Effects of QSYQ Drop Pills on Secondary Treatment of Myocardialinfarction and the Mechanism
|Course||Traditional Chinese Medicine|
|Keywords||Myocardial infarction QiShenYiQi drop pills Simvastatin Vunerable plaque eNOS Astragalus polysaccharides lipopolysaccharide human aortic endothelial cells|
BackgroundAcute cardiovascular diseases are common and multi-onsct diseases,which have serious effects on the hcalth of human beings.It has been reported that 20 million patients died of acute cardiovascular events,and over ten thousands patients occurred acute myocardial infarction（AMI）.With the development of aging,the incidence of AMI in our country was at the increased tendency and got close to thc international average level.In recent years,although the advances in monitoring and therapy have improved the survival of patients who cxpcrience myocardial infarction,patients who survive an MI are still at increased risk of subsequent re-infarction,congestive heart failure,and sudden death.So much more attention should be paid to the secondary treatment of MI.The secondary treatment of MI is thc prevention of re-infarction and sudden death after MI.Several pharmacological agents arc known to improve prognosis in these patients,i.e.beta-blockers,antiplatelet agents,statins,and angiotensin converting enzyme inhibitors（ACEI）.Statins is the basis of preventive treatment since the first trial which demonstrated that statins could decrease the risk and the total mortality of coronary incidences of the patients who survived from MI. Improving the blood lipid could decrease the rate of recurrence and mortality of MI despite it only reduced the coronary stenosis by 1%-4%because the most AMI occurred at slight or moderate stenotic coronary artery,and the lesion which induced myocardiolysis is the rupture and hemorrhage of plaques and subsequent thrombus formation and occlusive blood vessel not the fixed stenosis.The secondary treatment of MI for statins is based on the prevention of plaques ruptures and hemorrhage.The plaques unstable or prone to rupture and thrombosis were at increased risk of reinfarction,so the stablization of vulnerable plaques has more clinical values compared with the prevention of thrombis,and it is the final target of secondary treatment of MI.Recently,many studies have discovered that there are multiple vulnerable piques in patients with ACS.So the general drug therapy is the basis of stabilizing vulnerable plques.Many basic researchs have demonstrated statins could significantly stabilize the vulnerable plaques by improving the blood lipid,inhibiting the inflammatory reaction in plaques and thickening the fibrous caps of plaques. However,the Prove-It Trial showed that 22.4%of patients experienced acute coronary event in 2 years despite an intensive statin therapy.Moreover,some patients withdraw from statin treatment because of liver dysfunction.In addition,the drug combination could have better effect compared with one kind of medicine,so the "polypill" was prefered in secondary prevent treatment by two British scholars in 2004.Although the manuscription met with the doubt,the change of mode has important meaning.The inflammatory reaction plays an important role in the formation and rupture M of unstable plaque.Chinese herb medicine could protect vascular endothelial cells, improve the lipid metabolism,inhibit the inflammatory reaction to stabilize the vulnerable plaques and might have potential therapeutic effect on stabilization of vulnerable plaques.In addition,Chinese drugs pharmaceutics is moderate and have less toxicant and more effect when compatibility with other Chinese drugs,so it could be used for a long time in secondery treatment of MI.In recent years,the researchs on MI were much and made rapid advancement,however,there were very few studies in the relationship between the secondary treatment of MI and plaque stabilization. QiShenYiQi drop pills（QSYQ drop pills）made by TianShiLi Group has the effects of benefiting vital energy and promoting blood flow and has been used in cardiovascular system diseases with positive effects,however the effects on stabilizing unstable plaques is unknown.The present study was aimed to observe the therapeutic effects QSYQ drop pills on vulnerable plaques through the molecular biology and imageology technology and to elucidate the possible mechanism.Objective（1）To establish an animal model of vulnerable plaques that is mimic to human pathological changes and convenient for intervention;（2）To observe the imaging feature of vulnerable plaques using intravascular ultrasound technique and to assess the value of this technique in evaluating the development of atherosclerotic plaques;（3）To compare the therapeutic effects of QSYQ drop pills and Simvastatin on stabilizing vulnerable plaques and approach the possible mechanism.Methods1.Establishing the animal model:70 rabbits were randomly divided into the normal group and the experiment group.10 rabbits in the normal group were fed normal diet,60 rabbits in the experiment group were fed