摘要
目的 探讨橙皮素(HES)对心肌缺血再灌注损伤(MIRI)大鼠细胞自噬与凋亡的影响,并初步分析其机制.方法 将SD大鼠分为假手术(Sham)组、MIRI组、低剂量HES(HES-L,15 mg/kg)组、高剂量HES(HES-H,30 mg/kg)组和HES-H+Toll样受体4(TLR4)抑制剂(HES 30 mg/kg+复合物C 0.2 mg/kg)组.连续给药7 d,除Sham组外,大鼠采用冠脉结扎法构建MIRI模型,24 h后检测左心室血流动力学参数[包括左室舒张期末压(LVEDP)、左室内压最大上升速率(dp/dtmax)、左室内压最大下降速率(-dp/dtmax)];采用原位末端转移酶标记技术(TUNEL)检测心肌细胞凋亡,氯化三苯基四氮唑(TTC)染色检测心肌梗死(心梗)面积,酶联免疫吸附试验(ELISA)检测心肌组织中丙二醛(MDA)、超氧化物歧化酶(SOD);Western blotting检测大鼠心肌组织中微管相关蛋白1-轻链3(LC3)Ⅱ、LC3Ⅰ、自噬效应蛋白(Beclin1)、TLR4、磷酸化(p)-TLR4、雷帕霉素靶蛋白(mTOR)、p-mTOR、unc-51样自噬激活激酶1(ULK1)、p-ULK1蛋白表达.结果 与Sham组比较,MIRI组大鼠心肌细胞线粒体损伤、自噬空泡形成;LVEDP[mmHg(1 mmHg≈0.133 kPa):14.22±2.12比 5.44±0.83]、心肌细胞凋亡率[(31.52±4.01)%比(2.41±0.26)%]、心梗面积(mm2:43.68±3.86 比 0.00)、心肌组织MDA(ng/L:8.25±1.03 比 3.71±0.65)、p-mTOR/mTOR(0.76±0.08 比 0.33±0.04)、p-ULK1/ULK1(0.69±0.08比0.22±0.03)均显著升高,dp/dtmax(mmHg/s:3 441.43±289.25比4 910.57±350.12)、-dp/dtmax(mmHg/s:2 588.96±260.23比3 845.94±364.19)、心肌组织LC3Ⅱ/LC3Ⅰ(1.13±0.14比2.35±0.21)、Beclin1(0.35±0.06 比 0.87±0.09)、p-TLR4/TLR4(0.40±0.05 比 0.84±0.10)均显著降低(均P<0.05).与MIRI组比较,HES-L、HES-H组大鼠线粒体损伤缓解,自噬空泡少见;LVEDP(mmHg:11.56±1.60、7.88±1.21 比14.22±2.12)、心肌细胞凋亡率[(25.52±3.11)%、(17.54±1.89)%比(31.52±4.01)%]、心梗面积(mm2:36.67±3.58、29.58±3.04比43.68±3.86)、心肌组织MDA(ng/L:6.98±0.65、4.12±0.48比8.25±1.03)、p-mTOR/mTOR(0.60±0.07、0.45±0.05 比 0.76±0.08)、p-ULK1/ULK1(0.54±0.05、0.32±0.04 比 0.69±0.08)均显著降低,dp/dtmax(mmHg/s:3 972.82±310.17、4 442.97±396.24 比 3 441.43±289.25)、-dp/dtmax(mmHg/s:3 152.30±312.18、3 430.57±324.24比2 588.96±260.23)、心肌组织中LC3Ⅱ/LC3Ⅰ(1.76±0.18、2.01±0.20比1.13±0.14)、Beclin1(0.56±0.07、0.79±0.08比0.35±0.06)、p-TLR4/TLR4(0.55±0.06、0.73±0.08比0.40±0.05)均显著升高(均P<0.05).结论 HES可通过抑制TLR4-mTOR-ULK1信号通路促进MIRI大鼠心肌细胞自噬,抑制凋亡.
