ACE2在脑缺血损伤中的作用及灯盏乙素的神经保护作用研究
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摘要
缺血性脑血管病(ischemia cerebral vascular disease,ICVD)是严重危害人类健康的疾病,具有发病率、致残率和死亡率高的特点,是中老年人致死和致残的主要疾病。为了对脑缺血损伤进行有的放矢的治疗,世界各国的科研人员对其发病机制进行了广泛的研究,提出了能量代谢障碍、钙超载、兴奋性氨基酸毒性、毒性氧自由基、炎性反应和损伤等学说,为解释脑缺血的病理生理机制奠定了基础。一些治疗脑缺血的药物如钙通道阻滞药、自由基清除剂、兴奋性氨基酸拮抗剂等应运而生,但迄今尚无理想的治疗药物。因此研究缺血性脑血管病的发生发展机制及寻找有效的治疗药物具有重要的理论价值和应用价值。本课题包括两部分研究内容:1.研究血管紧张素转化酶2(ACE2)在脑缺血损伤中的作用及其调控机制,探讨ACE2表达与脑缺血损伤的关系;2.研究灯盏乙素对缺氧所致PC12细胞凋亡的作用及其机制,探讨灯盏乙素防治脑缺血损伤的潜在应用价值。
一、ACE2在脑缺血损伤中的作用与机制研究
肾素-血管紧张素系统(renin-angiotensin system,RAS)是机体重要的体液调节系统,血管紧张素Ⅱ(AngⅡ)是RAS的主要活性物质,研究表明,AngⅡ在脑缺血损伤过程中发挥着重要作用,抑制AngⅡ已成为目前治疗缺血性脑血管病的一个重要途径。2000年,研究发现一种新型羧基肽酶血管紧张素转化酶2(ACE2),可将AngⅡ转化为Ang(1-7)。目前认为,ACE2对RAS可能起着负调控作用,其功能主要通过两方面产生,一方面灭活AngⅡ而限制其作用,另一方面生成Ang(1-7),Ang(1-7)具有促进血管舒张、抗生长、抗增殖等作用,可以抵消AngⅡ的作用。由于脑中存在有ACE2和Ang(1-7)的分布,且AngⅡ在脑缺血发病过程中的重要作用已经得到证实,因此,我们推测ACE2可能在脑缺血发病过程中发挥重要的调节作用。本研究利用行为学、形态学、生物化学和分子生物学等方法,从整体动物和体外培养细胞两方面研究ACE2在脑缺血损伤中的作用及机制。
采用大脑中动脉内栓线阻断法(MCAO)制备大鼠局灶性脑缺血损伤模型。随机分为假手术组、缺血6h组、缺血12h组、缺血6h+氯沙坦(5mg/kg)组、缺血6h+卡托普利组(5mg/kg),分别检测下列指标:神经功能学评分;TTC染色法测定脑梗死范围;RT-PCR法检测ACE2 mRNA表达变化;免疫组织化学法检测脑组织ACE2蛋白表达变化。与假手术组相比,缺血组出现明显神经功能障碍和显著的脑梗死范围,与缺血时间呈正相关关系。卡托普利组和氯沙坦组均可明显改善神经功能障碍和减低脑梗死面积。RT-PCR结果显示,与假手术组相比,缺血组大鼠缺血区脑组织ACE2 mRNA表达明显增加,氯沙坦和卡托普利均可增加ACE2 mRNA表达,二者相比,氯沙坦作用更为明显。免疫组化结果显示,与假手术组相比,缺血组大鼠缺血区皮质神经元细胞、海马神经元细胞和星形胶质细胞ACE2蛋白表达均明显增加;与缺血组相比,氯沙坦和卡托普利均可进一步增加ACE2蛋白表达,二者相比,氯沙坦作用更为明显。以上结果表明,脑缺血损伤可使脑组织ACE2表达增加,AT1受体阻断剂氯沙坦和血管紧张素转化酶抑制剂卡托普利均可增加ACE2表达并发挥脑缺血损伤保护作用,提示ACE2高表达与保护脑缺血损伤可能存在关联性,并且AngⅡ可能参与了ACE2的表达过程。为进一步证实上述推理,我们进行了以下实验:
分离、培养大鼠脑皮质星形胶质细胞,构建缺氧(95%N_2+5%CO_2)致细胞损伤模型,实验分组:正常对照组、缺氧2h组、缺氧4h组、缺氧8h组、缺氧4h+10~(-7)mol/L氯沙坦组、缺氧4h+10~(-7)mol/L PD123319组。通过细胞活性染色、LDH漏出率测定、ATP生成量等指标检测细胞活性,RT-PCR法检测ACE2 mRNA表达变化;Western blot法检测ACE2蛋白质表达变化。与正常对照组相比,缺氧组细胞存活率和ATP生成量明显降低,与缺氧诱导时间呈负相关关系;缺氧组细胞培养液LDH含量明显升高,与缺氧诱导时间呈正相关关系。与缺氧4h组相比,氯沙坦可提高细胞存活率和ATP生成量,降低细胞培养液LDH含量,PDl23319对细胞存活率、ATP生成量和细胞培养液LDH含量无影响。RT-PCR结果显示,与正常对照组相比,各缺氧组细胞ACE2 mRNA表达均明显增加,与缺氧诱导时间呈正相关关系;与缺氧组相比,氯沙坦可增加ACE2 mRNA表达,PD123319对ACE2 mRNA表达无影响。Western blot结果显示,与正常对照组相比,各缺氧组细胞ACE2蛋白表达均明显增加,与缺氧诱导时间呈正相关关系;与缺氧组相比,氯沙坦可增加ACE2蛋白表达,PD123319对ACE2蛋白表达无影响。以上结果表明,缺氧损伤可使细胞内ACE2表达增加,氯沙坦可增强ACE2表达并发挥抗缺氧损伤作用,而AT2受体阻断剂PD123319则无此作用,进一步提示ACE2高表达与抗缺氧损伤存在关联性,且ACE2表达与AT1受体有关,与AT2受体无关。
利用ACE2高表达质粒pcDNA-ACE2,分别转染大鼠脑皮质星形胶质细胞12h、24h、48h,RT-PCR法和Western blot法进行鉴定,结果表明,pcDNA-ACE2转染效率与转染时间呈正相关关系,转染48h时效率最高。实验分组:pcDNA3.1空白对照组、pcDNA3.1+缺氧4h组、pcDNA-ACE2+缺氧4h组,检测细胞活性染色、ATP生成量和细胞培养液LDH含量,观察ACE2高表达对缺氧(95%N_2+5%CO_2)所致细胞损伤的作用。结果表明,与pcDNA3.1空白对照组相比,缺氧(95%N_2+5%CO_2)4h可明显降低细胞存活率和ATP生成量,并显著增加细胞培养液LDH含量;与缺氧组相比,ACE2高表达可明显提高细胞存活率和ATP生成量,并显著降低培养液LDH含量。结果表明,ACE2高表达对缺氧所致细胞损伤具有保护作用,证实了ACE2高表达和抗缺氧损伤之间的关联性。
为进一步研究调控ACE2表达的机制,我们构建了AngⅡ致大鼠脑皮质星形胶质细胞损伤模型,实验分组:正常对照组、10~(-8)mol/L AngⅡ组、10~(-7)mol/L AngⅡ组、10~(-6)mol/L AngⅡ组、10~(-7)mol/L AngⅡ+10~(-7)mol/L氯沙坦组、10~(-7)mol/LAngⅡ+10~(-7)mol/L PD123319组。通过细胞活性染色、LDH漏出率测定、ATP生成量等指标检测细胞活性,RT-PCR法检测ACE2 mRNA表达变化;Western blot法检测ACE2蛋白质表达变化。与正常对照组相比,AngⅡ组细胞存活率和ATP生成量明显降低,细胞培养液LDH含量明显升高。与10~(-7)mol/L AngⅡ组相比,氯沙坦可明显提高细胞存活率和ATP生成量,降低细胞培养液LDH含量,而PD123319无明显作用。与正常对照组相比,AngⅡ组细胞ACE2 mRNA和蛋白表达均明显降低,且与AngⅡ诱导浓度呈负相关关系,氯沙坦可明显增加ACE2 mRNA和蛋白表达,PD123319无明显作用。结果表明,AngⅡ可抑制ACE2表达并导致细胞损伤,此作用通过AT1受体介导。
