological changes in synaptic plasticity. The high glucose activiated TSP-1and TGF-β1signaling pathway, then caused the multiple organ damage in patients with diabetes, brought to our concern. Thrombospondin (Thrombospondin, TSP), especially TSP-1, is an important physiological activator. TSP-1is an extracellular glycoprotein molecules, initially in platelets stimulated by thrombin product of a-granule release was found, after study has confirmed the TSP is not a platelet-specific, can be mesangial cells, fibroblasts and other cells, many organizations such as the kidney, heart, cartilage and brain are the product of the TSP gene expression, many different organizations, an important component of the extracellular matrix. Study found that TSP-1peptide by the three chains of the same homologous matrix glycoprotein trimer, each peptide chain from the N-terminal, C-terminal globular domain, procollagen homology region, typel repeats, type2repeats, type3repeat sequences. One typel repeats (thrombospondin typel repeats, TSRs) prepared by the three solution-like motif (TSR1, TSR2, TSR3) composition. Studies have shown that, TSP-1molecule KRFK sequence with L-TGF-β1of the LAP area, thereby changing the spatial configuration of the LAP, and exposure TGF-β1receptor binding site, then become activated TSP-1/L-TGF-β1. The research also showed that thrombospondin in hippocampus are closely related to the establishment of synaptic activation, plasticity the morphology and function. Therefore, we propose the following hypothesis:the diabetic state (such as high sugar stimulation) conditions caused by various types of body cells TSP-1ang TGF-β1activation, thus promoting proliferation of these cells and collagen fibers, leading to fibrosis, and changes of synaptic plasticity, which leads to diabetic multiple organ injury. Recent studies have revealed that interference in the mammalian small RNA (siRNA) through the conservative structure of biological RNA interference to inhibit the expression of the corresponding target genes. RNA interference is highly sequence-specific, can shut down the expression of target proteins. Many studies have confirmed that RNA interference can block many types of mammalian virus replication, inhibition of cancer growth. RNA interference has been developed as a powerful reverse genetics tool and has shown broad prospects for therapeutic application on.In this study, we investigated the expression of TSP-1and TGF-β1changes under high glucose on hippocampal neurons, design of siRNA on silencing expression of TSP-1gene, and to assess the reduction of TSP-1expression on neuronal morphology and synapse formation. It would provide the in vitro experimental evidence on a target gene therapy of TSP-1in diabetic learning and memory ability injury. PART Ⅰ TSP-1and TGF-β1expression in penis, myocardium, and hippocampus of diabetic rat1ObjectiveInvestgating the TSP-1and TGF-(31express in penis, myocardium, and hippocampus of diabetic rat, proved TSP-1and TGF-β1was involved in diabetic above-mentioned organ injury under high glucose.2Materials and Methods2.1Preparation of diabetic ratsSD rats were randomly divided into two groups, diabetes rat's model were induced by intraperitoneal injection of streptozotocin (STZ)65mg/kg, regular testing urine, blood glucose and body weight in order to ensure the maintenance of high blood sugar status. While the normal control group rats were injected with normal saline, with same feeding conditions for six weeks.2.2The organ function tests2.2.1Behavioral testing: The learning and memory changes in each group of rats were to detect by Morris water maze test (recording the escape latency and search strategy).2.2.2Erectile function testing: Using subcutaneous injection of apomorphine hydrochloride (APO) solution (150ug/kg) about1ml, observation30min, recording its erection situation.2.2.3Heart function testing: The rats were killed after full anesthesia thoracotomy, the heart quickly removed, placed in4°C KH solution to remove blood, then quickly transferred and fixed on the Langendorff perfusion device with a modified KH solution by conventional retrograde perfusion constant (76mmHg), observed the end of perfusion balance the amount of cardiac work changes (rate-pressure product, RPP), that is left ventricular developed pressure*heart rate (HR*LVDP).2.3Pathological changes of organs observationThe rats were killed after anesthesia, penis, heart and brain were taken out and making paraffin slice. Pathological changes of these organs were observed by HE staining, while heart and hippocampus tissue slices were produced embedding and using electron microscope to observe the ultra-micro-structure changes.2.4Immunohistochemistry and real-time quantitative PCRThe rats were killed after anesthesia, penis, heart and brain were taken out. TSP-1and TGF-β1protein expression were examed by immunohistochemical ABC method, while TSP-1and TGF-β1mRNA expression was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).2.5Statistical analysisValues are mean±SD. The differences in multiple groups were analysised by statistical software SPSS16.0one-way analysis of variance, while the differences between the two groups were analysised by/t test.3Results3.1The body weight, blood and urine glucoseBefore diabetes model induction, the body weight, blood and urine glucose were no significant differences between the two groups (P>0.05). After6weeks, the diabetic group body weight251.6±27.4g, blood glucose were22.6±3.6mmol/L, urine for the++-+++. Normal rats weighing397.4±21.3g, blood glucose5.1±0.5mmol/L, urine glucose (-). There are significant differences between the two groups (P<0.01). 