on an atherogenic diet containing 1%cholesterol for 12 weeks after abdominal aortic wall injuries were induced using an intravascular balloon.2.Intervention methods:Two rabbits died from the anesthetic accident in the operation of abdominal aortic wall injuries induced by an intravascular balloon and one rabbit died of diarrhea.Eight rabbit were used to prove the establishment of the animal model.At the end of 12thweek,49 rabbits were randomly divided into three groups and given drugs for another 12 weeks.15 rabbits in the control group were fed with a normal diet plus 20ml distilled water.17 rabbits in QSYQ Drop pills group were given the same diet as in the first group plus QSYQ Drop pills（1.5g/kg/d）.17 rabbits in Simvastatin group of rabbits were fed the same diet as the other two groups but supplemented with Simvastatin（5mg/kg/d）.3.Gene transfection:At the end of the 24thweek,laparotomy was conducted in all rabbits after having been anesthetized with an intravenous injection of sodium pentobarbital（30mg/kg）and an adenoviral vector containing recombinant p53 was transfected into the location of plaques of these rabbits.These rabbits were maintained on a normal diet for additional 2 weeks. 4.Pharmacological triggering:At the end of the 26thweek 26,plaque rupture was induced by pharmacological triggering using Chinese Russell’s Viper Venom and histamine.Rabbits were euthanized 24-48 hours after the above procedure.5.Body weight:Body weights of all rabbits were measured at baseline,the end of the 12thand 26thweek.6.Lipid measurement:blood samples were collected from all rabbits at the baseline,the end of the 12thand 26thweek.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.Biochemical studies:The blood samples were collected from all rabbits at the baseline,the end of the 12thand 26thweek.Plasma fibrinogen was measured by rate-nephelometry.Serum high sensitivity C-reactive protein（hsCRP）,soluble vascular cell adhesion molecule-1（sVCAM-1）and oxidized low-density lipoprotein （ox-LDL）were assayed using highly sensitive enzyme-linked immunosorbent assay （ELISA）kits.8.High frequency ultrasound:At the baseline,end of the 12thand 24thweek,the abdominal aorta was scanned using a high frequency duplex ultrasonographic system. The intima-media thickness（IMT）,the end-diastolic luminal diameters（Dd）,the end-systolic luminal diameter（Ds）,the mean velocity（Vm）,peak velocity（Vp）, velocity integral（VTI）and the average image intensity（AⅡ）of the abdominal aorta were measured.9.Intravascular ultrasound（IVUS）:IVUS studies were performed at the baseline, the end of the 12thand 26thweek.The external elastic membrane area（EEMA）,lumen area（LA）,plaque area（PA）and vessel diameter（VD）were measured.The EI and RI were calculated.10.Histopathological analysis:the abdominal aorta was processed and examined by hematoxylin and eosin,Masson and oil red O staining.11.Immunohistochemical staining:The expression of RAM11,a-actin,p53, VCAM-1,MCP-1,MMP-9 and TIMP-1 were determined.12.Electron microscope:Scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of the abdominal aorta.13.Real-time PCR:The mRNA expressions of p53,VCAM-1,MCP-1,MMP-9 and TIMP-1 in the abdominal aorta tissue were analyzed.14.Western blot:The protein expressions of p53,VCAM-1,MCP-1,MMP-9 and TIMP-1 in the abdominal aorta tissue were analyzed.15.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:15 rabbits in the control group,16 rabbits in QSYQ Drop pills group and Simvastatin group completed the study.2.Body weight:Body weights in the three groups were essentially equal. However,body weight in QSYQ Drop pills group was significantly lower than that in the control group（P＜0.05）.But there was no difference between control group and Simvastatin group.3.Incidence of Plaque Rupture:12 rabbits in the control group（80.00%）,6 in QSYQ Drop pills group（37.50%）and 7 in Simvastatin group（43.75%）had ruptured atherosclerotic plaques in the abdominal aorta.The incidence of rupture in QSYQ Drop pills group and Simvastatin group was significantly lower than that in the control group（both P＜0.05）,while there were no differences in the plaque rupture rates between QSYQ Drop pills group and Simvastatin group.4.Lipid measurement:Compared with the control group,the levels of LDL-C decreased significantly in QSYQ Drop pills group and Simvastatin group（both P＜0.01）,and the LDL-C levels were lower in Simvastatin group than those in QSYQ Drop pills group.In addition,Simvastatin decreased the levels of TC（P＜0.01）and QSYQ Drop pills decreased TG levels significantly（P＜0.01）.5.Inflammatory markers:Compared with the control group,the serum hs-CRP levels decreased significantly in QSYQ Drop pills group and Simvastatin group at the end of the 26thweek（both P＜0.01）,and the hs-CRP levels in Simvastatin group were significantly lower than those in QSYQ Drop pills group（P＜0.05）.The plasma fibrinogen increased in QSYQ Drop pills group and Simvastatin group compared with the control group（P＜0.05 and P＜0.01）,and the fibrinogen levels in Simvastatin group were significantly