抗自由基中药对小鼠脑片及PC12细胞脑缺血损伤模型的保护作用及作用机制研究
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摘要
脑卒中是导致全球人类致死致残的主要病因之一,缺血性脑卒中是卒中的最常见类型,占80%-85%。脑缺血再灌注损伤是缺血性脑卒中损伤的主要原因,而自由基的过度形成是造成脑缺血再灌注损伤的重要因素。过量自由基的产生主要是由于在脑缺血时氧化应激反应中的关键酶如环氧合酶、脂氧合酶、NADPH氧化酶等的激活使氧化-抗氧化系统失衡。因此抗自由基药物或抑制氧化应激反应中的关键酶如环氧合酶等以减轻脑缺血再灌注损伤成为近年来研究重点。抗自由基药物依达拉奉现已作为抗脑缺血药物在日本与中国上市,若能筛选到同时具有抑制氧化应激反应中的关键酶如环氧合酶、NADPH氧化酶、一氧化氮合酶、基质金属蛋白酶等作用又有抗自由基作用的多靶点作用的药物,有望成为一种更有效的抗脑缺血药物,而抗自由基中药,更可能具有多靶点作用机制,是我们筛选的主要目标。
本课题在细胞和脑片水平上建立高效、稳定的抗脑缺血药物筛选模型,首先通过评价环氧合酶抑制剂在这些模型中的作用,完善药物筛选模型；然后选择一些文献报道有神经保护作用的抗自由基中药,并在我们建立的模型中筛选出对脑缺血再灌注损伤保护作用较强的中药,进行下一步的深入研究,考察其主要作用机制,是否具有多靶点作用机制,分析其在临床的应用前景。本论文主要研究内容为：
1.在细胞、脑片模型上评价环氧合酶抑制剂塞来昔布、依托考昔及吲哚美辛对脑缺血再灌注损伤的保护作用。
2.在细胞、脑片模型上筛选抗自由基中药,如：人参皂苷Rgl、丹皮酚、槲皮素、黄芪甲苷、白黎芦醇、绿原酸、黄芩昔、穿心莲内酯、羟基红花黄色素A和黄连解毒汤对脑缺血再灌注损伤的保护作用。
3.在上述药物中选择一个在细胞、脑片模型中对损伤保护作用较明显的中药有效成分,探讨其对脑缺血再灌注损伤保护作用的机制,分析其临床应用前景。
第一部分OGD/RP诱导的小鼠离体脑片及PC12细胞损伤模型的建立与完善
目的在离体水平上,建立与完善缺氧缺糖再灌注(OGD/RP)诱导的脑片及PC12细胞损伤模型,并在这两种模型上考察COX抑制剂对损伤的保护作用及机制。
方法以小鼠离体脑片建立OGD/RP模型,TTC染色检测小鼠离体脑片的活性；同时以大鼠肾上腺嗜铬细胞瘤细胞株(PC12细胞)OGD/RP损伤模型模拟脑缺血损伤,采用光镜观察细胞形态学变化,MTT法检测细胞存活率,乳酸脱氢酶(LDH)法检测细胞死亡率,免疫细胞化学方法检测环氧合酶-2(COX-2)的表达。
结果在离体小鼠脑片上,COX-2选择性抑制剂塞来昔布(Cel)及依托考昔(Eto)能减少OGD/RP引起的损伤面积,增加吸光度；而COX非选择性抑制剂吲哚美辛(Ind)未见显著性差异。在PC12细胞OGD/RP模型上,Cel、Eto及Ind均能增加OGD/RP损伤后PC12细胞存活率。其中,Eto在10-160μmol/L能显著性增加OGD/RP诱导后PC12细胞的存活率,并降低LDH释放,减少PC12细胞死亡率。同时免疫组织化学检测显示Eto能抑制OGD/RP诱导的COX-2蛋白的表达。
结论本实验建立的OGD/RP诱导的小鼠离体脑片及PC12细胞损伤模型可试用于抗脑缺血药物的筛选；COX-2选择性抑制剂对脑缺血损伤具有保护作用,其中新型一代的Eto在离体小鼠脑片及PC12细胞OGD/RP模型上均有显著性的神经保护作用,其机制可能与抑制COX-2蛋白的诱导表达有关。
第二部分抗自由基中药对OGD/RP诱导的小鼠脑片及PC12细胞损伤的保护作用
目的在离体水平上,筛选抗自由基中药对缺氧缺糖再灌注(OGD/RP)诱导的脑片及PC12细胞缺氧缺糖再灌注(OGD/RP)损伤的保护作用。
方法以小鼠离体脑片建立OGD/RP模型,TTC染色检测小鼠离体脑片的活性；同时以大鼠肾上腺嗜铬细胞瘤细胞株PC12细胞OGD/RP诱导细胞损伤模拟脑缺血损伤模型,MTT法检测细胞存活率。以这几个指标为标准,在细胞和脑片两种损伤模型上考察文献报道有神经保护作用的抗自由基中药,如：人参皂苷Rgl、丹皮酚、槲皮素、黄芪甲苷、白黎芦醇、绿原酸、黄芩苷、穿心莲内酯、羟基红花黄色素A和黄连解毒汤等对损伤保护作用的差别。
