西洋参茎叶总皂苷减轻心肌缺血/再灌注损伤的研究
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摘要
尽早恢复组织血供,是目前防治心肌缺血损伤最有效的措施。但研究发现,缺血一定时间的心肌在重新恢复血液供应后,损伤反而加重,出现心肌顿抑、心功能低下、恶性心律失常等,即心肌缺血/再灌注(ischemia/reperfusion, I/R)损伤。随着急性心肌梗死(AMI)溶栓、经皮穿刺冠状动脉腔内血管成型术和冠状动脉旁路手术等再灌注疗法的广泛应用,心肌再灌注损伤成为心血管领域研究的热点之上世纪九十年代以来,我国对西洋参茎叶化学成分和药理学作用进行了大量研究,表明其化学成分包括皂苷类、氨基酸类、糖类、挥发油类、无机元素类和脂肪酸类等。西洋参茎叶总皂苷(panax quinquefolium saponins, PQS)是从西洋参茎叶中提取出的活性成分。研究显示,PQS具有抗I/R心肌细胞凋亡、维持细胞内Ca2+稳态等作用。
内质网(endoplasmic reticulum, ER)为细胞中调控蛋白质折叠、Ca2+稳态的细胞器之一,对应激非常敏感,缺血缺氧、葡萄糖/营养物质匮乏、三磷酸腺苷(adenosine triphosphate, ATP)耗竭、大量自由基产生及Ca2+稳态破坏等均可引起内质网功能障碍,触发内质网应激(endoplasmic reticulum stress, ERS)。一定程度的ERS通过上调葡萄糖调节蛋白类(glucose-regulated proteins, GRPs)、钙网蛋白(calreticulin, CRT)、蛋白质折叠酶等促进内质网功能恢复,持续或严重的ERS则破坏细胞Ca2+稳态、上调促凋亡因子CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein, CHOP)及caspase12的表达和活化,诱导ERS相关细胞凋亡,加重I/R损伤。
本研究采用大鼠心肌I/R模型及心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)模型,模拟在体心肌I/R损伤,研究西洋参茎叶总皂苷保护心肌I/R损伤的作用,并从内质网应激方面,探讨可能的分子机理。研究分为以下三个部分：研究一西洋参茎叶总皂苷对于心肌缺血/再灌注损伤的影响目的：观察西洋参茎叶总皂苷(PQS)对心肌I/R损伤的作用。方法：采用SD大鼠在体心脏I/R模型,检测血流动力学及血清心肌肌钙蛋白T(cardiac troponin T, cTnT)含量,以TTC和伊文思蓝双染法检测心梗面积,采用HE和TUNEL法分别评估心肌病理损伤和心肌细胞凋亡程度,采用Westernblot法检测心肌组织凋亡调节因子的表达。采用乳鼠心肌细胞缺氧／复氧(H/R)损伤模型,以台盼蓝排斥实验、乳酸脱氢酶活性以及流式细胞术检测细胞损伤及凋亡情况,以RT-PCR和Western blot方法,检测凋亡相关蛋白Bcl-2、Bax的表达。
结果：(1)动物实验结果：与Sham组比较,I/R组平均动脉压升高55.0%(P<0.05),左室收缩与舒张功能(±dp/dtmax)分别降低42.0%和43.0%(P<0.05),血清cTnT含量为Sham组的3.9倍(P<0.05),梗死区心肌／缺血区面积(area of necrosis/area at risk, AN/AAR)百分比为(44.2±1.4)%,心肌细胞凋亡指数是Sham组的7.0倍(P<0.05)。病理观察表明,心肌纤维结构紊乱,局部呈断裂、坏死融合。抗凋亡蛋白Bcl-2表达降低54.9%(P<0.05),促凋亡蛋白Bax表达是Sham组的3.9倍(P<0.05)；与I/R组比较,PQS+I/R组平均动脉压降低32.0%(P<0.05),左室±dp/dtmax分别升高64.0%和35.0%(P<0.05),血清cTnT含量降低53.3%(P<0.05), AN/AAR百分比降低65.5%(P<0.05),心肌细胞凋亡率降低54.9%(P<0.05)；心肌组织病理损伤程度减轻；抗凋亡蛋白Bcl-2表达升高110.0%(P<0.05),促凋亡蛋白Bax表达降低47.8%(P<0.05)。
(2)细胞实验结果：与对照组比较,H/R组心肌细胞凋亡率升高5.0%(P<0.05),细胞存活率降低21.7%(P<0.05),LDH活性较对照组高约6.0倍(P<0.05)。Bcl-2mRNA和蛋白表达分别降低44.5%和58.3%(P<0.05), Bax mRNA和蛋白表达分别升高86.9%和167.0%(P<0.05)；和H/R组比较,PQS+H/R组细胞凋亡率降低4.2%(P<0.05),存活率升高21.2%(P<0.05), LDH活性降低66.6%(P<0.05)。 Bcl-2mRNA和蛋白的表达分别升高30.9%和48.0%(P<0.05), Bax mRNA和蛋白的表达分别降低39.7%和48.4%(P<0.05)。
