The Effects And Mechanism Of Tongluo Formula Or Herbs On Lipid-Lowering, Inhibiting Inflammation And Anticongulation

PAPERⅠTHE EFFECTS AND MECHANISM OF TONGXINLUO SUPERFINE ON LIPID-LOWERING,INHIBITING INFLAMMATION AND ANTICONGULATIONCoronary heart disease is the commonly encountered and frequently-occurring disease which harms health seriously.With the elevation of living standard and ageing of population,the morbidity and mortality rise year by year and the pathology change is atherosclerosis(AS).AS is the pathologic processes including many factors and the pathogenesis is very complex and the research about it is multiply.Because the AS is easy to happen and serious,so people have realized that is necessary to prevent and cure as soon as possible for the past few years.However,the AS patients are common and the effective intervention about its treatment is lacking.Therefore,to aim directly at the pathogenesis and make use of reasonable and effective drugs is the basic principle of delaying the development of AS.The role of lipid metabolic disorder inducing the AS is generally acknowledged and the relation of lipid and CHD is confirmed.Lot's of epidemiological data showed the severity and death rate of AS aggravates with the increasing of serum cholesterol level.It is previously believed the serum cholesterol is the primary cause but now the low density lipoprotein(LDL-C) is deemed more important.The oxidative damage of LDL-C occurs on artery vascular intima and there is special receptor of oxidized low density lipoprotein(ox-LDL) in endothelial cell,and the cell would express more harmful substance because of sustained injuring,even which can bring the vicious cycle of tunica intima due to injured continually.The lipids about AS are from plasma lipoprotein like free cholesterol(TC),Triglyceride(TG),et al.The increasing of LDL-C level is the capital risk factors of AS or other cardiovascular and cerebrovascular diseases about AS.Conversely,the high density lipoprotein cholesferol(HDL-C) is beneficial to anti AS and the low level of HDL-C is one of risk factors of AS.Although the Cochrane doesn't recognize the HDL-C as simple curing target point,to increase the level of HDL-C really can slow down the process of AS.The correlation of low level of HDL-C and cardiovascular diseases is more significant in aged and female group.Therefore,to reduce the LDL-C and raise HDL-C level is the important target of curing AS.Although the "lipid invasion theory" explains the pathologic mechanism of AS well,many researchers found that the form of plaque is not only because of lipid deposition but also the bioprocess after vascular injuring in 1970s.The hypothesis of "endothelium injured" and "thrombosis" were suggested by researchers on the base of "lipid invasion theory".For the past few years,the "inflammatory reaction theory" of Ross is becoming the major idea.Ross thought the form of AS had two pathways:(1) the injuring of endothelial cell because of various inflammation stimulus or lipoprotein and the platelet aggregation,activation,expression of platelet-derived growth factor(PDGF) and the proliferate of smooth muscle cell(SMC) since the injuring happened;(2) once the endothelium was injured the endothelial cell and macrophage would secrete kinds of factors and SMC also bring the PDGF,and then, the interaction of them will lead to the form and development of fibrous plaque.The AS is the chronic inflammation diseases which the inflammatory reaction penetrates in the all process of AS including the rupture of plaque and thrombosis.In the process of inflammatory reaction,the white blood cell activated by cellular adhesion molecule combines with endothelial cell surface and migrates to the inflammatory tissue out of vascular.In the development of AS,the adhesion of monocyte and endothelial cell is mediated by intercellular adhesion molecules(ICAM-1) and vascular cellular adhesion molecules(VCAM-1) chiefly.Wallen found the increase of serum ICAM-1 and VCAM-1 level in angina pectoris patients positively correlate to the incidence rate of cardiac death or acute myocardial infarction,which supplied the evidence for the adhesion molecule participating to evaluate the prognosis of AS patients. The final result of atherosclerotic heart disease is the thrombosis of vascular and the collateral circulation isn't setup timely that result in myocardial ischemia and induce acute cardiovascular event.However,the form of thrombus relates with the change of blood