| BackgroundWith the improvement of people’s living standards and the accelerated pace of life,ischemic heart disease has become the main cause of the increase in cardiova-scular mortality in China,and it is also a major chronic non-communicable public health problem in China.Percutaneous coronary intervention(PCI)is the main measure for the treatment of acute myocardial infarction in coronary heart disease.It can directly act on the infarct site,so that the crimed coronary artery can be recanalized and myocardial blood supply could be improved.However,with the deepening of the understanding of the pathogenesis of ischemic heart disease,Chinese and foreign scholars have found that in addition to the traditional chronic obstructive coronary disease(CAD),coronary microvascular dysfunction is also important for myocardial ischemia.Cardiac reperfusion of the epicardial large vessels do not mean the recovery of reperfusion at myocardial level.In some patients,even if the infarct related arteries(IRA)were opened,the injured myocardium did not receive effective blood perfusion.This phenomenon is called no reflow(NR).In fact,the incidence of no-reflow after PCI is between 5% and 25% per year in our country.Once there is no reflow,often accompanied by extensive and severe myocardial damage,patients may have left ventricular dilatation,decreased cardiac function and malignant arrhythmia,and ultimately increase the risk of cardiovascular adverse events such as sudden death and congestive heart failure.It can be seen that early detection and diagnosis of microcirculatory disorders after PCI is necessary.A large number of epidemiological and clinical studies have found many risk factors for coronary atherosclerosis,but traditional risk factors can only partially explain the occurrence of coronary heart disease.Some studies have found that some patients with coronary heart disease do not have traditional risk factors.It is closely related to the increase of plasma homocysteine(Hcy)level.The high homocysteine level was positively correlated with the mortality rate of coronary heart disease.Forevery 5 umol/L increase in Hcy,the mortality rate of coronary heart disease increased by 1.52 times.Why are coronary heart disease patients with hyperhomocysteinemia,whether it is vascular lesions(multiple vessel disease),or vascular stenosis,even in-stent restenosis,and mortality after myocardial infarction are higher than the patients with normal homocystine levels.The mechanism has not been studied in depth.Whether Hcy aggravates the coronary microcirculation disorder and causes a poor prognosis has not been reported.Nicorandil acts as an ATP-sensitive potassium channel opener(KATP),which can open the vascular smooth muscle on the ATP-sensitive potassium channel opener,effectively expanding the tiny coronary artery and increasing oxygen supply in ischemic area;can also simulate ischemic preconditioning by opening the KATP channel on the mitochondrial membrane of the myocardium to reduce myocardial damage.Therefore,it is commonly used clinically to treat patients with angina pectoris.The meta-analysis also confirmed that the application of nicorandil during the PCI period significantly reduced slow/no-reflow,reduced the incidence of heart failure after myocardial infarction,improved cardiac function and long-term prognosis.Therefore,whether nicorandil can reverse the severity of coronary heart disease in patients with coronary heart disease with hyperhomocysteinemia(HHcy),improve coronary microcirculation dysfunction.To make a breakthrough in the treatment of coronary heart disease,especially those with HHcy coronary heart disease.The purpose of this study is to: 1)use HHcy+ myocardial infarction model to explore whether Hcy can damage coronary endothelial cells,leading to coronary microcirculation disturbance,lack of adequate blood supply to myocardial cells,and aggravate coronary heart disease lesions and poor prognosis;2)whether Nicorandil can reverse the microcirculatory disorder associated with HHcy coronary heart disease;3)To explore whether nicorandil participates in the mechanism of HHcy-induced coronary microcirculation disorder by NO production and inhibiting inflammation,so as to find a targeted therapy to improve the severe lesions and adverse prognosis of HHcy-induced coronary heart disease.Part Ⅰ:Hyperhomocysteinemia?aggravates coronarymicrocirculation damage in mice after myocardial infarction Purpose:To study the damage of hyperhomocysteinemia?on coronary microcirculation after myocardial infarction in mice.Method:1.After 7 weeks of adaptive feeding of C57BL/6 male mice,they were randomly divided into three groups: control group: normal full-price feed and drinking double distilled water;high homocysteine group: Containing(1.8g/L)homocysteine double distilled water feeding and common feed;folic acid group: high homocysteine group+ folic acid group,folic acid suspension,dosage is 0.071ug/g/day,intragastric administration,feeding time for 8 