结论：PQS可减轻心肌缺血／再灌注损伤,其机制与增加抗凋亡因子Bcl-2和降低促凋亡因子Bax的表达有关。
研究二西洋参茎叶总皂苷减轻心肌缺血／再灌注损伤致严重内质网应激的研究
目的：观察西洋参茎叶总皂苷(PQS)对心肌I/R损伤致严重ERS的影响。方法：采用SD大鼠在体心脏缺血／再灌注(I/R)模型和乳鼠心肌细胞缺氧/复氧(H/R)模型,以RT-PCR和Western blot方法,分别检测内质网应激相关因子的表达。
结果：(1)动物实验结果：与Sham组比较,I/R组GRP78、CRT蛋白表达分别为Sham组的3.1倍和3.0倍(P<0.05), CHOP蛋白表达为Sham组的3.4倍(P<0.05),剪切后的caspase12的蛋白表达是Sham组的2.5倍(P<0.05)；与I/R组比较,PQS+I/R组CRT蛋白表达降低43.4%(P<0.05), CHOP蛋白表达和剪切后的caspase-12蛋白表达分别降低38.6%和23.7%(P<0.05)。
(2)细胞实验结果：与对照组比较,H/R组GRP78mRNA和蛋白的表达分别升高380.0%和142.0%(P<0.05), CRT mRNA和蛋白的表达分别升高96.0%和312.0%(P<0.05); CHOP mRNA和蛋白的表达分别升高311.0%和219.0%(P<0.05)；剪切后的caspase-12蛋白表达升高180.0%(P<0.05)；与H/R组比较,PQS+H/R组GRP78mRNA和蛋白的表达分别降低61.6%和37.7%(P<0.05), CRT mRNA(?)口蛋白的表达分别降低35.7%和52.2%(P<0.05); CHOP mRNA和蛋白的表达分别降低57.0%和51.7%(P<0.05)；剪切后的caspase-12蛋白表达降低34.9%(P<0.05)。
结论：PQS可减轻心肌I/R诱导的严重ERS,表现为降低I/R后CRT的过表达,抑制CHOP、caspase-12等内质网凋亡通路的激活,减少过度ERS介导的细胞凋亡。
研究三西洋参茎叶总皂苷减轻心肌缺血/再灌注损伤致严重内质网应激的下游信号通路研究
目的：探讨西洋参茎叶总皂苷(PQS)减轻大鼠心肌I/R损伤致严重ERS的机制。
方法：采用SD大鼠在体心脏缺血/再灌注(I/R)模型,检测血流动力学及血清cTnT含量,以TTC和伊文思蓝双染法检测心梗面积,采用HE和TUNEL法分别评估心肌病理损伤和心肌细胞凋亡程度,按CaN测试盒步骤测定心肌CaN活性,采用Western blot法检测心肌组织CaN蛋白表达；采用乳鼠心肌细胞缺氧／复氧(H/R)损伤模型,转染pCDB-CaN质粒或CaN抑制剂FK506干扰CaN表达,以流式细胞术检测细胞凋亡,按CaN测试盒步骤测定心肌细胞CaN活性,以Western blot方法,检测心肌细胞CaN表达。
结果：(1)动物实验结果：与PQS+I/R组比较,CaN抑制剂(PQS+CaN+I/R)组平均动脉压、左室收缩舒张功能(±dp/dtmax)血清cTnT含量、心梗面积、心肌细胞凋亡率、抗凋亡蛋白Bcl-2和促凋亡蛋白Bax表达、CaN活性及蛋白表达无显著差异(P>0.05)。
(2)细胞实验结果：与PQS+H/R组比较,CaN抑制剂(FK506)组心肌细胞凋亡率、抗凋亡蛋白Bcl-2和促凋亡蛋白Bax表达、CaN活性及蛋白表达无显著差异(P>0.05)。
结论：PQS减轻心肌I/R致严重ERS的下游信号通路可能与CaN途径无关。
Early reperfusion in ischemic myocardium is the most effective measure to improve the prognosis of acute myocardial infarction (AMI). However, a great concern of ischemia/reperfusion (I/R) injury with reperfusion approach has raised for nearly30years. I/R injury mainly manifested as myocardial stunning, heart dysfunction, malignant ventricular arrhythmias and even sudden death. With the wide application of reperfusion therapy, such as thrombolysis, percutaneous coronary intervention and coronary artery bypass surgery, myocardial reperfusion injury has become a hot spot in the cardiovascular researches.
