| BackgroundAtherosclerosis(AS) is one of the most important reasons that induce cardiovascular disease.The traditional view of AS as a lipid storage disease crumbles in the face of extensive and growing evidence that inflammation participates centrally in all stages of this disease,from the initial lesion to the end-stage thrombotic complications.Monocytes,platelet and lymphocytes adhere to the vessel wall after its injury,and release a series of cytokines and growth factors connecting and trancducing with the specific receptor,then impacting on the phenotype and physeal signal of vascular smooth muscle cells(VSMCs) and adventitial fibroblasts(AF).It enhances the fibrohyperplasy.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. Interleukin-10(IL-10) is an anti-inflammatory molecule,which is one of the most potent homeostatic regulators of inflammation.It is attracting more and more attention recently.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.Consequently,it is fairly necessary to find a new path of atherosclerotic treatment by researching the series of potential functional capacity and related intervention effects of syndecan-4 in the development of AS.ObjectiveTo investigate the effects of recombinant human interleukin 10(rhIL-10) on cell proliferation and expression of syndecan-4 protein in rat aortic VSMCs and NIH/3T3 cells induced by TNF-αin vitro.The cell proliferation was evaluated by nonradioactive MTS/PMS assay and the expression of syndecan-4 protein was detected by Western blotting using anti-syndecan-4 antibody.We expect to explain the potential functional capacity of syndecan-4 in the development of AS at cell and molecular level.Methods1.The detection of cell's proliferation:Exp one:In vitro cultured rat aortic VSMCs were passed on 96 well assay and stimulated by 20ng/mL TNF-α,100ng/mL rhIL-10,200ng/mL rhIL-10,100ng/mL rhIL-10 with 20ng/mL TNF-α,200ng/mL rhIL-10 with 20ng/mL TNF-αfor 24 hours respectively.Each concentration has 6 holes and we repeat the experiment 6 times at the same condition,then we get 36 data in total.Take the same number of holes that have no drugs as control group,we also have 36 control data to observe if there was a change between drug post-stimulatory cell and non-irritant cell.The ratio of cell proliferation was determined by non-radioactive MTS/PMS assay and represented by the optical density.Exp two:In vitro cultured NIH/3T3 cells were passed on 96 well assay and stimulated by 20ng/mL TNF-α,100ng/mL rhIL-10,200ng/mL rhIL-10,100ng/mL rhIL-10 with 20ng/mL TNF-α,200ng/mL rhIL-10 with 20ng/mL TNF-αfor 24 hours respectively.Each concentration has 7 holes and we repeat the experiment 3 times at the same condition,then we get 21 data in total.Take the same number of holes that have no drugs as control group,we also have 21 control data to observe if there was a change between drug post-stimulatory cell and non-irritant cell.The ratio of cell proliferation was determined by non-radioactive MTS/PMS assay and represented by the optical density.2.The detection of syndecan-4 protein expressionExp one:In vitro cultured rat aortic VSMCs were exposed to treatment with 20ng/mL TNF-α,100ng/mL rhIL-10,200ng/mL rhIL-10,100ng/mL rhIL-10 with 20ng/mL TNF-α,200ng/mL rhIL-10 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 NIH/3T3 cells were exposed to treatment with 20ng/mL TNF-α,100ng/mL rhIL-10,200ng/mL rhIL-10,100ng/mL rhIL-10 with 20ng/mL TNF-α,200ng/mL rhIL-10 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 rhIL-10 on proliferation of rat aortic vascular smooth muscle cells induced by TNF-αThe proliferation rate(represented by the optical density) of VSMCs was 1.822±0.455 in the control group,2.130±0.270 in 20ng/mL TNF-αgroup, 1.989±0.309 in 100ng/mL rhIL-10 group,2.010±0.370 in 200ng/mL rhIL-10 group, 1.918±0.322 in 100ng/mL rhIL-10 with 20ng/mL TNF-αgroup,and 1.924±0.145 in 200ng/mL rhIL-10 with 20ng/mL TNF-αgroup.Statistical analysis showed that TNF-αcan significantly stimulate the proliferation of rat VSMCs with the concentration of 20ng/mL(F=12.208,t=-3.494,P=0.001).rhIL-10 alone had no obvious effect on VSMCs growth(F=2.636,P＞0.05),but significantly inhibited proliferation of VSMCs induced by TNF-α(F=7.964,P＜0.05).2.Effects of rhIL-10 on expression of syndecan-4 protein in rat aortic vascular smooth muscle cells induced by INF-αChoose the value of the control group's relative intensity as 1,the expression of syndecan-4 in rat aortic vascular smooth muscle cells after 24 hours was 1.287±0.070 in 20ng/mL TNF-αgroup,1.027±0.075 in 100ng/mL rhIL-10 group,1.017±0.067 in 200ng/mL rhIL-10 group,0.937±0.119 in 100ng/mL rhIL-10 with 20ng/mL TNF-αgroup,and 0.833±0.051 in 200ng/mL rhIL-10 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(F=49.971,t= -7.069,P=0.002).rhIL-10 alone had no effect on the expression of syndecan-4 protein(F=0.162,P＞0.05),but significantly inhibited the expression of syndecan-4 protein induced by TNF-α(F=23.304,P＜0.01).3,Effects of rhIL-10 on proliferation of NIH/3T3 cells induced by TNF-αThe proliferation rate(represented by the optical density) of NIH/3T3 cells was 0.577±0.052 in the control group,0.634±0.040 in 20ng/mL TNF-αgroup, 0.587±0.035 in 100ng/mL rhIL-10 group,0.575±0.052 in 200ng/mL rhIL-10 group, 0.571±0.061 in 100ng/mL rhIL-10 with 20ng/mL TNF-αgroup,and 0.575±0.068 in 200ng/mL rhIL-10 with 20ng/mL TNF-αgroup.Statistical analysis showed that TNF-αcan significantly stimulate the proliferation of NIH/3T3 cells with the concentration of 20ng/mL(F=15.872,t=-3.984,P＜0.001).rhIL-10 alone had no effect on NIH/3T3 cells growth(F=0.410,P＞0.05),but can significantly inhibit proliferation of NIH/3T3 cells induced by TNF-α(F=7.893,P＜0.01).4,Effects of rhIL-10 on expression of syndecan-4 protein in NIH/3T3 cells induced by TNF-αChoose the value of the control group's relative intensity as 1,the expression of syndecan-4 in NIH/3T3 cells after 24 hours was 2.103±0.220 in 20ng/mL TNF-αgroup,1.146±0.054 in 100ng/mL rhIL-10 group,1.235±0.215 in 200ng/mL rhIL-10 group,0.996±0.072 in 100ng/mL rhIL-10 with 20ng/mL TNF-αgroup,and 1.082±0.136 in 200ng/mL rhIL-10 with 20ng/mL TNF-αgroup.Statistical analysis suggested that the expression of syndecan-4 protein in NIH/3T3 cells was significantly enhanced by TNF-αwith the concentration of 20 ng/mL(F=75.69,t= -8.700,P=0.013).rhIL-10 alone had no effect on the expression of syndecan-4 protein(F=2.574,P＞0.05),but significantly inhibited the expression of syndecan-4 protein induced by TNF-α(F=47.459,P＜0.001). ConclusionsOur study suggests that rhIL-10 can significantly inhibit the proliferation of rat aortic vascular smooth muscle cells and NIH/3T3 cells induced by TNF-αin vitro, respectively,rhIL-10 can also significantly inhibit expression of syndecan-4 protein in rat aortic vascular smooth muscle cells and NIH/3T3 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.
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