| Coronary microvascular dysfunction(CMD)is defined as abnormalities in structure and function of the coronary microcirculation,which vessels diameter less than 300μm.Percutaneous coronary intervention(PCI)cannot benefit CMD patients,furthermore,despite the large number of novel therapies under basic scientific investigation,translation of strategies into the clinical setting to improve CMD patient outcomes has been disappointing.Novel therapeutics in CMD and translating cardioprotective strategies into clinical practice are needed.CMD animal models are usually established by coronary or intraventricular injection of embolism microspheres in large animals.The experimental conditions and the cost are very high.Recently,some CMD research were conducted using rat models.However,these reports significant deficits in knowledge and consensus on detailed of the establishment of rat CMD model In order to study the mechanism of CMD and the drug action,the methodological study of rat CMD disease model is urgently needed.Shuangshen Ningxin capsule(SSNX)is a traditional Chinese medicine,which is composed of the effective parts and components in ginseng,salvia miltiorrhiza and corydalis tuber.SSNX have the effect of invigorating Qi,activating blood circulation and relieving pain,has been developed for treatment of coronary heart diseases.Previous experimental studies have shown that SSNX capsule has a good therapeutic effect on ischemic heart disease,however the effect and mechanism of SSNX capsule on coronary microvascular dysfunction are still poorly understood.In the present study,we investigated the rat coronary microvascular dysfunction model establishment by different embolization material,and then carried out the research on the therapeutic effect of SSNX capsule.Base on the network pharmacology method,the possible therapeutic mechanism of the blood components of SSNX on CMD were predicted.The network pharmacology suggested vasodilatory effect of SSNX on CMD was also investigated.The present study is divided into four parts as follows.1.Exploration of the methods to establish coronary microvascular dysfunction(CMD)model by using microembolization1.1 A comparative study on the establishment of rat CMD model by chemical damage endothelium and microsphere embolizationAims To explore the differences between chemical damage and microspheres mechanical embolization in the establishment of CMD in rats.Methods Sprague-Dawley(SD)rats were randomly divided into sham group,sodium laurate group and microsphere embolization group.The animals were anesthetized,microspheres(42～120 m)or 1g/L sodium laurate was injected into left ventricle.Hemodynamic indexes,echocardiography,serum CK,CK-MB and LDH activity,and myocardial tissue were measured 24h after surgery.Results There was no significant difference in LV+dp/dtmax between the sodium laurate group and the sham group,the microsphere embolization group was significantly reduced.The echocardiographic EF value and FS of the microsphere embolization group decreased by about 30%and 23%respectively.Microsphere embolization significantly increased CK-MB and LDH activity.Microthrombus formation in myocardium was observed in the sodium laurate group with HE staining,and microvascular embolization microspheres were observed in the microsphere embolization group.Myocardial cell dissolution,rupture and neutrophil infiltration were observed in both groups.Summary Both intraventricular injection of sodium laurate and microspheres can cause CMD and have certain effects on cardiac function in rats.Compared with the endothelial of chemical injury model,microspheres mechanical embolization model was more obvious and stable.Microspheres embolization model hemodynamic and cardiac functions were significantly decreased and myocardial enzymes were increased after 24h.40～120μm microsphere ventricular injection is more suitable for the establishment of rat CME model.1.2 A comparative study on the preparation of rat CMD model by microsphere with different diametersAims To explore the differences between chemical damage and microspheres mechanical embolization in the establishment of CMD in rats.Methods SD rats were randomly divided into sham group,45～53μm,40～120μm,106-125μm microsphere group.The left ventricle was injected with microspheres of different diameters.The hemodynamic indexes,echocardiography,gross morphology and TTC staining of the myocardial group were measured 24h after the operation.Results Compared with the sham operation,the left ventricular ejection fraction(EF)of 45～53pm,40～120μm and 106～125μm microspheres were reduced by 8%,15%and 20%,left ventricular fractional shortening(FS)decreased by 11%,19%and 22%,respectively.SBP,DBP and MAP were decreased in 40～120μm and 106～125μm microspheres.All the three diameter microspheres can reduce LVSP.LV+dp/dtmax was reduced by 25%,27%and 30%,respectively,in the 45～53μm,40～120μm and 106-125μm microspheres groups.The three diameters of microspheres can also reduce LV-dp/dtmax.According to TTC staining of rat heart sections,only a small number of infarcted areas were observed in the 45～53μm microsphere group,while more scattered infarcted areas were observed in the 40～120μm microsphere group,and large areas of infarcted areas were fused in the 106～125μm microsphere group,almost covering the left ventricle and right ventricle.Summary All the three kinds of microspheres can cause different degrees of coronary microvascular dysfunction.The 45-53μm microsphere group was relatively slight injury,while the 106～125μm