| BackgroundAcute coronary syndrome(ACS),as an important component of coronary heart disease,has become an important cause of all-cause death in humans.Percutaneous coronary intervention(PCI)is currently recognized as an effective treatment for patients with acute coronary syndrome(ACS),which can effectively restore myocardial perfusion,relieve symptoms and improve clinical prognosis.However,despite the continuous maturity and improvement of PCI technology,some patients still fail to benefit from PCI.Instead,there are symptoms such as no reflow and hypoperfusion after PCI,which is myocardial no-reflow(MNR)phenomenon.The specific mechanism of MNR occurrence is unclear.At present,MNR phenomenon is considered to be a manifestation of microcirculation disturbance in myocardial ischemia/reperfusion injury.MNR is a strong predictor of poor long-term prognosis and major cardiovascular adverse events in patients with ACS.How to effectively prevent MNR is an urgent problem for clinical cardiologists.Clinical studies have found that intravascular coronary vasodilators nitroglycerin,nicorandil,nitropuna,verapamil,diltiazem,and so on,may reduce the occurrence of no reflow.Nicorandil has the dual effects of nitrate-like and ATP-sensitive potassium channel(KATP)opener,which can significantly improve coronary microcirculation.In the past decade,many researchers have tried intravenous or intracoronary application of nicorandil to improve coronary microcirculation and myocardial perfusion during PCI and reduce myocardial ischemia-reperfusion injury.Nicorandil before emergency PCI in AMI patients can significantly improve coronary blood flow,reduce the occurrence of ventricular arrhythmias,and improve left ventricular function.Although there is no large-scale,multi-center,double-blind controlled clinical research data,most clinical studies show that compared with nitroglycerin,it can better correct coronary no-reflow,increase myocardial perfusion,and improve cardiac function.In addition to dilating coronary microvasculature,does nicorandil improve PCI-related myocardial perfusion disorders through other mechanisms of action?In order to explore the possible mechanism of Nicorandil to improve myocardial perfusion after PCI,the presence study included patients with acute coronary syndrome who planned to undergo PCI.Nicorandil was given intracoronarily before PCI,compared with the control group.To observe the dynamic changes of the expression of inflammation-related indicators before and after PCI,and use proteomics analysis and screening for possible molecular mechanisms of nicorandil to improve myocardial perfusion.Objectives1.To evaluate the effect of intracoronary administration of nicorandil on the inflammatory response after PCI;2.To scan the potential mechanisms of intracoronary administration of nicorandil in improving myocardial perfusion after PCI by using Proteomic Analysis.MethodsStudy population:(1)Selection criteria:①Patients aged 30-80 years with acute coronary syndrome;②Coronary angiography confirmed moderate to severe coronary stenosis(>75%),and PCI was planned.(2)Exclusion criteria:① Hepatic impairment:the level of ALT or AST is three times or more above the upper limit of normal values;② Renal insufficiency:Serum creatinine>2.5mg/dl(221umol/L);③ Patients with malignant tumors;④ Primary or secondary thrombocytopenia or dysfunction;⑤ Patients with moderate to severe anemia:Hemoglobin<90g/L;⑥ A history of PCI or coronary artery bypass grafting;⑦Acute heart failure;⑧ Complicated coronary artery lesions(LM lesions,bifurcation lesions,Severe calcified lesions)⑨People who are allergic to Nicorandil.Research groups:Before coronary angiography,all ACS patients enrolled were informed of the purposeand protocol of the study.After obtaining consent and signing the informed consent form,basic information was collected and coronary angiography was performed in strict accordance with the operating procedures.According to the results of coronary angiography,the need for PCI was determined.Patients who planned to undergo PCI were included in this study and randomly divided into the nicorandil group and the control group according to the 1:1 principle:(1)Nicorandil group(n=30):2 mg bolus injection of Nicorandil in the coronary artery to be treated before PCI(2)Control group(n=30):before the PCI,an equal volume of normal saline was injected once;Detection indicators1.The expression of inflammatory factors before and after PCIVenous blood samples