| Effect of Astragalus on matrix metalloproteinase-2 secreted by rat vascular smooth muscle cellsObjective Percutaneus translumianl coronary angioplasty (PTCA) is now well established as a effective means of achieving revascularization in many patients with coronary artery disease. Despite high intial success rates, its long-term efficacy has been greatly limited by the development of restenosis at the site of angioplasty in up to 30-50% of cases within six months. Although the molecular mechanisms of restenosis are far elucidated, numerous studies have shown that excessive VSMC growth play a pivotal role in the pathophysiology of atherosclerosis and restenosis following PTCA. Recently , matrix metalloproteinases (MMPs) has been found and is supposed to play an important role in the pathophysiology of atherosclerosis and restenosis following PTCA. Under normal conditions, VSMC in the media are quiescent and are embedded in a network of different extracellular matrix (ECM) components that act as barriers to both VSMC migration and proliferation. Digestion and remodeling of the ECM occur early after vascular injury and are related to the activation of different proteases, including MMPs. It has been shown that MMP-2 is constitutively expressed in the rat carotid artery. Therefore, the time frame of the changes in MMP-2 are consistent with a role in the migration of VSMC into the intima. Nitric oxide (NO) is an important message molecular, nurmous evidences have beenfound that it can inhibite SMC proliferation. Because MMP-2 play an important role in cell migration and NO inhibits VSMC migration, we want to study the effect of the Chinese traditional herb-Astragalus on matrix metalloproteinase-2 secreted by SD rat vascular smooth muscle cell and NO levels and investigate their relationship. Methods Cell cultureVSMCs were routinely isolated from thoracic aorta of 150-250g male Sprague-Dawley rats and cultured in Dulbecco's modified Eagle's medium ( DMEM, Gibco) supplemented with 10 % fetal bovine serum (FBS) , 100U/ml penicillin and 100 μ g/ml streptomycin . Cells were characterized as smooth muscle cells by their typical "hill-and-valley" morphology and by positive immunostaining for a -smooth muscle actin. Cells between passages 3 to 6 were used for all experiments. After 24 hours serum free cultured, VSMC was treated with Astragalus 0 mg/ml, 0.5mg/ml, 5 mg/ml, 50mg/ml with 10%FBS DMEM respectively for 24 hours (n=4). On the other hand, After 24 hours serum free cultured, VSMC was treated with or without Astragalus 5 mg/ml with 10%FBS DMEM for 0, 6, 12, 24 hours respectively (n=4). Measurement of MMP-2 ActivatyAfter cultured with serum free for 24 hours, 10% FBS and different concentrations of Astragalus were added to stimulate VSMC. Conditioned media were obtained at the indicated times. Protein concentration was estimated by Lowry method. Then samples were denatured in SDS sample buffer, without boiling, and then subjected to electrophoresis on 10% SDS-polyacrylamide gels containing 0.1% (wt/vol) gelatin. Gels were washed with 2.5% Triton X-100 for 30 minutes at room temperature then incubated at 37 ℃ for 18 hours . Then gels were stained with Coomassie blue R-250, and MMP-2 produced clear areas of lysis in the gel. The digestive area were visualized with enhanced chemiluminescence reagents.Measurement of NOConditioned media were obtained, then NO concentration of media was detected according to the directions of the kit. ResultsAfter treated with Astragalus 0 mg/ml, 0.5mg/ml, 5 mg/ml, 50mg/ml respectively for 24 hours (n=4), the activity of MMP-2 was 100%, 81.75 ± 6.40%, 67.00±11.75%, 52.75±11.18%( P< 0.05); NO concentration of media was 15.32±4.38 μM/L, 51.82± 13.19uM/L, 120.57 ± 29.85 μM/L, 185.46 ± 37.23 uM/L respectively(P< 0.05); On the other hand, After treated with Astragalus 5 mg/ml for 0, 6, 12, 24 hours respectively (n=4), the activity of MMP-2 was control groups: 100%, 96.78 ± 4.78%, 97.59± 4.54%, 103.94± 3.36%; conditioning group...
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