木犀草素可减轻异丙肾上腺素诱导的心力衰竭大鼠心肌细胞线粒体的损伤

Luteolin attenuates myocardial mitochondrial injury in isoprenaline-induced heart failure rats

摘要:

目的 探讨木犀草素(Lu)对异丙肾上腺素诱导的心力衰竭(HF)大鼠心肌细胞线粒体损伤的影响。方法 30只雄性(6~7周)SD大鼠随机分为对照组、模型组(HF)和Lu干预组(HF+Lu),各组10例。HF组和HF+Lu组大鼠给予异丙肾上腺素50mg/(kg·d)腹腔注射建立HF模型,对照组给予等量生理盐水腹腔注射。注射药物同时HF+Lu组大鼠给予Lu 50mg/(kg·d)灌胃(体积3ml),对照组和HF组给予50g/L羧甲基纤维素钠3ml灌胃,均干预10d。第11天行超声心动图检测大鼠心脏功能[左室射血分数(LVEF)、左室短轴缩短率(LVFS)、左室收缩末期内径(LVEDs)及左室舒张末期内径(LVEDd)],透射电镜观察心肌线粒体超微结构改变,荧光酶标仪检测心肌细胞线粒体膜电位(MMP)、琥珀酸脱氢酶(SDH)及细胞色素C氧化酶(COX)活性；RT-PCR法检测凋亡相关基因Bax、caspase3、caspase9 mRNA的表达。应用SPSS 19.0统计软件分析数据。结果 超声心动图结果显示,与对照组比较,HF组大鼠LVEF[(68.0±3.1)%和(86.0±4.5)%,P=0.023]、LVFS[(32.0±3.7)%和(43.0±2.5)%,P=0.002]均明显降低,LVEDs[(4.10±0.29)和(3.20±0.27)mm,P=0.010]、LVEDd[(7.10±0.34)和(5.87±0.35)mm,P=0.034]均明显增加,差异具有统计学意义。与HF组相比,HF+Lu组大鼠LVEF[(78.0±5.8)%和(68.0±3.1)%,P=0.028]及LVFS[(39.0±1.5)%和(32.0±3.7)%,P=0.006]均明显增加,而 LVEDs[(3.40±0.23)和(4.10±0.29)mm,P=0.043]及LVEDd[(5.65±0.21)和(7.10±0.34)mm,P=0.019]均明显降低,差异亦有统计学意义。透射电镜下可见,对照组大鼠心肌线粒体结构清楚完整,Z线清晰,粗细肌丝排列整齐、有序,形状多为圆形或椭圆形。HF组大鼠心肌线粒体肿胀、大小不均一,膜完整性破坏,嵴断裂或溶解,空泡形成；与HF组比较,HF+Lu组大鼠心肌线粒体损伤明显减轻,嵴尚清晰,膜完整性较好,无空泡。与对照组比较,HF组大鼠MMP、SDH及COX活性显著降低(P＜0.05),凋亡相关基因Bax、caspase3及caspase9 mRNA表达显著增高(P＜0.05)；与HF组比较,HF+Lu组大鼠心肌MMP、COX、SDH活性显著增加 (P＜0.05),Bax、caspase3及caspase9 mRNA 表达显著降低(P＜0.05)。结论 Lu可通过减轻异丙肾上腺素诱导的HF大鼠心肌线粒体损伤及凋亡,发挥心肌保护作用。

Abstract:

Objective To explore the effect of luteolin (Lu) on myocardial mitochondrial injury in rats with heart failure (HF) induced by isoprenaline. Methods Thirty male SD rats (6-7 weeks old) were randomly divided into control group, model group (HF) and Lu intervention group (HF+Lu), with 10 animals in each group. Rat model of HF were established by intra-peritoneal injection of 50mg/(kg·d) isoproterenol in HF and HF+Lu group, while the rats in control group were intraperitoneally injected with normal saline equally. At the same time, the rats in HF+Lu group were also given 3ml Lu 50mg/(kg·d) by gavage, while the rats in the other 2 groups were given 3ml 50g/L sodium carboxymethyl cellulose by gavage. After intervention for 10d, echocardiography was performed to detect cardiac function; on the eleventh day transmission electron microscopy was used to observe the ultrastructural changes of myocardial mitochondria; and fluorescent enzyme labeling was employed to measure mitochondrial membrane potential (MMP), and activities of succinate dehydrogenase [JP+2](SDH) and cytochrome C oxidase (COX); RT-PCR was applied to detect theexpression of apoptosis-related genes Bax, caspase3 and caspase9. SPSS statistics 19.0 was used to analyze the data. Results Echocardiography showed that compared with control group, left ventricular ejection fraction [LVEF, (68.0±3.1)% vs (86.0±4.5)%, P=0.023] and left ventricular fraction shortening [LVFS, (32.0±3.7)% vs (43.0±2.5)%, P=0.002] were decreased significantly, while left ventricular end-systolic diameter [LVEDs, (4.10±0.29) vs (3.20±0.27)mm, P=0.010] and left ventricular end-diastolic diameter [LVEDd, (7.10±0.34) vs (5.87±0.35)mm, P=0.034] were increased markedly in the HF group. After Lu intervention, LVEF [(78.0±5.8)% vs (68.0±3.1)%, P=0.028] and LVFS [(39.0±1.5)% vs (32.0±3.7)%, P=0.006] were elevated while LVEDs [(3.40±0.23) vs (4.10±0.29)mm, P=0.043] and LVEDd [(5.65±0.21) vs (7.10±0.34)mm, P=0.019] were decreased in the HF+Lu group compared with HF group. Under a transmission electron microscope, the myocardial mitochondria in control group had clear and complete structure, clear Z line, well-arranged thick and thin filaments, and mostly round or oval shaped. Myocardial mitochondria in HF group were swollen and uneven in size, with damaged membrane integrity, broken or dissolved cristae, and formed vacuoles. Compared with HF group, the damage of myocardial mitochondria in HF+Lu group was significantly reduced, cristae was clear, membrane integrity was better, and no vacuoles were found. Compared with control group, the MMP and activities of SDH and COX in HF group were significantly decreased (P<0.05), and the mRNA levels of Bax, caspase3 and caspase9 were significantly increased (P<0.05); compared with HF group, the MMP and activities of COX and SDH in HF+Lu group were significantly increased (P<0.05), while the levels of above genes were significantly decreased (P<0.05). Conclusion Lu plays a protective role in HF rats by reducing isoprenaline-induced myocardial mitochondrial damage and apoptosis.