| Puerarin was extracted from Chinese herb Pueraria lobata (Wild), which has been widely used in treating cardiovascular diseases (such as acute hypertetion, myocardial infarction and cardiac arrhythmia ), cerebral insufficiency, insulin resistance and could even improve the function of endothelium or endothelial progenitor cells (EPC) . Recently, puerarin has been reported to have effects on ion channel with patch clamp, and show effects on bone metabolism and estrogen level. However, most of these results are based on the clinic evidence and the mechanisms of puerarin on these diseases are still not very clear. So we hypothesized that puerarin might have the similar effect on rat thoracic aorta and the underline mechanism. Also, We attempted to examine the role of puerarin on the proliferation of VSMC induced by insulin and the underline mechanisms such as cell cycle, which could account for its long-term advantage effects on VSMC and improves the clinical symptoms and prognosis of patients with coronary arterial deseases.Objective1. To investigate the vasorelaxant effect of puerarin in SHR and WKY rat aortic rings and the underlying mechanism.2. Testify whether puerarin have the antiproliferation effect on VSMC and the possible mechanism.Methods1. Organ Bath:Rats were killed by stunning and subsequent cervical dislocation. The main branch of the Aorta were rapidly removed and stored in cold Krebs solution. Aorta were cleaned of fat and connective tissues and cut into 3 mm rings. The isolated thoracic aortic rings were mounted on the organ bath containing 10 ml Krebs solution at 37℃ and the contractile responses of the vessel were recorded.2. Cell culture:Male Sprague-Dawley rats (150-160 g) were decapitated. Thoracic aorta was aseptically removed rapidly and placed in physiological balanced saline. The endothelium was removed by gently rubbing with a cotton swab moistened with PBS. Then, the aorta was cut into small pieces about 1-3 mm3 and plated in Dulbecco's Modified Essential Medium (DMEM) containing 20%FCS, 100 u ml-1 penicillin and 10'6 g ml-1 streptomycin. VSMC were cultured in a humidified atmosphere of 5%CO2 and 95% air at 37 ℃ and confirmed by the positive responses to antibody of smooth muscle a-actin.3. Cell count and MTT test:VSMC were cultured in 24 well plates, in which DMEM plus 10% fetal calf serum were present. The cell density was 106 cells/L. After 24 h, cells were arrested by 0.5%FCS for 24 h. Then, drugs were added to the medium (antagonists were added 0.5 h prior to agonists). After 24 h incubation, cellswere trypsinized and counted.The cells at density of l×104cells per well were seeded into 96-well plates and incubated at 37℃. After 24h, the confluent smooth muscle cells were exposed to insulin(100 nmol/L), with Pue at the final concentration ranging from 1.5× 10-5- 1.5× 10-3 mol/L except the control cells for 24h, respectively. The antiproliferation effect of Pue on VSMC induced by insulin were evaluated by MTT method.4. Determination of cell cycle and protein contents of VSMC:VSMC were arrested to growth by DMEM containing 0.5% fetal calf serum. After 24 h,VSMC were exposed to DMEM with or without puerarin at the final concentration of lxlO^mol/L for another 24h. Insulin 100×10-9 mol/l was added to the cells at designed groups. After 48h, the cells were digested by a mixture of 0.25% trypsin and centrifuged at 150×g for 10 min, the pellets were washed in PBS solution twice, and then cells were dyed and analyzed by flow cytometry (Becton Dickinson, USA), respectively.5. RT-PCR analysis of MKP-1 mRNA expression:Total RNA isolation and RT-PCR were performed as described previously. The cycling conditions used were: 1 cycle at 94 ℃ for 5 min; 30 cycles at 94 ℃ for 30 s, 55 ℃ for 30 s; 1 cycle at 72 ℃ for 1 min. Amplified products were analysed by electrophoresis through 2 % MetaPhor agarose (FMC BioProducts).ResultPart 1: Mechanism of vasorclaxant effect of puerarin on rat thoracic aorta1. Puerarin caused complete relaxation of endothelium-intact aortic rings o...
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