| Diabetes mellitus(DM)is a metabolic disorder mainly characterized by chronic hyperglycemia caused by multiple etiologies,which has become a major public health problem worldwide.DM often induces vascular complications and endangers many organs such as heart,brain,kidney,eye,foot and so on,among which cardiovascular disease is more likely.In recent years,the dysfunction of coronary microcirculation related to DM has gradually attracted the attention of researchers.Cardiac microvessels are mainly composed of cardiac microvascular endothelial cells(CMEC)located at the end of the circulation,which control myocardial perfusion and coronary artery reserve.In view of the direct contact between microvessels and blood flow,CMEC is more vulnerable to hyperglycemia than cardiomyocytes.With the occurrence and development of DM,the energy metabolism of CMEC is abnormal,which leads to the impairment of endothelial regulation that maintains the steady state of blood vessels,and finally leads to the dysfunction of microcirculation.The mechanism may be related to the increase of polyol pathway flux and excessive activation of hexosamine pathway,which leads to excessive generation of AGEs and ROS.At present,the treatment of DM is mainly based on hypoglycemia,but most anti-glycemic treatments fail to reduce cardiovascular risk.Therefore,finding a way to reduce cardiac microvascular damage under high glucose conditions is the key to delaying or alleviating cardiovascular complications of DM.Shuangshen Ningxin Capsule(SSNX)is an innovative component of traditional Chinese medicine independently developed by our research group,which is composed of the effective parts of Renshen,Danshen,and Yanhusuo in a certain proportion.It has the effect of Supplementing qi and activating blood circulation,in which Renshen can invigorate qi and tonify deficiency,Danshen can promote blood circulation to remove blood stasis,Yanhusuo can promote blood circulation,activate qi and relieve pain,and the three drugs are compatible to treat both symptoms and root causes,which can use for coronary heart disease,angina pectoris,coronary microcirculation disorders with qi deficiency and blood stasis syndrome.Previous studies in our laboratory have found that SSNX can reduce myocardial oxygen consumption and improve myocardial cell energy metabolism,which can improve acute myocardial ischemia and myocardial infarction.In addition,SSNX can also dilate blood vessels and increase coronary blood flow,which has a significant improvement effect on obstructive coronary microcirculation disfuncton.However,the effect and mechanism of SSNX on diabetic coronary microcirculation disfuncton have not been studied.Therefore,in vivo and in vitro experiments were conducted to study the effects and molecular mechanism of SSNX on diabetic coronary microcirculation function.Objective:In vivo and in vitro experiments were used to elucidate the effect and molecular mechanism of SSNX in improving diabetic coronary microcirculation dysfunction.Research contents and methods:1 Study on the effect and mechanism of Shuangshen Ningxin Capsule on diabetic coronary microcirculation dysfunction1.1 Effect of Shuangshen Ningxin Capsule on glucose and lipid metabolism in diabetic mice with coronary microcirculation dysfunctionThe mice model of diabetic coronary microcirculation dysfunction was established by feeding KKay mice with high sugar and fat diet.After 4 weeks of feeding,fasting blood glucose(FBG)and myocardial perfusion were detected.The mice with FBG≥11.1 mmol/L and myocardial perfusion ability significantly lower than the control group were used as the model replication success rate(81.2%).Model mice were randomly divided into model group,SSNX high dose group(SSNX,360 mg/kg),SSNX medium dose group(SSNX,180 mg/kg),SSNX low dose group(SSNX,90 mg/kg),Tongxinluo group(2 g/kg),Nicordil group(10 mg/kg),There were 13 mice in each group,and another 13 healthy C57BL/6J mice were used as the normal group,and the normal group was fed with conventional diet.The drug group was given 0.lml/10g by gavage.The normal group and the model group were given the same volume of distilled water by gavage once a day