Since1990s, there're extensive researches to chemical composition of American ginseng stem and leaf, indicating that its chemical constituents include saponins, amino acids, carbohydrates, essential oils, the class of inorganic elements and fatty acids. PQS (panax quinquefolium saponins) is the active ingredient extracted from Panax quinquefolius. Studies have shown that it can prevent cardiomyocyte apoptosis against ischemia/reperfusion injury and maintain intracellular Ca2+homeostasis.
Endoplasmic reticulum (ER) is the most important organelle in adjusting protein folding, Ca2+homeostasis and stress response in cells. ER is very sensitive to stress stimulation. Ischemia/hypoxia, glucose/nutrient deprivation, ATP depletion, a large number of free radicals production or disturbance of Ca2+homeostasis can cause dysfunction of endoplasmic reticulum and trigger ERS (endoplasmic reticulum stress). A certain degree of ERS improves the restoration of ER function through upregulation of ER molecular chaperones such as glucose-regulated proteins (GRPs), calreticulin (CRT) and folding enzymes. However excessive and prolonged ERS, as one of the mechanisms of ischemia/reperfusion injury, destroy Ca2+homeostasis and induce ER associated cell death, and result in tissue injury through inducing and activating of pro-apoptotic factors such as CHOP/GADD153and caspase-12.
The study used rat myocardial ischemia/reperfusion model and neonatal rat cardiomyocyte hypoxia/reoxygenation model to simulate in vivo cardiac ischemia/reperfusion injury. First to confirm cardioprotective effects of PQS in myocardial I/R injury; then on this basis, the detection of endoplasmic reticulum stress-related proteins to explore the impact of PQS for endoplasmic reticulum stress-mediated apoptosis; subsequently adopted by the CaN inhibitor or transfected plasmid CaN to interfere calcineurin phosphatase expression to further explore the cell signaling pathways. This study is divided into three parts:
Part I:The effects of panax quinquefolium saponins on myocardial ischemia/reperfusion injury
Objective:Investigate the protective effect of panax quinquefolium saponins against myocardial ischemia/reperfusion injury.
Methods:The model of myocardial I/R injury in vivo was made by occluding the left anterior descending artery for45min followed by24h of reperfusion in SD rats. Hemodynamics and serum content of cardiac tropoin T (cTnT) were measured. Myocardial infarct size was measured by evens blue and2,3,5-triphenyhetrazolium chloride (TTC) staining; cardiomyocyte apoptosis was detected using in situ TdT-mediated dUTP nick end labeling (TUNEL). Cultured cardiomyocytes of neonatal Sprague-Dawley rats were made into hypoxia/reoxygenation (H/R) model as to observe the cardioprotective effects of PQS. Morphological studies, lactate dehydrogenase (LDH) activity and Flow Cytometry were employed to assess cell injury and apoptosis. RT-PCR and Western blot were used to test the mRNA and protein expression of apoptosis-associated proteins Bax and Bcl-2.
Results:(1) The results in vivo showed that:Compared with the sham group, in the I/R group the mean arterial pressure was increased by55.0%, left ventricular±dp/dtmax were decreased by42.0%and43.0%, respectively (P<0.05); the serum content of cTnT was increased by290%(P<0.05); the percentage of AN/AAR value was (44.2±1.4)%and the apoptosis rate was increased by600.0%(P<0.05); Pathological observations suggest that the structural of cardiac muscle fibers disordered, the local was broken, motified and fused. Bcl-2protein expression was decreased by54.9%and that of Bax was increase by290.0%(P<0.05).Compared with the I/R group:in the PQS+I/R group the mean arterial pressure was decreased by32.0%, left ventricular±dp/dt max were increased by64.0%and35.0%, respectively (P<0.05); the serum content of cTnT was decreased by53.3%(P<0.05); the percentage of AN/AAR value was reduced by65.5%and the apoptosis rate was decreased by54.9%(P<0.05); the myocardial pathological changes were improved; Bcl-2protein expression was increased by110.0%and that of Bax was decreased by47.8%(P<0.05);
(2) The results in vitro showed that:Compared with the control group, in the H/R group the apoptosis rate and the LDH concentration in the culture medium were increased by5.0%and600.0%, respectively(P<0.05); the survival rate was decreased by21.7%(P<0.05). Bcl-2mRNA and protein expression of the cardiomyocytes were decreased by44.5%and58.3%(P<0.05), those of Bax were increased by86.9%and167.0%(P<0.05). Compared with H/R cardiomyocytes: The apoptosis rate and the LDH concentration in the culture medium were decreased by4.2%and66.6%, respectively in PQS+H/R cardiomyocytes (P<0.05); the survival rate was increased by21.2%(P<0.05). Bcl-2mRNA and protein expression of the cardiomyocytes were increased by30.9%and48.0%(P<0.05), those of Bax were decreased by39.7%and48.4%(P<0.05).
