| BackgroundAtherosclerosis (AS) is one of the most important reasons that induce cardiovascular disease. Growing evidence show that AS is a kind of excessive inflammatory response to vascular injury. Proliferation and phenotypic change of Vascular smooth muscle cells (VSMCs) is one of the main pathological basis of atherosclerosis (AS).Tumor necrosis factor-alpha (TNF-α) is one of glycosidoprotein mainly created by mononuclear macrophage. It is also one of criticality regulatory factors in inflammatory reaction that created by various inflammatory cells and playing an important role in the development of AS. Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecans family. It is the one of a major class of heparin sulphate proteoglycans in the vasculature. Current researches show that syndecan-4 is a kind of co-receptor linking with growth factor regulates many cell biological effects, as an important part in the cell expansion, recognition, adhesion, migration, multiplication regulation at equal pace mediates inflammatory reaction. Cordycepin or 3'-deoxy-adenosine, as nitrogen-containing glycoside nucleic acid derivatives, is one of the major pharmacologically active ingredient of Cordyceps sinensis.Past research had shown that cordycepin has anti-inflammatory and immunomodulatory effects and so on. Now statins(includes pravastatin)have become the cornerstone of treatment for coronary heart disease, they inhibited 3-hydroxy-3-methyl-coenzyme A reductase (HMG-CoA) competitively, thus reducing cholesterol synthesis.However, Statins' anti-inflammatory effect often does not depend on its cholesterol-lowering effect. Statins not only can suppress the expression of many inflammatory factors and reduce the level of serum inflammatory markers but also can inhibit various stages of the inflammationObjectiveTo respectively investigate the effects of cordycepin and pravastatin on cell proliferation and expression of syndecan-4 protein in rat aortic VSMCs induced by TNF-αin vitro. We hope to explain the potential role of syndecan-4 in the development of AS at cell and molecular level furtherly.Methods1. The detection of cell's proliferation:Exp one:rat VSMCs were cultured in 96 well assay in vitro and exposed to TNF-α(20ng/mL) or cordycepin (20μmol/mL,40μmol/mL) respectively, and then co-treated TNF-α(20ng/mL) with cordycepin (20μmol/mL,40μmol/mL) respectively. All the groups were cultured for 24 hours, in addition to the untreated control group established for comparison. Each concentration has 6 holes and we repeat the experiment 6 times at the same condition, then we get 36 data in total. The ratio of proliferation of VSMCs was determined by non-radioactive MTS/PMS assay.Exp two:rat VSMCs were cultured in 96 well assay in vitro and exposed to TNF-α(20ng/mL) or pravastatin (10μmol/mL,20μmol/mL) respectively, and then co-treated TNF-α(20ng/mL) with cordycepin (10μmol/mL,20μmol/mL) respectively. All the groups were cultured for 24 hours, in addition to the untreated control group established for comparison. Each concentration has 13 holes and we repeat the experiment 2 times at the same condition, then we get 26 data in total. The ratio of proliferation of VSMCs was determined by non-radioactive MTS/PMS assay 2. The detection of syndecan-4 protein expressionExp one:In vitro cultured rat aortic VSMCs were exposed to treatment with 20ng/ml TNF-α,20μmol/ml cordycepin,40μmol/mL cordycepin,20μmol/mL cordycepin with 20ng/ml TNF-α,40μmol/ml cordycepin with 20ng/ml TNF-αfor 24 hours respectively, and then the cell were lysed and the concentration of protein was evaluated using Coomassie brilliant blue G-250. The expression of syndecan-4 protein in rat vascular smooth muscle cells was detected by the immunoblotting technique using anti-syndecan-4 antibody. Each concentration was repeated 3 times and we set up drug non-irritant as control each time. The expression of syndecan-4 protein was represented by the intensity.Exp two:In vitro cultured VSMCS cells were exposed to treatment with 20ng/ml TNF-α,10μmol/ml pravastatin,20μmol/mL pravastatin,10μmol/mL pravastatin with 20ng/ml TNF-α,20μmol/ml pravastatin with 20ng/ml TNF-αfor 24 hours