weeks;2.The systolic/diastolic blood pressure of the mice was detected by non-invasive rat tail artery blood pressure meter;the serum Hcy level was detected by Hcy kit;3.Ligation of the left coronary artery in mice to establish an acute myocardial infarction model.TTC staining and electrocardiogram were used to evaluate the success of modelling a myocardial infarction model.4.The survival curves of mice were analyzed by SPSS19.0 to compare the relationship between the two groups.5.Small animal ultrasound detection of cardiac function in mice after myocardial infarction;All mice were sacrificed after 4 weeks,and angiogenesis of ischemic myocardial tissue was observed under a microscope by immunofluorescence staining.6.Tomato lectin staining to assess the number of microcirculation changes after myocardial infarction.Result:1.The serum Hcy concentration in mice fed Hcy for 8 weeks was significantly higher than that in the normal diet group(P<0.05),while the Hcy level in the folic acid group was significantly lower.At the same time,the systolic blood pressure and diastolic blood pressure of the HHcy group were higher than those of the normal group,but there was no statistical difference.2.Establish a mouse model of myocardial infarction,ligating the anterior descending branch vessels,the myocardium is changde from red to pale with the naked eye and the dynamic ST changes were seen on the electrocardiogram,and the gray-white myocardial infarction area was seen by TTC staining,which proved that the mouse myocardial infarction model was established successfully.3.The mice were divided into: sham operation group(normal diet group),myocardial infarction group(normal diet + myocardial infarction group),HHcy group(HHcy myocardial infarction group),folic acid group(HHcy myocardial infarction +folic acid group).Compared with the normal group,the survival rate of the mice in the myocardial infarction group was significantly lower than that in the normal group(P<0.01),while the HHcy group was lower than the normal diet group(P<0.05).Compared with the folic acid group,the survival rate of mice has increased.4.The effect of HHcy on cardiac function in mice after myocardial infarction,left ventricular ejection fractions(LVEF)and short-axis shortening rate(FS)were lower than non-HHcy myocardial infarction group(P<0.05),the left ventricular end-diastolic diameter(LVIDd)and the left ventricular end-systolic dimension(LVIDs)were higher than those in the non-Hcy myocardial infarction group(P<0.05).After the folic acid supplementation treatment,the Hcy level was decreased,LVEF,FS increased,and LVIDd,LVIDs decreased.5.The expression of CD31 in myocardial infarction group was significantly lower than that in sham operation group.The expression of CD31 was significantly decreased in HHcy myocardial infarction mice(P<0.05),and the expression of CD31 in the folic acid group was increased(P<0.05).6.The intensity of tomato lectin antibody in myocardial infarction group was significantly weaker than that in normal group.The decrease of HHcy group was more than that in myocardial infarction group(P<0.05),while the fluorescence intensity of folic acid group was enhanced(P<0.05).Conclusion:1.With HHcy can affect the angiogenesis and microcirculation function of mice after myocardial infarction,thereby affecting cardiac function and aggravating the mortality of mice after myocardial infarction.2.Folic acid supplementation can reduce Hcy,partially reverse the above changes,protect the microcirculatory disorder and angiogenesis function of mice after myocardial infarction,and promote the improvement of heart function and survival rate in mice.Part Ⅱ Nicorandil improves microcirculatory disturbance aftermyocardial infarction in HHcy mice ObjectiveA mice model of myocardial infarction was established to evaluate whether nicorandil preconditioning can improve microcirculatory dysfunction in mice with HHcy myocardial infarction.Method:The mice were randomly divided into 7 groups: 1)sham operation group(Sham group): no other treatment with normal feeding;2)regular diet + myocardial infarction group(MI group): normal feeding + mouse myocardial infarction group;3)HHcy +MI group: HHcy diet + myocardial infarction group;4)Nicorandil group 1(HHcy+MI group + low-dose nicorandil group,tail vein injection of nicorandil5mg/kg);5)Nicorandil 2 group(HHcy+MI group + medium dose nicorandil group,venalil injection 15 mg/kg);6)nicorandil group 3(HHcy+MI group + high dose nicorandil group,nicorandil was injected into the tail vein(45 mg/kg);7)in the Vehicle group(HHcy+MI+PBS control group,the same amount of PBS was injected through the tail vein).2.Pretreatment of nicorandil on the survival rate of patients with myocardial infarction and changes in cardiac color ultrasonography;3.HE staining of small blood vessels after myocardial infarction,changes of myocardial ischemic tissue in each group of HBFP,4.The angiogenesis of ischemic myocardial tissue was observed undermicroscope by CD31 