| Percutaneous transluminal coronary angioplasty has now become the most widely implemented method of heart revascularization in the treatment of patients with coronary artery disease. However, the rate of restenosis is still relatively high, which remains a major limitation and a challenging problem of percutaneous revascularization. The molecular mechanism involved a complex interplay of several different aspects, endothelial cells, vascular smooth muscle cells, vascular adventitial fibroblasts and extracellular matrix are involved in the pathological process. The adventitial layer surrounding the blood vessels which has long been exclusively considered as a supporting tissue may play an important role in vascular injury.Bad vascular remodeling might play an important role in the progress of restenosis. Substantial experimental evidence supports the idea that fibroblasts may play a significant role in vascular remodeling. Fibroblasts from the adventitia have the ability to rapidly respond to hypoxic stress and to modulate their function to adapt to local vascular needs. It has demonstrated that fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, migrate and synthesize extracellular matrix under injured conditions. Increased extracellular matrix formation and excessive collagen deposition appear to form the bulk of the hyperplasia tissue which eventually induce fibrosis and occurrence of restenosis.Chinese medical herbs have been promising in the inhibition of neointimal formation and occurrence of restenosis after PTCA. Many drugs targeting different mechanisms of restenosis have been studied previously including antiplatelet and anticoagulant agents, calcium antagonists, angiotensin-coverting enzyme inhibitors, antiproliferative of smooth muscle cells or immunosuppressive drugs. We hypothesize that new effective herbs targeting adventitia could be invested to prevent neointimal hyperplasia and restenosis with better curative effect and less side-effects.Considering of the important role of vascular adventitial fibroblasts in the restenosis, especially its biological behaviour including proliferation, migration, and matrix protein production, we used the fibroblasts as our study object and angiotensin II as the stimulation factor to explore the effects of active components of four traditional herbs including tetramethylpyrazine, puerarin, tanshinoneâ…¡A and paeonol on the proliferation and collagen synthesis of vascular adventitial fibroblasts in injured conditions.1 Primary culture rat vascular adventitial fibroblasts and biological behaviour studyThe purpose of this study was to explore the methods of isolation, cultivation and identification of rat vascular adventitial fibroblasts in vitro, meanwhile we observed its biological behaviour.Dulbecco's modified Eagle's medium(DMEM) was the basic culture medium, being supplemented with 20% fetal bovine serum using for culturing thoracic aortal adventitia tissue pieces, and with 10% fetal bovine serum using for culturing passage cells. The fibroblasts were incubated at 37℃in a humidified atmosphere of 5%CO2 and 95% air in the incubator. Growth kinetics was investigated by MTT. Cellular morphologies were observed, and the cultured cells were identified by cell cytoskeleton immunostaining.Cell cytoskeleton is the special protein which can be used as the marker of different cells or tissues. The endothelial cell, the smooth mucle cell and the fibroblast were identified with anti-vWF, anti-desmin, and anti-vimentin respectively. The results indicated that the immunostaining of vimentin was positive, while which of desmin and vWF were negative, so we can conclude that the cells we got were the right cells we want.The isolated fibroblasts could grow and proliferate in vitro. The exponential phase of growth was from the second to the fourth day after passage, and the cells can keep normal state until 7-8 passages.Rat vascular adventitial fibroblasts can be cultured in stable condition in vitro, and sufficient and reliable target cells can be obtained for the study of mechanisms of vascular stenosis at molecular level.2 Effects of tetramethylpyrazine, puerarin, tanshinoneâ…¡A and paeonol on the proliferation of rat vascular adventitial fibroblasts in normal condition and abnormal condition induced by angiotensinâ…¡We explored the effects of active components of four traditional herbs including tetramethylpyrazine, puerarin, tanshinoneâ…¡A and paeonol on the proliferation of vascular adventitial fibroblasts induced by angiotensinâ…¡, and detectd the effects of the four ingredients on the fibroblasts in normal condition.We used the third passage fibroblasts as our research target to determine the maximum of no toxicity concentration of the active components of four traditional herbs. The results determined by MTT indicated that tetramethylpyrazine, puerarin, tanshinoneâ…¡A and paeonol had no obvious toxicity at the concentration of 4．0×10-3mol·L-1,2．0×10-3mol·L-1,2．0×10-5mol·L-1,4．0×10-3mol·L-1 respectively.The effects of four components on the proliferation of fibroblasts at normal condition were investigated by MTT in the following concentrations: tetramethylpyrazine: 2．0×10-5,1．0×10-4,5．0×10-4,2．0×10-3,4．0×10-3mol·L-1ï¼›puerarin:5．0×10-6,2．0×10-5,1．0×10-4,5．0×10-4,2．0×10-3mol·L-1;tanshinoneâ…¡A:1．0×10-7,4．0×10-7,2．0×10-6,1．0×10-5,2．0×10-5mol·L-1ï¼›paeonol:2．0×10-5,1．0×10-4,5．0×10-4,2．0×10-3,4．0×10-3mol·L-1.The results indicated thatthey had no obvious impacts on the proliferation of fibroblasts in a short term.Angiotensinâ…¡was used as the stimulation factor. The proliferate rate of fibroblasts induced by angiotensinâ…¡were investigated by MTT. The results indicated that the proliferating of fibroblasts of the group pretreatmented with 1×10-7mol·L-1 angiotensinâ…¡for 24h had great significant difference (P<0.01) compared with control, so the concentration of 1×10-7mol·L-1 was used for our further research.To further study the effects of four components on the proliferation of fibroblasts induced by angiotensinâ…¡, we used the method of MTT. The results indicated that the four components could inhibit the excessive proliferation of fibroblasts under injured conditions, showing obvious dose-effect relationship.3 The effects of tetramethylpyrazine, puerarin, tanshinoneâ…¡A and paeonol on the collagen synthesis of rat vascular adventitial fibroblasts induced by angiotensinâ…¡We explore the effects of active components of four traditional herbs including tetramethylpyrazine, puerarin, tanshinoneâ…¡A and paeonol on the collagen synthesis of vascular adventitial fibroblasts induced by angiotensinâ…¡.Using angiotensinII as the stimulation factor with the concentration of 1×10-7mol·L-1, hydroxyproline chromatometry and western blotting were used for detecting collagen synthesis respectively.Through hydroxyproline chromatometry examinating, after 48h these four herbs significantly decreased the amount of hydroxyproline of fibroblasts in the following concentrations: tetramethylpyrazine: 2.0×10-5,1.0×10-4,5.0×10-4,2.0×10-3,4.0×10-3mol·L-1;puerarin:5.0×10-6,2.0×10-5,1.0×10-4,5.0×10-4,2.0×10-3mol·L-1;tanshinoneâ…¡A:1.0×10-7,4.0×10-7,2.0×10-6,1.0×10-5,2.0×10-5mol·L-1; paeonol:2.0×10-5,1.0×10-4,5.0×10-4,2.0×10-3,4.0×10-3mol·L-1.The results of western blotting indicated that these ingredients could depressthe expression of procollagenâ… in such concentrations respectively: 4.0×10-3mol·L-1,2.0×10-3mol·L-1,2.0×10-5mol·L-1,4.0×10-3mol·L-1.Active components of the four herbs could decrease the collagen synthesis of fiboblasts under injured conditions.Based on our findings, we can presume that these Chinese medical herbs may inhibit the neointimal formation and occurrence of restenosis through their inhibition effects on the proliferation and collagen synthesis of fibroblasts, it also provides the scientific evidence for wide clinical application. |
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