血管紧张素Ⅱ通过活性氧调控ACE和ACE2的表达

目的研究血管紧张素II(Angiotensin II,Ang II)是否通过活性氧(Reactive oxygen species,ROS)调控人肾小管上皮细胞(Human kidney tubular epithelial cell line,HK-2)中血管紧张素转换酶(Angiotensin I converting enzyme,ACE)及血管紧张素转换酶2(Angiotensin I converting enzyme 2,ACE2)的表达。方法首先给予HK-2细胞不同浓度的Ang II刺激以检测ACE及ACE2表达的变化;然后给予细胞不同浓度的Ang II,在不同时间点,用2’7’-二氯双乙酸盐(2’7’-Dichlorofluorescin diacetate,DCFH)标记结合细胞内的ROS,流式细胞仪检测ROS水平;最后,我们给予细胞ROS抑制剂二苯基氯化碘(diphenyleneiodonium chloride,DPI),分别检测ROS、ACE及ACE2的表达。结果Ang II诱导肾小管上皮细胞内ACE表达上调,ACE2表达下调。另外,Ang II会引起细胞内ROS产生增多,且与刺激浓度和刺激时间相关。DPI能抑制Ang II诱导的ROS产生,并且抑制Ang II对ACE及ACE2的调控作用。结论 Ang II通过诱导人肾小管上皮细胞产生ROS从而调控ACE以及ACE2的表达。     

组蛋白脱乙酰基酶2在血管紧张素Ⅱ致心肌细胞肥大中的表达

目的观察血管紧张素Ⅱ(AngⅡ)刺激心肌细胞,造成心肌细胞肥大过程中组蛋白脱乙酰基酶2(histonedeacetylase2,HDAC2)的表达。方法培养原代心肌细胞,AngⅡ刺激心肌细胞造成肥大,在不同时间取细胞进行反转录聚合酶链反应(RT-PCR),观察HDAC2mRNA表达,免疫组化法检测HDAC2蛋白表达,相差镜和电镜下观察细胞形态变化。结果经AngⅡ刺激后,相差镜下可见心肌细胞面积变大;电镜下见细胞内部结构亦发生明显的改变;HDAC2mRNA水平随着AngⅡ刺激时间延长而增高;HDAC2蛋白表达亦增加。结论AngⅡ致心肌细胞肥大过程中伴有HDAC2表达增加,HDAC2有可能参与心肌细胞的肥大机制。     

血管紧张素Ⅱ及其受体拮抗剂对心肌细胞肥大的影响

目的 观察血管紧张素Ⅱ（AngⅡ）、AT1受体拮抗剂氯沙坦和AT2受体拮抗剂PD123177对心肌细胞蛋白质合成速率和AT1受体mRNA表达的影响。方法 采用^3H－亮氨酸掺入法测定培养的心肌细胞蛋白质合成速率,RT－PCR方法检测心肌细胞AT1受体mRNA表达。结果 在培养的心肌细胞中加入AngⅡ可明显增加心肌细胞^3H－亮氨酸的掺入量,并呈剂量依赖性,氯沙旦可显著抑制AngⅡ引起的蛋白质合成增加,而PD123177对其无影响;AngⅡ上调AT1受体基因表达,氯沙坦抑制其上调,PD123177无影响。结论 AngⅡ可通过上调AT1受体引起心肌细胞肥大,氯沙坦下调AT1受体,抑制心肌细胞肥大。     

血管紧张素1-7对培养心肌细胞血管紧张素转换酶2基因表达的影响

目的探讨血管紧张素1-7(Ang1-7)对培养心肌细胞血管紧张素转换酶2(ACE2)基因表达的影响。方法将体外原代培养的新生SD大鼠心肌细胞,随机分为对照组和Ang1-7刺激组。测量心肌细胞直径和表面积,用RT-PCR方法检测心肌细胞ACE2mRNA表达,激光共聚焦显微镜检测ACE2蛋白表达部位。结果Ang1-7对心肌细胞直径和表面积没有影响(P>0.05);Ang1-7刺激组较对照组ACE2mRNA表达显著升高(P<0.01);激光共聚焦显微镜显示,ACE2在心肌细胞膜表达。结论外源性Ang1-7对心肌细胞形态无影响,Ang1-7刺激心肌细胞ACE2mRNA表达上调,其意义在于加速血管紧张素Ⅱ降解为Ang1-7,发挥心肌保护效应。     