environment and whether the function of fibrinolysis system is normal is important.Hoffmeiste reported the fibrinolysis activity of CHD patients decrease decreased and abnormal fibrinolysis activity might predict the occurring of coronary artery disorder.The plasma tissue plasminogen activator(t-PA) and plasminogen activator inhibitor 1(PAl-1) is the important active compound of fibrinolysis system and the physiologic equilibrium of them is fundamental to keep blood stream unobstructed and prevent thrombosis.The pathomechanism of AS is very complicated and the lipid deposition, inflammatory reaction and thrombosis correlate and affect each other.In the early period,much stimulating factor induce the inflammatory reaction on the vessel wall lipid deposited,cell adhesion,lipid plaque,proliferate and immigration of SMC;the inflammation also affects the metabolism of lipid and promotes the oxidation of LDL-C,meanwhile,which can decrease the stability of lipid plaque and induce the rupture of plaque.Therefore,people realize ideal strategy about the treatment of AS is that can combine with the reducing blood fat,inhibiting inflammation and anticoagulation and play the joint action.However,to find the target point of them will be shortcut to treat AS.The clinical research confirmed the statins could not only reduce LDL-C level but also inhibit the inflammation and improve outcomes by decreasing the inflammation markers like C reactive protein and adhesion molecules.Meanwhile, statins could reduce the plasma PAI-1 level and improve the function of fibrinolysis significantly.However,the clinical trial showed that 22.4%patients who summed the large dose of atorvastatin experienced acute coronary artery disease in 2 years. Moreover,some patients withdraw from statins treatment because of liver dysfunction. In addition,the drug combination could have better effect compared with one kind of medicine,so the "polypill" was preferred in secondary prevent treatment by two British scholars in 2004.Although the manuscript met with the doubt,the change of mode has important meaning.Traditional Chinese medicines have the characteristic of much way,much link and much target point and stable the AS plaque by protecting the vascular endothelial cell,regulating the lipid metabolism,inhibiting the inflammatory reaction and anticoagulation.Traditional Chinese medicines have the potential curative effect in delaying the development of AS and are suitable to long-time drug taking of secondary treatment because of the moderate potency,less side effect and compatibility.Formerly,the research of traditional Chinese medicines about treating AS usually aim directly at the simple cause or symptom and the systemic research of mechanism is necessary.Tongxinluo superfine is the representative Chinese drug which treats cardiovascular and cerebrovascular disease with theory of collateral disorders and dredging collaterals herbs.Tongxinluo has the effects of benefiting vital energy and promoting blood flow and has been used in CHD that show off deficiency of heart-energy and blood stasis.The present study adopts the simvastatin to be positive control and uses the AS animal model made by laboratory and be aimed to observe the therapeutic effects of Tongxinluo on AS by regulating lipid,inhibiting inflammation and anticoagulation and elucidate the possible mechanism and relationship of them through the molecular biology and imageology technology.Objective(1) To establish an animal model of AS that is mimic to human pathological changes and convenient for intervention;(2) To observe the changes of lipid,inflammation and clotting mechanism and find the functionary target point in common with serology histology Protocols in Molecular Biology.(3) To compare the therapeutic effects of Tongxinluo and Simvastatin on preventing and curing AS and approach the possible mechanism.Methods1.Establishing the animal model:75 rabbits were fed on an atherogenic diet containing 1%cholesterol for 10 weeks after abdominal aortic wall injuries were induced using an intravascular balloon.2.Intervention methods:Group A(control group):15 rabbits were fed normal diet for 8 weeks after the model was established.Group B(low dose):15 rabbits were fed normal diet and Tongxinluo super (0.15g/kg/d) for 8 weeks after the model was established.Group C(middle dose):15 rabbits were fed normal diet and Tongxinluo super (0.3g/kg/d) for 8 weeks after the model was established.Group D(high dose):15 rabbits were fed normal