| Objective:Cardiovascular disease is one of the major diseases threatening human health and life.In recent years,the morbidity and mortality of ischemic heart disease in China is still in a rising stage,which has brought a significant disease burden and economic pressure to the country and society.Atherosclerosis is a lipid-filled,multifocal,stasis immunoinflammatory pathological process that occurs in the large and middle arteries.It is also the most common pathological basis for acute myocardial ischemia.The evolution of atherosclerosis is a gradual development process.The instability of atherosclerotic plaques and plaque shedding caused by various reasons eventually lead to the formation of thrombus and trigger acute cardiovascular events.As a serious complication of atherosclerotic lesions,Acute myocardial ischemia,has very complicated etiology and pathological process been very complicated.It is believed that there is a clear causal relationship range from fine particulate matter’s exposure to atherosclerotic plaque instability and the incidence and mortality of acute ischemic heart disease.PM2.5 is considered a variable risk of cardiovascular disease factor.At the same time,the frequent emergence of ultra-high concentrations of air pollution particulate matter in our country and chronic long-term exposure to excessive concentrations have induced and aggravated cardiovascular disease and caused widespread concern.In summary,fine particulate matter is one of the key risk factors for the progression of atherosclerosis and eventually promotes cardiovascular disease,and oxidative stress is the core mechanism of cardiovascular disease induced and exacerbated by fine particulate matter,which is the prevention and treatment potential targets for cardiovascular disease.At present,research on the mechanism of fine particulate matter-induced and aggravated cardiovascular disease has become a hot topic at home and abroad,but there is still no effective prevention and treatment measures.This topic takes the traditional classical formula Shengmaiyin as the research object,and uses oxidative stress regulation as the core to open new indications for Shengmaiyin to explore the protective effect and mechanism on cardiovascular damage caused by fine particles.Methods:1.Network Pharmacological Study of Shengmaiyin（Dangshenfang）in the Treatment of Atherosclerotic Cardiovascular DiseaseUsing the SymMap database,TCMSP platform and BATMAN-TCM platform to obtain the main chemical components of Shengmaiyin（Dangshenfang）,SymMap and ETCM to search for compound targets,and DisGeNET and GeneCards databases to search for disease targets;compound targets and disease targets intersect Shengmaiyin（Dangshenfang）was used to predict the target of ASCVD.The STRING database was used to construct the target protein interaction（PPI）network map.Cytoscape 3.6.0 was used to obtain the key compounds and key targets of Shengmaiyin（Dangshenfang）acting on ASCVD.Finally,the key targets enriched by David were used for GO.Functional enrichment and KEGG pathway analysis.2.Effect of PM2.5 on Atherosclerotic Plaque Stability in ApoE-/-Mice and the Mechanism of SMAt the animal level,a model of atherosclerosis was established using ApoE receptor knockout mouse combined with high-fat diet for 12 weeks.（High-fat feed formula:food-grade egg yolk powder 10%,lard 10%,high-purity cholesterol 1%,maintenance feed 79%,frozen storage.）Intratracheal instillation of PM2.5 suspension for 4 weeks（1 mg/mL,50μl/animal,once/week for 4 weeks）,which lasted to the 16th week,high,medium and low doses of Shengmaiyin（6.24,3.12,1.56 mg/kg·d）,administered orally.Construction of PM2.5 exposure model in atherosclerotic mice.On the 16th week,animal ultrasound was used to detect the size and curvature of the aortic arch and the plaque area.Mice were sacrificed and unilateral eye blood was collected for blood cholesterol（TC）,triglyceride（TG）,high density lipoprotein（HDL）,and low density lipoprotein（LDL）.Aortic specimens were retained,and then the specimens were stained with HE,oil red O,Masson,and ROS.Immunohistochemical methods were used to detect the expression of matrix metalloproteinases（MMP9）in the aortic arch.3.Effect of PM2.5 in the myocardial ischemic injury of ApoE-/-mice and the mechanism of SMAt the animal level,after 12 weeks of feeding with