3.2The organs functionThe erectile function was detected in rats with diabetes and erectile erections per unit time and rate were1.4±0.5times/30min and40%. It was significantly lower than the normal control group (2.4±0.8times/30min and100%)(P<0.01).Heart function tests also showed that diabetic rats RPP was significantly lower than normal rats,(P<0.01).Morris water maze behavioral experiments showed that normal rats on day1of the escape latency of (40.1±26.0s);2days (24.8±14.2s);3days (13.4±4.8s); through platform number is (6.1±3.8); diabetic rats on day1of the escape latency of (71.6±33.7s);2days (43.4±13.8s);3days (26.9±19.1s), through platform number is (3.2±1.3), the difference was statistically significant (P<0.01).3.3Pathological changes in penis, myocardium and hippocampusHE staining of the tissues showed that the thickness of the albuginea penis increased, penis collagen fiber damaged, and lost its characteristic undulating appearance, but also degradation of endothelial and smooth muscle cells in diabetic rats. Diabetic rats heart have varying degrees of interstitial fibrosis and myocardial hypertrophy, myocardial fiber structural appeared sparse. Micro-structure of diabetic rat heart revealed that myocardial myofibril necrosis, dissolution, loss of normal sarcomere structure, and even myocardial apoptosis under electron microscope. Diabetic rat hippocampus shows that mitochondria within neurons disappear, transformed the synaptic structure between neurons, while the number of synapses per unit area of the hippocampus was significantly less than normal rats.3.4Immunohistochemistry and real-time quantitative PCR assayTSP-1and TGF-β1protein expression in penis, heart and hippocampus of diabetes rats were significantly up-regulated, in which the penis, heart and hippocampus of TSP-1and TGF-β1in unit area of positive cells were respectively higher than the control group rats (P<0.01). The real-time quantitative PCR showed that TSP-1mRNA expression in penis, myocardium and hippocampus were2.8folds,1.7folds,6.6folds and TGF-β1mRNA expression were6.9folds,4.3folds,1.4folds while compared with the comtrol group rats.4ConclusionSTZ-induced diabetic rats had increased blood sugar, urine sugar, and decreased the weight, sexual function, cardiac function and learning and memory ability, with the penis, the heart and hippocampus of different degrees of pathological changes. In addition, TSP-1and TGF-β1protein and mRNA expression in dibetes rats were significantly increased while comparing with the normal control rats. PART Ⅱ The protection of siRNA against TSP-1on high glucose cultured neurons form TSP-1expression changes1ObjectiveTo explore the siRNA targeting TSP-1can effectively improve the high glucose cultured neuronal morphology and expression of TSP-1, and the protection of neurons and synapses of the contact normal.2Materials and Methods2.1Cell cultureSD rats pregnant16days,0.5%sodium pentobarbital anesthesia, opening the uterus, remove the fetus, and isolated hippocampal brain to take. Organization and the purpose of grinding through200mesh, cells were collected, centrifuged and resuspended. Inoculated at1×Polylysine-coated6-well plates and a small round glass slides in48-well culture plates at37℃with5%CO2and95%air.2.2siRNA preparation and transfectionEach of the siRNA sequence is as follows. TSP-1sence5'-UACACUUGGCACCAGCAAAGCAGGG-3', LacZ primers upstream primer5'-ACCAGAAGCGGRGCCGGAAA-3'primer5'-CCACAGCGGATGGTTCGGAT-3', cell preparation, transfection, and then gently Shakers,5%carbon dioxide,37℃humidified air in the conventional culture for48hours. After transfected for48h, cells were harvested for the next experiments.2.3Scanning electron microscopy observationThe cell culture, the dry glass slides covered with small round48-well culture plates in culture medium,4℃saline wash1,2.5%glutaraldehyde1h,0.01M PBS washed2timesx15min,1%osmium tetroxide fixed1h,0.1M PBS solution, rinsed twice for15minutes. Gradient alcohol dehydration,100%acetone dehydration20min. With50%isoamyl acetate replacement of15min,100%amyl acetate replacement of15min, the critical point drying, sticky table, gold-plated, observed on the machine.2.4Immunohistochemistry and Western Blot assayImmunohistochemistry:0.01M PBS containing0.3%of the rest were washed TRITON X-100(pH value7.4, PBS-T), then immersed in PBS2%normal goat serum for2hours at37℃4℃overnight, TSP-1antibody (1:100) and PBS containing1%bovine serum albumin, washed in PBS (3×5minutes), biotinylated goat anti-rabbit IgG (1:200) were incubated in PBS wash, PBS-T (3x5minutes), at room temperature for2hours, the immune markers diaminobenzdine0.05%, plus0.3%H2O2in PBS, staining, endoscopic control of reaction time, and then dehydrated with graded ethanol and xylene. Which PBS was used instead of primary antibody as negative control.Western Blot asay:the same amount of protein extract loaded onto SDS/PAGE gel and was separated, then transferred to PVDF membrane, and then with5%skim milk TBST solution closed1hour, then using the antibody at room temperature incubated for2hours, then TBST membranes were washed4times, then use the anti-mouse IgG-HRP (DAKO) incubated for1hour at room temperature, the final protein with the ECL detection system. Protein relative expression=optical density of test protein/internal reference optical density value.2.5Statistical analysisValues are mean±SD. The differences in multiple groups were analysised by statistical software SPSS16.0one-way analysis of variance, while the differences between the two groups were analysised by ttest. 3Results3.1SEM observationThe cultured cells by scanning electron microscopy showed normal group of neurons surface microvilli structural integrity, neuronal links between broad and close, high glucose group of neurons in cell surface microvilli structure loss, the membrane slightly injury, and neurons contact sparse, while the RNA interference group of neurons showed less severe injury.3.2Immunohistochemistry and Western Blot assayDetection of neuronal morphology and TSP-1and TGF-β1protein expression to assess the impact of siRNA on neurons. TSP-1and TGF-β1protein expression was significantly reduced in the TSP-1siRNA group while compare with the normal control group (P<0.01). The TSP-1and TGF-β1protein expression of negative transfection group and control group, the difference was not statistically significant (P>0.05).4ConclusionOur study implied that interference by siRNA targeting TSP-1can effectively improve the high glucose neuronal morphological and synapses changes and reduce TSP-1and TGF-β1protein expression.