Akt/GSK-3β/eNOS通路在藏红花酸保护离体大鼠心肌缺血再灌注损伤中的作用

The effect of crocetin on Akt/GSK-3β/eNOS signaling pathway in myocardial ischemiareperfusion injury of isolated rat hearts

目的研究藏红花酸(CRO)对离体大鼠心肌缺血再灌注损伤的保护作用,探讨其与丝氨酸/苏氨酸蛋白激酶(Akt)/糖原合成酶激酶(GSK)-3β/一氧化氮合酶(eNOS)信号通路的关系。方法 SD大鼠40只,随机数字表法均分为正常(N)组、缺血再灌注(IR)组及5、10、15 mg/L加药(CRO_1、CRO_2、CRO_3)组,比较再灌注30 min时各组心率(HR),冠脉流量(CF),左心室内压测定(LVDP、LV+dp/dtmax、LV-dp/dtmax)。灌流结束TTC心肌染色评估梗死范围,分光光度法测定肌浆乳酸脱氢酶(LDH)和肌酸激酶(CK-MB);Western blot检测各组心肌组织内Akt、磷酸化的丝氨酸/苏氨酸蛋白激酶(p-Akt)、GSK-3β、磷酸化的糖原合成酶激酶(p-GSK)-3β、eNOS、磷酸化的一氧化氮合酶(p-NOS)。结果 IR组再灌注30 min时HR、CF、LVDP、LV+dp/dtmax、LV-dp/dtmax较N组及加药组降低,心肌梗死面积较加药组增大,LDH、CK-MB表达较N组及加药组增加(均P<0.05),CRO_3组再灌注指标较CRO_1组升高,梗死面积降低,LDH、CK-MB表达减少(P<0.05)。IR组Akt、GSK-3b及eNOS较N组及加药组的表达差异无统计学意义,但p-Akt、p-GSK-3β及p-NOS与N组及加药组降低,且CRO_3组较CRO_1组增加(均P<0.05)。结论藏红花酸能够减轻大鼠心肌缺血再灌注损伤,其作用机制可能与激活Akt/GSK-3β/eNOS通路并影响其磷酸化水平有关。

Objective To study the protective effects of crocetin on myocardial ischemia-reperfusion injury, and their correlation with the signaling pathway of serine/threonine protein kinase （Akt）/glycogen synthase kinase （GSK）-3β/nitric ox？ide synthase （eNOS）. Methods Forty healthy SD rats were divided into normal group （N）, ischemia reperfusion group （IR） and 5, 10 and 15 mg/L of crocetin groups （CRO1, CRO2 and CRO3） by random number table method. The values of heart rate （HR）, coronary flow （CF） and left ventricular pressure measurement （LVDP, LV＋dp/dtmax, LV-dp/dtmax） 30 minutes after reperfusion were compared between five groups. TTC staining was used to detect the infarct volume. Spectrophotometric method was used to determinate the expression of lactate dehydrogenase （LDH） and creatine kinase （CK-MB）. The levels of Akt, the phosphorylation of Akt （p-Akt）, GSK-3β, phosphorylation of GSK-3β（p-GSK-3β）, eNOS and phosphorylation of eNOS （p-NOS） were detected by Western blot assay. Results The HR, CF, LVDP, LV＋dp/dtmax and LV-dp/dtmax were significantly lower 30 min after reperfusion in IR group than those of N group and crocetin groups （P〈0.05）. The myocardial infarction area was bigger in IR group than that of crocetin groups. The expression levels of LDH and CK-MB were signifi？cantly higher in IR group than those of N group and crocetin groups （P〈0.05）. The reperfusion index was higher in CRO3 group than that of CRO1 group. The infarction area, LDH and CK-MB expressions were significantly decreased in CRO3 group than those of CRO1 group （P〈0.05）. There were no significant differences in expressions of Akt, GSK-3βand eNOS between IR group, N group and crocetin groups. But p-Akt, p-GSK-3βand p-NOS were significantly decreased in IR group than those of N group and crocetin groups. The p-Akt, p-GSK-3βand p-NOS were significantly increased in CRO3 group than those of CRO1 group （P〈0.05）.Conclusion Crocetin has protective effects on myocrdial ischemia reperfusion injury in rats, which may be involved in the enhancing the phosphorylation of signalling pathway of Akt/GSK-3β/eNOS.