ApoE-/-mice combined with high-fat diet in Chapter 2,PM2.5 suspension was instilled by Intratracheal instillation（1 mg/mL,50μL/animal,once/week for 4 weeks）.which lasted to the 16th week,high,medium and low doses of Shengmaiyin（6.24,3.12,1.56 mg/kg·d）,administered orally.24 hours after the last exposure,the mice were subjected to ligation of the left anterior descending coronary artery to construct an animal model of myocardial ischemia in the ApoE-/-mice.The ECG of the mouse was recorded at the same time,and the changes of the ECG wave form and the height of the ST segment of the mouse before and after LAD ligation were recorded.24 hours after operation,LVEF,LVFS,SV,CO,LVID,S,LVID,d,IVS,S,IVS,d,LVPW,S,LVPW,d,AV peak Vel,Coronary peak Vel were detected by ultrasound.Mice were sacrificed and myocardial tissue samples were taken,and myocardial infarction area was evaluated by TTC staining.Transmission electron microscope was used to observe the ultrastructure of myocardial tissue,mitochondria and autophagosomes;ROS staining was used to detect changes in reactive oxygen species in myocardial tissue;immunohistochemical methods were used to detect the expression of Nrf2 and downstream genes NQO1.Results:1.Network Pharmacological Study of Shengmaiyin（Dangshenfang）in the Treatment of Atherosclerotic Cardiovascular DiseaseShengmaiyin（Dangshenfang）has 33 key compounds for ASCVD and 25 key targets.The GO analysis results show that the biological functions of Shengmaiyin（Dangshenfang）in treating ASCVD are mainly related to the regulation of apoptotic process,oxidative stress response,inflammation response,regulation of nitric oxide synthesis,regulation of insulin secretion Process;KEGG pathway is mainly enriched in 20 signal pathways including TNF signal pathway,PI3K-Akt signal pathway,apoptotic signal pathway,and estrogen signal pathway.2.Effect of PM2.5 on Atherosclerotic Plaque Stability in ApoE-/-Mice and the Mechanism of SM（1）Effect of PM2.5 on lipid metabolism of ApoE-/-mice and the efficacy of SM:Compared with the normal control group,the serum levels of TC,TG,and LDL in AS model group were significantly increased（P<0.01,P<0.05,P<0.01）,and HDL levels were significantly reduced（P<0.01）.Compared with the AS model group,the levels of TC and LDL in the serum of mice were significantly increased（P<0.05,P<0.01）,the HDL level was significantly decreased（P<0.01）,and the TG level was increased after PM2.5 tracheal instillation.But there was no statistical difference.Compared with the compound model group,the serum TC levels of mice in each dose group of Shengmai San were significantly reduced（P<0.01,P<0.01,P<0.01）,and the TG levels were significantly reduced（P<0.05,P<0.05,P<0.01）,the LDL levels of Shengmai San in the middle and high dose groups were significantly reduced（P<0.01,P<0.01）,and the LDL in the low dose group had a downward trend,but there was no statistical difference.The levels of HDL in Shengmai San’s low,medium and high dose groups were significantly increased（P<0.01,P<0.01,P<0.01）.（2）Effect of PM2.5 on the aortic wall thickness of ApoE-/-mice and the efficacy of SM:Compared with the normal control group,the thickness of the vascular wall of the aortic arch region,the small curvature region and the innominate artery region of the AS model group mice was significantly increased（P<0.01,P<0.05,P<0.01）.After PM2.5 tracheal instillation,compared with the AS model group,the wall thickness of the aortic arch in the large,small and innominate arteries increased more significantly（P<0.05,P<0.05,P<0.05）.Compared with the AS+PM2.5 model group,the thickness of the vascular wall in the aortic arch area,the small curvature area,and the innominate artery area of the mice in each dose group of SM was significantly reduced（P<0.05,P<0.05,P<0.05）.（3）Effect of PM2.5 on aortic plaque in ApoE-/-mice and the efficacy of SM:Compared with the normal control group,the plaque area and lipid content in the AS group were significantly increased（P<0.01,P<0.01）,and the collagen fiber content was significantly decreased（P<0.01）.Compared with the AS group,the atherosclerotic plaque area and lipid content in the AS+PM2.5 model group were significantly increased（P<0.01,P<0.01）,and the collagen fiber content was significantly reduced（P<0.05）.Compared with the AS+PM2.5 model group,the arterial plaque area of mice in each dose group of Shengmai San decreased,and the difference was statistically significant（P<0.05,P<0.05,P<0.05）.The lipid content of the mice in the group was decreased,and the difference was statistically significant（P<0.05,P<0.05）.The collagen fiber content of the mice in each dose group of Shengmai Powder increased significantly（P<0.01,P<0.01,P<0.01）.