血管紧张素Ⅱ及其受体拮抗剂对心肌细胞肥大的影响

目的观察血管紧张素Ⅱ（AngⅡ）、AT1受体拮抗剂氯沙坦和AT2受体拮抗剂PD123177对心肌细胞蛋白质合成速率和AT1受体mRNA表达的影响。方法采用3H-亮氨酸掺入法测定培养的心肌细胞蛋白质合成速率,RT-PCR方法检测心肌细胞AT1受体mRNA表达。结果在培养的心肌细胞中加入AngⅡ可明显增加心肌细胞3H-亮氨酸的掺入量,并呈剂量依赖性,氯沙坦可显著抑制AngⅡ引起的蛋白质合成增加,而PD123177对其无影响;AngⅡ上调AT1受体基因表达,氯沙坦抑制其上调,PD123177无影响。结论AngⅡ可通过上调AT1受体引起心肌细胞肥大,氯沙坦下调AT1受体,抑制心肌细胞肥大。     

ACE及ACE2在慢性香烟烟雾诱导的大鼠肺动脉高压对血管紧张素Ⅱ的调控作用

目的 探讨慢性香烟暴露诱导的肺动脉高压形成过程中ACE、ACE2和血管紧张素Ⅱ（AngⅡ）的调控作用及特异性血管紧张素Ⅱ受体拮抗剂氯沙坦的治疗作用.方法 健康雄性SD大鼠60只,随机分为A组：对照组（Con）;B组：30 mg/kg单纯氯沙坦组（30 mg/kg Los）;C组：香烟诱导组（SM）;D组：香烟诱导＋10 mg/kg氯沙坦组（SM＋10 mg/kg Los）;E组：香烟诱导＋30 mg/kg氯沙坦组（SM＋30 mg/kg Los）.香烟暴露组和香烟暴露＋氯沙坦组在标准毒理香烟暴露箱中接受被动吸烟;对照组同时在同种毒理箱中暴露于新鲜空气;氯沙坦给药采用每天腹腔内注射;正常对照组给予等量生理盐水注射.建立香烟诱导的肺动脉高压大鼠模型后,用插入导管法检测右室收缩压（RVSP）;Western blotting法分析ACE2和ACE的蛋白表达量;放射免疫分析试剂药盒测定肺组织中的AngⅡ表达水平.结果 香烟暴露6个月后,慢性香烟暴露组大鼠RVSP较对照组明显升高,大鼠肺组织中AngⅡ表达水平显著增高.Western blotting结果显示,ACE表达水平增高,香烟暴露组大鼠肺组织ACE2蛋白表达水平降低,而ACE蛋白表达水平较对照组升高.氯沙坦干预治疗后,香烟暴露＋氯沙坦组大鼠RVSP和AngⅡ较香烟暴露组降低（P＜0.05）,肺组织ACE2蛋白表达水平较香烟暴露组增强,ACE蛋白表达水平较香烟暴露组减少（P＜0.05）.结论 慢性香烟暴露可导致肺动脉高压,还可刺激肺组织ACE2和ACE的蛋白表达变化,提示ACE2和ACE在慢性香烟暴露诱导的肺动脉高压中起一定作用.     