diet and Tongxinluo super (0.6g/kg/d) for 8 weeks after the model was established.Group E(simvastatin):15 rabbits were fed normal diet and simvastatin (5mg/kg/d) for 8 weeks after the model was established.3.Body weight:Body weights of all rabbits were measured at baseline,the end of the 10^th and 18^th week.4.High frequency ultrasound:At the baseline,end of the 10^th and 18^th week,the abdominal aorta was scanned using a high frequency duplex ultrasonographic system.The intima-media thickness (IMT) and peak velocity(Vp) were measured.5.Liver and kidney function test:Blood samples were collected from all rabbits at the baseline,the end of the 10^th and 18^th week.The serum ALT,GGT,TBIL,BUN and Cr were detected with automatic biochemical analyzer.6.Lipid measurement:Blood samples were collected from all rabbits at the baseline,the end of the 10^th and 18^th week.Serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C) were measured by enzymic method.7.Serum inflammatory factor:Blood samples were collected from all rabbits at the baseline,the end of the 10^th and 18^th week.The serum high sensitivity C-reactive protein(hs-CRP),ox-LDL,ICAM-1 and VCAM-1 were assayed using highly sensitive enzyme-linked immunosorbent assay(ELISA) kits.8.Plasma fibrinolysis detection:Blood samples were collected from all rabbits at the baseline,the end of the 10^th and 18^th week.The plasma PAI-1 and t-PA were assayed using ELISA kits.9.Histopathological analysis:The abdominal aorta was processed and examined by hematoxylin and eosin (HE),Movat and oil red O staining.10.Immunohistochemical staining:The expression of RAM11,VCAM-1,ICAM-1,CRP,cholesterol ester transfer protein(CETP) and low density lipoprotein receptor-related protein(LRP) were determined.11.Western blot:To detected the protein expression of CETP and LRP in liver.12.Scanning electron microscope:Scanning electron microscope was used to observe the the degree of endangium damage and lipids deposition.13.Statistical analysis:Data are expressed as mean±SD for continuous variables and by frequency count and percentage for qualitative variables.Incidence rate of plaque rupture was compared with Pearson Chi-Square test and other indexes were compared with One-Way ANOVA comparison test.The SPSS was version 13.0.P＜0.05 was considered statistically significant.Results1.General state of the experimental animals:Five rabbits died of diarrhea,respiratory tract infection and so on,and there were 70 rabbits finished the study:15 rabbits in group A,14 rabbits in group B,14 rabbits in group C,13 rabbits in group D,14 rabbits in group E.2.Body weight: Body weights of all rabbits were significantly higher at the end of 10^th week than that in begin(P＜0.01);there were no difference among the groups after treatment.3.High frequency ultrasound measurement:At the end of 10^th week,the maximum IMT of the rabbits were significantly higher(P＜0.01),and the Vp was also higher(P＜0.05);after treatment,the IMT of group C,D and E were lower than group A(P＜0.05),the Vp of group C and E was lower(P＜0.05).4.Detection of liver and kidney function:At the end of 18^th week,the ALT and GGT in group E were higher than other groups(P＜0.05).5.Lipid measurement:Intra-group comparison:at the end of 10^th week,except the HDL-C,the TC,TG and LDL-C level were higher(P＜0.01);at the end of 18^th week,except the HDL-C, the TC,TG and LDL-C level were lower(P＜0.01).Inter-group comparison:compared with group A,except the HDL-C,at the end of 18^th week,the lipid level in the drug groups decreased significantly especially in group D and E,but the serum HDL-C level of drug groups increased.6.Detection of serum inflammatory factor:Intra-group comparison:at the end of 10^th week,the serum ox-LDL,hs-CRP, ICAM-land VCAM-1 increased significantly(P＜0.01);at the end of 18^h week,the serum inflammatory factor of group C,D and E decreased markedly(P＜0.05).Inter-group comparison:at the end of 18^th week,compared with group A,the serum ox-LDL,hs-CRP,ICAM- 1 and VCAM- 1 of group A was higher than group C, D and E significantly(P＜0.01～0.05).7.Detection of plasma fibrinolysis factors:Intra-group comparison:except the group A,at the end of 10^th week,the plasma PAI-1 level increased and t-PA decreased significantly(P＜0.05～0.01).Inter-group comparison:at the end of 18^th week,compared with group A,the plasma PAI-1 level increased and t-PA decreased significantly in group D and E (P＜0.05～0.01).8.Pathologic staining: The H&E staining showed the obviously plaque in