respectively, then the cell were lysed and the concentration of protein was evaluated using Coomassie brilliant blue G-250. The expression of syndecan-4 protein in rat vascular smooth muscle cells was detected by the immunoblotting technique using anti-syndecan-4 antibody. Each concentration was repeated 3 times and we set up drug non-irritant as control each time. The expression of syndecan-4 protein was represented by the intensity.Results1. Effects of cordycepin on proliferation of rat aortic vascular smooth muscle cells induced by TNF-αThe proliferation rate (represented by the optical density) of VSMCs was 0.666±0.098 in the control group,1.013±0.150 in 20ng/mL TNF-αgroup, 0.660±0.097 in 20μmol/ml cordycepin group,0.676±0.085 in 40μmol/ml cordycepin group,0.662±0.107 in 20μmol/mL cordycepin with 20ng/ml TNF-αgroup, and 0.627±0.135 in 40μmol/mL cordycepin with 20ng/ml TNF-αgroup. Statistical analysis showed that TNF-αcan significantly stimulate the proliferation of rat VSMCs with the concentration of 20ng/mL (P<0.05). cordycepin alone had no obvious effect on VSMCs growth (P>0.05), but significantly inhibited proliferation of VSMCs induced by TNF-α(P<0.05).2. Effects of cordycepin on expression of syndecan-4 protein in rat aortic vascular smooth muscle cells induced by TNF-αTake the ratio of interest protein andβ-actin's gray scale of control group as 1, the expression of syndecan-4 in rat aortic vascular smooth muscle cells after 24 hours was 1.375±0.017 in 20ng/mL TNF-αgroup,1.009±0.120 in 20μmol/ml cordycepin group,1.002±0.050 in 40μmol/ml cordycepin group,0.962±0.057 in 20μmol/mL cordycepin with 20ng/ml TNF-αgroup, and 0.914±0.057 in 40μmol/mL cordycepin with 20ng/ml TNF-αgroup. Statistical analysis suggested that the expression of syndecan-4 protein in VSMCs was significantly enhanced induced by TNF-αwith the concentration of 20 ng/mL(P<0.05). cordycepin alone had no effect on the expression of syndecan-4 protein(P>0.05), but significantly inhibited the expression of syndecan-4 protein induced by TNF-α(P<0.05).3,Effects of pravastatin on proliferation of VSMCs induced by TNF-αThe proliferation rate (represented by the optical density) of VSMCs was 0.625±0.133 in the control group,1.069±0.146 in 20ng/mL TNF-αgroup, 0.635±0.134 in 10μmol/ml pravastatin group,0.651±0.134 in 20μmol/ml pravastatin group,0.684±0.128 in 10μmol/mL pravastatin with 20ng/ml TNF-αgroup, and 0.630±0.145 in 20μmol/mL pravastatin with 20ng/ml TNF-αgroup.Statistical analysis showed that TNF-a can significantly stimulate the proliferation of VSMCs with the concentration of 20ng/mL (P<0.05). pravastatin alone had no effect on VSMCS cells growth (P>0.05), but can significantly inhibit proliferation of VSMCs induced by TNF-α(P<0.05).4,Effects of pravastatin on expression of syndecan-4 protein in VSMCs induced by TNF-αTake the ratio of interest protein and P-actin's gray scale of control group as 1, the expression of syndecan-4 protein in VSMCs after 24 hours was 1.340±0.050 in 20ng/mL TNF-αgroup,1.020±0.035 in 10μmol/mL pravastatin group,1.010±0.072 in 20μmol/mL pravastatin group,0.967±0.038 in 10μmol/mL pravastatin with 20ng/mL TNF-αgroup, and 0.923±0.038 in 20μmol/mL pravastatin with 20ng/mL TNF-αgroup. Statistical analysis suggested that the expression of syndecan-4 protein in VSMCs was significantly enhanced by TNF-αwith the concentration of 20 ng/mL (P<0.05). Pravastatin alone had no effect on the expression of syndecan-4 protein (P>0.05), but significantly inhibited the expression of syndecan-4 protein induced by TNF-α(P<0.05).ConclusionsOur study suggests that cordycepin and pravastatin can significantly inhibit the proliferation of rat aortic vascular smooth muscle cells induced by TNF-αin vitro, respectively. Cordycepin and pravastatin can also significantly inhibit expression of syndecan-4 protein in rat aortic vascular smooth muscle cells induced by TNF-αin vitro, respectively. This founding may add to our understanding of the importance of homeostatic regulators of inflammation and represent an additional component of the anti-inflammatory pathways that are activated in response to changes in vascular wall. |
PDF Full Text Request