Conclusion:PQS alleviated myocardial ischemia/reperfusion injury by decreasing the expression of pro-apoptotic factor Bax and increasing the expression of anti-apoptotic factor Bcl-2.
Part Ⅱ:Panax quinquefolium saponins protect myocardium against ischemia/reperfusion injury through attenuating excessive endoplasmic reticulum stress
Objective:Investigate whether excessive endoplasmic reticulum stress was involved in the protective effect of panax quinquefolium saponins against ischemia/reperfusion injury in rat myocardium.
Methods:The model of myocardial I/R injury in vivo and cultured neonatal Sprague-Dawley cardiomyocytes of hypoxia/reoxygenation (H/R) injury in vitro were applied. RT-PCR and Western blot were used to test the mRNA and protein expression of glucose-regulated protein78(GRP78), calreticulin (CRT),(C/EBP) homologous protein (CHOP) and cleaved-caspase12.
Results:(1) The results in vivo showed that:Compared with the sham group, in the I/R group GRP78and CRT protein expression were increased by210.0%and200.0%(P<0.05); CHOP protein expression was increased by240.0%(P<0.05); cleaved-caspase12was increased by150.0%(P<0.05). Compared with the I/R group, in the PQS+I/R group CRT protein expression was decreased by43.4%(P<0.05); CHOP protein expression and the protein level of cleaved-caspasel2were decreased by38.6%and23.7%(P<0.05).
(2) The results in vitro showed that:Compared with the control group, in the H/R group GRP78mRNA and protein expression were increased by380.0%and142.0%(P<0.05); those of CRT were increase by96.0%and312.0%(P<0.05); CHOP mRNA and protein expression were increased by311.0%and219.0%(P<0.05); cleaved-caspase12protein expression was increased by180.0%(P<0.05). Compared with the H/R group, in the PQS+H/R group GRP78mRNA and protein expression of the cardiomyocytes were decreased by61.6%and37.7%(P<0.05), CRT mRNA and protein expression were decreased by35.7%and52.2%(P<0.05); CHOP mRNA and protein expression were decreased by51.0%and51.7%(P<0.05); the protein level of cleaved-caspase12was decreased by34.9%(P<0.05).
Conclusion:These results indicated that PQS could alleviate myocardium injury caused by I/R, its mechanism is probably related to inhibition of excessive ERS induced by I/R.
Part III:The machenism underlying panax quinquelium saponins attenuating excessive endoplasmic reticulum stress induced by I/R
Objective:Investigate the machenism of PQS supressing excessive ERS induced by myocardial ischemia/reperfusion.
Methods:The model of myocardial I/R injury in vivo was made by occluding the left anterior descending artery for45min followed by24h of reperfusion in SD rats. Hemodynamics and serum content of cardiac tropoin T (cTnT) were measured. Myocardial infarct size was measured by evens blue and2,3,5-triphenyhetrazolium chloride (TTC) staining; cardiomyocyte apoptosis was detected using in situ TdT-mediated dUTP nick end labeling(TUNEL). Cultured cardiomyocytes of neonatal Sprague-Dawley rats were made into hypoxia/reoxygenation (H/R) model. Transfect pCDB-CaN plasmid or apply the inhibitor of CaN (FK506) to interfere the CaN expression. Flow Cytometry was employed to assess apoptosis. The activity of CaN in myocardium and cardiomyocyte were detected according to the instructions. Western blot was used to test the protein expression CaN.
Results:(1) The results in vivo showed that:Compared with the PQS+I/R group, in the CaN inhibitor (PQS+CsA+I/R) group the mean arterial pressure, left ventricular±dp/dt max, the serum content of cTnT, AN/AAR value, the apoptosis rate, apoptosis protein Bcl-2and Bax expression, CaN activity and protein expression in myocardium were no significantly different (P>0.05).