（4）Effect of PM2.5 on aortic MMP-9 expression in ApoE-/-mice and SM efficacy:In the NC group,MMP-9 was hardly expressed on the inner wall of the aorta,and the expression on the inner wall of the blood vessel in the AS group was significantly higher than that in the NC group（P<0.01）.The expression of MMP-9 in the plaques of the AS+PM2.5 model group was significantly increased compared with the AS group（P<0.01）,the expression of MMP-9 in the plaques of SM low,middle and high dose groups was significantly lower than that of AS+PM2.5 model group（P<0.01,P<0.01,P<0.01）.（5）Effect of PM2.5 on ApoE-/-mice aortic Oxidative Stress and SM efficacy:Compared with the NC group,the ratio of red and blue fluorescence intensity of the AS group and the PM2.5-treated composite model group was significantly increased（P<0.01）,and the ratio of red and blue fluorescence intensity of the AS+PM2.5 model group was significantly higher than that of the AS group（P<0.01）.After treatment of SM low,medium and high dose groups,the red-green fluorescence intensity ratio was significantly reduced compared with the composite model group（P<0.05,P<0.01,P<0.05）.3.Effect of PM2.5 in the Myocardial Ischemic Injury of ApoE-/-mice and the Mechanism of SM（1）Effect of PM2.5 on ECG changes and SM efficacy in ApoE-/-mice after Myocardial Ischemia:Compared with the sham operation group,the ST segment of the mice in the MI group was significantly increased（P<0.01）,and a large deformed QRS fusion wave appeared.Compared with the MI group,the ST segment elevation in the MI+PM2.5 group was more significant（P<0.01）.The Shengmai medium-dose group can reduce the degree of ST segment elevation in rats（P<0.01）.（2）Effect of PM2.5 on myocardial infarction area and myocardial ultrastructure in ApoE-/-mice after Myocardial Ischemia and SM efficacy:Compared with the sham operation group,the area of myocardial infarction in the MI group was significantly increased（P<0.01）.Compared with the MI group,the MI+PM2.5compound model group increased the area of myocardial infarction,and the difference was statistically significant（P<0.05）.Compared with the MI+PM2.5 composite model group,the proportions of myocardial infarction in the low,middle and high dose groups were significantly smaller than those in the composite model group（P<0.05,P<0.01,P<0.05）.Compared with the sham group,the cardiomyocytes in the MI model group had worsened edema and the myofibrils were arranged disorderly.Compared with the MI group,the myocardial fibers in the MI+PM2.5 composite model group showed coagulative necrosis and the myofibrils were more disordered.Mitochondrial structure was more severely damaged,membrane damage,matrix dissociation,increased number of autophagy,and lipid droplets were present.Compared with the MI+PM2.5 composite model group,the myocardial cells in the low-medium-high-dose groups showed only moderate edema,and the Z line was only dissolved in a small area;and the local structure of the H-band remained,and the myofibrils were only dissolved in a small area.It was moderately swollen.Autophagy is present in small amounts.The myocardial ultrastructural lesions in the various dose groups of SM were alleviated to a different extent than the compound model group.（3）Effect of PM2.5 on ventricular structure and function changes of ApoE-/-mice after Myocardial Ischemia and SM efficacy:Compared with the Sham group,the IVS,d,IVS,s,and LVPWs in the MI group were significantly reduced（P<0.01,P<0.01,P<0.01）,and the LVID,d and LVID,s were significantly increased（P<0.05,P<0.01）There was a downward trend,but there was no statistical difference.Compared with the MI group,the left ventricle IVS,d and IVS,s in the MI+PM2.5 group were significantly reduced（P<0.01,P<0.05）,and the LVID,d and LVID,s were significantly increased（P<0.01,P<0.05）.Compared with the MI+PM2.5composite model group,the Shengmaiyin low,medium,and high dose groups had significantly increased IVS,d（P<0.01,P<0.01,P<0.01）,and IVS,s