Effects Of Polysaccharide Sulfate On Atherosclerosis Induced By Type 2 Diabetes

PrefaceThese days aging of the world population and changing of people life style, prevalence rate of Type 2 diabetes mellitus is gradually increasing. But disease of cardiovascular mainly result in death of it, occupied 80% . Atherosclerosos is the most popular. It is now clear that Type 2 diabetes mellitus commomly occur together with a collection of clinical and biochemical features which have been called metabolic syndrome X;hyperglycaemia, insulin resistance, dyslipidae-mia, and fibrinolytic system dysfunction. They are all the risk factors which can accelerate the processes of atherosclerosis. This experiment chooses to use with the rat model of Type 2 diabetes which is the similar with people. Observe te effect on the endothelium formation and function of atherosclerosis induced by Tppe 2 diabetes, activities of nuclear factor, expression of intercellular adhesion molecule and changes of activation of tissue - type plasminogen activator ( t -PA ) and plasminogen activator inhibitor type - 1 ( PAI - 1). We used the ocean medicine: polysaccharide sulfate exploring its effects and possible mechanism on atherosclerosis induced by Type 2 diabetes.Materiales and Methods1. Drugs and reagentsStreptozotocin (Sigma Co. , USA) ,Polysaccharide sulfate (qingdao medical factory) , One Touch Ultra blood - glucose device (LIFESCAN, USA) , Tissue -type plasminogen activator(t -PA) and Plasminogen activator inhibitor type - 1 ( PAI - 1) Test Kit ( Sunbiote, China) , Ultrasensitive? S - P Kit( Boster Co. , China) ,Western blotting reagents (Santa Cruze Co. , USA) .male Wister rats were obtained from animal experimental center, China medical university. Their weights were 150g or so.2. MethodsSeventy male Wister rats weight 150g or so. Randomly selecting ten rats fed normal chow diet is control group. The rests were given high -fat diet (2% cholesterol, 1% sodium cholate, 20% fat, 10%carbohydrate and 67% conventional chow) for eight weeks. Animals had free acess to food and water. Then they were injected with 0.3% STZ (0.1 ml/Kg, dissolved in citric acid buffer, PH 7. 2, Sigma,USA) into the tail veins. Three weeks later, the fat -fed rats which FBG increased above lOmmol/L were randomly and equally divided into four groups;model group, polysaccharide sulfate group, metformin group, lov-astatin group. Control group and model group were given o. 9% Nacl solution. Other groups were respectively treated with exact medicines: 1.8% polysaccharide sulfat solution, 2% metformin solution, 0. 04% lovastatin solution. Tre-ament does is 0. 1 ml/Kg and per oral gavage with vehicle. On the while all the groups are same. They were treated 8 weeks and during these days they had free common diet and water.At the beginning of this experiment and 9,12, 20 week, tail blood samples were taken and plasma glucose concentrations were detected by One Touch Ultra blood — glucose device. Animals were not anesthetized for this procedure. After twenty weeks blood samples were got from the venous plexus of the eyes. Serum was separated by centrifugation in serum separator tubes ( Becton Dickinson, British). Plasma lipid was detected by automatic biochemistry analyzer ( Hitachi, 7600 -020) . And the activities of tissue - type plasminogen activator (t -PA) and plasminogen activator inhibitor type - 1 ( PAI - 1 ) were measured by Test Kit (Sunbiote, China) .Twenty weeks later,rats in each group were sacrified after intraperitoneally (i. p. ) anesthetizing with pentobarbital sodium 40mg/kg and aortas were removed. About lcm of the aortas were fixed in 4% paraformaldehyde over night and embedded in paraffin by a routine procedure. Consecutive 4jjun paraffin section were made which were used for immunohistochemistry stains of NF - kB andICAM - 1 . The rests of aortas were preserved in liguid nitrogen which were fit for Western blotting of NF - kB and ICAM - 1. The same part of abdominal aortas were fixed in 3% glutaraldehyde overnight and used for doing the sections of transmission electron microscope, which were enlarged for observing and taking photographs.Results1. FBG concentrations and serum lipid (TC, TG, LDL) levels decreased in PSS group as compared to those of model group ( P < 0. 