(2) The results in vitro showed that:Compared with the PQS+H/R group, in the the CaN inhibitor (FK506) group cardiomyocyte apoptosis, the expression of Bcl-2and Bax, the activity of CaN and its protein expression were not significantly different (P>0.05).
Conclusion:The machenism of PQS supressing excessive ERS induced by myocardial ischemia/reperfusion may not be associated with the CaN signal pathway.
内质网(endoplasmic reticulum, ER)为细胞中调控蛋白质折叠、Ca2+稳态的细胞器之一,对应激非常敏感,缺血缺氧、葡萄糖/营养物质匮乏、三磷酸腺苷(adenosine triphosphate, ATP)耗竭、大量自由基产生及Ca2+稳态破坏等均可引起内质网功能障碍,触发内质网应激(endoplasmic reticulum stress, ERS)。一定程度的ERS通过上调葡萄糖调节蛋白类(glucose-regulated proteins, GRPs)、钙网蛋白(calreticulin, CRT)、蛋白质折叠酶等促进内质网功能恢复,持续或严重的ERS则破坏细胞Ca2+稳态、上调促凋亡因子CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein, CHOP)及caspase12的表达和活化,诱导ERS相关细胞凋亡,加重I/R损伤。
本研究采用大鼠心肌I/R模型及心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)模型,模拟在体心肌I/R损伤,研究西洋参茎叶总皂苷保护心肌I/R损伤的作用,并从内质网应激方面,探讨可能的分子机理。研究分为以下三个部分：研究一西洋参茎叶总皂苷对于心肌缺血/再灌注损伤的影响目的：观察西洋参茎叶总皂苷(PQS)对心肌I/R损伤的作用。方法：采用SD大鼠在体心脏I/R模型,检测血流动力学及血清心肌肌钙蛋白T(cardiac troponin T, cTnT)含量,以TTC和伊文思蓝双染法检测心梗面积,采用HE和TUNEL法分别评估心肌病理损伤和心肌细胞凋亡程度,采用Westernblot法检测心肌组织凋亡调节因子的表达。采用乳鼠心肌细胞缺氧／复氧(H/R)损伤模型,以台盼蓝排斥实验、乳酸脱氢酶活性以及流式细胞术检测细胞损伤及凋亡情况,以RT-PCR和Western blot方法,检测凋亡相关蛋白Bcl-2、Bax的表达。
结果：(1)动物实验结果：与Sham组比较,I/R组平均动脉压升高55.0%(P<0.05),左室收缩与舒张功能(±dp/dtmax)分别降低42.0%和43.0%(P<0.05),血清cTnT含量为Sham组的3.9倍(P<0.05),梗死区心肌／缺血区面积(area of necrosis/area at risk, AN/AAR)百分比为(44.2±1.4)%,心肌细胞凋亡指数是Sham组的7.0倍(P<0.05)。病理观察表明,心肌纤维结构紊乱,局部呈断裂、坏死融合。抗凋亡蛋白Bcl-2表达降低54.9%(P<0.05),促凋亡蛋白Bax表达是Sham组的3.9倍(P<0.05)；与I/R组比较,PQS+I/R组平均动脉压降低32.0%(P<0.05),左室±dp/dtmax分别升高64.0%和35.0%(P<0.05),血清cTnT含量降低53.3%(P<0.05), AN/AAR百分比降低65.5%(P<0.05),心肌细胞凋亡率降低54.9%(P<0.05)；心肌组织病理损伤程度减轻；抗凋亡蛋白Bcl-2表达升高110.0%(P<0.05),促凋亡蛋白Bax表达降低47.8%(P<0.05)。
(2)细胞实验结果：与对照组比较,H/R组心肌细胞凋亡率升高5.0%(P<0.05),细胞存活率降低21.7%(P<0.05),LDH活性较对照组高约6.0倍(P<0.05)。Bcl-2mRNA和蛋白表达分别降低44.5%和58.3%(P<0.05), Bax mRNA和蛋白表达分别升高86.9%和167.0%(P<0.05)；和H/R组比较,PQS+H/R组细胞凋亡率降低4.2%(P<0.05),存活率升高21.2%(P<0.05), LDH活性降低66.6%(P<0.05)。 Bcl-2mRNA和蛋白的表达分别升高30.9%和48.0%(P<0.05), Bax mRNA和蛋白的表达分别降低39.7%和48.4%(P<0.05)。