综上所述,脑缺血缺氧损伤可使ACE2表达增加,AngⅡ可抑制ACE2表达,氯沙坦可通过阻断AT1受体增加ACE2表达而发挥脑缺血保护作用。研究结果将为进一步理解脑缺血损伤的发生机制提供实验依据和理论基础。
二、灯盏乙素对氯化钻诱导PC12细胞凋亡的保护作用研究
ICVD发病机制复杂,但最终都会造成神经细胞的损伤,进而影响患者的预后。研究表明,神经细胞凋亡在ICVD中起着非常重要的作用,而缺氧所致氧化损伤是造成神经细胞凋亡的一个重要因素,因此,寻找新的抗氧化损伤和保护神经细胞的药物成为治疗缺血性脑血管病的研究热点之一。灯盏乙素是灯盏花提取物中的主要有效成分,以往研究表明,灯盏花提取物具有抗氧化作用。为了从细胞水平探讨灯盏乙素对神经细胞凋亡的保护作用,本研究采用氯化钴(CoCl_2)作为外源性化学缺氧剂,利用PC12细胞模拟神经细胞反应,建立体外培养的神经细胞凋亡模型,研究神经细胞的凋亡机制并进一步探讨灯盏乙素对神经细胞凋亡的可能保护作用。
无菌条件下进行PC12细胞培养。首先,利用MTT染色法确定CoCl_2诱导PC12细胞凋亡的最佳浓度和时间。然后,取同一代PC12细胞,待细胞生长至亚融合状态时,随机分为5组:正常对照组、CoCl_2诱导组、灯盏乙素低浓度组(0.1μmol/L)、灯盏乙素中浓度组(1μmol/L)、灯盏乙素高浓度组(10μmol/L)。MTT法检测PC12细胞存活率;倒置相差显微镜下进行一般形态学观察;透射电子显微镜下进行超微结构观察;流式细胞术测定PC12细胞凋亡率;化学发光法检测活性氧(ROS)生成和Caspase-3活性变化;Western Blot法测定Bax、Bcl-2、Bcl-X_L、p38、P-p38的表达变化。
结果显示,PC12细胞存活率与CoCl_2诱导浓度和诱导时间呈负相关,500μmol/L CoCl_2诱导24h可引起明显的PC12细胞凋亡,倒置相差显微镜下一般形态学观察及透射电子显微镜下超微结构观察可见明显的细胞凋亡形态,流式细胞术检测显示PC12细胞凋亡率明显升高,灯盏乙素各剂量组均可提高PC12细胞存活率,并明显改善CoCl_2所致PC12细胞形态损伤,降低PC12细胞凋亡百分率。500μmol/L CoCl_2诱导24h可致PC12细胞Caspase-3活性增加、Bcl-X_L表达下降、p38 MAPK磷酸化和ROS生成增加,灯盏乙素各剂量组均可抑制Caspase-3活性、促进Bcl-XL表达、抑制p38 MAPK磷酸化和降低ROS生成。
综上所述,灯盏乙素可能通过清除ROS、抑制p38 MAPK磷酸化、上调Bcl-X_L表达,进而降低Caspase-3活性,从而保护CoCl_2所致PC12细胞凋亡。结果提示,灯盏乙素对缺氧所致神经细胞凋亡具有保护作用,为灯盏乙素治疗相关疾病提供了理论依据和实验基础。
Stroke represents the first leading cause of death in China and the most important cause of chronic disability.Cerebral ischemia is caused by a deficiency in blood supply to a part of brain,which in turn triggers various pathophysiological changes.It has been accepted recently that ischemic cell injury arises from complex interactions between multiple electrophysiological, hemodynamic,and biochemical cascades,which include disturbances in energy metabolism,modifications in synaptic transmission,production of reactive oxygen species,stimulation of the inflammatory process,endothelial dysfunction, et al.All neuroprotective agents so far targeting a specific pathway in ischemic cascade failed to demonstrate clinical efficacy.Therefore,the search for novel mechanism and therapeutic approaches is even more critical.There are two parts in this study:one is to investigate the role of ACE2 in the brain ischemia injury, the other is to study the protective effect of scutellarin on the hypoxia-induced injury in PC12 cells.
PartⅠ:The role of ACE2 in rat brain ischemia injury
The renin-angiotensin system(RAS) is a very important regulator for the physiological function and angiotensinⅡ(AngⅡ) is the major component of RAS.It is reported that AngⅡacts important roles in brain ischemia injury and inhibiting AngⅡ is becoming an new approach in stroke therapy.In 2000,angiotensin converting enzyme 2(ACE2) was found.ACE2 can catelyze AngⅡto Ang(1-7) which has the opposite actions of AngⅡ.It is believed that ACE2 may act as a negative regulator in RAS through two ways:one is inactivating AngⅡand the other is producing Ang(1-7).Since both ACE2 and Ang(1-7) are distributed in brain and the action of AngⅡin brain ischemia injury is proved,we assume that ACE2 may acts important functions in brain ischemia injury.In this study,we studied the role of ACE2 in brain ischemia injury both in vivo and in vitro.
Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO).The rats were divided into 5 groups randomly:sham,ischemia 6h, ischemia 12h,ischemia 6h + 5mg/kg losartan,ischemia 6h + 5mg/kg captopril. The behavioral tests and the infarct area by 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the brain injury.RT-PCR was used to examine the ACE2 mRNA expression and immunohistochemistry staining was used to evaluate the ACE2 protein level.Comparing with the sham group,the obvious neurological disorders and the enlarged infarction areas were found in the ischemia group.Both ACE2 mRNA and ACE2 protein were increased after brain ischemia injury.Compared with the ischemia group,both losartan and captopril improved the neurological functions,decreased the infarction area and increased the expression of ACE2 in both mRNA and the protein level.These results show that the brain ischemia injury can increase ACE2 expression.Both the AT1 receptor blocker losartan and angiotensin converting enzyme inhibitor captopril can increase ACE2 expression and manage the protective action against brain ischemia injury.It point out that the high expression of ACE2 perhaps has relation to the anti-ischeimic action in brain and AngⅡmay be involved in the expression of ACE2. To further verify the reasoning,several experiments were carried in vitro.
Using the cultured rat cortical astrocyte,the hypoxia(95%N_2+5%CO_2) induced cell injury was examined.Cells were divided into 6 groups:control,hypoxia 2h, hypoxia 4h,hypoxia 8h,hypoxia 4h + 10~(-7)mol/L losartan,hypoxia 4h + 10~(-7)mol/L PD123319.Cell viability was assessed by methylthiazol tetrazolium(MTT) assay and cell injury by the rate of lactate dehydrogenase(LDH) release and adenosine triphosphate(ATP) production.RT-PCR was used to examine the mRNA expression of ACE2 and western blot analysis was used to evaluate the protein level of ACE2.Comparing with the control group,decrease of cell viability and ATP concentration and increase of LDH release were found in the hypoxia groups. Losartan increased the cell viability and ATP concentration and decreased the LDH release.PD123319 had no effect on cell viability and cell injury.Both the mRNA and the protein of ACE2 were increased after hypoxia injury.Losartan increased the expression of ACE2 in both mRNA and the protein level,while PD123319 had no effect on ACE2 expression.These results show that the hypoxia injury can increase ACE2 expression and the AT1 receptor,not the AT2 receptor is involved in the expression of ACE2.It further reminder that the high expression of ACE2 has relation to the anti-hypoxic injury.