microsphere model was too severe,and the 40～120μm microsphere group had significant effect on the cardiac function of rats,and showed the appropriate infarcted area,which was suitable for further study of mechanism and drug intervention.2.Protective effects of SSNX capsule on rat model of CMD2.1 Protective effects of SSNX capsule against CMD in ratAims To study the effects of SSNX capsule on the heart and its function in rats with CMD.Methods SD rats were randomly divided into sham operation group,coronary microembolism(CME)model group,Nicorandil tablet group,Tongxinluo capsule group,SSNX 180,90,45mg/kg group.The corresponding drugs were given by intragastric administration for 7d before operation,and the model was prepared 2h after the last administration.Left ventricular injection embolism microspheres(40～120μm),the animals were sacrificed 24 hours after the operation.The hemodynamic indexes,determination of echocardiography,cardiac function,serum biomarkers,serum vascular endothelial function index,Western bolt detection of myocardial tissue apoptosis protein expression,inflammatory cytokines,myocardial TTC staining the infarction area,HE and Heidenhain staining to observe the pathological changes,the ultrastructure was observed by transmission electron microscopy.Results SSNX capsule can increase EF by about 13～20%,increase FS by about 10-18%.and significantly reduce LVIDs.LVESV,increase LVEDV,stroke volume and cardiac output.SBP,DBP and MAP were increased.Can also increase LVSP by 14-18%,LV+dp/dtmax by 24-30%,and LV-dp/dtmax by 28-31%.Serum CK,LDH,CK-MB and cTnT were decreased.Serum ET-1,Ang Ⅱ were decreased and NO level and was increased.The expression of caspase-3 was decreased and the expression of Bcl-2 was increased.Reduced the area of myocardial infarction by about 40～50%.It alleviates the myocardial histopathological changes,reduces the number and diffusiveness of Heidenhain stained myocardial cells,and improves the ultrastructural changes of myocardium.Summary SSNX capsule can significantly improve the hemodynamics and cardiac function of rats with CMD,reduce the area of myocardial infarction,reduced the increase of myocardial injury markers,and increased the level of serum NO,reduced the ET-1 and Ang Ⅱ level,SSNX capsule can protect the myocardium by inhibiting apoptosis of myocardial cells.2.2 Effect of SSNX capsule on myocardial perfusion in rats with CMDAims To study the effect of SSNX capsule on myocardial microvascular perfusion in rats with CMD.Methods SD rats were randomly divided into sham operation group,model group and SSNX 90mg/kg group.Myocardial contrast echocardiography was performed 24h after operation,and local blood perfusion was determined by 15μm fluorescence microspheres.Results Compared with the model group,the reflecting the local myocardial blood volume A value of SSNX capsule increased by about 38%,the reflecting myocardial blood flow velocity β value increased by about 46%,and the reflecting the myocardial blood flow A·β value increased by about 93%.The local myocardial blood flow measured by fluorescence microspheres was about 35%higher than that of the model group.Summary SSNX capsule can significantly improve CMD rats blood perfusion,increase microvascular density,and increase local blood flow in myocardium.3.Study on the visualization of CMD rats based on diffraction enhanced imaging 3.1 Coronary microvascular cast in rats with CMDAims To study the effects of SSNX capsule on the heart and its function in rats with CMD.Methods SD rats were randomly divided into sham operation group,coronary microembolism(CME)model group,Nicorandil tablet group,Tongxinluo capsule group,SSNX 180,90,45mg/kg group.The corresponding drugs were given by intragastric administration for 7d before operation,and the model was prepared 2h after the last administration.Left ventricular injection embolism microspheres(40-120μm),the animals were sacrificed 24 hours after the operation.The hemodynamic indexes,determination of echocardiography,cardiac function,serum biomarkers,serum vascular endothelial function index,Western bolt detection of myocardial tissue apoptosis protein expression,inflammatory cytokines,myocardial TTC staining the infarction area,HE and Heidenhain staining to observe the pathological changes,the ultrastructure was observed by transmission electron microscopy.Results SSNX capsule can increase EF by about 13～20%,increase FS by about 10～18%,and significantly reduce LVIDs,LVESV,increase LVEDV,stroke volume and cardiac output.SBP,DBP and MAP were increased.Can also increase LVSP by 14-18%,LV+dp/dtmax by 24-30%,and LV-dp/dtmax by 28-31%.Serum CK,LDH,CK-MB and cTnT were decreased.Serum ET-1,Ang Ⅱ were decreased and NO level and was increased.The expression of caspase-3 was decreased and the expression of Bcl-2 was increased.Reduced the area of myocardial infarction by about 40～50%.It alleviates the myocardial histopathological changes,reduces the number and diffusiveness of Heidenhain stained myocardial cells,and improves the ultrastructural changes of myocardium.Summary SSNX capsule can significantly improve the hemodynamics and cardiac function of rats with CMD,reduce the area of myocardial infarction,reduced the increase of myocardial injury markers,and increased the level of serum NO,reduced the ET-1 and Ang Ⅱ level,SSNX capsule can protect the myocardium by inhibiting apoptosis of myocardial cells.3.2 Study on visualization of diffraction enhanced imaging in rats with CMDAims To study the coronary artery casting changes in rats with CMD and SSNX