结论：PQS可减轻心肌缺血／再灌注损伤,其机制与增加抗凋亡因子Bcl-2和降低促凋亡因子Bax的表达有关。
研究二西洋参茎叶总皂苷减轻心肌缺血／再灌注损伤致严重内质网应激的研究
目的：观察西洋参茎叶总皂苷(PQS)对心肌I/R损伤致严重ERS的影响。方法：采用SD大鼠在体心脏缺血／再灌注(I/R)模型和乳鼠心肌细胞缺氧/复氧(H/R)模型,以RT-PCR和Western blot方法,分别检测内质网应激相关因子的表达。
结果：(1)动物实验结果：与Sham组比较,I/R组GRP78、CRT蛋白表达分别为Sham组的3.1倍和3.0倍(P<0.05), CHOP蛋白表达为Sham组的3.4倍(P<0.05),剪切后的caspase12的蛋白表达是Sham组的2.5倍(P<0.05)；与I/R组比较,PQS+I/R组CRT蛋白表达降低43.4%(P<0.05), CHOP蛋白表达和剪切后的caspase-12蛋白表达分别降低38.6%和23.7%(P<0.05)。
(2)细胞实验结果：与对照组比较,H/R组GRP78mRNA和蛋白的表达分别升高380.0%和142.0%(P<0.05), CRT mRNA和蛋白的表达分别升高96.0%和312.0%(P<0.05); CHOP mRNA和蛋白的表达分别升高311.0%和219.0%(P<0.05)；剪切后的caspase-12蛋白表达升高180.0%(P<0.05)；与H/R组比较,PQS+H/R组GRP78mRNA和蛋白的表达分别降低61.6%和37.7%(P<0.05), CRT mRNA(?)口蛋白的表达分别降低35.7%和52.2%(P<0.05); CHOP mRNA和蛋白的表达分别降低57.0%和51.7%(P<0.05)；剪切后的caspase-12蛋白表达降低34.9%(P<0.05)。
结论：PQS可减轻心肌I/R诱导的严重ERS,表现为降低I/R后CRT的过表达,抑制CHOP、caspase-12等内质网凋亡通路的激活,减少过度ERS介导的细胞凋亡。
研究三西洋参茎叶总皂苷减轻心肌缺血/再灌注损伤致严重内质网应激的下游信号通路研究
目的：探讨西洋参茎叶总皂苷(PQS)减轻大鼠心肌I/R损伤致严重ERS的机制。
方法：采用SD大鼠在体心脏缺血/再灌注(I/R)模型,检测血流动力学及血清cTnT含量,以TTC和伊文思蓝双染法检测心梗面积,采用HE和TUNEL法分别评估心肌病理损伤和心肌细胞凋亡程度,按CaN测试盒步骤测定心肌CaN活性,采用Western blot法检测心肌组织CaN蛋白表达；采用乳鼠心肌细胞缺氧／复氧(H/R)损伤模型,转染pCDB-CaN质粒或CaN抑制剂FK506干扰CaN表达,以流式细胞术检测细胞凋亡,按CaN测试盒步骤测定心肌细胞CaN活性,以Western blot方法,检测心肌细胞CaN表达。
结果：(1)动物实验结果：与PQS+I/R组比较,CaN抑制剂(PQS+CaN+I/R)组平均动脉压、左室收缩舒张功能(±dp/dtmax)血清cTnT含量、心梗面积、心肌细胞凋亡率、抗凋亡蛋白Bcl-2和促凋亡蛋白Bax表达、CaN活性及蛋白表达无显著差异(P>0.05)。
(2)细胞实验结果：与PQS+H/R组比较,CaN抑制剂(FK506)组心肌细胞凋亡率、抗凋亡蛋白Bcl-2和促凋亡蛋白Bax表达、CaN活性及蛋白表达无显著差异(P>0.05)。
结论：PQS减轻心肌I/R致严重ERS的下游信号通路可能与CaN途径无关。
Early reperfusion in ischemic myocardium is the most effective measure to improve the prognosis of acute myocardial infarction (AMI). However, a great concern of ischemia/reperfusion (I/R) injury with reperfusion approach has raised for nearly30years. I/R injury mainly manifested as myocardial stunning, heart dysfunction, malignant ventricular arrhythmias and even sudden death. With the wide application of reperfusion therapy, such as thrombolysis, percutaneous coronary intervention and coronary artery bypass surgery, myocardial reperfusion injury has become a hot spot in the cardiovascular researches.