before PCI,24hrs and 7 days after PCI were obtained from all participants,and serums were collected after centrifugation.The expression levels of a serial of inflammatory factors were detected for each patients.2.Proteomics analysisTo scan the potential mechanisms involved in the protection effects of nicorandil on the coronary microcircution,serums of pre-PCI and 24 hrs after PCI from patients in the nicorandil and control groups(n=3 for each group for each time point)were analyzed by using Proteomics analysis.Statistical analysisMeasurement data are expressed as mean ± standard error.The independent t test was used to compare the relevant indicators at the same time between the two groups;the paired t test was used to compare the relevant indicators at the two time points in the same group;the one-way analysis of variance was used for the multi-sample comparison.Least significant difference(LSD)method,Dunnett T3 method is used when the variance is uneven.Counting data are expressed as percentages and analyzed by chi-square test.When n<40,Fisher’s Exact Test is used.P<0.05 was considered statistically significant.All statistical data were analyzed using SPSS 16.0(SPSS Inc.,Chicago,IL,USA)software package.Results1.Expression level of inflammatory factors before PCIBefore PCI,there was no significant difference between the control and nicorandil groups in the expression levels of pro-inflammatory factors and anti-inflammatory factors(P>0.05);2.Changes of the expression levels of inflammatory factors after PCI(1)For the control group:Compared with pre-PCI,the expression levels of pro-inflammatory factors,including TNF-α,IL-6,IL-1β,VCAM-1 and ICAM-1,in the control group were significantly increased 24 hours after PCI.The expression levels of anti-inflammatory factors,including IL-33,IL-10,and IL-19,were significantly decreased(P<0.05);7 days after PCI,the expression levels of IL-6,VCAM-1,and ICAM-1 remained higher the those of pre-PCI,while the expression levels of anti-inflammatory factors(IL-33,IL-10,IL-19)were still significantly reduced.(2)For the nicorandil group:24 hours after PCI,the expression levels of pro-inflammatory factors in the Nicorandil group had no significant change compared with those before PCI,while the expression level of anti-inflammatory factor IL-10 was significantly higher than before PCI(P<0.05).3.Comparison of expression levels of inflammatory factors between the two groups after PCIAt 24 hours after PCI,the expression levels of proinflammatory factors,invovled TNF-α,IL-6,IL-1β,VCAM-1,ICAM-1,CXCL-2 and MCP-1,in the nicorandil group were significantly lower than those in the control group,while the expression of IL-10 was higher than that in the control group(P<0.05).4.Results of proteomics analysis(1)Compared with before PCI,the expression of 53 protein showed significant changes in the control group after PCI,including 39 protein expressions were significantly up-regulated and 14 protein expressions were significantly down-regulated.Prophylactic application of nicorandil in the coronary artery can be reversed abnormal expression of these proteins.Biological information analysis was performed on the differentially expressed proteins between the two groups.Gene ontology(GO)analysis showed that these proteins were mainly distributed in the extracellular region,that is,mostly secreted proteins;KEGG enrichment analysis showed that these differentially expressed proteins were more concentrated in Inflammation-related signaling pathway.This suggested that the regulation of inflammation-immune response may be an important mechanism of nicorandil to prevents myocardial no-reflow and protect coronary microcirculation.Conclusions1.The expression levels of inflammatory factors is significantly increased after PCI,while the expression level of anti-inflammatory factors is significantly reduced,which indicated that the inflammatory response is enhanced after PCI;2.The application of nicorandil in the coronary artery before PCI significantly inhibited the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors,which suggested that the prophylactic application of nicorandil can regulate the level of inflammatory response after PCI;3.Proteomics analysis found that prophylactic intracoronary application of nicorandil can regulate the expression of inflammation-related pathway proteins after PCI.This suggested that nicorandil may regulate the inflammatory response after PCI,which may be an important mechanism of