significantly increased（P<0.05,P<0.01）.,P<0.01),LVPWs increased significantly（P<0.05,P<0.01,P<0.01）,LVID,d decreased significantly（P<0.01,P<0.01,P<0.01）,and LVID,s decreased significantly（P<0.05,P<0.01,P<0.01）.Compared with the sham operation group,the LVEF,LVFS,CO,and SV of the mice in the MI group were significantly reduced（P<0.01,P<0.01,P<0.01,P<0.01）.Compared with the MI group,the left ventricle LVEF,LVFS,CO,and SV in the MI+PM2.5 group were significantly reduced（P<0.01,P<0.01,P<0.01,P<0.01）.Compared with the MI+PM2.5 group,the LVEF,LVFS,CO,and SV of Shengmaiyin in each dose group were significantly higher than those in the MI+PM2.5 group（P<0.01,P<0.01,P<0.01,P<0.01）.（4）Effect of PM2.5 on aorta and coronary blood flow after myocardial ischemia in ApoE-/-mice and SM efficacy:Compared with the Sham group,the AV Peak Vel and Coronary Peak Vel of the mice in the MI group were significantly reduced（P<0.01,P<0.01）.Compared with the MI group,the peak velocity of the aortic outflow tract in the MI+PM2.5 group showed a downward trend,but there was no significant difference,and the Coronary Peak Vel value was significantly reduced（P<0.01）.Compared with the MI+PM2.5 composite model group,the AV Peak Vel of the Shengmaiyin medium-dose group was significantly higher than the composite model group（P<0.01）.Although there was an upward trend in the other administration groups,there was no significant statistical significance.Coronary Peak Vel in the low,medium,and high dose groups of Shengmai San increased significantly（P<0.01,P<0.01,P<0.01）.（5）Effect of PM2.5 on myocardial tissue oxidative stress after ApoE-/-mice Ischemia and SM efficacy:Compared with the Sham group,the red-blue fluorescence intensity ratio of the MI group increased significantly（P<0.01）,and the AS+MI composite model group significantly increased the red-blue fluorescence intensity ratio of the MI model group（P<0.01）.After treatment of SM low,medium and high dose groups,the red-green fluorescence intensity ratios were significantly reduced compared with the AS+MI model group（P<0.05,P<0.01,P<0.01）.（6）Effect of PM2.5 on myocardial tissue MMP-9,Nrf2,NQO1 expression and SM efficacy in ApoE-/-mice:MMP-9,Nrf2,and NQO1 were expressed only in a small amount in myocardial tissues of the Sham group.Compared with the Sham group,the expressions of MMP-9,Nrf2,and NQO1 were significantly increased in the MI group（P<0.01,P<0.01,P<0.01）.In the MI+PM2.5 group,the expression of MMP-9 in myocardial tissue increased compared with that in the MI model group alone（P<0.05）,and the expressions of Nrf2 and NQO1 did not increase significantly.The expression of MMP-9 in myocardial tissue of SM low,middle and high dose groups was significantly lower than that of the composite model group（P<0.01,P<0.01,P<0.01）.The compound model group increased significantly（P<0.01,P<0.01）,and the low-dose SM group had an increasing trend,but there was no statistical difference.The expression of NQO1 in myocardial tissue of the low-,medium-,and high-dose SM groups was significantly higher than that of the composite model group Increase（P<0.01,P<0.01,P<0.01）.Conclusion:Through the above work,this study confirms that fine particles have the effects of increasing atherosclerotic plaque area,changing plaque properties,increasing plaque vulnerability,increasing myocardial infarction area,reducing myocardial diastolic function,and aggravating myocardial ischemic damage.PM2.5 can increase the body’s oxidative stress response caused by external stimuli by increasing the ROS content of the cardiovascular system,which causes the balance move toward to the oxidation direction and cause nuclear translocation of Nrf2,which in turn affects the expression of the downstream phase II detoxifying enzyme NQO1,making The body’s antioxidant defense capacity is reduced,which in turn causes damage to the cardiovascular system.The traditional Chinese medicine and classical formula Shengmaiyin can reduce the thickness of the inner wall of blood vessels,reduce the increase of AS plaque area in mice under PM2.5,reduce plaque vulnerability,improve myocardial ultrastructure,increase cardiac diastolic function,and mediate through Nrf2 pathway The antioxidant defense pathway has a good protective effect on cardiovascular damage caused by PM2.5. |
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