all groups;oil red "O" staining showed the lipid in plaque of group A was higher than group C,D and E; Movat showed the ground substance in plaque of group A was higher than in group C, D and E;there was no difference among the group D and E.9.Inununohistochemical staining:Compared with group A,the expressions of LRP,RAM11,VCAM-1,ICAM-1 and CRP in plaque of group C,D and E decreased significantly,and no difference was found between group D and E.There was no significantly difference of CETP among the Tongxinluo groups.10.Western blot:There was high expression of CETP and LRP in liver in all groups.The expression of LRP protein in group C,D and E is lower than in group A,The expression of CETP protein in group E is lower than in other groups.11.Scanning electron microscope:The endothelial cell of normal group was clostridial form,integrate,lining up in order and the long axis of cell was concordant with direction of blood stream,the nucleolus swelled up and orientated toward the lumens and there was no cell adhesion; the endothelial cell of other group was injured and the cell was arranged in disorder, and much cell of intima ablated and exposed the collagen,there adhered much red blood cell,platelet and lipid droplet near the deciduo-as area.Under the lower power lens,the endodermis swelled up and partly mixed together and the plaque formed. The damage of group A and B was more serious than other groups and there was no significant difference between group C,D and E.Conclusions(1) The rabbit AS model,which is established with balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet is mimic to human disease and suitable for the observation of drug therapeutic effects.(2) The Tongxinluo increased the HDL-C level and protected the normal function of reverse cholesterol transport not by inhibiting the CETP.(3) The combination of Tongxinluo and simvastatin could effectively decrease the lipid,inhibit the inflammatory reaction and enhance the fibrinolysis function and the common target maybe was LRP. PAPERⅡINTERVENTION OF B.MACROSTEMI ON TF/TFPI OF HAECs OX-LDL ACTIVATEDBackgroundThe happen and development of AS run through the whole pathologic processes of CHD,however,from the lipid deposition to atheromatous plaque and even the plaque rupture and thrombosis inducing the danger incidence,the endothelial cell play the important role.Many professors consent the "injuring theory" is the initiate mechanism of AS.The oxidative damage of LDL-C betide artery vascular intima and there is specific receptor of ox-LDL in endothelial cell,and the cell would express more harmful substance because of sustained injuring,even which can bring the vicious cycle of tunica intima due to injured continually.Ox-LDL decrease the activity of glutathione peroxides and the generate prostacyclin of cultural HUVEC and increase the lipid peroxides that showed the ox-ldl can injure the endothelium directly.Meanwhile,the lectin-like oxidized LDL receptor-1(lox-1) that endothelial cell expressed increase and that promote the endothelial cell to swallow the senile and apoptosis cell,and activate much inflammatory reaction signal pathway,for example, p44/42MAPK,p38MAPK,CD40/CD40L,et al..The endothelial cell release more procoagulant factors like tissue factor(TF) and less anticoagulant substance like tissue factor pathway inhibitor(TFPI) when those pathways was activated,then,the clotting mechanism enhanced and thrombus form.The important pathological basis of high incidence cardiovascular and cerebrovascular diseases of old people is atherosclerosis(AS),and the high risk factor of AS is too much lipid deposition in endarterium due to high fat diet.For the past few years,lots of epidemiological study showed the increase of serum cholesterol especially the low-density lipoprotein(LDL-C) promote the development of AS,and the oxidized low density lipoprotein(ox-LDL) more enhance the degree of pathological changes that it has become one of the initiating agent of plaque rupture and thrombosis.With the development of aging,the incidence of coronary heart disease(CHD) in our country was at the increased tendency and got close to the international average level.In spite of many patients have recognized the serious of CHD,the AS patients have been ill or other reason induced(smoking,heredity,and menopause) are still ubiquitous.Therefore,to find the effective drugs that can inhibit the injure of endothelial cell by ox-LDL,to decrease the release of procoagulant factor and keeping the