结果在离体小鼠脑片上,黄芪甲苷(1μmol/L)、白黎芦醇(1.10μmol/L)、绿原酸(100μmol/L)、黄芩苷(10,100μmol/L)、羟基红花黄色素A (1,10,100μmol/L和FE50(1μmol/L)均能减少OGD/RP引起的损伤面积,增加吸光度；而人参皂苷Rgl、丹皮酚、槲皮素、穿心莲内酯和FE30未见显著性差异。在PC12细胞OGD/RP模型上,人参皂苷Rgl (1μmol/L)、白黎芦醇(10μmol/L)、黄芩苷(0.1,1,10μmol/L)、穿心莲内酯(1μmol/L)、羟基红花黄色素A (0.01,0.1,1,10μmol/L).FE30(1,10μmol/L)和FE50(1,10μmol/L)均能显著性增加OGD/RP诱导后PC12细胞的存活率,减少PC12细胞死亡率；黄芪甲苷、丹皮酚、槲皮素、绿原酸未见显著性差异。
结论黄芩苷和羟基红花黄色素A对两种模型的OGD/RP损伤的保护作用均较强,因此我们考虑可进一步在PC12细胞上进行试验考察其作用机制。
第三部分羟基红花黄色素A对PC12细胞缺氧缺糖再灌注损伤的保护作用及机制研究
目的探讨羟基红花黄色素A(HSYA)对缺氧缺糖再灌注(OGD/RP)诱导肾上腺嗜铬细胞瘤细胞(PC12细胞)损伤的保护作用及对基质金属蛋白酶-9(MMP-9)表达的影响。
方法以PC12细胞OGD/RP损伤模拟脑缺血再灌注损伤,MTT法检测PC12细胞的存活率,乳酸脱氢酶(LDH)检测PC12细胞死亡率,Hoechst33342检测PC12细胞凋亡,细胞免疫化学法和免疫印迹法检测PC12细胞经OGD/RP诱导后COX-2表达,细胞免疫化学法检测MMP-9表达。
结果OGD/RP诱导PC12细胞损伤,与正常对照组相比,OGD4h-R2h组细胞存活率为(75.5士3.7)%,明显降低(P<0.01)。MTT、LDH和Hoechst33342染色结果显示1μmol/L HSYA预处理能增加OGD/RP诱导后PC12细胞存活率(90.8土7.6)%,降低细胞LDH释放率(109.5士7.0)%,同时减少PC12细胞凋亡率(19.7土7.5)%,与模型组(OGD4h-R2h)相比,均具有显著性差异(P<0.01)。COX-2和MMP-9经OGD/RP处理后均能产生诱导表达,而HSYA能抑制MMP-9表达而不抑制COX-2表达。
结论HSYA在OGD/RP时预先给药对OGD/RP诱导的PC12细胞损伤具有明显保护作用,能减少细胞凋亡,其机制可能是通过抑制MMP-9的诱导表达有关。HSYA作为活血化瘀药预防给药可能减少中风的发生减轻神经损伤。
Stroke is one of the leading causes of mortality and morbidity. Ischaemic stroke accounts for approximately80-85%of all cases and it is characterised by the disruption of cerebral blood flow and lack of oxygen to the affected area. Ischemia/reperfusion injury (IRI) is crucial in the pathology of ischaemic stroke. Oxidative stress has emerged as a key deleterious factor in brain ischemia and reperfusion. Reactive oxygen species are implicated in a number of disease processes. They mediate damage to cell structures, including lipids, membranes, proteins, and DNA. Blood-brain barrier (BBB) leakage and brain edema are critical parts of stroke pathophysiology. Oxidative stress closely relates with the active of matrix metallo proteinase (MMPs), which will damage brain microvascular endothelial cells and their tight junctions to cause BBB dysfunction such as edema, hemorrhage and inflammation.