To further confirm the the action of high expression of ACE2 on the hypoxic injury,we transfected the rat brain astrocytes with a plasmid pcDNA-ACE2 which highly expressed ACE2.The efficiency of transfection was examined by RT-PCR and western blot analysis.Cells were divided into 3 groups:pcDNA3.1(control), pcDNA3.1+hypoxia 4h,pcDNA-ACE2+hypoxia 4h.Cell viability was assessed by MTT assay and cell injury by the rate of LDH release and ATP production. Comparing with the control group,decrease of cell viability and ATP concentration and increase of LDH release were found in the hypoxia groups. Compared with the hypoxia group,both the cell viability and ATP production were increased and the LDH release was decreased in pcDNA-ACE2 group.These results show that the high expression of ACE2 can protect against the hypoxia injury in rat brain astrocytes.It confirmed the relation between the high ACE2 expression and the anti-hypoxic ation.
To further explore the regulation of ACE2 expression,the AngⅡ-induced brain astrocyte injury was examined.Cells were divided into 6 groups:control,10~(-8)mol/L AngⅡ,10~(-7)mol/L AngⅡ,10~(-6)mol/L AngⅡ,10~(-7)mol/L AngⅡ+10~(-7)mol/L losartan, 10~(-7)mol/L AngⅡ+10~(-7)mol/L PD123319.Cell viability was assessed by MTT assay and cell injury by the rate LDH release and ATP production.RT-PCR was used to examine the mRNA expression of ACE2 and western blot analysis was used to evaluate the protein level of ACE2.Comparing with the control group,decrease of cell viability and ATP production and increase of LDH release were found in the hypoxia groups.Losartan increased the cell viability and ATP concentration and decreased the LDH release,but PD123319 had no effect on it.Both the mRNA and the protein of ACE2 was decreased after AngⅡinjury.Losartan increased the expression of ACE2 in both mRNA and the protein level,but PD123319 has no effect on the expression of ACE2.These results show that AngⅡcan inhibit the ACE2 expression via AT1 receptor.
In conclusion,ACE2 expression was increased after the brain ischemia injury in vivo and the hypoxia injury in vitro.AngⅡcan inhibit the expression of ACE2,while losartan can antagonized its function through an AT1 pathway.All the results provide a deep understanding on RAS in the brain ichemia injury.
PartⅡ:Effects of scutellarin on apoptosis induced by CoCl_2 in PC12 cells
The mechanisms of ischemia cerebral vascular disease(ICVD) are complex,and it can result in the injury of neurons.Recent research suggests that the apoptosis of neurons play a major role in ICVD,and hypoxia is one of the important factors which can induce apoptosis.Many researchs suggested that scutellarin has the ability of anti-oxidation.This investigation was preformed to study the detailed characterization of PC12 cells responses to the presence of cobalt chloride(CoCl_2) and evaluate the protective effects of scutellarin on the hypoxia-induced apoptosis in PC12 cells.
PC12 cells were cultured and passaged in DMEM.Experiments were performed with cells from the same passage.As grown to 80%confluence in DMEM with 10% FBS,PC12 cells were divided into 5 groups:control group,500μmol/L CoCl_2 group and scutellarin 0.1,1,10μmol/L groups.The drug-treated groups were pre-incubated with scutellarin for 2 hours.Other groups with the exception of the normal one were stimulated by CoCl_2(500μmol/L) simultaneously for 24 hours.All the above steps were carried out under sterile conditions.The viability of cells was detected by MTT assay. Morphological changes of PC12 cells were visualized under light and electron microscopes.Flow cytometry was employed to observe the occurrence of apoptosis. Both the reactive oxygen species(ROS) generation and Caspase-3 activity were quantified by their reagents and the absorbance was assessed by fluorescence spectrophotometer.The expression of Bax、Bcl-2、Bcl-X_L、p38 and P-p38 was examined by western blot.
The results show that CoCl_2 triggers neuronal PC12 cells apoptosis in a dose- and time-dependent manner,and the best concentration and time of CoCl_2-induced PC12 apoptosis are 500μmol/L and 24 h,respectively.Incubation of PC12 cells with 500μmol/L CoCl_2 for 24 h resulted in significant apoptosis as evaluated by the MTT assay,electron microscopy and flow cytometry assays.The increase of Caspase-3 activity,decrease of Bcl-X_L expression,phosphorylation of p38 mitogen-activated protein kinase(MAPK) and accumulation of intracellular ROS were also seen in CoCl_2-treated PC12 cells.Scutellarin at 0.1,1 and 10μmol/L significantly protected against the apoptotic cell death induced by CoCl_2.Scutellarin decreased Caspase-3 activity,increased Bcl-X_L expression,inhibited p38 phosphorylation and attenuated ROS production.These results demonstrate that scutellarin can protect PC12 cells from CoCl_2 induced apoptosis by scavenging ROS,inhibiting p38 phosphorylation, up-regulating Bcl-X_L expression and decreasing Caspase-3 activity,and may reduce the cellular damage in pathological conditions associated with hypoxia-mediated neuronal injury
This research shows that scutellarin can protect PC12 cells from CoCl_2-induced apoptosis which indicates scutellarin has remarkable ability on rescuing ICVD.
