Puerarin To Regulate The Mechanism Of The Inflammatory Response In-stent Stenosis

Objective and BackgroundThe clinical use of stent is the landmark of percutanenous coronary intervention (PCI) in 1987.Coronary stent implantation had become an important treatment of coronary heart disease (CHD) since the following10 years, but the high rate of in-stent restenosis (ISR)(10 %⁵0 %)confined the development of coronary stent implantation.With the application of drug eluting stent (DES),which can slowly release the drug of inhibiting proliferation of vascular smooth muscle cells, the rate of ISR has been decreased markedly and re-PCI has been delayed .At present, Rapamycin eluting stent and Paclitaxel eluting stent are used frequently in clinic, but there are a lot of problems in the use of DES still, such as the long - term curative effects, expensive price and so on. It is necessary to find a kind of more effective and cheaper DES.As the eluting drug of the stent, Puerarin has the five advantages: (1) It is a fat-soluble isoflavone, so it can stay in the tissues for a long time. (2)It can inhibit proliferation of vascular smooth muscle cells and promote the apoptosis of vascular smooth muscle cells. (3)It can promote the repair of Endothelial cells. (4)It has the effect of anti- inflammation. (5)it can inhibit the aggregation of platelet. Because of all characteristics above, Puerarin has the possibility of preventing and curing the ISR.We did the animal experiment using Puerarin eluting stent and compared it with the bare stent, PC eluting stent and Rapamycin eluting stent(Firebird^TM)made in china. We observed the curative effect of Prerarin after the stent implantating into Rabbit Iliac Artery ,in order to make clear whether Puerarin eluting stent could effectively inhibit the endometrial hyperplasia and the happen of ISR;Through experiments in vitro, we tested whether Puerarin can protect the vascular endothelial cell and inhibit inflammation and primary discuss whether Puerarin can block the arise and development of ISR at the early phase and its anti-inflammation mechanism on preventing the ISR.Methods and ResultsThis study included three parts:1.The research of reaparing and proetcting effect of Puerarin on HUVEC damaged by IL-1β.Methods: (1)The isolating culture and identity of HUVEC;(2) Determing the effect of Puerarin on the proliferation and activity of HUVEC cultured in vitro by MTT chromatometry; (3)Detecting the effect of Puerarin on the content of nitric oxide(NO) in HUVEC culture supernatant by the method of nitrore ductase.(4) Determining the ET-1 content in HUVEC culture supernatant pretreated by IL-1βwith radioimmunoassay Results:The experiment was divided into three medicine groups: high-dose, medium-dose and low-dose. The whole three medicine groups can protect EC and improve the activity of HUVEC. Especially the high-dose group that the content of Puerarin is 10^-3mol·L^-1,can significantly improve the activity of HUVEC(P<0.01);Compared with control group,IL-1βcould increase the expression of ET-1 and decrease the content of NO in HUVEC culture supernatant., the difference was very significant(P<0.01). The secretion of NO in the whole three medicine groups was increased, the difference between medicine groups and model group was very significant(P<0.01).While the expression of ET-1 in the high-dose, medium- dose groups was decreased, the difference was very significant(P<0.01).2.The effect of Puerarin on the cytokines'expression of HUVEC injuried by inflammation and on the adhesion and migration of inflammatory cell.Methods: (1)Testing the content of MCP-1,IL-6,TNF-α,E-selectin in HUVEC culture supernatant by ELISA. (2)Testing the effect of Puerarin on the adhesion between HUVEC and U937 monocyte by adhesion experiment. (3)Testing the effect of Puerarin on the migration of U937 monocyt by Transwell. (4)Testing the effect of Puerarin on the activity of the nuclear factor-κB and the P-IκBαof the endothelial cells by Westernblot.Results: The results of ELISA showed: After activiated by IL-1β,the expression of E-selectin,MCP-1,IL-6 and TNF-αof HUVEC was increased and Puerarin could reduce these factors'expression. The results of adhesion experiment showed:after activiated by IL-1β,the adhesion between HUVEC and U937 monocyte was enhanced,and Puerarin could inhibit this effect. The results of migration experiment showed: after being activiated by IL-1β, endothelial cell's chemotaxis effect on U937 monocyte was enhanced, and Puerarin could inhibit this effect. The results of westen blot experiment showed: instimulation of IL-1βcould induce the phosphonation of IκBαand elevate the activity of NF-κB in HUVEC, and Puerarin could inhibit this effect.3.Observating of the curative effect of Puerarin eluting stent after the implantation of stent into the iliac artery of rabbitMethods:After implantating the stents into the iliac arteries of rabbits, the rabbits were executed 4 weeks later and the vascular slices were obtained from the blood vessels where the stents were implanted. Previous study had confirmd that Puerarin could inhibit the hyperplasia of rabbit iliac artery's neointimal, and the results of immunohistochemistry showed Puerarin could increase the expression of endothelial growth factors(VEGF) and decrease the expression of IL-6 in tissues. On the basis of the previous study, we further detected the expression of NF-κBP65 and MCP-1 in the tissue through immunohistochemistry.Results:compared with the bare stent group, Puerarin eluting stent could decrease the expression of MCP-1 and NF-κB in the restenosis model of rabbit iliac artery(P<0.05). Conclusions:Formation mechanism of ISR is complex. Being a complex network reaction, inflammation plays an important role on the development of ISR.The results of the three part experiments above proved that:1. Puerarin could protect the vascular endothelial out of injury, and regulate the secretion of endothelial cells'active material;2. Through inhibiting the expression of endothelial cell's transcription factors NF-κB which is closely related with inflammation, could inhibit the expression of its downstream expressive product such as monocyte chemokine, adhesion molecules and inflammatory factors .By furthermore inhibiting the adhesion and migration between monocyte and vascular endothelial, Puerarin inhibited the further development of inflammation;3. Animal experiment indicated Puerarin eluting stent could inhibit the hyperplasia of endangium in the restenosis model of rabbit iliac artery. Its mechanism maybe connect with that Purerain could improve repair function of endothelial cell and inhibit the inflammation.