Therefore, these findings suggest that antioxidants or inhibition of the pathway of cytoplasmic oxidases, such as cyclooxygenase, may be a possible therapeutic strategy of cerebral ischemia. Anti-ischemia drugs, such as Edaravone, have already been marketed in Japan and China. If we can screen out a drug which not only have the effect of antioxidant but also the inhibition effect of the cytoplasmic oxidases, such as cyclooxygenase, NADPH, NOS, MMPs, those drug may be more effective. We aim at screening out the antioxidant from Traditional Chinese Medicine which may be effective on one or more target pathways of ischemia/reperfusion injury.
This study aims at establishing the model of brain ischemia in mouse brain slices and PC12cells. These models will be used to investigate the following questions on oxygen-glucose deprivation/reperfusion induced injury and their possible mechanism.
1. To investigate the protective effects of selective COX-2inhibitors on oxygen-glucose deprivation/reperfusion induced injury and possible mechanism in mouse brain slices and PC12cells.
2. To investigate the protective effects of antioxidantsts such as Ginsenoside Rgl, Paeonol, Quercetin, Astragaloside, Andrographolide, resveratrol, chlorogenicacid, Baicalin and Huanglian-Jie-Du-Tang on oxygen-glucose deprivation/reperfusion induced injury in mouse brain slices and PC12cells.
3. To investigate the neuroprotective effects of hydroxysafflor yellow A (HSYA) on oxygen-glucose deprivation/reperfusion induced injury, and and possible mechanism in PC12cells.
Part1Establishment and improvement of mouse brain slices and injury model of PC12induced by oxygen-glucose deprivation and reperfusion
Objective To establish the model of brain ischemia in mouse brain slices and PC12cells. To investigate the protective effects of selective COX inhibitors on oxygen-glucose deprivation/reperfusion induced injury and possible mechanism in mouse brain slices and PC12cells.
Methods In mouse brain slices, the activity after OGD and reperfusion was assessed by the formation of formazn, a red product of2,3,5-trihenylterzolium chloride (TTC). In PC12cells, the cell viability and damage after OGD and reperfusion were assessed by MTT reduction and LDH. The expression of COX-2protein was assessed by immunocytochemisry.
Results In mouse brain slices, selective COX-2inhibitors (Cel and Eto) significantly attenuated OGD injury while non-selective COX inhibitor Ind had no influence. In PC12cells, Cel and Eto protected the cells from OGD-induced injury at10-160μmol/L. Ind also protected PC12cells at1-10μmol/L. The latest selective COX-2inhibitor Eto increased cell viability and attenuated LDH release obviously in OGD-induced PC12cells. Expression of COX-2protein on OGD PC12cells was also inhibited by the treatment of Eto.
Conclusion The model of oxygen-glucose deprivation/reperfusion induced injury in mouse brain slices and PC12can be used to screen out anti-ischemia drug effectively. Selective COX-2inhibitors, especially the latest selective COX-2inhibitor Eto, have concentration-dependent protective effect on OGD injury in vitro. The possible mechanism may be mediated by the inhibition of COX-2protein expression.
Part2Protective effect of free radical scavenger from Traditional Chinese Medicine on oxygen-glucose deprivation induced injury in mouse brain slices and PC12cells
Objective To investigate the protective effects of antioxidants from traditional chinese medicine on oxygen-glucose deprivation induced injury in mouse brain slices and PC12cells.