一、ACE2在脑缺血损伤中的作用与机制研究
肾素-血管紧张素系统(renin-angiotensin system,RAS)是机体重要的体液调节系统,血管紧张素Ⅱ(AngⅡ)是RAS的主要活性物质,研究表明,AngⅡ在脑缺血损伤过程中发挥着重要作用,抑制AngⅡ已成为目前治疗缺血性脑血管病的一个重要途径。2000年,研究发现一种新型羧基肽酶血管紧张素转化酶2(ACE2),可将AngⅡ转化为Ang(1-7)。目前认为,ACE2对RAS可能起着负调控作用,其功能主要通过两方面产生,一方面灭活AngⅡ而限制其作用,另一方面生成Ang(1-7),Ang(1-7)具有促进血管舒张、抗生长、抗增殖等作用,可以抵消AngⅡ的作用。由于脑中存在有ACE2和Ang(1-7)的分布,且AngⅡ在脑缺血发病过程中的重要作用已经得到证实,因此,我们推测ACE2可能在脑缺血发病过程中发挥重要的调节作用。本研究利用行为学、形态学、生物化学和分子生物学等方法,从整体动物和体外培养细胞两方面研究ACE2在脑缺血损伤中的作用及机制。
采用大脑中动脉内栓线阻断法(MCAO)制备大鼠局灶性脑缺血损伤模型。随机分为假手术组、缺血6h组、缺血12h组、缺血6h+氯沙坦(5mg/kg)组、缺血6h+卡托普利组(5mg/kg),分别检测下列指标:神经功能学评分;TTC染色法测定脑梗死范围;RT-PCR法检测ACE2 mRNA表达变化;免疫组织化学法检测脑组织ACE2蛋白表达变化。与假手术组相比,缺血组出现明显神经功能障碍和显著的脑梗死范围,与缺血时间呈正相关关系。卡托普利组和氯沙坦组均可明显改善神经功能障碍和减低脑梗死面积。RT-PCR结果显示,与假手术组相比,缺血组大鼠缺血区脑组织ACE2 mRNA表达明显增加,氯沙坦和卡托普利均可增加ACE2 mRNA表达,二者相比,氯沙坦作用更为明显。免疫组化结果显示,与假手术组相比,缺血组大鼠缺血区皮质神经元细胞、海马神经元细胞和星形胶质细胞ACE2蛋白表达均明显增加;与缺血组相比,氯沙坦和卡托普利均可进一步增加ACE2蛋白表达,二者相比,氯沙坦作用更为明显。以上结果表明,脑缺血损伤可使脑组织ACE2表达增加,AT1受体阻断剂氯沙坦和血管紧张素转化酶抑制剂卡托普利均可增加ACE2表达并发挥脑缺血损伤保护作用,提示ACE2高表达与保护脑缺血损伤可能存在关联性,并且AngⅡ可能参与了ACE2的表达过程。为进一步证实上述推理,我们进行了以下实验:
分离、培养大鼠脑皮质星形胶质细胞,构建缺氧(95%N_2+5%CO_2)致细胞损伤模型,实验分组:正常对照组、缺氧2h组、缺氧4h组、缺氧8h组、缺氧4h+10~(-7)mol/L氯沙坦组、缺氧4h+10~(-7)mol/L PD123319组。通过细胞活性染色、LDH漏出率测定、ATP生成量等指标检测细胞活性,RT-PCR法检测ACE2 mRNA表达变化;Western blot法检测ACE2蛋白质表达变化。与正常对照组相比,缺氧组细胞存活率和ATP生成量明显降低,与缺氧诱导时间呈负相关关系;缺氧组细胞培养液LDH含量明显升高,与缺氧诱导时间呈正相关关系。与缺氧4h组相比,氯沙坦可提高细胞存活率和ATP生成量,降低细胞培养液LDH含量,PDl23319对细胞存活率、ATP生成量和细胞培养液LDH含量无影响。RT-PCR结果显示,与正常对照组相比,各缺氧组细胞ACE2 mRNA表达均明显增加,与缺氧诱导时间呈正相关关系;与缺氧组相比,氯沙坦可增加ACE2 mRNA表达,PD123319对ACE2 mRNA表达无影响。Western blot结果显示,与正常对照组相比,各缺氧组细胞ACE2蛋白表达均明显增加,与缺氧诱导时间呈正相关关系;与缺氧组相比,氯沙坦可增加ACE2蛋白表达,PD123319对ACE2蛋白表达无影响。以上结果表明,缺氧损伤可使细胞内ACE2表达增加,氯沙坦可增强ACE2表达并发挥抗缺氧损伤作用,而AT2受体阻断剂PD123319则无此作用,进一步提示ACE2高表达与抗缺氧损伤存在关联性,且ACE2表达与AT1受体有关,与AT2受体无关。
利用ACE2高表达质粒pcDNA-ACE2,分别转染大鼠脑皮质星形胶质细胞12h、24h、48h,RT-PCR法和Western blot法进行鉴定,结果表明,pcDNA-ACE2转染效率与转染时间呈正相关关系,转染48h时效率最高。实验分组:pcDNA3.1空白对照组、pcDNA3.1+缺氧4h组、pcDNA-ACE2+缺氧4h组,检测细胞活性染色、ATP生成量和细胞培养液LDH含量,观察ACE2高表达对缺氧(95%N_2+5%CO_2)所致细胞损伤的作用。结果表明,与pcDNA3.1空白对照组相比,缺氧(95%N_2+5%CO_2)4h可明显降低细胞存活率和ATP生成量,并显著增加细胞培养液LDH含量;与缺氧组相比,ACE2高表达可明显提高细胞存活率和ATP生成量,并显著降低培养液LDH含量。结果表明,ACE2高表达对缺氧所致细胞损伤具有保护作用,证实了ACE2高表达和抗缺氧损伤之间的关联性。
为进一步研究调控ACE2表达的机制,我们构建了AngⅡ致大鼠脑皮质星形胶质细胞损伤模型,实验分组:正常对照组、10~(-8)mol/L AngⅡ组、10~(-7)mol/L AngⅡ组、10~(-6)mol/L AngⅡ组、10~(-7)mol/L AngⅡ+10~(-7)mol/L氯沙坦组、10~(-7)mol/LAngⅡ+10~(-7)mol/L PD123319组。通过细胞活性染色、LDH漏出率测定、ATP生成量等指标检测细胞活性,RT-PCR法检测ACE2 mRNA表达变化;Western blot法检测ACE2蛋白质表达变化。与正常对照组相比,AngⅡ组细胞存活率和ATP生成量明显降低,细胞培养液LDH含量明显升高。与10~(-7)mol/L AngⅡ组相比,氯沙坦可明显提高细胞存活率和ATP生成量,降低细胞培养液LDH含量,而PD123319无明显作用。与正常对照组相比,AngⅡ组细胞ACE2 mRNA和蛋白表达均明显降低,且与AngⅡ诱导浓度呈负相关关系,氯沙坦可明显增加ACE2 mRNA和蛋白表达,PD123319无明显作用。结果表明,AngⅡ可抑制ACE2表达并导致细胞损伤,此作用通过AT1受体介导。
综上所述,脑缺血缺氧损伤可使ACE2表达增加,AngⅡ可抑制ACE2表达,氯沙坦可通过阻断AT1受体增加ACE2表达而发挥脑缺血保护作用。研究结果将为进一步理解脑缺血损伤的发生机制提供实验依据和理论基础。
二、灯盏乙素对氯化钻诱导PC12细胞凋亡的保护作用研究