for 8 weeks.The body mass and FBG of mice in each group were measured before administration(0W),2W,4W,6W and 8W respectively;Glycosylated hemoglobin(HbA1c)was detected before administration(0W)and 8W after administration.At the end of the experiment,four parameters of blood lipid(TC、TG、LDL-C、HDL-C)were measured by biochemical method,the carbohydrate level in myocardial tissue of mice in each group was detected by PAS staining,and the level of fatty acid transporter(CD36)in myocardial tissue of mice in each group was detected by immunohistochemistry.1.2 Effect of Shuangshen Ningxin Capsule on diabetic coronary microcirculation functionModel establishment and group administration are the same as above.After the experiment,HE staining to observe the myocardial tissue morphology,Masson staining to detect myocardial fibrosis,ultra-high resolution of animal diastolic function and myocardial ultrasonic instrument testing mice irrigation situation,immunohistochemical method to detect myocardial tissue alpha smooth muscle actin(a-SMA)levels,myocardial microvascular density(MVD),transmission electron microscope to observe the myocardial ultrastructure of capillary,Serum creatine kinase isoenzyme(CK-MB)and brain natriuretic peptide(BNP)levels were detected by ELISA.1.3 Molecular mechanism of Shuangshen Ningxin Capsule on diabetic coronary microcirculation dysfunctionAccording to the previous comprehensive results,SSNX high dose group has the best effect on improving diabetic coronary microcirculation.In this part,the left ventricular tissues(2 in each group)of mice in the normal group,model group and SSNX high dose group were used to analyze the potential mechanism of SSNX based on single-cell sequencing technology.1.4 Effect of Shuangshen Ningxin Capsule on PI3K/AKT/AMPK/eNOS pathway in diabetic mice with coronary microcirculation dysfunctionModel establishment is the same as above.The model mice were randomly divided into model group,SSNX high dose group(SSNX,360 mg/kg),Tongxinluo group(2 g/kg)and Nicodil group(10 mg/kg),with 13 mice in each group,and 13 healthy C57BL/6J mice as the normal group,which were fed with conventional diet.The drug group was given 0.1 ml/10g by gavage.The normal group and the model group were given the same volume of distilled water by gavage once a day for 8 weeks.After the experiment,the protein levels of p-PI3K,PI3K,p-AKT,AKT,p-AMPK,AMPK,HK1,PFK-1,PFKFB3 and eNOS in the myocardial tissue of mice in each group were detected by Western blotting,and detected by nitric acid reduction method.Serum nitric oxide(NO)content,and serum endothelin-1(ET-1),adenosine triphosphate(ATP)and adenosine diphosphate(ADP)levels were detected by ELISA.2 Study on the effect and molecular mechanism of Shuangshen Ningxin capsule and its active components Rg1,SAA and DHC on myocardial microvascular endothelial cell injury induced by high glucose2.1 Shuangshen Ningxin capsule and its active components Rg1,SAA and DHC were selected for optimal protective concentration and protective components against myocardial microvascular endothelial cell injury induced by high glucoseMouse myocardial microvascular endothelial cell line(MMVEC)was used to detect the cell survival rate of MMVEC cultured in complete medium with different glucose concentrations by CCK8,and a suitable glucose concentration was selected to establish a model of MMVEC damage induced by high glucose.CCK8 was used to screen the best protective concentration of SSNX,Rg1,SAA and DHC against MMVEC damage induced by high glucose.The protective effects of SSNX,Rg1,SAA and DHC on high glucose-induced MMVEC damage were screened out by nitric acid reduction method to detect cellular NO content and ELISA to detect cellular ATP content.2.2 Effect of SAA on PI3K/Akt/AMPK/eNOS pathway in myocardial microvascular endothelial cells injured by high glucoseUsing the above high glucose induced MMVEC damage model,the experiment was divided into normal group(Con,glucose concentration 5.5 mmol/L),model group(HG,glucose concentration 250 mmol/L),SAA low dose group(HG+SAA 25μM),SAA high dose group(HG+SAA 50μM),SAA+inhibitor group(HG+SAA 50μM+LY),inhibitor group(LY,glucose concentration was 