丹参酮ⅡA对血管紧张素Ⅱ诱导的心肌细胞肥大、凋亡的影响

目的 研究丹参酮Ⅱ A(1、SN)对血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大、凋亡的影响。方法 培养乳鼠心肌细胞分别暴露于AngⅡ(10^-6mol／L)、TSN(10^-6mol／L、AngⅡ(10^-6mol／L) TSN(10^-5mol／L)36h，检测心肌细胞的凋亡率、心肌细胞直径和^3H亮氨酸掺入率。结果 TSN可抑制AngⅡ所诱导的心肌细胞的凋亡率、心肌细胞直径和^3H亮氨酸掺入率的显著增高。结论 TSN可抑制AngⅡ所诱导的心肌细胞的凋亡和肥大。     

丹参素和川芎嗪对血管紧张素Ⅱ诱导乳鼠心肌细胞凋亡的影响

目的研究活血药丹参素和川芎嗪对血管紧张素Ⅱ（AngⅡ）诱导的心肌细胞凋亡的影响,探讨其抑制心肌肥大的作用机制.方法收集各组心肌培养细胞,PI染液,于流式细胞仪上进行检测.结果 AngⅡ能诱导心肌细胞凋亡,AngⅡ组与空白对照组相比,心肌细胞凋亡率显著增加（P〈0.01）,并随着时间的延长其凋亡率不断增高,于第7天达到高峰;洛沙坦明显抑制AngⅡ所诱导各个时间段的心肌细胞凋亡率（P〈0.01）,而活血药丹参素和川芎嗪也明显抑制AngⅡ所诱导各个时间段的心肌细胞凋亡率（P〈0.05或P〈0.01）.结论丹参素和川芎嗪能通过抑制AngⅡ诱导的心肌细胞凋亡而产生心肌保护作用,具有防止心肌细胞肥大作用,从而防治心室肥厚.     

缬沙坦对大鼠主动脉内皮球囊损伤后血管紧张素转化酶与血管紧张素转化酶Ⅱ表达的影响

目的观察大鼠主动脉内皮球囊损伤后血管重塑发生以及血管紧张素转换酶Ⅱ、血管紧张素转换酶表达的变化,探讨缬沙坦对其干预作用及作用机制。方法建立球囊损伤大鼠主动脉内皮损伤模型,并将大鼠随机分为对照组、手术组和缬沙坦治疗组。每组分别于术后14天和30天取主动脉组织,通过逆转录酶连反应技术检测血管紧张素转换酶Ⅱ、血管紧张素转换酶的mRNA水平,应用免疫组化方法检测血管紧张素转换酶Ⅱ、血管紧张素转换酶的蛋白表达。结果主动脉球囊损伤大鼠术后14天和30天血管内膜较对照组均显著增厚,给予缬沙坦干预14天和30天大鼠主动脉血管内膜较手术组明显减轻。手术组较对照组血管紧张素转换酶的mRNA及蛋白表达均显著升高(P<0.05),血管紧张素转换酶Ⅱ的蛋白表达显著降低(P<0.05)。缬沙坦能够降低血管紧张素转换酶的mRNA与蛋白表达(P<0.05),增加血管紧张素转换酶2的蛋白表达(P<0.05)。结论缬沙坦逆转主动脉内皮球囊损伤后血管重塑可能与增加血管紧张素转换酶Ⅱ表达和降低血管紧张素转换酶表达有关。     

SK-7041对血管紧张素Ⅱ致大鼠心肌细胞肥大的干预作用

目的探讨SK-7041在血管紧张素Ⅱ(AngⅡ)致心肌细胞肥大过程中的抑制作用。方法常规方法培养大鼠原代心肌细胞,分为3组:对照组、肥大组、SK-7041组。利用AngⅡ刺激心肌细胞造成肥大模型,并给予SK-7041进行干预。反转录聚合酶链反应观察β-肌球蛋白重链(β-MHC)mRNA表达;相差显微镜和电镜观察心肌细胞的表面积和超微结构变化;免疫组织化学法检测c-fos蛋白的表达。结果大鼠原代心肌细胞在AngⅡ作用下表面积增加,超微结构发生改变;β-MHC mRNA和c-fos蛋白表达增加(P<0.05)。给予SK-7041干预后,上述变化显著缓解(P<0.05)。结论 SK-7041可抑制AngⅡ刺激引起的心肌细胞肥大,为临床上治疗心肌肥厚提供一条新的思路。     