nicorandil to prevent myocardial reflow and improve coronary microcirculation.Background Clinical studies have shown that the prophylactic application of nicorandil can significantly reduce the occurrence of coronary reflow after percutaneous coronary intervention(PCI).However,the mechanisms of nicorandil improving coronary microcirculation is not yet clear.As shown in Paper I,we found that the application of nicorandil before PCI significantly reduced the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors.The potential mechanisms were further screened by use of proteomics analysis.We found the expression of 53 protein expression changed significantly in the control group after PCI,including 39 proteins up-regulated and 14 proteins down-regulated.Prophylactic application of nicorandil in the coronary artery before PCI can reverse the abnormal expression of these proteins.Biological information analysis of the differentially expressed proteins between the two groups showed that these proteins were mostly secreted proteins and were mostly enriched in inflammation-related signaling pathways.This suggests that the regulation of inflammation-immune response may be an important mechanism by which nicorandil prevents myocardial no-reflow and protects coronary microcirculation.So,what is the specific molecular mechanism of nicorandil to regulate the inflammatory response after ischemia-reperfusion?Myocardial no-reflow(MNR)is a manifestation of microcirculation disorder of myocardial ischemia / reperfiision injury.Coronary microvascular structure or dysfunction,local metabolic changes such as oxygen free radicals,inflammation and microcirculatory mechanical embolism,coronary spasm,etc.are involved.The inflammation-oxidative stress mechanism plays a key role in the development of MNR.During myocardial ischemia,the fimction of the oxygen free radical scavenging system weakens or even disappears.After reperfUsion,reactive oxygen species(ROS)are exploded through multiple channels,exceeding the scavenging ability of the antioxidant defense system,resulting in a large amount of ROS accumulation.ROS can lead to MNR through multiple pathways,such as promoting the expression of adhesion factors and stimulating inflammation.Vascular endothelial cells cover the inner surface of blood vessels,and the integrity and function of endothelium play an extremely important role in the development and outcome of coronary microcirculation.Therefore,interfering with the inflammation-oxidative stress response and protecting the vascular endothelial function may be an important link to prevent and manage the MNR phenomenon during ischemia-reperfusion.The mitochondrial respiratory chain is the main source of ROS,accounting for more than 90% of ROS sources.Normally,the generation of free radicals in the body is strictly regulated by the anti-oxidative stress system and is controlled at a low level.During ischemia-reperfusion,a large amount of ROS is generated through multiple pathways,and the endogenous clearance system is difficult to load,resulting in accumulation of reactive oxygen species in the body,causing oxidative damage.This suggests that mitochondria are at the core of oxidative stress.In recent years,studies have found that there is an ATP-sensitive potassium channel(mitoKATp)on the mitochondrial membrane involved in regulating the generation of oxygen free radicals.Nicorandil has an open effect on cell membrane and mitochondrial ATP-sensitive potassium channels.It acts on myocardial cells mitoKATP,reduces mitochondrial Ca2+ overload,regulates the generation of oxygen free radicals,mitochondrial matrix swelling and respiratory chain optimization,inhibits cell apoptosis and other pathways,reduces ischeraia-reperfusion myocardial injury,and has myocardial protection with drug preconditioning effect.Therefore,we propose the following hypothesis: Nicorandil regulates the oxidative stress-inflammatory response via the opening of mitoKATP after ischemia-reperfusion in the vascular endothelial cells.This may be the important mechanisms of nicorandil in preventing MNR and protecting coronary microcirculation after ischemia-reperfusion.Objectives We sought to elucidate the effect of prophylactic nicorandil on cell function,oxidative stress-inflammatory response after hypoxia / reoxygenation of vascular endothelial cells,and explore its potential molecular mechanism.Methods L Culture human umbilical vein endothelial cells in vitro;2.Using hypoxia / reoxygenation