Abstract
Objective To investigate the effect and mechanism of hesunate(HES)on cell autophagy and apoptosis in rats with myocardial ischemia reperfusion injury(MIRI).Methods SD rats were randomly grouped into sham operation group(Sham group),MIRI group,low-dose HES(HES-L,15 mg/kg)group,high-dose HES(HES-H,30 mg/kg)group and HES-H+Toll like receptor 4(TLR4)inhibitor(HES 30 mg/kg+Compound C 0.2 mg/kg)group,and were administered continuously for 7 days.The MIRI model was established in rats other than Sham group using coronary ligation method,after 24 hours,left ventricular hemodynamic parameters[including left ventricular end diastolic pressure(LVEDP),maximal left ventricular pressure rising rate(dp/dtmax)and maximal left ventricular pressure decreasing rate(-dp/dtmax)]were measured,TdT-mediated dUTP nick end labeling(TUNEL)method was applied to detecting cardiomyocyte apoptosis,myocardial infarction area was measured by triphenyltetrazolium chloride(TTC)staining,the levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in myocardial tissue were detected by enzyme linked immunosorbent assay(ELISA),and Western blotting was applied to detecting the expression of microtubule associated protein 1-light chain 3(LC3)Ⅱ,LC3Ⅰ,autophagy effector protein(Beclin1),TLR4,phosphorylated(p)-TLR4(p-TLR4),mammalian target of rapamycin(mTOR),p-mTOR,unc-51-like autophagy activated kinase 1(ULK1)and p-ULK1 in rat cardiac tissue.Results Compared with Sham group,MIRI group showed mitochondrial damage and autophagic vacuole formation.The levels of LVEDP[mmHg(1 mmHg≈0.133 kPa):14.22±2.12 vs.5.44±0.83],myocardial cell apoptosis rate[(31.52±4.01)%vs.(2.41±0.26)%],myocardial infarction area(mm2:43.68±3.86 vs.0.00),myocardial tissue MDA(ng/L:8.25±1.03 vs.3.71±0.65),p-mTOR/mTOR(0.76±0.08 vs.0.33±0.04)and p-ULK1/ULK1(0.69±0.08 vs.0.22±0.04)showed significant increase,while dp/dtmax(mmHg/s:3 441.43±289.25 vs.4 910.57±350.12),-dp/dtmax(mmHg/s:2 588.96±260.23 vs.3 845.94±364.19),myocardial tissue LC3Ⅱ/LC3Ⅰ(1.13±0.14 vs.2.35±0.21),Beclin1(0.35±0.06 vs.0.87±0.09),and p-TLR4/TLR4(0.40±0.05 vs.0.84±0.10)showed significant decrease(all P<0.05).Compared with MIRI group,the mitochondrial damage in HES-L and HES-H groups was alleviated,and autophagic vacuoles were rare.The levels of LVEDP(mmHg:11.56±1.60,7.88±1.21 vs.14.22±2.12),myocardial cell apoptosis rate[(25.52±3.11)%,(17.54±1.89)%vs.(31.52±4.01)%],myocardial infarction area(mm2:36.67±3.58,29.58±3.04 vs.43.68±3.86),myocardial tissue MDA(ng/L:6.98±0.65,4.12±0.48 vs.8.25±1.03),p-mTOR/mTOR(0.60±0.07,0.45±0.05 vs.0.76±0.08),p-ULK1/ULK1(0.54±0.05,0.32±0.04 vs.0.69±0.08)were significantly reduced,and dp/dtmax(mmHg/s:3 972.82±310.17,4 442.97±396.24 vs.3 441.43±289.25),-dp/dtmax(mmHg/s:3 152.30±312.18,3 430.57±324.24 vs.2 588.96±260.23),LC3Ⅱ/LC3Ⅰ in myocardial tissue(1.76±0.18,2.01±0.20 vs.1.13±0.14),Beclin1(0.56±0.07,0.79±0.08 vs.0.35±0.06)and p-TLR4/TLR4(0.55±0.06,0.73±0.08 vs.0.40±0.05)were significantly elevated(all P<0.05).Conclusion HES may promote autophagy and inhibit apoptosis of myocardial cells in MIRI rats by activating TLR4-mTOR-ULK1 signaling pathway.
基金项目
山东省青岛市医药卫生科研指导项目(2022-EJZD060)
维普
万方数据