Angiotensin II activates myostatin expression in cultured rat neonatal cardiomyocytes via p38 MAP kinase and myocyte enhance factor 2 pathway

Angiotensin II (AngII) plays a critical role in cardiac remodeling and promotes cardiac myocyte hypertrophy. Myostatin, a negative regulator of muscle growth, is increased in hypertrophied and infarcted heart. The direct effect of AngII on cardiac myocyte myostatin expression has not been previously investigated. We hypothesized that myostatin may act as a cardiac endocrine inhibitor for AngII. AngII-induced myostatin protein expression in cultured rat neonatal cardiomyocytes was dose-dependent. AngII significantly increased myostatin protein and mRNA expression in a time-dependent manner. Addition of losartan, SB203580, or p38 siRNA 30 min before AngII stimulation significantly blocked the increase of myostatin protein by AngII. AngII significantly increased phosphorylation of p38 while SB205380 and losartan attenuated the phosphorylation of p38 induced by AngII. AngII increased, while myostatin-Mut plasmid, SB203580, losartan, and myocyte enhance factor 2 (MEF-2) antibody abolished the myostatin promoter activity. Co-stimulation with myostatin and AngII significantly inhibited the protein synthesis induced by AngII. In conclusion, AngII enhances myostatin expression in cultured rat neonatal cardiomyocytes. The AngII-induced myostatin is mediated through p38 MAP kinase and MEF-2 pathway.     

Plasma Angiotensin I Determines the Rate of Angiotensin II Generation Even during ACE Inhibition

ABSTRACT

After single or multiple administration of an angiotensin converting enzyme(ACE) inhibitor, plasma angiotensin(ANG) II recovers more rapidly than plasma ACE-activity. To investigate the dynamics of the biochemical changes induced by ACE inhibition, 6 normal volunteers received in a randomized order one dose of enalapril (E, 20mg), an ACE-inhibitor with a 35 hour terminal half-life, or benazepril (B, 20 mg), an ACE-inhibitor with a 23 hour terminal half-life. Biochemical effects of the 2 agents were compared at 0-4 hours and 14-30 hours after drug intake. ACE-inhibition was estimated in vivc by the ratio between plasma ANG II and blood ANG I (ANG II/ANG I) and in vitro by a radioenzymatic assay. At peak inhibition (4 hours after E, 2 hours after B), plasma ACE-activity was inhibited with E by 94% in vivo and by 98% in vitro, compared to 98% and 97%, respectively, with B. Twenty-four hours post drug, plasma ACE-activity was inhibited with E by 84% in vivo and by 83% in vitro, compared to 64% and 94%, respectively, with B. Plasma ACE-inhibition largely exceeded 24 hours and was independent of terminal half-lives. ANG II levels were initially suppressed by 88% with E and by 95% with B, but they returned to their initial values within 24 hours. Active renin measured by IRMA was increased to 183% and 313% 4 and 24 hours after E, and to 342% and 337% after B. Corresponding ANG I levels were 240% and 319% for E, and 329% and 295% for B; they were highly correlated to active renin (r=0.82, n=252, p<0.001). Thus, 24 hours post-drug, ANG II/ANG I was still reduced but the biologically effective product of the renin-angiotensin system(RAS), ANG II, has returned to its initial level, as a consequence of the increase in renin secretion and ANG I production. The RAS is reset at a time when ACE-inhibition is still effective. Feed-back increase in renin release seems to be an important factor contributing to the reappearance of ANG II in plasma in the presence of ACE-inhibition.     

