| BackgroundCoronary artery disease (CAD, also called ischemic heart disease, IHD),of which mortality rate ranks only after stroke, contributes to about 90% of deaths in cardiovascular diseases with stroke together. In results, the prevention and control of cardiovascular diseases is a big problem and challenge to the National Public Health Service. Acute coronary syndrome(ACS)is a spectrum of conditions compatible with acute myocardial ischemia and/or infarction due to an abrupt reduction in coronary blood flow. ACS is the initial presentation of coronary artery disease. ACS is divided into non-ST segment elevated myocardial infarction(NSTEMI), ST segment elevated myocardial infarction(STEMI) and unstable angina (UA).In the ACS patients, an important manifestation on electrocardiogram (ECG) is that ST-segment (ST elevation) or new left bundle-branch block, which indicates immediate coronary angiography to determine if there is an indication for reperfusion therapy to open a likely completely occluded coronary artery. Percutaneous coronary intervention(PCI)is an effective and important treatment for ACS. More and more ACS patients were able to get PCI therapy. However, after PCI, some patients did not get the expected fully reperfusion thus inability to reperfuse regions of the myocardium despite removal of a large epicardial coronary artery occlusion due primarily to microvascular occlusion. This phenomenon in the heart is termed as no-reflow. And in angiographic, the no-reflow phenomenon substantial coronary antegrade flow reduction (less than Thrombolysis in Myocardial Infarction [TIMI]flow grade 3) without mechanical obstruction.As a therapeutic strategy for no-reflow, glycoprotein Ⅱb/Ⅲa antagonist tirofiban has arouse a lot of attention. Tirofiban as known, plays an important role in antiplatelet and anti-coagulation, is pervasively used in patients undergoing PCI before PCI. And it is reported that tirofiban could improve no-reflow by increasing micovascular flow, but the mechanisms remains unclear. Recent studies imply that tirofiban could reverse endothelial dysfunction and restoration of impaired nitric oxide activity. Nitric oxide has already been investigated a lot as a classic vascular relaxing factor, which is produced by three synthases: eNOS, nNOS and iNOS.PI3Ks are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phophatidylinositol(Ptdlns). There is a close relationship between PI3K and Akt, thus forming the PI3K/Akt signaling pathway. It has been demonstrated that PI3K/Akt plays a pivotal role in the regulation of eNOS phosphorylation. Whether PI3K/Akt plays a key role in the treatment of tirofiban on no-reflow remains unknown.As the receptor of NO, soluble guanylyl cyclase(sGC)can be activated by NO, and then catalysed cGTP to cGMP. cGMP is an important second messenger, which transduces intercellular signals into cells. Moreover cGMP regulates the coronary vasorelaxation via different ion channels, especially big-conductance calcium-activated potassium channels(BKCa), small-conductance calcium-activated potassium channels(SKca) and ATP- sensitive potassium channels.Based above discoveries, we supposed that tirofiban could regulate the function of arterial endothelial NOS, inducing the release of NO to attenuate no-reflow. The present study proved that tirofiban induces vasorelaxation of the coronary artery via an endothelium-dependent NO-cGMP signaling by activating the PI3K/Akt/eNOS pathway.Part I: The effect of tirofiban on isolated rat coronary arteryObjectives1. To observe the effect of tirofiban on isolated rat coronary artery.2. To study the association between the vasorelaxation induced by tirofiban and the interity of endothelium.Methoeds1. Isolated the Wistar rats’ coronary artery, normalize the isolated artery.2. Pretreat the isolated coronary artery with 5-HT(10-6 M) then add tirofiban accumulatively, record the effect of tirofiban on the artery.3. Remove the endothelium by gently rubbing the luminal surface of the artery with a human hair, and then add tirofiban accumulatively after pretreated with 5-HT. Record the vascular tone change.Results1. Tirofiban had a vasorelaxation on isolated coronary artery preconstricted with 5-HT in a concentration(10-7-0-5 M)-dependent manner,and reached the maximal relaxation 51.5±8.9% at the concentration of 10-5 M.2. After the endothelium removal, the maxiamal vasorelation induced by tirofiban decreased to 12.3±2.6% at the concentration 10-5 M.Part Ⅱ: Tirofiban induced the vasorelaxation of coronary artery by increased NO