| BackgroundAtherosclerosis(AS)is a chronic inflammation overactive to the injury of blood vessels.Proliferation and phenotypic change of vascular smooth muscle cells(VSMCs)is one of the main pathological basis of AS.Tumor necrosis factor-alpha(TNF-?)is one of critical regulatory factors in inflammatory reaction that plays an important role in the development of AS.Syndecan-4 is a member belonging to the syndecans family which are type ? transmembrane proteins having a core protein modified with glycosaminoglycan chains,most commonly heparin sulphate.Syndecan-4 is recognized as a "co-receptor" in conjunction with other receptors,such as growth factor,regulating the behavior of cells.It plays an important part in the cell expansion,recognition,adhesion,migration and proliferative regulation,and mediates inflammatory reaction.The signal pathway of mitogen-activated protein kinase(MAPK)is recognized as the co-pathway of intracellular signal transduction originating from the cell proliferation caused by extracellular signal.p44/42 MAPK is the most important one of the MAPK family,and is a critical pathway of cell proliferation induced by cell factor and growth factor.Curcumin is the active ingredient of rhizoma curcumae longae.Modern scientific research has demonstrated its potential effects in chronic inflammation diseases.The mechanism of the curcumin in the protective of atherosclerosis is worth of further exploration.An appreciation of extensively use in the primary and secondly prevention of the ischemic heart disease and stroke has made Acetylsalicylic acid(aspirin,ASA)the cornerstone of anti-platelet treatment.Although platelet-independent effects of aspirin inhibiting VSMCs proliferation is appealing,the underlying mechanism evolved remains to be fully elucidated.ObjectiveTo investigate the effects of curcumin and aspirin on cell proliferation and expression of syndecan-4 protein and p44/42 mitogen-activated protein kinase phosphorylation in rat vascular smooth muscle cells(VSMCs)induced by tumor necrosis factor-?(TNF-?)in vitro.Methods1.The detection of cell's proliferationExperiment one:rat VSMCs were cultured in 96 well assay in vitro and stimulated by 20 ng/mL TNF-?,20 ?mol/L curcumin and 20 ng/mL TNF-? with 20?mol/L curcumin respectively.All the groups were cultured for 24 hours,in addition to the untreated control group established for comparison.We repeated the experiment 4 times under the same conditions,then we got 215 data in total.The non-radioactive MTS/PMS assay was adopted to detect the ratio of proliferation in rat VSMCs.Experiment two:rat VSMCs were cultured in 96 well assay in vitro and stimulated by 20 ng/mL TNF-?,1 mmol/L aspirin,2 mmol/L aspirin,20 ng/mL TNF-? with 1 mmol/L aspirin,20 ng/mL TNF-? with 2 mmol/L aspirin respectively.All the groups were cultured for 24 hours,in addition to the untreated control group established for comparison.We repeated the experiment 3 times under the same conditions,then we got 180 data in total.The non-radioactive MTS/PMS assay was adopted to detect the ratio of proliferation in rat VSMCs.2.The detection of syndecan-4 protein expressionExperiment one:rat VSMCs were cultured in vitro and stimulated by 20 ng/mL TNF-?,20 ?mol/L curcumin and 20 ng/mL TNF-? with 20 ?mol/L curcumin for 24 hours respectively,the untreated control group established for comparison.The cells were lysed and the concentration of protein was evaluated by BCA Kit.The expression of syndecan-4 protein in rat VSMCs was detected by Western blot using syndecan-4 antibody.The experiment was repeated 3 times and we got 12 data in total.The expression of syndecan-4 protein was represented by the intensity.Experiment two:rat VSMCs were cultured in vitro and stimulated by 20 ng/mL TNF-??1 mmol/L aspirin?2 mmol/L aspirin?1 mmol/L aspirin with 20 ng/mL TNF-??2 mmol/L aspirin with 20 ng/mL TNF-? for 24 hours respectively,the untreated control group established for comparison.The cells were lysed and the concentration of protein was evaluated by BCA Kit.The expression of syndecan-4 protein in rat VSMCs was detected by Western blot using syndecan-4 antibody.The experiment was repeated 3 times and we got 18 data in total.The expression of syndecan-4 protein was represented by the intensity.3.The detection of phosph-p44/42 MAPK expressionExperiment one:rat VSMCs cultured in vitro and exposed to 20 ng/mL TNF-??20 ?mol/L curcumin?20 ?mol/L curcumin with 20 ng/mL TNF-? for 24 hours respectively.Control group were established for comprision.The cells were lysed and the concentration of protein was evaluated by BCA Kit.The expression of phosph-p44/42 MAPK in rat vascular smooth muscle cells was detected by Western blot using phosph-p44/42 MAPKinase antibody.The experiment was repeated 3 times and we got 12 data in total.The expression of phosph-p44/42 MAPK was represented by the intensity.Experiment two:rat VSMCs cultured in vitro and exposed to 20 ng/ml TNF-??1 mmol/L aspirin?2 mmol/L aspirin?1 mmol/L aspirin with 20ng/mL TNF-??2 mmol/L aspirin with 20 ng/mL TNF-? for 24 hours respectively.Control group were established for comprision.Then the cells were lysed and the concentration of protein was evaluated by BCA Kit.The expression