A New Houttuyfonate Derivative Inhibits Expression Of Syndecan-4Protein In Rat Vascular Smooth Muscle Cells And NIH/3T3 Cells Induced By Tumor Necrosis Factor-alpha In Vitro

BackgroundAtherosclerosis(AS) is one of the most important reasons that induce cardiovascular disease.Lots of proof shows that AS is an inflammatory overaction to the injury of blood vessels.Monocytes,platelet and lymphocytes adhere to the vessel wall after 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 factorα(TNF-α) is one of critical regulatory factors in inflammatory reaction that created by various inflammatory cells and plays an important role in the development of AS.Syndecan-4 is a heparan sulphate proteoglycan that is able to bind to some growth factors.It plays important roles in cell expansion,cell recognition,cell adhesion,cell migration and cell proliferation.It can also mediate inflammation simultaneously.Houttuynia cordata is a traditional Chinese herbal medicine.Houttnin which is an important component of houttuynia cordata has the function of anti-inflammation,antibiosis and immunoregulation. ObjectivesTo investigate the effects of a new houttnin derivative(NHD) on cell proliferation and expression of syndecan-4 protein in rat VSMCs and NIH/3T3 cells induced by TNF-αin vitro.We hope to find some new biological activities to widen the clinical usage of NHD.Methods1.The detection of cell proliferation:Experiment one:rat VSMCs were cultured in 96 well assay in vitro and exposed to TNF-α(20ng/mL) or NHD(4μg/mL,8μg/mL) respectively,and then co-treated TNF-α(20ng/mL) with NHD(4μg/mL,8μg/mL) respectively.All the groups were cultured for 24 hours,in addition to the untreated control group established for comparison.The ratio of proliferation of VSMCs was determined by non-radioactive MTS/PMS assay.Experiment two:NIH/3T3 cells were cultured in 96 well assay in vitro and exposed to TNF-α(20ng/mL) or NHD(4μg/mL,8μg/mL) respectively,and then co-treated TNF-α(20ng/mL) with NHD(4μg/mL,8μg/mL) respectively.All the groups were cultured for 24 hours,in addition to the untreated control group established for comparison.The ratio of proliferation of NIH/3T3 cells was determined by non-radioactive MTS/PMS assay.2.The detection of syndecan-4 protein expressionExperiment one:rat VSMCs were cultured in vitro and exposed to TNF-α(20ng/mL) or NHD(4μg/mL,8μg/mL) respectively,and then co-treated TNF-α(20ng/mL) with NHD(4μg/mL,8μg/mL) respectively.In addition to the untreated control group established for comparison.All the groups were cultured for 24 hours. Then the cells were lysed and the concentration of protein was measured using Coomassie brilliant blue G-250.The expression of syndecan-4 protein in VSMCs was evaluated by Western blotting technique using anti-syndecan-4 protein antibody.Set up non-irritant group as control.Experiment two:NIH/3T3 cells were cultured in vitro and exposed to TNF-α(20ng/mL) or NHD(4μg/mL,8μg/mL) respectively,and then co-treated TNF-α(20ng/mL) with NHD(4μg/mL,8μg/mL) respectively.In addition to the untreated control group established for comparison.All the groups were cultured for 24 hours. Then the cells were lysed and the concentration of protein was measured using Coomassie brilliant blue G-250.The expression of syndecan-4 protein in NIH/3T3 cells was evaluated by Western blotting technique using anti-syndecan-4 protein antibody.Set up non-irritant group as control.Results1.Effects of NHD on cell proliferation of rat VSMCs induced by TNF-αThe ratio of cell proliferation was 1.262±0.105 in control