lower than those in QSYQ Drop pills group（P＜0.01）.The serum sVCAM-1 levels in QSYQ Drop pills group were significantly lower than those in the control group（P＜0.05）.6.High frequency ultrasound measurement:The maximum IMT of the abdominal aorta in QSYQ Drop pills group and Simvastatin group were significantly lower than that in the control group（both P＜0.01）,and the value of the maximum IMT in Simvastatin group was lower than that in QSYQ Drop pills group（P＜0.05）.In addition,AIIc%values were increased in QSYQ Drop pills group and Simvastatin group compared with the control group（both P＜0.01）.7.IVUS measurement:The values of PA in QSYQ Drop pills group and Simvastatin group were significantly lower than those in the control group（both P＜0.01）,and the values of PA in Simvastatin group were lower than those in QSYQ Drop pills group.The LAS%and EEMA values decreased in Simvastatin group compared with the control group（P＜0.05 and P＜0.01）.8.Pathologic staining:Most rabbits in the control group developed plaque rupture and intravascular thrombosis with a dense infiltration of inflammatory cells. In contrast,the atherosclerotic lesions in QSYQ Drop pills group and Simvastatin group were similar in that plaque thickness was diminished,foam cells decreased and collagen proliferation present.9.Inununohistochemical staining:The expressions of RAM11,VCAM-1, MCP-1,MMP-9 and TIMP-1 in plaques were lower in the two drug group than those in the control group.The expressions of a-actin in control group were lower than those in the two drug groups.10.Electron microscopy examination:Most rabbits in the control group developed plaque rupture and intravascular thrombosis.Transmission electron microscopy showed karyopyknosis,heavy dyed-cytochylema,vacuolar degeneration chondriosome,and chaoticly alined muscle fiber in the control group and less disorderly-alined SMC,mainly normal structure of SMC and collagenous fibers in QSYQ Drop pills group and simvastatin group.11.Real-time PCR:The mRNA expression levels of VCAM-1,MCP-1,MMP-9 and TIMP-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups were significantly lower than those in the control group（P＜0.01 or P＜0.05）. The expression levels of VCAM-1 in QSYQ Drop pills group were lower than those in Simvastatin group（P＜0.01）,and the expression levels of MMP-9 in Simvastatin group is lower than those in QSYQ Drop pills group（P＜0.05）.12.Western blot:The protein expression levels of MCP-1,MMP-9 and TIMP-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups were significantly lower than those in the control group（both P＜0.01）.Compared with the control group,the protein expression levels of VCAM-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups decreased（P＜0.01 or P＜0.05）,and the expression levels of VCAM-1 in QSYQ Drop pills group than those in Simvastatin group（P＜0.01）.Conclusions（1）The rabbit vulnerable plaque model,which is established with balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet,gene transfection and drug triggering,is mimic to human disease and suitable for the observation of drug therapeutic effects.（2）Intravascular unltrasound technique is a realiable technique which can be used evaluate vulnerable plaques quantificationally to monitor the atherosclerotic plaque progression and evaluate the therapeutic effects.（3）QSYQ Drop pills and Simvastatin could stabilize the vulnerable plaque by improving the lipid metabolism,decreasing the levels of blood inflammatory factors, inhibiting the oxidation of lipid,decreasing the lipid contents in plaques and inhibiting the inflammatory reaction in plaques.The effects of Simvastatin about improving the lipid metabolism and anti-inflammation are better,and the anti-oxidation of QSYQ Drop pills is powerful.The combination of the two drugs might have better therapeutic effects on the secondary prevention of MI. BackgroundEndothelial dysfunction was associated with cardiovascular disease such as atherosclerosis（AS）.The vascular endothelial cell damage is the critical stage of AS, and the continuous endothelial cell injury led to the regulation dysfunction.In addition,the endothelial functional disorder was also in patients with acute coronary syndrome（ACS）.Endothelium-derived nitric oxide（NO）is synthesized from L-arginine by endothelial nitric oxide synthase（eNOS）and plays a critical role in the regulation of endothelial cell function.NO possesses complex cardiovascular actions such as regulating vascular tone,protecting the intima from platelet aggregation and mononuclear macrophage adhesion,and inhibiting vascular smooth muscle cell proliferation.A relative deficiency in vascular NO influences the vascular tone, increases vascular wall inflammation,plaque instability and thrombosis.Endothelial cells line the inner wall of blood vessels,so it is sensitive to injury by the stimulus in the blood such as endotoxin（i.e.lipopolysaccharide,LPS）,mediators of inflammation, free radical.LPS was paid close attention for its extensive biological effect.Many investigations have demonstrated that the