model to simulate ischemia-reperfusion in vitro Inoculate HUVECs cells in a 6-well plate for 24 hours,replace with 950 ml /L N2,50 ml / L C〇2 saturated D-hanks solution,and place in a three-gas incubator(containing 94% N25 5% C〇2 and 1% 〇2,37 °C)is hypoxia,simulated ischemia,set the hypoxia time to 2 hours(2hrs).After 2 hours,remove the culture plate from the three-gas incubator,and put it back into the normal incubator(20% O2,5% CO2,75% N25 37 0 C)5 which is reoxygenation,that is,hypoxia 2hrs / reoxygenation 4hrs.3.Cell grouping:? Normal control group(Control group);② Hypoxia-reoxygenation control group(H/R group): hypoxia 2hrs /reoxygenation 4hrs;?Nicodil group(Nic group): give Nicordil 100|iM incubation for 2hrs before the start of hypoxia,then hypoxia 2hrs / reoxygenation 4hrs;?Nicodil + 5-hydroxyquinic acid group(Nic + 5-HD group): Incubate with5-HD 500|j.M for 2 hours before the start of hypoxia,then add nicorandil 100|j,M to continue incubation for 2hrs,and then proceed Hypoxia 2hrs / reoxygenation 4hrs;?PI3K inhibitor LY294002 + nicoril group(Nic + LY): Pre-treat cells with PI3 K specific inhibitor LY294002(10^M)for 2 hours,then add nicorandil lOOjaM to continue incubation for 2hrs,and then perform hypoxia 2hrs / recover Oxygen for4 hrs,observe the change of corresponding detection index.After the experiment,the cell supernatant and cells were collected separately and stored frozen at-80 0 C for subsequent testing.4.Parameters(1)Detection of endothelial cell viability: using CCK-8 cell viability detection method;(2)Endothelial cell permeability: the size of the endothelial cell monolayer to albumin permeability is measured,and the endothelial cell monolayer to albumin permeability is expressed by the permeability coefficient Pa;(3)Detection of oxidative stress level: collect the cell supernatant of each group of cells;use the thiobarbituric acid method to determine the content of malondialdehyde(MDA)in the cell supernatant,and determine the activity of Superoxide dismutase(SOD)in the supernatant by xanthine oxidase method;(4)Detection of the expression of inflammatory factors including Tumor necrosis factor-a(TNF-a),interleukin-ip(IL-1|3)and IL-6,by using the enzyme-linked immunosorbent assay(ELISA);(5)Molecular detection of PI3 K / Akt signaling pathway: collect cells and extract proteins after the intervention of cells in each group,and detect the expression levels of PI3 K,Akt total protein and phosphorylated protein by western blot.Statistical analysis Data are expressed as mean ± standard error.Single-factor analysis of variance was used for multi-sample comparison.The difference between groups was compared in pairs.When the variances were uniform,the LSD(least significant difference)method was used.When the variances were uneven,the Dunnett T3 method was used.All statistical data were analyzed using SPSS 16.0(SPSS Inc.,Chicago,IL,USA)software package.P<0.05 was considered statistically significant.Results The effect of nicorandil on the survival rate of HUVECs after hypoxia /reoxygenation The cell survival rate of the H/R group was significantly lower than that of the normal control group.Pretreatment with nicorandil can significantly improve the cell survival rate(P <0.05,vs.H/R).The effects of nicorandil on cell survival after hypoxia / reoxygenation was significantly inhibited by the pretreatment with 5-HD?indicating that Nicorandil?s protective effect on HUVECs1 cell viability after hypoxia/ reoxygenation is mainly mediated by the opening of mitoKATP.The effect of nicorandil on the permeability of HUVECs endothelial cells after hypoxia / reoxygenation Compared with the control group,the permeability of endothelial cells to albumin in the H/R group was significantly increased.Pretreatment with different doses of nicorandil(50(aM-200^iM)for 2hrs can significantly reduce HUVECs1 permeability to albumin.The effect of nicorandil on improving cell permeability after H/R can be inhibited by pretreatment with 5-HD,indicating that the protective effect of Nicorandil on the permeability of HUVECs after hypoxia / reoxygenation is mainly mediated by the opening of mitoKATP.The effect of nicorandil on oxidative stress after hypoxia / reoxygenation of HUVECs Compared with the normal control group,the expression level of MDA in the cell supernatant increased and SOD decreased significantly after 2 hours of hypoxia.After reoxygenation,the MDA concentration in the cell supernatant increased more than that during