A Preliminary Study On The Interaction And Gap Junctions Between AF-SMC And The Intervention Of Yiqi Huoxue Jiedu Decoction

Vascular remodeling is the adaptive changes of vascular wall thickness and/or lumen size and the function of blood vessel caused by vascular endothelial injury,proliferation and migration of vascular cells and matrix precipitation.Vascular remodeling diseases include atherosclerosis,restenosis after angioplasty,hypertension and so on.The function of smooth muscle cells(SMC)and adventitial fibroblasts(AF)was studied deeply in the process of understanding the pathological mechanisms of vascular remodeling.However,recent researches suggest that there may exist certain relationship between the two cells.AF stimulated by foreign antigens or inflammatory mediators secretes a variety of cytokines regulating the function of blood vessels.In addition,the gap junctions(GJ)are important in the mutual regulation of the vascular endothelial cells(VEC)and VSMC.After intimal injury,with the adventitia of the early inflammatory response,such a non-specific inflammatory promotes activation,migration,proliferation and secretion of the adventitial fibroblast.In vascular adventitial fibroblasts by film arrives in the process,whether through gap junctions in smooth muscle cell function?The experiment simulates AF-SMC structure to build a gap junction channels between AF and SMC.Stimulated by the non-specific immuneagent,fibroblasts' roles to SMC through gap junctions were observed.To clarify the impact of AF on the SMC,both of which are involved in the pathogenesis of vascular remodeling.According to TCM surgical wound repair ideas,Yiqihuoxuejiedu Formula prevents and treats the vascular remodeling on the basis of Yiqihuoxue,plus antidote.The preparatory work shows that Yiqihuoxuejiedu Formula has the anti-inflammatory function,inhibiting vascular adventitial fibroblast migration and secretion to prevent restenosis after angioplasty.In this study,freeze dried powder of Yiqihuoxuejiedu Formula was studied to validate the pharmacological mechanism that traditional Chinese medicine inhibits the activation and proliferation of fibroblasts by inhibiting the inflammatory response,further inhibits the activation,prevention of SMC.This will provided a new method for treatment of vascular remodeling disease.ObjectiveThe experiment simulates to build a gap junction channels between AF and SMC clarifying the two jointly participation in the pathogenesis of vascular remodeling.Stimulated by the non-specific immuneagent,fibroblasts' roles to SMC through gap junctions were observed directly.We observed that the Yiqihuoxuejiedu Formula can inhibit the AFs,further inhibiting smooth muscle cells,so as to achieve the objective of controlling vascular remodeling.Methods1.Primary culture of rat thoracic aortic adventitial fibroblasts and smooth muscle cells and cell identification:culturing primary fibroblasts and smooth muscle cells by attached method and establishing stable cell experiment platform by purification,identification,resuscitation and training.2.Observation that the influence of lipopolysaccharide to fibroblasts proliferation:lipopolysaccharide as inflammatory stimulating factor,CCK-8 and ELISA method for measuring the changes of proliferation of fibroblasts and secretion of cytokines.3.Establishing AF-SMC co-culture model,first activated AF stimulated by LPS,then remove LPS,and contact the AF-SMC co-culture directly.We observe that AF-SMC cell proliferation,changes of cell cycle and cell secretion;CCK-8 method direct observation by activation of fibroblasts on the effect of AF-SMC cell proliferation;Observation on AF-SMC by flow cytometry cell cycle;ELISA method for the determination of changes in the secretion of MMP-2 and TIMP-2 in AF-SMC co-culture.4.Establishing AF-SMC co-culture model,using transmission electron microscopic observation of AF-SMC co-cultured after two cell morphology and gap junctions between two cell structures.5.Observation of Yiqihuoxuejiedu Formula on the proliferation of fibroblast cells induced by LPS effects and the effect of AF-SMC gap junction:determination of Yiqihuoxuejiedu Formula on CCK-8 effect of AF after LPS model;flow cytometric detection of Yiqihuoxuejiedu Formula on AF-SMC effect of cell cycle;ELISA method for determination of Yiqihuoxuejiedu Formula on the secretion of TIMP-2 in AF-SMC co-culture.Results1.The primary fibroblasts are polygonal,radial or reticulocyte-like growth.Cells were uncolored in the immunocytochemistry of Desmin,but they were brownish-yellow in the immunocytochemistry of Vimentin.These showed that cells were vascular adventitial fibroblasts,not the smooth muscle cells.The long spindle primary smooth muscle cells were"peak and valley"-like growth.They were brownish-yellow in the immunocytochemistry of Desmin and Vimentin.2.LPS at a concentration of 5 ?g/ml and 10?g/ml for 24 h influences the proliferation of fibroblasts.Enzyme-linked immunosorbent assay shows that the concentration of IL-6 increased significantly in the group of 5 ?g/ml lipopolysaccharide(P<0.05),but no significant difference in 10?g/ml group.So we choose a concentration of 5?g/ml of LPS,24h as the experiment conditions.3.Compared with normal AF-SMC,activation of AF co-cultured with SMC(AF-LPS-SMC group),can promote the proliferation of both cells;Cell cycle analysis showed that the percentage of cells in the S phase(DNA synthesis phase)in AF-LPS-SMC group increased(P<0.05),AF after LPS stimulation and proliferation of cultured cells become more active.ELISA assay results show that AF-SMC culture MMP-2 no significant change in each group,in the supernatant of activated AF TIMP-2 markedly reduced(P<0.01),compared with the AF-SMC group,LPS+AF-SMC group TIMP-2 content increased significantly(P<0.05).4.Transmission electron microscope,LPS-induced activated AF nucleus becomes large,wider and nuclear atypia.AF-AF and SMC-SMC are the presence of gap junctions,between activation of the AF and SMC have also formed gap junction.5.Yiqihuoxuejiedu Formula can significantly reduce the proliferation of AFs,on the secretion of AF-SMC co-cultured cell cycle and TIMP-2 had no effect.Yiqihuoxuejiedu Formula Group we observed that there have no gap junctions between SMCs and AFs.Conclusion1.LPS can induce the proliferation of fibroblasts in vitro,suggesting that non-specific inflammatory reaction of the outer membrane may stimulate the activation and proliferation of AF,making it possible to observe the effect of AF on the SMC.2.The activated AFs can promote AF-SMC co-culture on proliferation and DNA synthesis and increase of TIMP-2,prompted to activate AF and there is interaction between SMC,two cells after exposure to each other,their function maybe positive adjustment or negative adjustment..3.The activated AFs form gap junctions with the SMC,indicating that the AF by signaling through gap junction and smooth muscle cells may be the structural basis of the interaction between two kinds of cells,but the types and functions of gap junction need further study:4.Yiqihuoxuejiedu Formula can significantly inhibit the proliferation of AF,may be the main mechanism of vascular remodeling.Under the experimental conditions,we failed to observe the effect of prescriptions of AF-SMC co-culture cells.Effects of Yiqihuoxuejiedu Formula on effect of gap junctions between AF and SMC needs further confirmation.