endotoxin was involved in the AS formation especially the initial procedure of the vascular inflammation.The expression and activity of eNOS directly influence NO production and affect endothelial function,eNOS activity is regulated by the phosphorylation of eNOS at many sites,among these sites,the phosphorylation at Ser1177 and Thr495 were most important.Phosphorylation on Ser1177 activates eNOS and increases its sensitivity to Ca2+/calmodulin,leading to enhanced NO production in endothelial cells.In contrast to Ser1177,Thr495 acts on eNOS as a negative regulatory site,i.e.,phosphorylation of this site is associated with a decrease in eNOS activity.In physiology,NO synthesized by eNOS plays a crucial role in the maintenance of vascular homeostasis by relaxation of the vascular smooth muscle,protection of the intima from platelet aggregation and leukocyte adhesion and inhibition of vascular smooth muscle cell proliferation.Endothelial dysfunction led to the decrease of NO production and release,and activate the inflammatory cells by combining with various kinds of leukocyte through adhesion molecules expressed on the surface of endothelial cells, and the active inflammatory cells ingress the artery wall and release hydrolytic enzyme,cytokines,chemokines,growth factor,and so on.These changes induced the plaque formation.So eNOS dysfunction plays an important role in the formation and progression of AS.The improvement of endothelial function and the prevention of endothelial dysfunction are new development tendency in the area of coronary heart disease treatment.Since coronary heart disease is associated with a decreased bioavailability of NO,enhancement of eNOS activity and upregulation of functional eNOS in the long-run may protect the cardiovascular system.Our previous study has proved that QiShenYiQi drop pills（QSYQ drop pills）could regulate the blood lipid and has anti-inflammatory effect.Radix Astragali,the principal drug in this prescription,can protect endothelial function,and the effect was associated with inhibiting apoptosis, changing permeability,anti-inflammatory effect and regulating vasomotion. Astragalus polysaccharides（APS）,the major component of radix astragali,can improve endothelial cell function.However,it has not been fully elucidated whether the molecular mechanisms responsible for these effects are associated with the expression and activity of eNOS.Previous researches paid more attention to the role of iNOS on endothelial dysfunction,eNOS and Phospho-eNOS were less mentioned. In addition,intravenous endothelial cells or bovine aortic endothelial cells were adopted usually in these researches,moreover,these studies focus on iNOS,thus,in the present study,in order to elucidate the prevention therapeutic effect on the endothelial cell damage induced by LPS,human aortic endothelial cells（HAECs） were cultured in vitro,and we observed the effect of LPS on the expression of eNOS and Phospho-eNOS and the protective effect of APS on the endothelial cells damage induced by LPS.Objective（1）To establish the model of human aortic endothelial cells damage;（2）To investigate whether APS attenuates endothelial dysfunction in LPS-activated HAECs and approach the possible mechanism.MethodsTo establish a model of endothelial cell damage,we observe the effect of LPS with different concentration and different time on the expression of eNOS and Phospho-eNOS.The cultured HAECs were pretreated with or without APS（0.025mg/ml、0.25mg/ml、2.5mg/ml）for 12h and were observed under inverted microscope.The effects of APS with different concentration on eNOS mRNA expression were investigated by real-time PCR and the effects of APS on the protein expression of eNOS and Phospho-eNOS 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 LPS damage group,and mainly normal structure of endothelial cells in APS groups.Based on the aim of the present study and the effect of LPS with different concentration and different time on the expression of eNOS and phosphated eNOS, 100ng/ml LPS for 24h is the optimized stimulus for the present study.In cultured HAECs,100ng/ml LPS significantly decreased eNOS expressions at protein and mRNA levels.In addition,100ng/ml LPS significantly decreased the phosphorylation of eNOS on Ser1177 and enhanced the phosphorylation of eNOS on Thr495. Cultured HAECs pretreated with APS for 12h,0.25mg/ml and 2.5mg/ml APS significantly unregulated eNOS expressions at protein and mRNA levels,and enhanced phosphorylation of eNOS on Ser1177,but no effect on Thr495.Conclusion（1）100ng/ml LPS for 24h not only decreased the eNOS expression but also inhibited the eNOS activity by decreasing the phosphorylation of eNOS on Ser1177 and enhancing the phosphorylation of eNOS on Thr495.So 100ng/ml for 24h is the optimized stimulus for the present study.（2）0.25mg/ml and 2.5mg/ml APS significantly upregulated eNOS expressions at protein and mRNA levels,and enhanced the increased phosphorylation of eNOS on Ser1177.APS enhanced the expression and activity of eNOS to improve the endothelial function.