5.5 mmol/L).The cell survival rate was detected by CCK8,the content of NO was detected by nitric acid reduction method,the content of ATP was detected by ELISA,the level of glycolysis was detected by Seahorse XFp bioenergy analyzer,and the protein levels of p-PI3K,PI3K,p-AKT,AKT,p-AMPK,AMPK,eNOS,HK1,PFK-1and PFKFB3 were detected by western blotting.The levels of PI3K mRNA,AKT mRNA,AMPK mRNA,eNOS mRNA,HK1 mRNA,PFK-1 mRNA and PFKFB3 mRNA in cells of each group were detected by RT-qPCR.Results:1 Study on the effect and mechanism of Shuangshen Ningxin Capsule on diabetic coronary microcirculation dysfunction1.1 Effect of Shuangshen Ningxin Capsule on glucose and lipid metabolism in diabetic mice with coronary microcirculation dysfunctionSSNX can reduce the body weight and increase the grasping power of DM mice with coronary microcirculation dysfunction,and improve the general signs of model mice.SSNX can also reduce the contents of TC and LDL-C in serum and the levels of sugars and CD36 in myocardial tissue,which can improve the glucose and lipid metabolism of DM mice with coronary microcirculation dysfunction.1.2 Effect of Shuangshen Ningxin Capsule on diabetic coronary microcirculation functionSSNX can effectively improve the myocardial morphology,reduce myocardial fibrosis,improve the ultrastructure of myocardial microvessels and increase the density of myocardial microvessels in DM mice with coronary microcirculation dysfunction.SSNX can also reduce the contents of CK-MB and BNP in serum,and restore cardiac diastolic function and coronary microcirculation function.The above results show that the effect of high dose of SSNX is better than that of medium and low dose.1.3 Molecular mechanism of Shuangshen Ningxin Capsule on diabetic coronary microcirculation dysfunctionThe results showed that a total of 41723 high quality cells were obtained after quantitative quality control of each heart sample,and 16 optimal cell groups were obtained after dimensionless clustering,which were identified into 7 types of cells:Fibroblasts(cluster 1),Cardiomyocytes(cluster 2,3,10,11,15),endothelial/fibroblasts(cluster 5),Macrophage(cluster 6,7,16),Endothelial cells(cluster 4,8,13),Pericytes(cluster 9,12),Smooth muscle cells(cluster 14).SSNX can improve the transformation of cardiac endothelial cells into fibroblasts,reduce the proportion of fibroblasts,and alleviate myocardial fibrosis in model group mice.The endothelial cells were identified as five EC subgroups:capillary endothelial cells(capillary EC),large vein endothelial cells(large vein EC),arterial capillary endothelial cells(cap.arterial EC),lymphatic endothelial cells(lymphatic EC)and arterial endothelial cells(artery EC).These five endothelial cell subgroups have their own specific genes and functions,but they also share the expression of other genes and are functionally related to each other.It is worth noting that the specifically expressed genes of capillary EC are mainly related to the proliferation,survival and migration of endothelial cells,and the pathway of capillary EC enrichment is mainly related to substance metabolism,so we speculate that the changes of substance metabolism and function in capillary EC play an important role in the function of coronary microcirculation in DM.The differential pathway enrichment scores of normal group,model group and SSNX high dose group in capillary EC showed that SSNX could up-regulate complement and coagulation cascade,glycolysis/gluconeogenesis,pentose phosphate pathway,citric acid cycle,oxidative phosphorylation,AMPK signal pathway,fatty acid metabolism,actin cytoskeleton regulation,cell adhesion molecules,gap junctions,tight junctions,etc.Down-regulate arginine biosynthesis,macromolecular glycan biosynthesis,necrotizing apoptosis,iron death,P53 signal pathway,TGF-β signal pathway,AGE-RAGE signal pathway,leukocyte transendothelial migration and so on.The main target cell of SSNX is capillary EC,which is related to energy metabolism,cell death and inflammatory response.The analysis of genes related to energy metabolism in capillary EC groups showed that the target genes regulated by SSNX were mainly genes related to glycolysis,including