Since1990s, there're extensive researches to chemical composition of American ginseng stem and leaf, indicating that its chemical constituents include saponins, amino acids, carbohydrates, essential oils, the class of inorganic elements and fatty acids. PQS (panax quinquefolium saponins) is the active ingredient extracted from Panax quinquefolius. Studies have shown that it can prevent cardiomyocyte apoptosis against ischemia/reperfusion injury and maintain intracellular Ca2+homeostasis.
Endoplasmic reticulum (ER) is the most important organelle in adjusting protein folding, Ca2+homeostasis and stress response in cells. ER is very sensitive to stress stimulation. Ischemia/hypoxia, glucose/nutrient deprivation, ATP depletion, a large number of free radicals production or disturbance of Ca2+homeostasis can cause dysfunction of endoplasmic reticulum and trigger ERS (endoplasmic reticulum stress). A certain degree of ERS improves the restoration of ER function through upregulation of ER molecular chaperones such as glucose-regulated proteins (GRPs), calreticulin (CRT) and folding enzymes. However excessive and prolonged ERS, as one of the mechanisms of ischemia/reperfusion injury, destroy Ca2+homeostasis and induce ER associated cell death, and result in tissue injury through inducing and activating of pro-apoptotic factors such as CHOP/GADD153and caspase-12.
The study used rat myocardial ischemia/reperfusion model and neonatal rat cardiomyocyte hypoxia/reoxygenation model to simulate in vivo cardiac ischemia/reperfusion injury. First to confirm cardioprotective effects of PQS in myocardial I/R injury; then on this basis, the detection of endoplasmic reticulum stress-related proteins to explore the impact of PQS for endoplasmic reticulum stress-mediated apoptosis; subsequently adopted by the CaN inhibitor or transfected plasmid CaN to interfere calcineurin phosphatase expression to further explore the cell signaling pathways. This study is divided into three parts:
Part I:The effects of panax quinquefolium saponins on myocardial ischemia/reperfusion injury
Objective:Investigate the protective effect of panax quinquefolium saponins against myocardial ischemia/reperfusion injury.
Methods:The model of myocardial I/R injury in vivo was made by occluding the left anterior descending artery for45min followed by24h of reperfusion in SD rats. Hemodynamics and serum content of cardiac tropoin T (cTnT) were measured. Myocardial infarct size was measured by evens blue and2,3,5-triphenyhetrazolium chloride (TTC) staining; cardiomyocyte apoptosis was detected using in situ TdT-mediated dUTP nick end labeling (TUNEL). Cultured cardiomyocytes of neonatal Sprague-Dawley rats were made into hypoxia/reoxygenation (H/R) model as to observe the cardioprotective effects of PQS. Morphological studies, lactate dehydrogenase (LDH) activity and Flow Cytometry were employed to assess cell injury and apoptosis. RT-PCR and Western blot were used to test the mRNA and protein expression of apoptosis-associated proteins Bax and Bcl-2.
Results:(1) The results in vivo showed that:Compared with the sham group, in the I/R group the mean arterial pressure was increased by55.0%, left ventricular±dp/dtmax were decreased by42.0%and43.0%, respectively (P<0.05); the serum content of cTnT was increased by290%(P<0.05); the percentage of AN/AAR value was (44.2±1.4)%and the apoptosis rate was increased by600.0%(P<0.05); Pathological observations suggest that the structural of cardiac muscle fibers disordered, the local was broken, motified and fused. Bcl-2protein expression was decreased by54.9%and that of Bax was increase by290.0%(P<0.05).Compared with the I/R group:in the PQS+I/R group the mean arterial pressure was decreased by32.0%, left ventricular±dp/dt max were increased by64.0%and35.0%, respectively (P<0.05); the serum content of cTnT was decreased by53.3%(P<0.05); the percentage of AN/AAR value was reduced by65.5%and the apoptosis rate was decreased by54.9%(P<0.05); the myocardial pathological changes were improved; Bcl-2protein expression was increased by110.0%and that of Bax was decreased by47.8%(P<0.05);
(2) The results in vitro showed that:Compared with the control group, in the H/R group the apoptosis rate and the LDH concentration in the culture medium were increased by5.0%and600.0%, respectively(P<0.05); the survival rate was decreased by21.7%(P<0.05). Bcl-2mRNA and protein expression of the cardiomyocytes were decreased by44.5%and58.3%(P<0.05), those of Bax were increased by86.9%and167.0%(P<0.05). Compared with H/R cardiomyocytes: The apoptosis rate and the LDH concentration in the culture medium were decreased by4.2%and66.6%, respectively in PQS+H/R cardiomyocytes (P<0.05); the survival rate was increased by21.2%(P<0.05). Bcl-2mRNA and protein expression of the cardiomyocytes were increased by30.9%and48.0%(P<0.05), those of Bax were decreased by39.7%and48.4%(P<0.05).