Methods In mouse brain slices, the activity after OGD was assessed by the formation of formazn, a red product of2,3,5-trihenylterzolium chloride (TTC). In PC12cells, the cell viability and damage after OGD were assessed by MTT reduction.
Results In mouse brain slices, antioxidants (Astragaloside, resveratrol, chlorogenic acid, Baicalin and safflor yellow A) significantly attenuated OGD injury whereas antioxidants (Ginsenoside Rgl, Paeonol, Quercetin and Andrographolide) had no influence. In PC12cells, Ginsenoside Rgl and Andrographolide protected the cells from OGD-induced cell injury at1μmol/L. Baicalin and safflor yellow A protected PC12cells at0.1-10μmol/L. Resveratrol also protected PC12cells at10μmol/L. Antioxidants (Astragaloside, Paeonol, quercetin and chlorogenic acid) had no influence.
Conclusion Antioxidants (Baicalin and hydroxy safflor yellow A) significantly attenuated OGD induced injury both in mouse brain slices and PC12cells.
Part3Protective effect of hydroxy safflor yellow A on oxygen-glucose deprivation and reperfusion induced injury and its mechanism
Objective To investigate the neuroprotective effects of hydroxyl safflor yellow A (HSYA) on oxygen-glucose deprivation and reperfusion induced injury, and its effect on the expression of cyclooxygenase and metrix metalloproteinase-9(MMP-9) in PC12cells.
Methods The PC12cells were exposed to oxygen-glucose deprivation (OGD), and reperfusioned for different times. The PC12cell viability and lactate dehydrogenase (LDH) release were seperately detected by MTT assays and LDH detecting kits. The apoptotic cells were double stained by propidumiodid (PI) and Hoechst33342. The expression of COX-2was detected by immunocytochemistry and western blot. The expression of MMP-9was detected by immunocytochemistry.
Results Compared to the control group, in OGD4h-R2h group, cell viability significantly decreased to (75.5±3.7)%(P<0.01). HSYA increased cell viability (90.8±7.6)%and reduced releasing of LDH to (109.5±7.0)%(P<0.01) at the concentration of10μmol/L.The result of PI and Hoechst33342showed that HSYA also markedly inhibited the apoptosis of PC12cell induced by OGD/RP, which decreased to (19.7±7.5)%in HSYA1μmol/L group (P<0.01). Meanwhile, the expression of active MMP-9induced by OGD/RP decreased after the treatment of HSYA, similar to positive control, while the expression of active COX-2induced by OGD/RP was not affected.
Conclusion It is proved that HSYA protects the injury and inhibits the apoptosis induced by OGD/RP in PC12cells, and the mechanism may be related to the inhibition of active MMP-9expression.
