Based On The NF-?B Pathway, Explore The Mechanism Of Yiqi Liangxue Shengji Decoction In Intervention Of Rabbit Carotid Artery Balloon Injury Repair

Background:With the in-depth study of coronary artery disease(CAD)in modern medicine,percutaneous coronary intervention(PCI)has become an important treatment of CAD.However,PCI also brings new problems and challenges,such as ischemia-reperfusion injury,late in-stent thrombosis and in-stent restenosis(ISR),which seriously reduce the benefits of patients.Inflammatory reaction is one of the key mechanisms of major adverse cardiac event(MACE)after PCI.Local mechanical injury of blood vessels is caused by the implantation of stents after PCI.The repair process after injury involves chronic inflammatory reaction of vascular intima,including vascular endothelial injury,platelet and leukocyte aggregation,chemokines and inflammatory factor production.Poor or delayed endothelial repair is the main cause of MACE.As the first found membrane receptor in TLR family of mammalian,Toll-like receptor 4(TLR4)connects innate immunity with acquired immunity.Activation of TLR4 can activate nuclear factor ?B(NF-?B),then induce a series of inflammatory reactions and release various proinflammatory cytokines and chemokines,leading to adverse events.Objective:To establish a rabbit common carotid artery balloon injury endothelial denudation model,and to explore the effect and mechanism of Yiqi Liangxue Shengji recipe on improving vascular endothelial function and reducing inflammatory reaction of rabbit carotid artery after balloon injury based on NF-?B pathway.Methods:1.Animal model:6-month-old male New Zealand white rabbits were selected to establish the model of common carotid artery balloon injury.2.Grouping and administration:except for normal control group(group N)and sham operation group(group S),postoperative rabbits were randomly divided into model group(group M,normal saline),low concentration group of Yiqi Liangxue Shengji recipe(group YL,equivalent to crude drug 1.5 g/kg/d),middle concentration group of Yiqi Liangxue Shengji recipe group(group YM,equivalent to crude drug 3.0 g/kg/d),high concentration group of Yiqi Liangxue Shengji recipe group(group YH,equivalent to crude drug 6.0 g/kg/d),western medicine group(group P,Attovastatin calcium tablets).3.Detection methods and indicators:(1)the body weight and physiological status of animals were recorded every week.(2)4 weeks after operation,vascular blood flow was measured by ultrasound,including intima-media thickness(IMT),peak systolic velocity(PSV),end-diastolic velocity(EDV)and resistance index(RI).(3)4 weeks after operation,vascular morphological and structural changes were evaluated by HE staining and EVG staining,including intimal thickness(IT),media thickness(MT),lumen area(LA),intimal area(IA)and media area(MA).(4)blood samples were collected before operation and 1 day,3 days,7 days,14 days and 28 days after operation for Elisa detection,including NO,ET-1,IL-6,TNF-? and hs-CRP levels.(5)4 weeks after operation,the proportion and distribution of vascular endothelial cell(VEC)and vascular smooth muscle cell(VSMC)were detected by flow cytometry and immunofluorescence.(6)4 weeks after operation,the mRNA and protein expression of TLR4,MyD88,NF-?B,TNF-? and.ICAM-1 were detected by RT-PCR and Western blot.(7)4 weeks after operation,the ultrastructure was observed by transmission electron microscope.Results:1.The health status of animals in the experiment was normal,and there was no significant difference in body weight among the 7 groups(P<0.05).2.Comparison of ultrasonic testing results:4 weeks after operation,compared with group N,IMT,PSV and RI increased and EDV decreased in group M(P<0.05);compared with group M,IMT,PSV and RI decreased and EDV increased in group YL,YM,YH and P(P<0.05);and the decrease of IMT in group YM and YH was more significant than that in group P(P<0.05).3.Comparison of the results of pathological analysis:(1)comparison of HE staining results:4 weeks after operation,the vascular morphology of group N and S was almost normal;in group M,YL,YM,YH and P,the vascular morphology was tortuous and degenerated,the lumen inner wall of each group was not smooth and irregular,and the lumen area decreased significantly,among which group M was the most serious,and all treatment groups were improved in varying degrees compared with group M,among which group YM improved most obviously.