Study On The Mechanism Of Shexiang Tongxin Dropping Pill In Treating Coronary Microcirculatory Disturbance In Diabetic Cardiomyopath

BACKGROUND:The diabetic cardiomyopathy(DCM)is a common cardiovascular complication caused by diabetes,which is independent of coronary artery disease and hypertensive heart disease,and it is one of the important complications leading to the death of diabetic patients.Coronary microcirculation dysfunction(CMD)is an early pathological change in DCM and an important pathophysiological basis for myocardial ischemia or heart failure in diabetic patients.Coronary microcirculation optimizes the nutrient and oxygen supply to the heart by coordinating the resistance within different microvascular domains to regulate myocardial blood supply.In the diabetic state,coronary microcirculation is affected by a series of such as hyperglycemia,inflammatory response,and cardiac autonomic dysfunction,leading to myocardial ischemia.There are no effective drugs or treatments for diabetic cardiomyopathy CMD.Capillary density changes are an important marker of CMD.Capillary thinning results in insufficient blood supply to meet cardiac demand,leading to reduced myocardial perfusion and cardiac ischemia,resulting in cardiovascular complications in diabetic patients.At the same time,impaired endothelial barrier integrity is also a major pathological alteration of CMD.Meanwhile,factors such as high glucose status,ischemia and inflammation disrupt the intercellular connections of the vascular endothelium and impair endothelial barrier integrity,leading to increased endothelial permeability,which affects the myocardial microcirculatory system.Therefore,promoting myocardial angiogenesis and improving endothelial intercellular junctions may be an effective way to improve cardiac function therapy in diabetic patients.Shexiang Tongxin Dripping Pill(STDP)is a listed proprietary Chinese medicine for the treatment of stable exertional angina pectoris in coronary artery disease,and has clear therapeutic effects on ischemic heart disease such as myocardial ischemia and ischemic heart failure,and has positive effects on streptozotocin(STZ)-induced DCM,as well as it also has a therapeutic effect on CMD after myocardial ischemia-reperfusion,,so we hypothesize that STDP can treat diabetic coronary microcirculatory disorders.Nicorandil has the effect of dilating microvasculature and improving microcirculation,which has certain efficacy in diabetic cardiomyopathy,and is commonly used in clinical practice to improve coronary ischemia,so we choose nicorandil as a positive control drug.In this study,DCM mice induced by STZ were used as a model to observe the therapeutic effect of STDP on diabetic cardiomyopathy CMD,and to explore its mechanism from the perspective of promoting myocardial angiogenesis and improving endothelial cell connection.OBJECTIVE:To establish a mouse model of DCM and observe the efficacy of the Chinese medicine compound STDP in the treatment of CMD in diabetic cardiomyopathy,and to investigate its pharmacological mechanism from the perspective of promoting angiogenesis and improving interendothelial cell junctions in a preliminary manner.METHODS:1.SPF grade male C57BL/6 mice,6-8 weeks old,were randomly divided into 6 control and 30 diabetic groups after 3 days of acclimatization.After overnight fasting for 12 h,the diabetic group was injected intraperitoneally with STZ and the control group was given the same volume of sodium citrate buffer for 5 consecutive days,calculated on the day the injection was completed,and measured 7 days later After 7 days,fasting blood glucose was measured,and a blood glucose value>16.7 mmol/L was considered as successful modeling.The mice with blood glucose reaching the standard were randomly divided into 5 groups accordin g to the random number table method:model group,STDP low-dose group,STDP medium-dose group,STDP high-dose group and nicorandil group.2.After the animals were grouped,the drug intervention was started,in which the STDP low,medium and high dose groups were administered by gavage with 15,30 and 60 mg/kg/d STDP suspension,respectively,and the nicorandil group was administered by gavage with 15 mg/kg/d suspension,while the control and model groups were administered with the same volume of distilled water for 12 weeks.During the treatment period,body weight was monitored every 2 weeks,fasting blood glucose was measured every 4 weeks,and serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL-C)and high-density lipoprotein(HDL-C)levels were measured by automatic biochemical analyzer in