本课题在细胞和脑片水平上建立高效、稳定的抗脑缺血药物筛选模型,首先通过评价环氧合酶抑制剂在这些模型中的作用,完善药物筛选模型；然后选择一些文献报道有神经保护作用的抗自由基中药,并在我们建立的模型中筛选出对脑缺血再灌注损伤保护作用较强的中药,进行下一步的深入研究,考察其主要作用机制,是否具有多靶点作用机制,分析其在临床的应用前景。本论文主要研究内容为：
1.在细胞、脑片模型上评价环氧合酶抑制剂塞来昔布、依托考昔及吲哚美辛对脑缺血再灌注损伤的保护作用。
2.在细胞、脑片模型上筛选抗自由基中药,如：人参皂苷Rgl、丹皮酚、槲皮素、黄芪甲苷、白黎芦醇、绿原酸、黄芩昔、穿心莲内酯、羟基红花黄色素A和黄连解毒汤对脑缺血再灌注损伤的保护作用。
3.在上述药物中选择一个在细胞、脑片模型中对损伤保护作用较明显的中药有效成分,探讨其对脑缺血再灌注损伤保护作用的机制,分析其临床应用前景。
第一部分OGD/RP诱导的小鼠离体脑片及PC12细胞损伤模型的建立与完善
目的在离体水平上,建立与完善缺氧缺糖再灌注(OGD/RP)诱导的脑片及PC12细胞损伤模型,并在这两种模型上考察COX抑制剂对损伤的保护作用及机制。
方法以小鼠离体脑片建立OGD/RP模型,TTC染色检测小鼠离体脑片的活性；同时以大鼠肾上腺嗜铬细胞瘤细胞株(PC12细胞)OGD/RP损伤模型模拟脑缺血损伤,采用光镜观察细胞形态学变化,MTT法检测细胞存活率,乳酸脱氢酶(LDH)法检测细胞死亡率,免疫细胞化学方法检测环氧合酶-2(COX-2)的表达。
结果在离体小鼠脑片上,COX-2选择性抑制剂塞来昔布(Cel)及依托考昔(Eto)能减少OGD/RP引起的损伤面积,增加吸光度；而COX非选择性抑制剂吲哚美辛(Ind)未见显著性差异。在PC12细胞OGD/RP模型上,Cel、Eto及Ind均能增加OGD/RP损伤后PC12细胞存活率。其中,Eto在10-160μmol/L能显著性增加OGD/RP诱导后PC12细胞的存活率,并降低LDH释放,减少PC12细胞死亡率。同时免疫组织化学检测显示Eto能抑制OGD/RP诱导的COX-2蛋白的表达。
结论本实验建立的OGD/RP诱导的小鼠离体脑片及PC12细胞损伤模型可试用于抗脑缺血药物的筛选；COX-2选择性抑制剂对脑缺血损伤具有保护作用,其中新型一代的Eto在离体小鼠脑片及PC12细胞OGD/RP模型上均有显著性的神经保护作用,其机制可能与抑制COX-2蛋白的诱导表达有关。
第二部分抗自由基中药对OGD/RP诱导的小鼠脑片及PC12细胞损伤的保护作用
目的在离体水平上,筛选抗自由基中药对缺氧缺糖再灌注(OGD/RP)诱导的脑片及PC12细胞缺氧缺糖再灌注(OGD/RP)损伤的保护作用。
方法以小鼠离体脑片建立OGD/RP模型,TTC染色检测小鼠离体脑片的活性；同时以大鼠肾上腺嗜铬细胞瘤细胞株PC12细胞OGD/RP诱导细胞损伤模拟脑缺血损伤模型,MTT法检测细胞存活率。以这几个指标为标准,在细胞和脑片两种损伤模型上考察文献报道有神经保护作用的抗自由基中药,如：人参皂苷Rgl、丹皮酚、槲皮素、黄芪甲苷、白黎芦醇、绿原酸、黄芩苷、穿心莲内酯、羟基红花黄色素A和黄连解毒汤等对损伤保护作用的差别。
结果在离体小鼠脑片上,黄芪甲苷(1μmol/L)、白黎芦醇(1.10μmol/L)、绿原酸(100μmol/L)、黄芩苷(10,100μmol/L)、羟基红花黄色素A (1,10,100μmol/L和FE50(1μmol/L)均能减少OGD/RP引起的损伤面积,增加吸光度；而人参皂苷Rgl、丹皮酚、槲皮素、穿心莲内酯和FE30未见显著性差异。在PC12细胞OGD/RP模型上,人参皂苷Rgl (1μmol/L)、白黎芦醇(10μmol/L)、黄芩苷(0.1,1,10μmol/L)、穿心莲内酯(1μmol/L)、羟基红花黄色素A (0.01,0.1,1,10μmol/L).FE30(1,10μmol/L)和FE50(1,10μmol/L)均能显著性增加OGD/RP诱导后PC12细胞的存活率,减少PC12细胞死亡率；黄芪甲苷、丹皮酚、槲皮素、绿原酸未见显著性差异。
结论黄芩苷和羟基红花黄色素A对两种模型的OGD/RP损伤的保护作用均较强,因此我们考虑可进一步在PC12细胞上进行试验考察其作用机制。
第三部分羟基红花黄色素A对PC12细胞缺氧缺糖再灌注损伤的保护作用及机制研究
目的探讨羟基红花黄色素A(HSYA)对缺氧缺糖再灌注(OGD/RP)诱导肾上腺嗜铬细胞瘤细胞(PC12细胞)损伤的保护作用及对基质金属蛋白酶-9(MMP-9)表达的影响。
方法以PC12细胞OGD/RP损伤模拟脑缺血再灌注损伤,MTT法检测PC12细胞的存活率,乳酸脱氢酶(LDH)检测PC12细胞死亡率,Hoechst33342检测PC12细胞凋亡,细胞免疫化学法和免疫印迹法检测PC12细胞经OGD/RP诱导后COX-2表达,细胞免疫化学法检测MMP-9表达。
结果OGD/RP诱导PC12细胞损伤,与正常对照组相比,OGD4h-R2h组细胞存活率为(75.5士3.7)%,明显降低(P<0.01)。MTT、LDH和Hoechst33342染色结果显示1μmol/L HSYA预处理能增加OGD/RP诱导后PC12细胞存活率(90.8土7.6)%,降低细胞LDH释放率(109.5士7.0)%,同时减少PC12细胞凋亡率(19.7土7.5)%,与模型组(OGD4h-R2h)相比,均具有显著性差异(P<0.01)。COX-2和MMP-9经OGD/RP处理后均能产生诱导表达,而HSYA能抑制MMP-9表达而不抑制COX-2表达。
结论HSYA在OGD/RP时预先给药对OGD/RP诱导的PC12细胞损伤具有明显保护作用,能减少细胞凋亡,其机制可能是通过抑制MMP-9的诱导表达有关。HSYA作为活血化瘀药预防给药可能减少中风的发生减轻神经损伤。
