Studies About Effects Of Puerarin On Expressions Of Cathepsin S In Extracellular Matrix Remodeling During Restenosis After Arterial Balloon Injury

OBJECTIVEIt has been believed that percutenious translumen coronary angioplasty (PTCA) is one of the best methods for coronary revascularization and is available approach to treat coronary atherosclerotic heart disease and widely applied to clinics.But restenosis rate after PTCA six months later is as high as 30%～50%.Athough implantations of intracoronary stents have reduced restenosis,restenosis rate is still as high as 15%～35% by implanting bare metal stents stents(BMS),which has been a big problem for clinic applications.Restenosis after PTCA is a complex process.Inflammatory reaction occurs after blood vessels injuried,then migration of vascular smooth muscle cells(VSMCs)from the tunica media to the intima and proliferation,along with neointimal lesion formation hyperplasia and extracellular matrix (ECM) remodeling.Extracellular matrix (ECM) remodeling is a key physiopathologic basis of restenosis and a course of precise regulation,which relies on activity of extracellular proteinases.The extracellular proteases are a complex and heterogeneous superfamily of enzymes.They include metalloproteinases (matrix metalloproteinases),serine proteases,and the cysteine proteases (such cathepsins),etc.MMPs are key extracellular proteases to stabilize extracellular matrix and collagen contents. Lately report confirmed: cysteine proteases-Cathepsin S(CatS) and its inhibitor Cystatin C(CysC) are closely connected with ECM remodeling during restenosis. CatS can digest laminnin,fibronection,type I or III collagen and many other ECM ingredients,which attributes to VSMCs migration from the tunica media to the intima.Cathepsin S is closely related to aggragations of macrophages,foam cells formation.CysC can suppress the acticity of CatS.So CatS/CysC system plays a role in the progress of athero sclerosis,stabilities of atherosclerotic plaques and restenosis.It has been confirmed that Puerarin can prevent and control restenosis.But molecular mechanism is not clear.Restenosis models are to be established after balloon overstretch injury in iliac arteries of rabbits in order to investigate expressions of CatS/CysC during restenosis and investigate effects of Puerarin on expression changes of CatS/CysC,changes of ECM collagen contents and migrations of VSMCs.Aim of this study is to discuss that effects of Puerarin on influences of extracellular matrix remodeling and disclose the molecular mechanism in order to provide new ideas and methods to prevent and control restenosis.METHODS1. Division of experiment:Forty male New Zealand rabbits were divided into four groups: control group(n=8):no hypercholesterol feed,no operation or drugs;hyper cholesterol group:hypercholesterol feed;injury group: hypercholesterol feed and right iliac artery injury by balloon overstretch; hypercholesterol group and injury group were the same rabbit(n=16),left iliac artery was hypercholesterol group(no injury)and right iliac artery(jury)was injury group.Puerarin group(n=16):hypercholesterol feed,balloon overstretch injury and Puerarin intravenous injection.Hypercholesterol feed was fed two weeks ago before operation excluding control group.2. Balloon injury models established:3% sodium perborate was injected by ear edge vein before balloon overstretch injury in order to anaesthetize the rabbits, puncture needle was successfully dipped into right iliac artery, through 0.014 guiding wire(3.5mm×15mm) ,balloon catheter was dipped into abdomen aortic artery about 20cm.Eight atm pressure was filled with and reverse drawing to iliac artery,all around three times.3. Puerarin intervention and artery tissues disposed: Puerarin was given to Puerarin group.Puerarin was injected by ear edge vein 80mg·kg^-1,at different time point week one,week two,week four and week eight respectively.Triglyceride (TG),Total cholesterol (TCh),LDL-Ch and HDL-Ch of blood plasma were measured using routine biochemical methods respectively.Intima,atherosclerotic plaque,lumen diameters of iliac arteries were detected by ultrasound equipment,respectively.After the experimental animals were scarcificed at different time point,week one,week two,week four and week eight,the injured blood vessels were fixed and dewax.HE staining,expressions of Cathepsin S and Cystatin C were identified by immnohistochemisty staining,VSMCs migrations and VSMCs contents in neointima were detected by SM a-actin staining.Changes of collagen contents were detected by VG staining.RESULTS1.Blood lipids No significant difference exits in control group, hypercholesterol group,injury group,Puerarin group at week one or week two(P>0.05);Blood TCh at week four,week eight and LDL-Ch at week eight is lower in Puerarin group than hypercholesterol group and injury group,significant difference (P<0.05).2.Ultrasound Detection No significant difference exits in control group or hypercholesterol group one week before and after operation(P>0.05),vessels diameters are wider in Puerarin group than injury group.3.HE Staining Comparing with control group,in hypercholesterol group,VSMCs in artery media began to arrange turbulently at week two,foam cells formed at week four and much even serious than before,and part inner elastin was injuried at week eight.In injury group and Puerarin group,neointima formed at week one.VSMCs in artery media arranged turbulently,much foam cells formed and inner elastin was injuried at week two and week four,total occlusion came into being at week eight in injury group. Comparing with injury group,hyperplasia of artery inner elastin and media is gently,vascular smooth muscle cells and foam cells were less and lumen diameters were wider.4. Specific Staining(VG staining)Collagen fibers staining is red,staining of vascular smooth muscle cells and red blood cells is yellow and much red staining is found in neointima.Comparing with hypercholesterol group,much more red collagen was found in injury group and Puerarin group at week one.Red collagen contents was less in Puerarin group than injury group at week two and week four.Lumen total occlusion occurred and no significant changes of collagen contents existed in injury group than in Puerarin group at week eight.5. Immnohistochemisty StainingLittle brown positive cellular grains can be found in cytoplasm of normal arterial vascular smooth muscle cells.Comparing with Puerarin group,CatS positive cellular grains are more in injury group and significant changes exist at week two,week four and week eight(P<0.05).Comparing with hypercholesterol group,CysC positive cellular grains are more in Puerarin group and injury group and significant changes exist at week two,week four and week eight(P<0.05).No significant changes exist between Puerarin group and injury group at anytime.SM a-actin staining:SM-α-actin positive cellular grains can be found in media of normal arterial.Comparing with Puerarin group,brown positive grains in neointima is more in injury group at week four.Lumen total occlusion occurred,and in spite of much positive cellular grains in it,no significant changes of SM-α-actin positive cellular grains exist in injury group than in Puerarin group at week eight.CONCLUSIONS1. Restenosis models were established successfully after balloon overstretches injury in iliac arteries of New Zealand rabbits.2. During the course of extracellular matrix remodeling of restenosis aftert balloon overstretch injury,expressions of Cathepsin S were up-regulated while expressions of Cystatin C were remarkable in early stage than late stage.3. During the course of extracellular matrix remodeling of restenosis aftert balloon overstretch injury,Puerarin can intervene expressions of CatS and suppress over-expression of extracellular matrix, such as collagen.VSMCs migrations were limited,but expressions of CysC were not remarkable.INNOVATION AND LIMITATION1. Innovation Previous studies focused on the relationships between CatS/CysC system and progress of atherosclerosis or stabilities of atherosclerotic plaques.CatS/CysC system participate in extracellular matrix remodeling during restenosis.This study first discussed that effects of Puerarin on expressions of CatS during the course of restenosis and influences of extracellular matrix remodeling,disclosed molecular mechanism for preventing and controlling restenosis with Puerarin and provided a new idea to prevent and control restenosis.2. Limitation Beacuse time and fund for scientific research is limited,expression changes of MMPs/TIMP were not detected in the experiment at the same time. Changes of collagen contents were just identified by VG staining.Analysis of gene and protein is not applied.