each group at the end of the experiment.3.After 12 weeks of drug administration,echocardiography was performed to determine left ventricular ejection fraction(EF),left ventricular short-axis shortening(FS)and coronary flow reserve(CFR)in mice.After sampling,HE staining,Masson staining,CD31 staining,WGA staining and immunofluorescence of endothelial intercellular junctional protein were performed for pathological examination.4.The expression of VEGF,VEGFR2,p-VEGFR2,PI3K3,p-PI3K,AKT,p-AKT,eNOS,p-eNOS in myocardial tissues of each group of mice was detected by Western blot protein immunoblotting to evaluate the angiogenesis;the expression of endothelial intercellular junction proteins ZO-1,Occludin,VE-Cadherin,and CFR were detected in myocardial tissues of each group of mice,VE-Cadherin,Claudin-5,and JAM-A expression for evaluation of endothelial intercellular junctional changes.RESULTS:1.STZ induced DCM mouse model.Compared with the control group,the mice in the model group had a lean body shape,yellowish fur,dryness and lack of luster,poorer spirit,unmotivated and unresponsive,significantly increased food intake and water intake,and significantly increased urine output;blood glucose and body weight were significantly increased(P<0.01),indicating the successful replication of the diabetic model.STDP had no obvious improvement on the body weight and fasting blood glucose level of DCM mice(P>0.05);Compared with the control group,TC,TG and LDL-C levels in the model group were significantly increased(P<0.01)and HDL-C levels were significantly decreased(P<0.01),and STDP significantly reduced serum TC,TG and LDL-C levels and increased HDL-C levels(P<0.01);2.Compared with the control group,EF and FS values in the model group were significantly decreased(P<0.01),consistent with the pathological manifestations of reduced cardiac function in DCM,with uneven myocardial tissue staining,disorganized myofascicular arrangement,massive connective tissue hyperplasia with inflammatory cell infiltration,significant collagen fiber deposition(P<0.01),significantly increased CVF(P<0.01),increased cardiomyocyte area(P<0.01),decreased CFR values(P<0.01),and decreased capillary density(P<0.01),while the cardiac endothelial intercellular junctional protein ZO-1 and VE-Cadherin were irregularly dispersed in the cell periphery and fluorescence staining was dark;STDP significantly increased EF and FS values(P<0.01),improved cardiac function,and repaired disturbed myocardial structure,significantly STDP significantly improved EF and FS values(P<0.01),improved myocardial function and repaired disordered myocardial structure,significantly reduced CVF(P<0.01),reduced myocardial cell area(P<0.01),increased capillary density(P<0.01),increased CFR level(P<0.01),and increased the distribution of endothelial intercellular junction protein ZO-1 and VE-Cadherin in a more regular and continuous manner with enhanced fluorescence intensity.3.Myocardial tissue vascular endothelial growth factor VEGF expression was significantly reduced in the model group(P<0.01),and p-VEGFR2/VEGFR2,p-PI3K/PI3K,p-AKT/AKT,and p-eNOS/eNOS ratios were significantly reduced(P<0.01).STDP could significantly increase VEGF expression,promote VEGFR2,PI3K,AKT,and eNOS phosphorylation(P<0.01,P<0.05).4.The expression of endothelial intercellular junctional proteins ZO-1,Occludin,VE-cadherin,Claudin-5,and JAM-A were significantly lower in the model group compared with the control group(P<0.01),and STDP could significantly increase the expression of the above mentioned proteins(P<0.01).CONCLUSION:1.DCM mice showed reduced cardiac systolic function,myocardial fibrosis,cardiomyocyte hypertrophy and other lesions,suggesting the presence of cardiomyopathy in mice;at the same time,the myocardium of DCM mice showed reduced capillary density,reduced CFR and disrupted endothelial intercellular junctions,suggesting the presence of CMD in DCM mice.2.STDP significantly improved cardiac function in DCM mice,with no significant therapeutic effect on body weight or fasting glucose.The drug was able to increase CFR,increase capillary density,and improve endothelial cell junctions.The drug has a clear therapeutic effect on diabetic cardiomyopathy CMD,and its mechanism of action is partly related to promote angiogenesis and improvement of endothelial cell junctions.SIGNIFICANCE:This study clarified the efficacy of STDP in the treatment of CMD in DCM mouse model and preliminarily revealed its pharmacological mechanism of promoting angiogenesis and improving endothelial intercellular connections,thereby improving myocardial CMD.