Experimental Study On Effects Of Yishenhuoxue Capsule On Proliferative Rabbits' Vascular Smooth Muscle Cells Induced By HCY

Objectives1.To investigate the effects of Yishenhuoxue Capsule on proliferative rabbits' vascular smooth muscle cells induced by HCY.2.To investigate the effects of Yishenhuoxue Capsule on NF-κB activation,the expression of TGF-β₁,MCP-1,VCAM-1,PCNA and cell cycles.3.To explore the potential mechanism of modulating the NF-κB signal transduction pathway on proliferative rabbits'vascular smooth muscle cells induced by HCY.MethodsVSMCs were cultured by tissue explants in vitro and pured by the difference of adherent rate.Celluar morphology and growth nature were observed with inverted microscope.After identified with a-SM-actin by SP,the purity of cells was greater than 95%,then cells in logarithmic phase from 3 generation to 6 were used to test. Proliferative cells were induced by HCY(1mM),next they were divided into 6 groups-the control group(no HCY),the HCY group and four treatment groups(5000,500,50mg/L YSHXC,5mM folic acid respectively),and then tested after 24 hours culture.The proliferation of cells and cell cycles were determined by MTT and flow cytometry;the expressions of TGF-β₁,MCP-1,VCAM-1,PCNA and NF-κB activation were detected and analyzed with immunocytochemistry and RT-PCR.All tests were repeated for three times.The data was analyzed by statistics. Results1.YSHXC of different concentration could inhibit cells'proliferation.The inhibition and cell cycles detection by MTT and flow cytometry showed that,in treatment groups the number of cells at G₀/G₁ stage increased greatly,while the number at S and G₂/M stages decreased;which was different from the HCY group(P＜0.01).And it also presented dose-dependent.2.The positive products of NF-κB activation and PCNA showed the brown particles located in cytoplasm and/or nucleus,the ones of TGF-β₁,MCP-1,VCAM-1 expression were mainly located in cytoplasm,which showed brown particles as well. They were downregulated after YSHXC treating for 24 hours.Downregulation of TGF-β₁,MCP-1,VCAM-1 expression were closely correlated with inhibition of NF-κB activation(P＜0.01).3.RT-PCR analysis showed that,compared with the control group,VSMCs treated with YSHXC at different concentration for 24 hours indicated the decline of TGF-β₁,MCP-1,VCAM-1 expression respectively.And the decline of TGF-β₁,MCP-1,VCAM-1 expression of the YSHXC higherdose and middledose groups was superior to that of the folic acid group 0.46±0.04,0.45±0.05,0.42±0.04(P＜0.01).Conclusions1.YSHXC can effectively and dose-dependently inhibit the NF-κB activation, reduce the abnormal proliferation of VSMCs,and adjust the cell cycles.2.TGF-β₁,MCP-1,VCAM-1,PCNA expression will downregulate accompanied with the inhibition of NF-κB activation,which were closely correlated.3.Inhibition of NF-κB activation by YSHXC may affect the expressions of TGF-β₁,MCP-1,VCAM-1,PCNA through interfering the gene transcription action.