ICVD发病机制复杂,但最终都会造成神经细胞的损伤,进而影响患者的预后。研究表明,神经细胞凋亡在ICVD中起着非常重要的作用,而缺氧所致氧化损伤是造成神经细胞凋亡的一个重要因素,因此,寻找新的抗氧化损伤和保护神经细胞的药物成为治疗缺血性脑血管病的研究热点之一。灯盏乙素是灯盏花提取物中的主要有效成分,以往研究表明,灯盏花提取物具有抗氧化作用。为了从细胞水平探讨灯盏乙素对神经细胞凋亡的保护作用,本研究采用氯化钴(CoCl_2)作为外源性化学缺氧剂,利用PC12细胞模拟神经细胞反应,建立体外培养的神经细胞凋亡模型,研究神经细胞的凋亡机制并进一步探讨灯盏乙素对神经细胞凋亡的可能保护作用。
无菌条件下进行PC12细胞培养。首先,利用MTT染色法确定CoCl_2诱导PC12细胞凋亡的最佳浓度和时间。然后,取同一代PC12细胞,待细胞生长至亚融合状态时,随机分为5组:正常对照组、CoCl_2诱导组、灯盏乙素低浓度组(0.1μmol/L)、灯盏乙素中浓度组(1μmol/L)、灯盏乙素高浓度组(10μmol/L)。MTT法检测PC12细胞存活率;倒置相差显微镜下进行一般形态学观察;透射电子显微镜下进行超微结构观察;流式细胞术测定PC12细胞凋亡率;化学发光法检测活性氧(ROS)生成和Caspase-3活性变化;Western Blot法测定Bax、Bcl-2、Bcl-X_L、p38、P-p38的表达变化。
结果显示,PC12细胞存活率与CoCl_2诱导浓度和诱导时间呈负相关,500μmol/L CoCl_2诱导24h可引起明显的PC12细胞凋亡,倒置相差显微镜下一般形态学观察及透射电子显微镜下超微结构观察可见明显的细胞凋亡形态,流式细胞术检测显示PC12细胞凋亡率明显升高,灯盏乙素各剂量组均可提高PC12细胞存活率,并明显改善CoCl_2所致PC12细胞形态损伤,降低PC12细胞凋亡百分率。500μmol/L CoCl_2诱导24h可致PC12细胞Caspase-3活性增加、Bcl-X_L表达下降、p38 MAPK磷酸化和ROS生成增加,灯盏乙素各剂量组均可抑制Caspase-3活性、促进Bcl-XL表达、抑制p38 MAPK磷酸化和降低ROS生成。
综上所述,灯盏乙素可能通过清除ROS、抑制p38 MAPK磷酸化、上调Bcl-X_L表达,进而降低Caspase-3活性,从而保护CoCl_2所致PC12细胞凋亡。结果提示,灯盏乙素对缺氧所致神经细胞凋亡具有保护作用,为灯盏乙素治疗相关疾病提供了理论依据和实验基础。
Stroke represents the first leading cause of death in China and the most important cause of chronic disability.Cerebral ischemia is caused by a deficiency in blood supply to a part of brain,which in turn triggers various pathophysiological changes.It has been accepted recently that ischemic cell injury arises from complex interactions between multiple electrophysiological, hemodynamic,and biochemical cascades,which include disturbances in energy metabolism,modifications in synaptic transmission,production of reactive oxygen species,stimulation of the inflammatory process,endothelial dysfunction, et al.All neuroprotective agents so far targeting a specific pathway in ischemic cascade failed to demonstrate clinical efficacy.Therefore,the search for novel mechanism and therapeutic approaches is even more critical.There are two parts in this study:one is to investigate the role of ACE2 in the brain ischemia injury, the other is to study the protective effect of scutellarin on the hypoxia-induced injury in PC12 cells.
PartⅠ:The role of ACE2 in rat brain ischemia injury
The renin-angiotensin system(RAS) is a very important regulator for the physiological function and angiotensinⅡ(AngⅡ) is the major component of RAS.It is reported that AngⅡacts important roles in brain ischemia injury and inhibiting AngⅡ is becoming an new approach in stroke therapy.In 2000,angiotensin converting enzyme 2(ACE2) was found.ACE2 can catelyze AngⅡto Ang(1-7) which has the opposite actions of AngⅡ.It is believed that ACE2 may act as a negative regulator in RAS through two ways:one is inactivating AngⅡand the other is producing Ang(1-7).Since both ACE2 and Ang(1-7) are distributed in brain and the action of AngⅡin brain ischemia injury is proved,we assume that ACE2 may acts important functions in brain ischemia injury.In this study,we studied the role of ACE2 in brain ischemia injury both in vivo and in vitro.
Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO).The rats were divided into 5 groups randomly:sham,ischemia 6h, ischemia 12h,ischemia 6h + 5mg/kg losartan,ischemia 6h + 5mg/kg captopril. The behavioral tests and the infarct area by 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the brain injury.RT-PCR was used to examine the ACE2 mRNA expression and immunohistochemistry staining was used to evaluate the ACE2 protein level.Comparing with the sham group,the obvious neurological disorders and the enlarged infarction areas were found in the ischemia group.Both ACE2 mRNA and ACE2 protein were increased after brain ischemia injury.Compared with the ischemia group,both losartan and captopril improved the neurological functions,decreased the infarction area and increased the expression of ACE2 in both mRNA and the protein level.These results show that the brain ischemia injury can increase ACE2 expression.Both the AT1 receptor blocker losartan and angiotensin converting enzyme inhibitor captopril can increase ACE2 expression and manage the protective action against brain ischemia injury.It point out that the high expression of ACE2 perhaps has relation to the anti-ischeimic action in brain and AngⅡmay be involved in the expression of ACE2. To further verify the reasoning,several experiments were carried in vitro.
Using the cultured rat cortical astrocyte,the hypoxia(95%N_2+5%CO_2) induced cell injury was examined.Cells were divided into 6 groups:control,hypoxia 2h, hypoxia 4h,hypoxia 8h,hypoxia 4h + 10~(-7)mol/L losartan,hypoxia 4h + 10~(-7)mol/L PD123319.Cell viability was assessed by methylthiazol tetrazolium(MTT) assay and cell injury by the rate of lactate dehydrogenase(LDH) release and adenosine triphosphate(ATP) production.RT-PCR was used to examine the mRNA expression of ACE2 and western blot analysis was used to evaluate the protein level of ACE2.Comparing with the control group,decrease of cell viability and ATP concentration and increase of LDH release were found in the hypoxia groups. Losartan increased the cell viability and ATP concentration and decreased the LDH release.PD123319 had no effect on cell viability and cell injury.Both the mRNA and the protein of ACE2 were increased after hypoxia injury.Losartan increased the expression of ACE2 in both mRNA and the protein level,while PD123319 had no effect on ACE2 expression.These results show that the hypoxia injury can increase ACE2 expression and the AT1 receptor,not the AT2 receptor is involved in the expression of ACE2.It further reminder that the high expression of ACE2 has relation to the anti-hypoxic injury.