productionObjectives1. To investigate the effect of tirofiban on the production and time of HUVECs2. To study the effect of tirofiban on the expression of eNOS in HUVECs3. To observe the effects of different NOS inhibitors on the vasorelaxation of tirofiban on coronary artery and to study the effect of NOS types on the vasorelaxation of tirofiban on coronary artery.Methods1. Add tirofiban(10-7-10-5 M) to the HUVECs and collect the medium after culturing for different time(5, 10, 15, 30, 45 min). Detect the NO production between different groups by using an ELISA kit.2. Collect HUVECs treated by different periods of tirofiban and extract protein. Use western blotting to detect the expression of eNOS and phospho-eNOS..3. Add different NOS inhibitors: L-NAME(10-4 M) for eNOS inhibition, SMT(10-5 M)for iNOS inhibition and L-NMMA(10-5 M) for NOS inhibition. Then add tirofiban and record the vascular tone change.Results1. The effect of tirofiban on HUVEVs NO production is related with its concentration and treating time. And at the concentration 10-5 M) treated for 10 min significantly increased the NO production.2. Isolated coronary artery pretreated with different NOS inhibitors(L-NMMA 10-5 M,L-NAME 10-4 M and SMT 10-5 M) indicated that the NOS inhibitor L-NMMA and the eNOS inhibitor L-NAME attenuated the vasorelaxation induced by tirofiban.3. The effect of tirofiban on the expression of HUVECs phospho-eNOS is related with the treating time, and treated 10 min with tirofiban(10-5 M) significantly increased the expression of phospho-eNOS.Part III: Tirofiban induces isolated coronary artery vasorelaxation by activating the PI3K/Akt pathwayObjective1. To ascertain the effect of tirofiban on the expression of Akt and phospho-Akt in HUVEC.2. To study the effects of tirofiban on the expression of eNOS and phospho-eNOS by using the PI3K/Akt pathway inhibitor.Methods1. Collect HUVECs treated by different periods of tirofiban and extract protein. Use western blotting to detect the expression of Akt and phospho-Akt.2. Pretreat HUVECs with PI3K inhibitors wortmannin(10-7) and LY294002(5×10-5 M).Then decreased the tirofiban induced phospho-eNOS and phospho-Akt expression as well as the NO production. Then collect HUVECs treated by tirofiban and extract protein. Use western blotting to detect the expression of eNOS and phospho-eNOS.3. Pretreat HUVECs with Akt inhibitor SH-5(10-5 M). Then collect HUVECs treated by tirofiban and extract protein. Use western blotting to detect the expression of eNOS and phospho-eNOS.Results1. The effect of tirofiban on the expression of HUVECs phospho-Akt is related with the treating time, and treated 10 min with tirofiban(10-5 M) significantly increased the expression of phospho-Akt.2. Pretreat HUVECs with wortmannin(10-7 M) and LY294002(5×10-5 M) decreased the tirofiban induced phospho-eNOS expression as well as the NO production.3. Pretreat HUVECs with SH-5(10-5 M) decreased the tirofiban induced phospho-eNOS expression.Part IV: The effect of tirofiban on the potassium channels in isolated rat coronary arteryObjective1. To study the role of cGMP in the coronary vasorelaxation induced by tirofiban with or without endothelium.2. To observe the effect of tirofiban on isolated rat coronary artery after using the sGC inhibitor ODQ.3. To study the role of different potassium channels in the effect of tirofiban on the isolated rat coronary artery.Methods1. Pretreat the coronary artery with 5-HT(10-6 M) and ODQ(10-5 M) with or without endothelium, then record the vascular tone change.2. Extract the protein from the tirofiban treated isolated coronary artery with or without endothelium, then using a cGMP ELISA kit to detect the expression of cGMP.2.Pretreat the isolated coronary artery with BKCa inhibitors iberiotoxin(10-7 M),SKca inhibitor apamin(10-7 M) and KATP inhibitor glibenclamide(10-5 M), then add tirofiban and record the vascular tone change.Results1. Tirofiban increased the cGMP expression and the effect is related to the endothelium integrity.2. ODQ blocked the vasorelaxation of tirofiban on isolated coronary artery.3. The BKCa inhibitor iberiotoxin significantly blocked the vasorelaxation of tirofiban.ConclusionIn summary, our findings suggest that tirofiban relaxes the coronary artery via an endothelium-dependent NO-cGMP signaling as confirmed by the activation of PI3K/Akt/eNOS. The present data also shows that the big-conductance calcium-activated potassium channel is involved in the tirofiban-induced vasorelaxation. |
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