| BACKGROUND:Coronary revascularization has been well-established as the most effective treatment of limiting the eventual infarct size to coronary artery diseases. However, coronary revascularization can also elicit serious ischemia reperfusion (IR) injury which affects the treatment effect. The research on protective measures to IR injury has always been the focus of clinical study. Recent study demonstrated that activation of the prosurvival kinase signaling cascade phosphatidylinositol3-kinase (PI3K)/Akt at the time of reperfusion confers powerful cardioprotection. It promotes myocardial cell survival and limits the infarct size, and is a new target to prevent IR injury. Ligustrazine (tetramethylpyrazine, TMP), an alkaloid extracted from Ligusricum wallichii Franchat (Apiaceae), has been widely used in traditional Chinese medicine for the treatment of cardiovascular and neurovascular diseases. It has extensive effect, including inhibiting platelet aggregation, relaxing vessels, and relieving stasis. Because of its wide variety of sources, safe utilization and multiple mechanisms, TMP has a perspective application in the treatment of cardiovascular disease. However, the protective mechanism of TMP remains poorly understood. Previous study on the protective effect of TMP focused mainly on its impact on the cardiac function and antioxidant indicators. There have not been a complete and systematic evaluation and an investigation on whether the protective effect of TMP is dose-related. More importantly, the signaling pathway mediating the cardioprotective effect of TMP and the role of endothelial nitric oxide synthase (eNOS) with subsequent nitric oxide (NO) production have not been investigated.OBJECTIVES:Through establishing the rat model of IR in vivo, this study aims to:1. Observe the protective effect of ligustrazine to hearts subject to IR injury (through evaluating myocardial enzyme, infarct size, electron microscopic structure, inflammatory reaction and apoptosis) and whether the protective effect of ligustrazine is dose-related.2. Discuss the protective mechanism of ligustrazine:the role of phosphatidylinositol3-kinase (PI3K)/Akt pathway in IR injury protection, the role of phosphorylation of the downstream target of PI3K7Akt-eNOS and production of NO in the anti-apoptotic and anti-inflammatory effect of TMP.METHODS:Seventy-eight Sprague-Dawley rats, weighing250-280g, were randomly divided into sham, IR, low-, middle-and high-dose of TMP groups (TMP-L,-M,-H group:TMP pre-treatment by intravenous injection5min before ligation,5mg/kg,10mg/kg and30mg/kg, respectively), TMP+wortmannin group (TMP+Wort group:TMP pre-treatment by intravenous injection,10mg/kg,5min before ligation and wortmannin, a PI3K inhibitor, by intravenous injection,15μg/kg,15min before reperfusion), NG-nitro-L-arginine methyl ester group (L-NAME group:L-NAME, a NOS inhibitor, administered at30mg/kg by intravenous injection15min before reperfusion) and TMP+L-NAME group (TMP pre-treatment by intravenous injection,10mg/kg,5min before ligation and L-NAME by intravenous injection,30mg/kg,15min before reperfusion). The rat heart model of IR in vivo was produced by35min of regional ischemia followed by120min of reperfusion. Electron and light microscopic evaluation was conducted and the activities of creatine kinase MB fraction (CK-MB) and lactate dehydrogenase (LDH), infarct size, myeloperoxidase (MPO), interleukin-1β (IL-1β) and NO were measured. Apoptosis of myocardial cell was measured by TUNEL staining and caspase-3activity. The expression of eNOS was detected by reverse transcription polymerase chain reaction. Expression of phosphorylated Akt and eNOS and total eNOS were detected by Western blot. All values are expressed as mean±SD. Statistical analysis was performed with one-way ANOVA followed by Student-Neuman-Keuls t-test.RESULTS:1. After the medium and high dose TMP pretreatment (10and30mg/kg, respectively), the areas of myocardial infarction induced by IR process were significantly reduced [(38.7±4.5)%,(36.3±4.3)%vs. IR(50.4±4.6)%, P<0.05]. This protective effect of TMP was not obvious in the low dose TMP group and TMP+Wort group [(46.9±3.1)%and (49.8±4.7)%, vs. IR,P>0.05, respectively]. Pretreatment with TMP10and30mg/kg markedly decreased CK-MB and LDH in plasma and MDA content in myocardium, increased SOD activity and attenuated ultrastructure lesion.2. TMP treatment caused a marked increase in NO production (0.40±0.04μmol/g vs IR0.30±0.03μmol/g, P<0.05). TMP markedly attenuated myocardial apoptosis, as evidenced by a decrease in the apoptotic index (15.0±2.9%vs IR25.2±3.3%, P<0.05) and reduced caspase-3activity (4.03±1.14nmol/mg vs IR9.54±2.69nmol/mg, P<0.05). Co-treatment with wortmannin or L-NAME completely blocked the TMP-induced NO increase.3. Compared to the IR group, the myocardial tissues of the TMP group showed a decrease in the MPO activity (6.237±0.901U/mg, vs IR8.670±1.260U/mg, P<0.05) and the level of IL-10(0.140±0.021pg/mg, vs IR0.235±0.026pg/mg,,P<0.05). TMP increased eNOS mRNA and protein expression in the myocardium. However, these effects could be significantly reversed by L-NAME which abolished the increase of eNOS mRNA expression brought by TMP.4. TMP induced Akt phosphorylation (1.61±0.18in TMP-M group and1.65±0.18in TMP-H group vs.0.79±0.10in IR control group, P<0.05) and eNOS phosphorylation (1.69±0.41in TMP-L group,1.87±0.33in TMP-M group and3.21±0.62in TMP-H group vs.0.94±0.22in IR control group, P<0.05). However, when wortmannin, a PI3K inhibitor, was given15min before reperfusion, the TMP-induced phosphorylation was ceased:Akt with wortmannin (0.54±0.15, P>0.05vs. IR group) and eNOS with wortmannin (0.53±0.14, P>0.05vs. IR group) and the cardioprotection of TMP was abolished.CONCLUSIONS:1. These findings suggest that TMP can decrease myocardial infarct size, reduce myocardial necrosis, attenuate ultrastructure lesion and oxidative stress induced by myocardial IR injury and have cardioprotective effects by a dose-related manner.2. The cardioprotective effect of TMP is mediated through activating the PI3K/Akt-eNOS pathway.3. Pretreatment of TMP attenuated apoptosis and inflammation of myocardial IR in rats. The cardioprotective mechanism of TMP may relate to its effects of increasing the expression and phosphorylation of eNOS and the level of subsequent NO. |
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