(2)comparison of EVG staining results:4 weeks after operation,compared with group N,LA in group M decreased significantly,while IT,MT,IT/MT,IA,MA and IA/MA increased significantly in group M(P<0.05).Compared with group M,LA in group YL increased significantly,while IT,IT/MT,IA,MA and IA/MA decreased significantly(P<0.05),LA in group YM increased significantly,while IT,MT,IT/MT,IA,MA and IA/MA decreased significantly(P<0.05),LA in group YH increased significantly,while IT,MT,IA,MA and IA/MA decreased significantly(P<0.05),LA in group P increased significantly,while IT,IT/MT,IA,MA and IA/MA decreased significantly(P<0.05).Compared with group P,the improvement of MA in group YM was better(P<0.05).4.Comparison of Elisa results:there was no significant difference in the levels of NO,ET-1,IL-6,TNF-? and hs-CRP among the 7 groups before operation,but on the first day after operation,compared with group N,the level of NO in group M was significantly lower,while the levels of ET-1,IL-6,TNF-? and hs-CRP in group M were significantly higher(P<0.05).On the 3rd,7th,14th and 28th day after operation,compared with group M,the levels of NO in group YL,YM,YH and P were significantly higher,while the levels of ET-1,IL-6,TNF-? and hs-CRP in group YL,YM,YH and P were significantly lower(P<0.05).5.Comparison of flow cytometry results:4 weeks after operation,compared with group N,the proportion of VEC decreased and the proportion of VSMC increased in group M(P<0.05).Compared with group M,the proportion of VEC in group YL,YM,YH and P increased and the proportion of VSMC decreased(P<0.05).Compared with group P,the proportion of VEC in group YM and YH increased and the proportion of VSMC decreased(P<0.05).6.Comparison of immunofluorescence detection:4 weeks after operation,compared with group N,VWF fluorescence decreased and ?-actin fluorescence increased in M group,while VWF fluorescence enhanced and ?-actin fluorescence decreased in group YL,YM,YH and P compared with group M,especially in group LM.7.Comparison of RT-PCR results:4 weeks after operation,compared with group N,the expression of TLR4,MyD88,NF-?B,TNF-? and ICAM-1mRNA in group M increased(P<0.05),while the expression of TLR4,MyD88,NF-?B,TNF-? and ICAM-1 mRNA in group YL,YM,YH and P decreased compared with group M(P<0.05).8.Comparison of Western blot results:4 weeks after operation,compared with group N,the protein expression of TLR4,MyD88,NF-?B,TNF-? and ICAM-1 in group M increased(P<0.05),while the protein expression of TLR4,TNF-? and ICAM-1 in group YM and P decreased and the protein expression of MyD88 and NF-?B in group YM,YH and P decreased(P<0.05).9.Comparison of transmission electron microscope:four weeks after operation,the intima of carotid artery in group N and S showed normal shape,smooth and intact,normal shape of VSMC,uniform distribution of chromatin in the cell body,endoplasmic reticulum and mitochondria and other organelles with different contents,and no lipid droplets were found.In group M,the VEC was obviously damaged and number of VEC was decreased,which was difficult to observe.The number of organelles in the cytoplasm of VSMC increased,filled with dilated and swollen mitochondria and rough endoplasmic reticulum,and a large number of lipid droplets were seen.The VEC in group YL,YM,YH and P were damaged in varying degrees,the cell morphology were irregular,the morphological deformation in group YM and P were mild,the number of organelles in VSMC was slightly increased,the number of endoplasmic reticulum,mitochondria and Golgi apparatus was less than that of M group,and a small amount of lipid droplets could be seen.Conclusion:1.Yiqi Liangxue Shengji recipe can promote the repair of rabbit carotid artery after balloon injury,promote the proliferation and repair of VEC,inhibit the proliferation and migration of VSMC,and improve the injured vascular endothelial function.2.Yiqi Liangxue Shengji recipe can reduce the inflammatory reaction of rabbit carotid artery after balloon injury,and its mechanism may be related to the inhibition of TLR4/NF-?B pathway.