Conclusion:PQS alleviated myocardial ischemia/reperfusion injury by decreasing the expression of pro-apoptotic factor Bax and increasing the expression of anti-apoptotic factor Bcl-2.
Part Ⅱ:Panax quinquefolium saponins protect myocardium against ischemia/reperfusion injury through attenuating excessive endoplasmic reticulum stress
Objective:Investigate whether excessive endoplasmic reticulum stress was involved in the protective effect of panax quinquefolium saponins against ischemia/reperfusion injury in rat myocardium.
Methods:The model of myocardial I/R injury in vivo and cultured neonatal Sprague-Dawley cardiomyocytes of hypoxia/reoxygenation (H/R) injury in vitro were applied. RT-PCR and Western blot were used to test the mRNA and protein expression of glucose-regulated protein78(GRP78), calreticulin (CRT),(C/EBP) homologous protein (CHOP) and cleaved-caspase12.
Results:(1) The results in vivo showed that:Compared with the sham group, in the I/R group GRP78and CRT protein expression were increased by210.0%and200.0%(P<0.05); CHOP protein expression was increased by240.0%(P<0.05); cleaved-caspase12was increased by150.0%(P<0.05). Compared with the I/R group, in the PQS+I/R group CRT protein expression was decreased by43.4%(P<0.05); CHOP protein expression and the protein level of cleaved-caspasel2were decreased by38.6%and23.7%(P<0.05).
(2) The results in vitro showed that:Compared with the control group, in the H/R group GRP78mRNA and protein expression were increased by380.0%and142.0%(P<0.05); those of CRT were increase by96.0%and312.0%(P<0.05); CHOP mRNA and protein expression were increased by311.0%and219.0%(P<0.05); cleaved-caspase12protein expression was increased by180.0%(P<0.05). Compared with the H/R group, in the PQS+H/R group GRP78mRNA and protein expression of the cardiomyocytes were decreased by61.6%and37.7%(P<0.05), CRT mRNA and protein expression were decreased by35.7%and52.2%(P<0.05); CHOP mRNA and protein expression were decreased by51.0%and51.7%(P<0.05); the protein level of cleaved-caspase12was decreased by34.9%(P<0.05).
Conclusion:These results indicated that PQS could alleviate myocardium injury caused by I/R, its mechanism is probably related to inhibition of excessive ERS induced by I/R.
Part III:The machenism underlying panax quinquelium saponins attenuating excessive endoplasmic reticulum stress induced by I/R
Objective:Investigate the machenism of PQS supressing excessive ERS induced by myocardial ischemia/reperfusion.
Methods:The model of myocardial I/R injury in vivo was made by occluding the left anterior descending artery for45min followed by24h of reperfusion in SD rats. Hemodynamics and serum content of cardiac tropoin T (cTnT) were measured. Myocardial infarct size was measured by evens blue and2,3,5-triphenyhetrazolium chloride (TTC) staining; cardiomyocyte apoptosis was detected using in situ TdT-mediated dUTP nick end labeling(TUNEL). Cultured cardiomyocytes of neonatal Sprague-Dawley rats were made into hypoxia/reoxygenation (H/R) model. Transfect pCDB-CaN plasmid or apply the inhibitor of CaN (FK506) to interfere the CaN expression. Flow Cytometry was employed to assess apoptosis. The activity of CaN in myocardium and cardiomyocyte were detected according to the instructions. Western blot was used to test the protein expression CaN.
Results:(1) The results in vivo showed that:Compared with the PQS+I/R group, in the CaN inhibitor (PQS+CsA+I/R) group the mean arterial pressure, left ventricular±dp/dt max, the serum content of cTnT, AN/AAR value, the apoptosis rate, apoptosis protein Bcl-2and Bax expression, CaN activity and protein expression in myocardium were no significantly different (P>0.05).
(2) The results in vitro showed that:Compared with the PQS+H/R group, in the the CaN inhibitor (FK506) group cardiomyocyte apoptosis, the expression of Bcl-2and Bax, the activity of CaN and its protein expression were not significantly different (P>0.05).
Conclusion:The machenism of PQS supressing excessive ERS induced by myocardial ischemia/reperfusion may not be associated with the CaN signal pathway.
引文
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