05 ) , and showed no difference compared to Lov group of serum lipid and to Met group of FBG .2. The activities of t - PA and PAI - 1 in PSS group were remarkably different to those of model group .3. Nuclear factor kappa B (NF - kB) activation and intercellular adhesion molecule (ICAM - 1) expression level of the aortas in the model group were significantly higher than PSS group (P <0. 05) .4. Damage to the arterial wall in PSS group is not serious than that in model group .DiscussionType 2 diabetes mellitus is the third serious disease after tumor and cardiovascular disease especially in developing countries. In addition, diabetic patients have a high incidence of microvascular and macrovascular complications mediated by atherosclerosis. However, the United Kingdom Prospective Diabetes Study ( UKPDS) and other similar studies indicated that intensive control of blood glucose in diabetic patients prevents and/or slows the progression of microvascular complications ,but has little effect on macrovascular complications. A number of studies showed that metabolic syndrome X of Type 2 diabetes : hy-perglycaemia, hyperinsulinaemia, dyslipidemia, and fibrinolytic system dysfunction were contributed to the forming and developing of atherosclerosis.It is well known that atherosclerosis is a pathegenesis process involved inthe inflammatory and proliferative responses of cells in which endothelial cells have an important function in the regulation of variety of genes. A variety of molecules have been identified in the atherosclerotic environment induced by Type 2 diabetes that are able to activate NF - kB or able to be activated by NF -kB.Nuclear factor kappa B is multidominance transcription factor which is widely existed in cytoplasm. It has the effect on regulation of gene transcription especial about inflammatory correlation genes such as genes of adhesion molecules, cytokines, growth factors and immunologic receptors. In inactivation state NF - kB binds its inhibition protein I - kB which form compound and is confined in cytoplasm. When some signals activate NF - kB , it is released from the compound through I - kB phosphorylation and being degradated by protease. Subsequently activated NF - kB enters into the nucleus and mediates gene transcription. Hyperglycaemia, hyperinsulinaemia and dyslipidemia of Type 2 diabetes enhance the oxidation and damage endothelia cells. In addition such reasons all can activate NF — kB and then activated NF - kB entering into nucleus promoting the transcriptions and expressions of ICAM - 1, VCAM - 1, MCP - 1 and so on. These factors facilitate the monocytes adhering to the vascular wall and infiltrating into the intima of the aortas.At the same time fibrinolytic system function disorders in Type 2 diabetes patients denoting that activities of t — PA decreases and PAI - 1 increases. The bodies are in hypercoagulable state which promotes the leucocytes, monocytes and platelets moving and adhering to the damaged endothelium. Simultaneously changes of activities of t - PA and PAI - 1 activate NF - kB and cause a series of biochemical reactions. Above all promote the atherosclerosis forming and developing.At present there are many medicines treating for Type 2 diabetes. But some of them are expensive, or some of them have single effect , or have serious side effects. Therefore we are exploring a new type of medicine using for Type 2 diabetes. Polysaccharide sulfate is a kind of ocean medicines which is negative ion polymer. It has the effect like heparin. This experiment confirmed that Polysaccharide sulfate had effects on decreasing the blood glucose, plasma lipid and in-sulin level. It improved the function of fibrinolytic system (increasing the activity of t - PA and decreasing the activity of PAI - 1) . Furthermore polysaccharide sulfate can alleviate the damage to the arterial wall and inhibit the NF - kB activating , ICAM - 1 expressing and the monocytes infiltrating into the arterial wall. In conclusion, polysaccharide sulfate can inhibit occurring and developing of atherosclerosis induced by Type 2 diabetes mellitus from a lot of aspects.Conclusion1. PSS can decrease the FBG concentrations.2. PSS can decrease serum lipid levels.3. PSS can adjust the activities of t -PA and PAI - 1.4. PSS can inhibiting NF - kB activation and ICAM - 1 expression.