group,1.505±0.137 in TNF-α20ng/mL group,1.282±40.110 in NHD 4μg/mL group,1.217±0.108 in NHD 8μg/mL group,1.325±0.122 in TNF-α20ng/mL combinated with NHD 4μg/mL group,and 1.228±0.111 in TNF-α20ng/mL combinated with NHD 8μg/mL group respectively.Statistical analysis showed that TNF-α20ng/mL group could stimulate cell proliferation of rat VSMCs compared with control group(t=-6.894,P＜0.001), TNF-α20ng/mL combinated with NHD 4μg/mL group could inhibit cell proliferation of rat VSMCs compared with TNF-α20ng/mL group(P＜0.001),and TNF-α20ng/mL combinated with NHD 8μg/mL group could inhibit cell proliferation of rat VSMCs compared with TNF-α20ng/mL group(P＜0.001).2.Effects of NHD on expression of syndecan-4 protein in rat VSMCs induced by TNF-αTake the ratio of interest protein andβ-actin's gray scale of control group as 1, the expression of syndecan-4 protein in VSMCs was 1.321±0.052 in TNF-α20ng/mL group,1.004±0.111 in NHD 4μg/mL group,0.902±0.667 in NHD 8μg/mL group, 1.029±0.070 in TNF-α20ng/mL combinated with NHD 4μg/mL group,and 0.991±0.046 in TNF-α20ng/mL combinated with NHD 8μg/mL group respectively. Statistical analysis showed that TNF-α20ng/mL group could stimulate expression of syndecan-4 protein in rat VSMCs compared with control group(t=-10.766,P=0.009), TNF-α20ng/mL combinated with NHD 4μg/mL group could inhibit expression of syndecan-4 protein in rat VSMCs compared with TNF-α20ng/mL group(P=0.001), and TNF-α20ng/mL combinated with NHD 8μg/mL group could inhibit expression of syndecan-4 protein in rat VSMCs compared with TNF-α20ng/mL group (P<0.001).3.Effects of NHD on cell proliferation of NIH/3T3 cells induced by TNF-αThe ratio of cell proliferation in 24 hours was 0.577±0.052 in control group, 0.621±0.041 in TNF-α20ng/mL group,0.514±0.149 in NHD 4μg/mL group, 0.490±0.155 in NHD 8μg/mL group,0.52±0.112 in TNF-α20ng/mL combinated with NHD 4μg/mL group,and 0.420±0.149 in TNF-α20ng/mL combinated with NHD 8μg/mL group respectively.Statistical analysis showed that TNF-α20ng/mL group could stimulate cell proliferation of NIH/3T3 cells compared with control group (t=-3.098,P=0.004),TNF-α20ng/mL combinated with NHD 4μg/mL group could inhibit cell proliferation of NIH/3T3 cells compared with TNF-α20ng/mL group (P=0.003),and TNF-α20ng/mL combinated with NHD 8μg/mL group could inhibit cell proliferation of NIH/3T3 cells compared with TNF-α20ng/mL group(P<0.001).4.Effects of NHD on expression of syndecan-4 protein in NIH/3T3 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 protein in NIH/3T3 cells was 1.305±0.042 in TNF-α20ng/mL group,0.988±0.038 in NHD 4μg/mL group,0.963±0.021 in NHD 8μg/mL group,1.0134±0.027 in TNF-α20ng/mL combinated with NHD 4μg/mL group,and 0.990±0.016 in TNF-α20ng/mL combinated with NHD 8μg/mL group respectively. Statistical analysis showed that TNF-α20ng/mL group could stimulate expression of syndecan-4 protein in NIH/3T3 cells compared with control group(t=-12.706, P＜0.001),TNF-α20ng/mL combinated with NHD 4μg/mL group could inhibit expression of syndecan-4 protein in NIH/3T3 cells compared with TNF-α20ng/mL group(P＜0.001),and TNF-α20ng/mL combinated with NHD 8μg/mL group could inhibit expression of syndecan-4 protein in NIH/3T3 cells compared with TNF-α20ng/mL group(P＜0.001).ConclusionsOur finding suggests that a certain dose of NHD can inhibit proliferation and expression of syndecan-4 protein in rat VSMCs and NIH/3T3 cells induced by TNF-αrespectively.As a result of this research,it is necessary to make use of NHD as a drug primer to study its correlated cure effect of angiemphraxis disease.