褪黑素对心肌细胞抗过氧化氢损伤的实验研究

目的 :观察 H2 O2 对心肌细胞内活性氧 （ROS）和游离钙 （[Ca2 + ]i）的影响及褪黑素 （MT）的保护作用。方法 :酶消化法原代分离培养乳鼠心肌细胞 ,分别用荧光探针 DCFH- DA和 Fluo- 3- AM标记 ,H2 O2 和 MT处理后 ,流式细胞仪检测细胞荧光强度的变化。结果 :低浓度的 H2 O2 （10 0 μm ol/ L）随着时间的延长可使心肌细胞内的 ROS呈上升趋势 ,30 m in,6 0 m in与 0 min或对照组相比均明显增高 （P<0 .0 1） ;MT干预后上升趋势均明显减弱 ,在 30 ,6 0 min两组均差异显著 （P<0 .0 1）。 [Ca2 + ]i 亦呈显著上升趋势 ,30 min时与 0 min或对照组相比有差异 （P<0 .0 5 ） ,6 0min时与 30 m in,0 min或对照组相比差异显著 （P<0 .0 1） ;MT干预后其上升明显减少 ,两者差异显著 （P<0 .0 1）。结论 :MT有很好的抗氧化效果 ,能起到保护心肌的作用。     

Angiotensin II increases expression and secretion of cathepsin F in cultured human monocyte-derived macrophages: an angiotensin II type 2 receptor-mediated effect

Increasing evidence suggests that cathepsins and angiotensin II (AngII) participate in atherosclerosis, particularly in remodeling of the extracellular matrix of the inflamed arterial intima. Here, we show that AngII induces mRNA expression of cathepsin F, a member of the cysteine protease family, in human monocyte-derived macrophages. AngII did not affect the amount of intracellular cathepsin F protein, but significantly enhanced its secretion by the treated cells. The stimulatory effect of AngII was mediated by the AngII type 2 (AT(2)) receptor, as demonstrated by the ability of the AT(2)-receptor antagonist PD123319 to block the AngII-induced increase in cathepsin F secretion. Our present data demonstrate a novel proatherogenic role for AngII, namely its ability to enhance secretion of lysosomal cathepsin F by monocyte-derived macrophages.     

内皮素对大鼠培养心肌细胞肥大及肌球蛋白基因表达影响的研究

为探讨内皮素（ET）在大鼠培养心肌细胞肥大及肌球蛋白（MHC）基因表达中的作用，采用心肌细胞培养及斑点杂交的方法，同时测定心肌细胞直径、数目及3H亮氨酸（3H－Leu）掺入率，观察ET耐心肌细胞肥大及肌球蛋白基因表达的影响。结果显示：ET（10-7mol／L）作用培养心肌细胞24h后，细胞直径明显增大（P＜0．01），细胞数目无明显增加（P＞0．05）；不同浓度ET（10－10～10－7mol／L）可刺激心肌细胞3H－Leu掺入率呈明显剂量依赖性增加；随ET浓度（10－9～10－7mol／L）增高，β-肌球蛋白重链（β-MHC）mRNA表达呈剂量依赖性增加，α－MHCmRNA表达相应减少。提示：①ET可促进心肌细胞肥大，而不诱导心肌细胞增殖;②ET促使心肌细胞由α-MHC向β-MHC转化，说明ET促心肌细胞肥大是病理性的，此作用发生在翻译前水平。     

促红细胞生成素对培养的乳鼠心肌细胞肥大的影响

目的:观察促红细胞生成素(EPO)对乳鼠心肌细胞肥大的影响.方法:体外分离培养乳鼠心肌细胞,用血管紧张素Ⅱ(AngⅡ)刺激产生肥大的心肌细胞,加入不同浓度的EPO对肥大心肌细胞进行干预,以细胞表面积和心钠素(ANF)mRNA表达作为细胞肥大的观察指标,采用Western blot方法检测细胞内信号分子转化生长因子β1(TGF-β1)、Smad2及p-Smad2蛋白表达.结果:20U/ml的EPO能有效逆转AngⅡ诱导的心肌细胞肥大,低于此浓度未见明显效果;EPO能减弱促心肌细胞肥大信号分子TGF-β1、Smad2 及p-Smad2蛋白表达.结论:EPO具有抗心肌细胞肥大作用,且这一作用与TGF-β1-Smad2信号途径有关.