immunofluorescence staining.5.Tomato lectin evaluates microcirculation function in mice after myocardial infarction Result:1.As the concentration of nicorandil increased,the survival rate of mice in the HHcy+MI group improved(P<0.05).2.After HHcy myocardial infarction,the heart function of the mice decreased,LVEF and LVFS group decreased significantly,and LVIDd and LVIDs were enlarged compared with the normal group.The pretreatment of nicorandil could improve the values of LVEF and LVFS after myocardial infarction.At the same time,the LVIDd and LVIDs in HHcy myocardial infarction groups were smaller(P<0.05).3.HE staining results showed that normal myocardial tissue stained myocardium with clear horizontal stripes,intact cell membrane,no inflammatory cell infiltration;In the MI group,myocardial tissue of myocardial infarction group showed red blood cell exudation and inflammatory cell infiltration;HHcy myocardial infarction group mice in addition to inflammatory cell infiltration,but also seen a large number of fibrosis tissue proliferation;In the nicorandil pretreatment group,myocardial tissue pathological changes were obvious,muscle fiber and interstitial edema reduced,inflammatory cell infiltration decreased..4.The results of HBFP staining in ischemic myocardium showed that the ischemic area of HHcy+MI group was higher than that of MI group(P<0.05),while nicorandil could reduce the ischemic area of HHcy+MI group,but the statistical difference was only seen in Nic2 group and Nic3 group(P<0.05).5.The experimental results of tomato lectin were realistic.Compared with MI group,the microcirculation injury of HHcy+MI group was more serious(P<0.05).The nicorandil 2 and 3 groups could improve the microcirculation function of mice after myocardial infarction.The fluorescence intensity after myocardial infarction was significantly higher than that of myocardial infarction group(P<0.05).6.The CD31 staining of microvascular endothelial cells in mice with myocardial ischemia and the fluorescence intensity of myocardial tissue were observed under microscope.The counts of HHcy group were significantly lower than those of othergroups,while the concentrations of nicorandil 2 and 3 increased.The ability of angiogenesis was enhanced(P<0.05).Conclusion:1.Nicorandil can improve cardiac function and 28-day survival rate in HHcy myocardial infarction mice.2.Nicorandil pretreatment can effectively improve the vascular injury of myocardial tissue in mice after myocardial infarction and reduce the area of myocardial ischemia.3.Nicorandil improves the angiogenic ability of myocardium after myocardial infarction and improves microcirculation function.Part Ⅲ The protective mechanism of nicorandil on microcirculation disturbance after myocardial infarction in HHcy mice Objective:In this study,the in vivo model of HHcy mice with myocardial infarction and the in vitro model of HHcy/ischemia-hypoxia injury of human coronary artery endothelial cells were used to explore the mechanism of microcirculation disturbance in nicordil HHHcy mice after myocardial infarction.The PI3K/Akt signal transduction pathway was observed to determine whether nicordil regulated NO and inhibited inflammation,and to play a protective role in microcirculation injury.Method:In vivo model,animal part1.Mice were randomly divided into 5 groups: 1)sham-operated group(Sham group): normal feeding without other treatment;2)HHcy + MI group: HHcy diet feeding + myocardial infarction group;3)nicorandil group(HHcy + MI group +medium dose nicorandil group,15 mg/kg by tail vein injection);4)LY294002 group(0.3 mg/kg PI3 K specific inhibitor LY294002 was slowly injected through tail vein before ligation).02,the same as Nicorandil group;5)L-NAME group(before ligation,0.3 mg/kg of NO synthase inhibitor L-NAME was slowly injected through tail vein,the same as Nicorandil group).2.Changes of survival rate and cardiac color Doppler echocardiography in mice after myocardial infarction in different treatment groups;3.The angiogenesis of ischemic myocardium was observed under microscope by CD31 immunofluorescence staining.4.Tomato agglutinin assesses microcirculation in mice after myocardial infarction5.The contents of IL-1β、IL-6,NO、ET-1 in heart homogenate were determined by enzyme-linked immunosorbent assay(ELISA).6.The expression of PI3K/AKt/eNOS in ischemic myocardium was detected by Western Blot.In vitro model,Cell part:1.Establishment of HHcy ischemia/hypoxia model: different Hcy concentra-tion and hypoxia time.The 24-hour Hcy concentration was 0.5mmol/L,1.0mmol/L,1.5mmol/L and 2.0mmol/L,and the hypoxia time was 3h,6h and 9h,respectively.2.The effects of different concentrations of Hcy and hypoxia time on human coronary artery endothelial cells growth were detected by MMT.Lactate dehydrogenase(LDH)detection kit was used to detect the LDH leakage rate of endothelial cells in each group.3..Western-blotting was used to detect the expression of PI3K/Akt/eNOS protein in each group.4.The protective effects of different