balance of intravascular environment is good for reducing the danger incident.Now the treatment of AS more often focus on regulating lipid,besides statins, there are antiplatelet drugs,β-receptor inhibitor,angiotensin converting enzyme inhibitor and so on that can improve clinic symptoms.The drugs play the positive therapeutic effect with regard to delay the process of AS.Moreover,because hyperlipidemia is the high risk of cardiovascular diseases,the statins have become the basic drugs to treat CHD.Much clinical trial indicated that those drugs which can protect endothelial cell,improve endothelium function and drugs have the role of anti-lipid peroxidation become choice drug.But most drugs like statins,although they have good survivability and safety,there are much side effects like dysfunction of liver,digestive system diseases and so on.There is much study about herbs for the past few years,the component of herbs is natural and they are effective in antioxidation,reducing blood fat, anti-inflammatory and anticoagulation.Furthermore,because of little side effect of herbs,in the range of safe dose there is tiny dysfunction of liver and kidney.Bulbus allii macrostemonis(B.macrostemi) was often used in compound preparation in the past and it also showed noticeable effect.In the aspect of cellular level research,most researchers took their attention on the study of essential oil.But the essential oil is difficulty to preserve because of unstable component and B.macrostemi contain lots of saponins and polyoses compound,therefore,the more deeply systematic research about B.macrostemi is necessary.Although there are lots of research about anti-oxidation or anticoagulation of essential oil,the mechanism about that is absent. In addition,intravenous endothelial cells or bovine aortic endothelial cells were adopted usually in these researches.In order to simulate clinical diseases,the human aortic endothelial cells(HAECs) were cultured in vitro and ox-LDL was applied for inducing factor of oxidative damage to endothelial cell.We observed the effect of ox-LDL on expression of Lox-1,TF and TFPI and the protective effect of B. macrostemi on the endothelial cells damage induced by ox-LDL.Objective(1) To establish the model of human aortic endothelial cells oxidative damage;(2) To investigate the effect and mechanism of different dose B.macrostemi inhibiting the expression of pro-coagulant factor in ox-LDL activated by HAECs.MethodsTo establish the model of human aortic endothelial cells oxidative damage,we observe the effect of ox-ldl with different concentration and different time on the expression of lox-1,TF and TFPI.The cultured HAECs were pretreated with or without B.macrostemi(0.25ug/ml,0.5ug/ml,1ug/ml) for 12h and were observed under inverted microscope.Meanwhile, the control group that cell were pretreated with blocking agent of lox-1-Poly(I:C) for 2h was designed.The effects of B.macrostemi with different concentration on lox-1,TF and TFPI protein expression were investigated by western blot.ResultsInverted microscope showed that the normal endothelial cells were at good condition,adherence stable,fusiform or round,tight cell-cell junction,more cleavage phase,less suspension cells;cell shrinkage,intercellular space broaden and more suspension cells in ox-LDL damage group;the shape and structure of endothelial cell in blocking agent and B.macrostemi 1ug/ml group was similar with normal cell.Based on the aim of the present study and the effect of ox-LDL with different concentration and different time on the expression of Lox-1,TF and TFPI,50ug/ml ox-LDL for 24h is the optimized stimulus for the present study. The Lox-1 protein expression of 250ug/ml Poly(I:C) group was less than ox-ldl group.The Lox-1 and TF protein expression were markedly inhibited by B.macrostemi with dose of 0.5 and 1ug/ml,and the Lox-1 level of 1ug/ml group was similar with blocking agent group;comparing with ox-LDL group,the TFPI level increased obviously.Conclusion(1) 50ug/ml ox-LDL for 24h not only increased the Lox-1 and TF expression but also inhibited the TFPI expression.So 50ug/ml for 24h is the optimized stimulus for the present study.(2) 250ug/ml Ploy(I:C) or inhibited lox-1 expression significantly and the effect of blocking was suitable for the present study.(3) B.macrostemi with dose of 0.5 and 1ug/ml could decrease the oxidative damage of endothelial cell and the pro-coagulant factor level and keep the balance of TF/TFPI by inhibiting the expression of Lox-1.