hypoxia,while the SOD level was less than that during hypoxia.This suggested that the oxidative stress response of HUVECs was increased after hypoxia / reoxygenation,and the antioxidant capacity decreased.Pretreatment of HUVECs with different doses of nicorandil(50|iM-200|iM)can significantly reduce the expression level of MDA in the cell supernatant and increase the expression level of SOD.This suggests that Nicorandil can reduce the oxidative stress level of HUVECs after hypoxia / reoxygenation and improve the antioxidant capacity.After pretreatment of cells with mitoKATP specific blocker 5-HD? the effects of nicorandil on reducing MDA expression and increasing SOD expression were significantly inhibited.This suggests that nicorandil reduces the oxidative stress of HUVECs mainly through its open mitoKATP.Effect of nicorandil on the expression of inflammatory factors after hypoxia / reoxygenation of HUVECs Compared with the normal control group,the expression levels of inflammatory factors,including TNF-a,IL-lp and IL-6,in the cell supernatant were significantly increased after 2hrs of hypoxia;the expression of the three inflammatory factors in the cell supernatant 1 hour after reoxygenation.The concentrations were higher than those in the non-reoxygenation group,and the expression level showed a further upward trend within 5 hours as the reoxygenation time increased.This suggests that after hypoxia / reoxygenation,the inflammation of HUVECs is enhanced.Pretreatment of HUVECs with different concentrations of nicorandil(50|iM-200(iM)can significantly reduce the expression of inflammatory factors after hypoxia / reoxygenation of HUVECs.Pretreatment with 50(iM nicorandil can significantly reduce the expression levels of TNF-a and IL-ip,while lOOpM can significantly reduce the expression level of IL-6 in the cell supernatant of HUVECs after hypoxia / reoxygenation.This suggests that Nicorandil significantly inhibits the expression of inflammatory factors after hypoxia / reoxygenation of HUVECs.In the Nic+5-HD group,the expression levels of TNF-a,IL-lp,and IL-6were significantly increased compared with the Nicorandil group,and there was no statistical difference compared with the hypoxia / reoxygenation group.This suggests that Nicorandirs inhibitory effect on the expression of inflammatory factors after hypoxia / reoxygenation of HUVECs is mainly mediated by opening mitoKATP.Effect of Nicorandil on PI3 K / Akt phosphorylation after hypoxia /reoxygenation of HUVECs Compared with the normal control group,the phosphorylation levels of PI3 K and Akt in cells were significantly inhibited after reoxygenation(P <0.05).Pretreatment with nicorandil significantly increased the phosphorylation levels of PI3 K and Akt after H/R? which could be inhibited by 5-HD.This suggests that PI3K/ Akt may be an important way for nicorandil to regulate oxidative stress-inflammatory response in HUVECs after hypoxia / reoxygenation.The role of PI3 K / Akt pathway in the regulation of oxidative stress inflammation after hypoxia/reoxygenation of HUVECs by Nicorandil Compared with the Nic group,the cells pretreated with PI3 K inhibitor LY294002 showed significantly higher MDA levels and lower SOD levels in the cell supernatant.The expression levels of the inflammatory factors including TNF-a,IL-lp and IL-6 were also increased(P<0.05).This suggests that Nicorandil regulates the oxidative stress-inflammatory response of HUVECs after hypoxia /reoxygenation through the PI3 K / Akt pathway.Conclusions1.Hypoxia / reoxygenation can significantly reduce the vitality of HUVECs,and increase cell permeability.Pretreatment with nicorandil can significantly increase the survival rate and cell permeability of HUVECs after hypoxia-reoxygenation;2.The oxidative stress level of HUVECs after hypoxia / reoxygenation is significantly increased,and the antioxidant capacity is reduced.Pretreatment with nicorandil can significantly reduce the oxidative stress level of HUVECs after hypoxia / reoxygenation and improve the antioxidant capacity;3.The expression of inflammatory factors in HUVECs increases significantly after hypoxia / reoxygenation.Nicorandil pretreatment can significantly inhibit the expression of inflammatory factors after hypoxia / reoxygenation in HUVECs;4.Nicorandil regulated the cell fimction and inhibited oxidative stress-inflammation in HUVECs after H/R via the opening mitoKATP and PI3K/Akt phosphorylation. |
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