concentrations of nicorandil on HHcy ischemia/hypoxia model.The experimental groups were as follows: 1)control group,2)HHcy+ Hypoxia6 h group,3)Nic 0 group(Hcy+Hypoxia 6h+PBS group),4)Nic 50 u M(HHcy+Hypoxia 6h+Nicorandil 50 u Mol/L),5)Nic100u M(HHcy+Hypoxia 6h+Nicorandil 100 u Mol/L),6)Nic125u M(HHcy+Hypoxia 6h+Nicorandil 125 u Mol/L).5.The expression of PI3K/Akt/eNOS protein in each group was detected byWestern-blotting,and the NO level was measured by extracting cell supernatant.6.The protective effect of nicorandil was changed after pretreatment with PI3 K inhibitor Ly294002 and eNOS inhibitor L-NAME.The changes of P-PI3K/PI3 K,P-Akt/Akt and P-eNOS/eNOS were examined by Western-blotting.7.Scratch test was used to observe the migration function of nicorandil on endothelial cells,and tubuloplasty was used to observe the effect of nicorandil on angiogenesis.8.The expression of m RNA IL-1β and IL-6 was detected by q RT-PCR.Result:1.After nicorandil treatment,the survival rate of mice with HHcy myocardial infarction increased(P <0.05),but under the action of PI3 K inhibitor and eNOS inhibitor,the protective effect of nicorandil on the survival rate of mice with HHcy myocardial infarction disappeared(P < 0.05).2.Compared with the normal group,the mice in HHcy group showed a decrease in LVEF and LVFS,and the LVIDd and LVIDs were enlarged.The pretreatment with nicorandil could increase the LVEF and LVFS values of the mice after myocardial infarction,and decrease the LVIDd and LVIDs(P < 0.05).However,under the action of PI3 K inhibitor and eNOS inhibitor,LVEF,LVFS,LVIDd and LVIDs did not improve significantly in HHcy group(P > 0.05).3.CD31 fluorescence staining of myocardial tissue showed that the number of HHcy myocardial infarction group was significantly less than that of other groups,while the fluorescence intensity of nicorandil treatment group was increased(P <0.05),but in LY294002 group and L-NAME group,the fluorescence intensity was not increased compared with that of Hcy myocardial infarction group.4.The results of tomato agglutinin experiment showed that nicorandil group could improve the microcirculation function of mice after myocardial infarction,and the fluorescence intensity after myocardial infarction was significantly higher than that of myocardial infarction group(P <0.05),but there was no significant difference between LY294002 group,L-NAME group and Hcy myocardial infarction group.5.Compared with Sham group,the levels of IL-1β and IL-6 in HHcy myocardial infarction group increased significantly(P <0.05),while nicorandil could decrease thelevels of IL-1β and IL-6 in plasma of mice.6.After HHcy myocardial infarction,NO and ET-1 levels in plasma of mice decreased,while nicorandil could increase NO and decrease ET-1 levels(P <0.05).However,in LY294002 and L-NAME groups,the expression of phosphorylated e NOs,NO and ET-1 levels were not significantly different from those in HHcy myocardial infarction group(P > 0.05).7.Compared with Sham group,phosphorylated PI3 K,Akt,eNOS decreased in myocardial ischemic tissue after HHcy myocardial infarction(P<0.05);P-PI3K/PI3 K,P-Akt/Akt,P-eNOS/eNOS ratio increased after nicorandil treatment(P <0.05);but in LY294002 group,P-PI3K/PI3 K,P-Akt/Akt and P-eNOS had no difference between HHcy myocardil infarction group;in L-NAME group,the P-eNOS/eNOS decreased with the increase of P-Akt/Akt and P-PI3k/PI3k(P<0.05).8.In vitro experiments,compared with the normal group,the cell viability of Hcy-induced HCAECs gradually decreased with the increase of Hcy concentration,and the ratio of p-eNOS/eNOS in protein level also decreased(P<0.05).Moreover,with the increase of hypoxia time,the cell viability also showed a downward trend(P<0.05).After nicorandil treatment,endothelial cell viability increased and lactate dehydrogenase activity decreased(P<0.05).9.Compared with the normal group,the NO level in the model group decreased significantly,the ratio of phosphorylated PI3 K,Akt and eNOS of endothelial cell also decreased(P<0.05),while the levels of IL-1β and IL-6 increased significantly.Nicorandil increased the ratio of P-PI3K/PI3 K,P-Akt/Akt,P-eNOS/eNOS(P<0.05),increased the level of NO and decreased the levels of IL-1β and IL-6(P<0.05).10.When nicorandil was combined with PI3 K inhibitors,the regulation of Akt and eNOS in cells was significantly reduced(P<0.05),while when nicorandil was combined with eNOS inhibitors,the regulation of PI3 K and Akt remained,but its regulation of eNOS was weakened.11.Nicorandil can improve cell migration and angiogenesis in model group(P<0.05).However,when combined with PI3 K inhibitor and eNOS inhibitor,the change of endothelial function disappeared.Conclusion:1.Nicorandil may improve the microcirculation function of HHcy in mice after myocardial infarction by increasing NO level and reducing inflammation.2.Nicorandil protects against ischemia and hypoxia by activating PI3K/Akt/eNOS signaling pathway,thereby improving endothelial cell function. |
PDF Full Text Request