To further confirm the the action of high expression of ACE2 on the hypoxic injury,we transfected the rat brain astrocytes with a plasmid pcDNA-ACE2 which highly expressed ACE2.The efficiency of transfection was examined by RT-PCR and western blot analysis.Cells were divided into 3 groups:pcDNA3.1(control), pcDNA3.1+hypoxia 4h,pcDNA-ACE2+hypoxia 4h.Cell viability was assessed by MTT assay and cell injury by the rate of LDH release and ATP production. Comparing with the control group,decrease of cell viability and ATP concentration and increase of LDH release were found in the hypoxia groups. Compared with the hypoxia group,both the cell viability and ATP production were increased and the LDH release was decreased in pcDNA-ACE2 group.These results show that the high expression of ACE2 can protect against the hypoxia injury in rat brain astrocytes.It confirmed the relation between the high ACE2 expression and the anti-hypoxic ation.
To further explore the regulation of ACE2 expression,the AngⅡ-induced brain astrocyte injury was examined.Cells were divided into 6 groups:control,10~(-8)mol/L AngⅡ,10~(-7)mol/L AngⅡ,10~(-6)mol/L AngⅡ,10~(-7)mol/L AngⅡ+10~(-7)mol/L losartan, 10~(-7)mol/L AngⅡ+10~(-7)mol/L PD123319.Cell viability was assessed by MTT assay and cell injury by the rate LDH release and ATP production.RT-PCR was used to examine the mRNA expression of ACE2 and western blot analysis was used to evaluate the protein level of ACE2.Comparing with the control group,decrease of cell viability and ATP production and increase of LDH release were found in the hypoxia groups.Losartan increased the cell viability and ATP concentration and decreased the LDH release,but PD123319 had no effect on it.Both the mRNA and the protein of ACE2 was decreased after AngⅡinjury.Losartan increased the expression of ACE2 in both mRNA and the protein level,but PD123319 has no effect on the expression of ACE2.These results show that AngⅡcan inhibit the ACE2 expression via AT1 receptor.
In conclusion,ACE2 expression was increased after the brain ischemia injury in vivo and the hypoxia injury in vitro.AngⅡcan inhibit the expression of ACE2,while losartan can antagonized its function through an AT1 pathway.All the results provide a deep understanding on RAS in the brain ichemia injury.
PartⅡ:Effects of scutellarin on apoptosis induced by CoCl_2 in PC12 cells
The mechanisms of ischemia cerebral vascular disease(ICVD) are complex,and it can result in the injury of neurons.Recent research suggests that the apoptosis of neurons play a major role in ICVD,and hypoxia is one of the important factors which can induce apoptosis.Many researchs suggested that scutellarin has the ability of anti-oxidation.This investigation was preformed to study the detailed characterization of PC12 cells responses to the presence of cobalt chloride(CoCl_2) and evaluate the protective effects of scutellarin on the hypoxia-induced apoptosis in PC12 cells.
PC12 cells were cultured and passaged in DMEM.Experiments were performed with cells from the same passage.As grown to 80%confluence in DMEM with 10% FBS,PC12 cells were divided into 5 groups:control group,500μmol/L CoCl_2 group and scutellarin 0.1,1,10μmol/L groups.The drug-treated groups were pre-incubated with scutellarin for 2 hours.Other groups with the exception of the normal one were stimulated by CoCl_2(500μmol/L) simultaneously for 24 hours.All the above steps were carried out under sterile conditions.The viability of cells was detected by MTT assay. Morphological changes of PC12 cells were visualized under light and electron microscopes.Flow cytometry was employed to observe the occurrence of apoptosis. Both the reactive oxygen species(ROS) generation and Caspase-3 activity were quantified by their reagents and the absorbance was assessed by fluorescence spectrophotometer.The expression of Bax、Bcl-2、Bcl-X_L、p38 and P-p38 was examined by western blot.
The results show that CoCl_2 triggers neuronal PC12 cells apoptosis in a dose- and time-dependent manner,and the best concentration and time of CoCl_2-induced PC12 apoptosis are 500μmol/L and 24 h,respectively.Incubation of PC12 cells with 500μmol/L CoCl_2 for 24 h resulted in significant apoptosis as evaluated by the MTT assay,electron microscopy and flow cytometry assays.The increase of Caspase-3 activity,decrease of Bcl-X_L expression,phosphorylation of p38 mitogen-activated protein kinase(MAPK) and accumulation of intracellular ROS were also seen in CoCl_2-treated PC12 cells.Scutellarin at 0.1,1 and 10μmol/L significantly protected against the apoptotic cell death induced by CoCl_2.Scutellarin decreased Caspase-3 activity,increased Bcl-X_L expression,inhibited p38 phosphorylation and attenuated ROS production.These results demonstrate that scutellarin can protect PC12 cells from CoCl_2 induced apoptosis by scavenging ROS,inhibiting p38 phosphorylation, up-regulating Bcl-X_L expression and decreasing Caspase-3 activity,and may reduce the cellular damage in pathological conditions associated with hypoxia-mediated neuronal injury
This research shows that scutellarin can protect PC12 cells from CoCl_2-induced apoptosis which indicates scutellarin has remarkable ability on rescuing ICVD.
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