Hkl,Pfkm/PFK1,Pfkfb3,Gapdh,Ldhb and Pkm,and SSNX could up-regulate the expression of these genes in the model group,promote energy metabolism of capillary EC and maintain normal EC function.In conclusion,the target cell of SSNX regulation is capillary EC,and the target genes are glycolysis related genes:Hkl,Pfkm/PFK1,Pfkfb3,Gapdh,Ldhb and Pkm.AMPK pathway is one of the target pathways of SSNX.AMPK can regulate the rate-limiting enzymes in glycolysis pathway to participate in energy metabolism,and also regulate eNOS to maintain endothelial function,which is regulated by PI3K/Akt pathway.Therefore,we speculate that SSNX can regulate energy metabolism of capillary EC,improve EC function and further improve the diabetic coronary microcirculation function,which may be related to PI3K/AKT/AMPK/eNOS.1.4 Effect of Shuangshen Ningxin Capsule on PI3K/AKT/AMPK/eNOS pathway in diabetic mice with coronary microcirculation dysfunctionBy regulating PI3K/AKT/AMPK/eNOS pathway,SSNX can not only improve the energy metabolism of EC,promote the expression of glycolysis key rate-limiting enzymes HK1,PFK1 and glycolysis pathway regulator PFKFB3,increase the content of serum ATP,but also improve the function of EC,promote the secretion of NO,and then improve the function of coronary microcirculation in diabetes.2 Study on the effect and molecular mechanism of Shuangshen Ningxin capsule and its active components Rg1,SAA and DHC on myocardial microvascular endothelial cell injury induced by high glucose2.1 Shuangshen Ningxin capsule and its active components Rg1,SAA and DHC were selected for optimal protective concentration and protective components against myocardial microvascular endothelial cell injury induced by high glucoseWhen the concentration of glucose was 33mmol/L,it began to damage MMVEC cells,and the degree of damage to MMVEC cells increased with the increase of glucose concentration.In this experiment,MMVEC cells with glucose concentration of 250mmol/L were selected as the cell injury model induced by high glucose.SSNX 20,40,80μg/ml could effectively improve the survival rate of MMVEC cells induced by high glucose,and 80 μg/ml had the best effect.Rg1 25,50,100,200,400 μmol/L could effectively improve the survival rate of MMVEC cells induced by high glucose,and 200 μmol/L had the best effect.SAA 6.25,12.5,25,50 μmol/L could effectively improve the survival rate of MMVEC cells induced by high glucose,and the effect of 50 μmol/L was the best.DHC 16 μmol/L can effectively improve the survival rate of MMVEC cells induced by high glucose.SAA can effectively improve the contents of NO and ATP in MMVEC cells induced by high glucose.2.2 Effect of SAA on PI3K/Akt/AMPK/eNOS pathway in myocardial micro vascular endothelial cells injured by high glucoseBy regulating PI-K/AKT/AMPK/eNOS pathway,SAA can not only improve the level of glycolysis of MMVEC,promote the protein expression and mRNA expression of key rate-limiting enzymes HK1,PFK1 and glycolysis pathway regulator PFKFB3,increase the content of ATP,but also improve the function of MMVEC,promote the secretion of NO.Conclusions:1 Shuangshen Ningxin Capsule can increase the myocardial microvessel density,improve the ultrastructure of myocardial microvessels,and enhance the coronary microcirculation function in mice with diabetic coronary microcirculation dysfunction.2 The target cell of Shuangshen Ningxin Capsule affecting myocardial microvascular endothelial cells is mainly capillary EC,the target gene is glycolysis related genes:HK1,pfk1,PFKFB3,and the target pathway is AMPK pathway.3 Shuangshen Ningxin Capsule can not only improve the energy metabolism of myocardial microvascular endothelial cells,but also improve the function of myocardial microvascular endothelial cells by regulating the PI3K/AKT/AMPK/eNOS pathway,thus improving the diabetic coronary microcirculation dysfunction.In conclusion,Shuangshen Ningxin Capsule and its active ingredients SAA can regulate the PI3K/AKT/AMPK/eNOS pathway,target the glycolysis and function of myocardial capillary endothelial cells,which can improve the diabetic coronary microcirculation dysfunction. |
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