Stroke is one of the leading causes of mortality and morbidity. Ischaemic stroke accounts for approximately80-85%of all cases and it is characterised by the disruption of cerebral blood flow and lack of oxygen to the affected area. Ischemia/reperfusion injury (IRI) is crucial in the pathology of ischaemic stroke. Oxidative stress has emerged as a key deleterious factor in brain ischemia and reperfusion. Reactive oxygen species are implicated in a number of disease processes. They mediate damage to cell structures, including lipids, membranes, proteins, and DNA. Blood-brain barrier (BBB) leakage and brain edema are critical parts of stroke pathophysiology. Oxidative stress closely relates with the active of matrix metallo proteinase (MMPs), which will damage brain microvascular endothelial cells and their tight junctions to cause BBB dysfunction such as edema, hemorrhage and inflammation.
Therefore, these findings suggest that antioxidants or inhibition of the pathway of cytoplasmic oxidases, such as cyclooxygenase, may be a possible therapeutic strategy of cerebral ischemia. Anti-ischemia drugs, such as Edaravone, have already been marketed in Japan and China. If we can screen out a drug which not only have the effect of antioxidant but also the inhibition effect of the cytoplasmic oxidases, such as cyclooxygenase, NADPH, NOS, MMPs, those drug may be more effective. We aim at screening out the antioxidant from Traditional Chinese Medicine which may be effective on one or more target pathways of ischemia/reperfusion injury.
This study aims at establishing the model of brain ischemia in mouse brain slices and PC12cells. These models will be used to investigate the following questions on oxygen-glucose deprivation/reperfusion induced injury and their possible mechanism.
1. To investigate the protective effects of selective COX-2inhibitors on oxygen-glucose deprivation/reperfusion induced injury and possible mechanism in mouse brain slices and PC12cells.
2. To investigate the protective effects of antioxidantsts such as Ginsenoside Rgl, Paeonol, Quercetin, Astragaloside, Andrographolide, resveratrol, chlorogenicacid, Baicalin and Huanglian-Jie-Du-Tang on oxygen-glucose deprivation/reperfusion induced injury in mouse brain slices and PC12cells.
3. To investigate the neuroprotective effects of hydroxysafflor yellow A (HSYA) on oxygen-glucose deprivation/reperfusion induced injury, and and possible mechanism in PC12cells.
Part1Establishment and improvement of mouse brain slices and injury model of PC12induced by oxygen-glucose deprivation and reperfusion
Objective To establish the model of brain ischemia in mouse brain slices and PC12cells. To investigate the protective effects of selective COX inhibitors on oxygen-glucose deprivation/reperfusion induced injury and possible mechanism in mouse brain slices and PC12cells.