of phosph-p44/42 MAPK in rat vascular smooth muscle cells was detected by Western blot using phosph-p44/42 MAPKinase antibody.The experiment was repeated 3 times and we get 18 data in total.The expression of phosph-p44/42 MAPK was represented by the intensity.Results1.Effects of curcumin on proliferation of rat VSMCs induced by TNF-?The proliferation rate of VSMCs was represented by the optical density,the date in each group are:1.418±0.172 in the control group,1.615±0.178 in 20 ng/mL TNF-?group,1.438±0.251 in 20 pmol/L curcumin group,1.484±0.160 in 20 ?mol/L curcumin with 20 ng/mL TNF-? group.Statistical analysis showed that compared to the control group,20 ng/mL TNF-? can significantly stimulate the proliferation of rat VSMCs(P<0.001).Curcumin alone had no effect on VSMCs growth(P=0.612),but significantly inhibited proliferation of VSMCs induced by TNF-?(P<0.001).2.Effects of curcumin on expression of syndecan-4 protein in VSMCs induced by TNF-?Take the ratio of interest protein and ?-actin's gray scale of control group as 1,the expression of syndecan-4 in rat VSMCa after 24 hours was 1.353±0.003 in 20 ng/mL TNF-? group,1.047±0.035 in 20 ?mol/L curcumin group,1.008±0.011 in 20±mol/L curcumin with 20 ng/mL TNF-? group.Statistical analysis suggested that compared to the control group,the expression of syndecan-4 protein in VSMCs was significantly enhanced induced by 20 ng/mL TNF-a(P<0.001).Curcumin alone had no effect on the expression of syndecan-4 protein(P=0.079),but significantly inhibited the expression of syndecan-4 protein induced by TNF-?(P<0.001).3.Effects of curcumin on expression of phosph-p44/42 MAPK in rat VSMCs induced by TNF-?Take the ratio of phosph-p44/42 MAPKinase and total-p44/42 MAPKinase's gray scale of control group as 1,the expression of phosph-p44/42 MAPK in rat aortic vascular smooth muscle cells after 24 hours was 1.297±0.090 in 20 ng/mL TNF-? group,1.010±0.046 in 20 ?mol/L curcumin group,0.950±0.059 in 20?mol/L curcumin with 20 ng/mL TNF-? group.Statistical analysis suggested that compared to the control group,the expression of phosph-p44/42 MAPK in VSMCs was significantly enhanced induced by TNF-? at the concentration of 20 ng/mL(P=0.005).Curcumin alone had no effect on the expression of phosph-p44/42 MAPK(P=0.715),but significantly inhibited the expression of phosph-p44/42 MAPK induced by TNF-?(P=0.005).4.Effects of aspirin on proliferation of VSMCs induced by TNF-?The proliferation rate of VSMCs was represented by the optical density,the date in each group are:1.088±0.086 in the control group,1.363±0.101 in 20 ng/mL TNF-?group,1.103±0.094 in 1 mmol/L aspirin group,1.087±0.063 in 2 mmol/L aspirin group,1.182±0.064 in 1 mmol/L aspirin with 20 ng/mL TNF-? group,and 1.164±0.060 in 2 mmol/L aspirin with 20 ng/mL TNF-? group.Statistical analysis showed that compared to the control group,20 ng/mL TNF-? can significantly stimulate the proliferation of VSMCs(P<0.001).Aspirin alone had no effect on VSMCs growth(P=0.530,P=0.971,respectively),but significantly inhibited the proliferation of VSMCs induced by TNF-?(both P<0.001).5.Effects of aspirin on expression of syndecan-4 protein in VSMCs induced by TNF-?Take the ratio of interest protein and ?-actin's gray scale of control group as 1,the expression of syndecan-4 protein in VSMCs after 24 hours was 1.449±0.048 in 20 ng/mL TNF-a group,1.002±0.021 in 1 mmol/L aspirin group,1.005±0.022 in 2 mmol/L aspirin group,1.041±0.004 in 1mmol/L aspirin with 20 ng/mL TNF-a group,and 1.054±0.011 in 2 mmol/L aspirin with 20 ng/mL TNF-a group.Statistical analysis suggested that compared to the control group,20 ng/mL TNF-?can significantly enhance the expression of syndecan-4 protein in VSMCs(P<0.001).Aspirin alone had no effect on the expression of syndecan-4 protein(P=0.866,P=0.701,respectively),but significantly inhibited the expression of syndecan-4 protein induced by TNF-?(both P<0.001).6.Effects of aspirin on expression of phosph-p44/42 MAPK in rat VSMCs induced by TNF-?Take the ratio of phosph-p44/42 MAPKinase and totol-p44/42 MAPKinase's gray scale of control group as 1,the expression of phosph-p44/42 MAPK in rat aortic vascular smooth muscle cells after 24 hours was 1.341±0.004 in 20 ng/mL TNF-?group,1.004±0.004 in 1 mmol/L aspirin group,0.995±0.062 in 2 mmol/L aspirin group,0.951±0.065 in 1 mmol/L aspirin with 20 ng/ml TNF-? group,and 0.969±0.107 in 2 mmol/L aspirin with 20 ng/ml TNF-? group.Statistical analysis suggested that the expression of phosph-p44/42 MAPK in VSMCs was significantly enhanced induced by TNF-? at the concentration of 20 ng/mL(P<0.001).Aspirin alone had no effect on the expression of phosph-p44/42 MAPK protein(P=0.253,P=0.889,respectively),but significantly inhibited the expression of phosph-p44/42 MAPK induced by TNF-?(P=0.009,P=0.026,respectively).ConclusionsOur study suggests that curcumin and aspirin both can significantly bring down the proliferation and the expression of syndecan-4 protein and phosph-p44/42 MAPK in cultured rat VSMCs induced by TNF-?,respectively.Consequently,we should make further researches into a wider potential effect of curcumin and aspirin on the treatment of atherosclerosis. |
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