capsule interventionMethods SD rats were randomly divided into sham operation group,coronary microembolism(CME)model group and SSNX capsule 90mg/kg group.Model establishment and administration of drug are the same as in part Ⅱ.The DEI peak map was collected at Beijing synchrotron radiation device 4W1A imaging experimental station.The 3d reconstruction of the processed fault map was conducted with Avizo9.0 software to observe the shape and spatial morphology of coronary micro vessel.Results The acquisition conditions of coronary X-ray diffraction enhanced imaging in rats with CMD were established.Vascular resolution can be up to 10μm level.coronary microvascular density in the model group was significantly lower than that in the sham group,and the coronary microvascular density could be increased after the intervention of SSNX capsule.Summary The coronary microvessels in rats can be observed clearly by X-ray diffraction enhanced imaging,and the 3d visualization model of the bleeding network can be constructed.The abnormality of coronary microvascular dysfunction caused by coronary microembolism in rats can be detected by this method,and the effect of SSNX capsule on increasing coronary microvascular density can also be reflected by this method.4.Study on the mechanism of intervention of SSNX capsule in CMD based onnetwork pharmacological prediction4.1 The prediction effects of SSNX capsule on CMD based on network pharmacologyAims To explore the potential target and mechanism of SSNX capsule in the treatment of CMD through network pharmacology technology.Methods According to the results of SSNX capsule pharmacokinetic in rats in the previous research,29 blood components were selected.The SEA database,STITCH database and Swiss TargetPrediction platform were used to predict the target of SSNX capsule in the treatment of CMD.Results SSNX capsule may play a role in the treatment of CMD through biological processes such as drug reaction,positive regulation of phosphorylation of phosphoprotein,calcium-mediated signal regulation,adenylate cyclase-activated adrenergic receptors and nitric oxide synthesis.The mechanism of action may be related to the regulation of calcium ion signaling pathway,vascular smooth muscle contraction,cAMP signaling pathway and myocardial adrenergic signaling pathwaySummary The blood components of SSNX capsule play a role in the intervention of CMD through the multi-target and multi-pathway synergia,among which the regulation of vascular smooth muscle contraction may be one of the key mechanisms.4.2 Effects of SSNX capsule on thoracic aorta rings isolated from SD ratsAims To verify the prediction results of network pharmacology and explore the vasodilation mechanism of SSNX.Methods The effect of SSNX capsule on endothelial integrity and endothelial vascular ring pre-constriction by potassium chloride(KCl)and norepinephrine(NE)was observed in isolated thoracic aortic rings of SD rats.Treating the aorta rings with nitric oxide synthase inhibitor L-NAME,guanylate cyclase inhibitor ODQ,epoxy synthase inhibitor indomethacin(Indo),voltage dependent potassium channel blocker 4-aminopyridine(4-AP)and calcium activated potassium channel blockers tetraethylamine chloride(TEA),ATP sensitive potassium channel blocker glibenclamide(Gli),inward rectifier type channel blocker barium chloride(Bacl2),beta blockers propranolol(Prop).Observing the effect of SSNX capsule on vascular ring tension.The effect of SSNX capsule on intracellular Ca2+release and in extracellular Ca2+influx in arterial ring was observed.Results SSNX capsule showed time-dependent and dose-dependent diastolic effects on the tension of KCl and NE pre-constriction aorta rings.When the vascular endothelium is removed,its diastolic effect is inhibited,and the vascular ring diastolic effect in L-NAME and ODQ treatment is also inhibited.There is no difference between the Indo and Prop treatment group and the untreated group.The vasodilation of the aorta rings treated with TEA and Bacl2 was inhibited,and there was no difference between the Gli and 4-AP treatment group and the untreated group.SSNX capsule can inhibit intracellular Ca2+release and in extracellular Ca2+influx in aorta rings.Summary SSNX capsule can not only inhibit the calcium flow induced by the voltage dependent calcium channel and receptor-operated calcium channel in the arterial smooth muscle membrane,but also inhibit the intracellular receptor-operated calcium channel.The endothelium-dependent eNOS-NO-cGMP diastolic pathway can also play a role in vasodilation.Vascular diastolic regulation can also be achieved by opening smooth muscle calcium activates potassium channel and inward rectifier potassium channel.These action mechanisms are in good agreement with the predicted results of network pharmacologyConclusion The CMD model established by embolization microspheres with diameters of 40～120 m is characterized by stability and appropriate degree of injury,which can be used for the study of pathogenesis and therapeutic intervention of CMDCoronary vessel casting model and X-ray diffraction enhanced imaging can be used to evaluate the spatial morphological changes of CMD microvessels,and the resolution of X-ray diffraction enhanced imaging can be close to that of capillaries.SSNX capsule can improve CMD vascular endothelial function,regulate vasomotor contraction,improve myocardial blood perfusion,reduce myocardial necrosis and apoptosis,and increase cardiac systolic and diastolic functions. |
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