Methods In mouse brain slices, the activity after OGD and reperfusion was assessed by the formation of formazn, a red product of2,3,5-trihenylterzolium chloride (TTC). In PC12cells, the cell viability and damage after OGD and reperfusion were assessed by MTT reduction and LDH. The expression of COX-2protein was assessed by immunocytochemisry.
Results In mouse brain slices, selective COX-2inhibitors (Cel and Eto) significantly attenuated OGD injury while non-selective COX inhibitor Ind had no influence. In PC12cells, Cel and Eto protected the cells from OGD-induced injury at10-160μmol/L. Ind also protected PC12cells at1-10μmol/L. The latest selective COX-2inhibitor Eto increased cell viability and attenuated LDH release obviously in OGD-induced PC12cells. Expression of COX-2protein on OGD PC12cells was also inhibited by the treatment of Eto.
Conclusion The model of oxygen-glucose deprivation/reperfusion induced injury in mouse brain slices and PC12can be used to screen out anti-ischemia drug effectively. Selective COX-2inhibitors, especially the latest selective COX-2inhibitor Eto, have concentration-dependent protective effect on OGD injury in vitro. The possible mechanism may be mediated by the inhibition of COX-2protein expression.
Part2Protective effect of free radical scavenger from Traditional Chinese Medicine on oxygen-glucose deprivation induced injury in mouse brain slices and PC12cells
Objective To investigate the protective effects of antioxidants from traditional chinese medicine on oxygen-glucose deprivation induced injury in mouse brain slices and PC12cells.
Methods In mouse brain slices, the activity after OGD was assessed by the formation of formazn, a red product of2,3,5-trihenylterzolium chloride (TTC). In PC12cells, the cell viability and damage after OGD were assessed by MTT reduction.
Results In mouse brain slices, antioxidants (Astragaloside, resveratrol, chlorogenic acid, Baicalin and safflor yellow A) significantly attenuated OGD injury whereas antioxidants (Ginsenoside Rgl, Paeonol, Quercetin and Andrographolide) had no influence. In PC12cells, Ginsenoside Rgl and Andrographolide protected the cells from OGD-induced cell injury at1μmol/L. Baicalin and safflor yellow A protected PC12cells at0.1-10μmol/L. Resveratrol also protected PC12cells at10μmol/L. Antioxidants (Astragaloside, Paeonol, quercetin and chlorogenic acid) had no influence.
Conclusion Antioxidants (Baicalin and hydroxy safflor yellow A) significantly attenuated OGD induced injury both in mouse brain slices and PC12cells.
Part3Protective effect of hydroxy safflor yellow A on oxygen-glucose deprivation and reperfusion induced injury and its mechanism
Objective To investigate the neuroprotective effects of hydroxyl safflor yellow A (HSYA) on oxygen-glucose deprivation and reperfusion induced injury, and its effect on the expression of cyclooxygenase and metrix metalloproteinase-9(MMP-9) in PC12cells.
Methods The PC12cells were exposed to oxygen-glucose deprivation (OGD), and reperfusioned for different times. The PC12cell viability and lactate dehydrogenase (LDH) release were seperately detected by MTT assays and LDH detecting kits. The apoptotic cells were double stained by propidumiodid (PI) and Hoechst33342. The expression of COX-2was detected by immunocytochemistry and western blot. The expression of MMP-9was detected by immunocytochemistry.
Results Compared to the control group, in OGD4h-R2h group, cell viability significantly decreased to (75.5±3.7)%(P<0.01). HSYA increased cell viability (90.8±7.6)%and reduced releasing of LDH to (109.5±7.0)%(P<0.01) at the concentration of10μmol/L.The result of PI and Hoechst33342showed that HSYA also markedly inhibited the apoptosis of PC12cell induced by OGD/RP, which decreased to (19.7±7.5)%in HSYA1μmol/L group (P<0.01). Meanwhile, the expression of active MMP-9induced by OGD/RP decreased after the treatment of HSYA, similar to positive control, while the expression of active COX-2induced by OGD/RP was not affected.
Conclusion It is proved that HSYA protects the injury and inhibits the apoptosis induced by OGD/RP in PC12cells, and the mechanism may be related to the inhibition of active MMP-9expression.
引文
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