Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion

Endogenous levels of angiotensin II (Ang II) are increased in the cortex and hypothalamus following stroke, and Ang II type 1 receptor blockers (ARBs) have been shown to attenuate the deleterious effects in animal stroke models using middle cerebral artery (MCA) intraluminal occlusion procedures. However, the endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO) model of cerebral ischaemia is thought to more closely mimic the temporal events of an embolic stroke. This method provides rapid occlusion of the MCA and a gradual reperfusion that lasts for 16–22 h. The aim of the present study was to evaluate whether systemic administration of an ARB prior to ET-1-induced MCAO would provide cerebroprotection during this model of ischaemic stroke. Injection of 3 μl of 80 μ m ET-1 adjacent to the MCA resulted in complete occlusion of the vessel that resolved over a period of 30–40 min. Following ET-1-induced MCAO, rats had significant neurological impairment, as well as an infarct that consisted of 30% of the ipsilateral grey matter. Systemic pretreatment with 0.2 mg kg⁻¹ day⁻¹ candesartan for 7 days attenuated both the infarct size and the neurological deficits caused by ET-1-induced MCAO without altering blood pressure. This study confirms the cerebroprotective properties of ARBs during ischaemic stroke and validates the ET-1-induced MCAO model for examination of the role of the brain renin–angiotensin system in ischaemic stroke.     

Evidence that angiotensin-(1-7) is an intermediate of gonadotrophin-induced oocyte maturation in the rat preovulatory follicle

Several studies have shown the presence of components of the renin-angiotensin system in mammalian ovaries and their involvement in ovarian physiology. We have previously shown the presence of angiotensin-(1-7) [Ang-(1-7)], an important biologically active component of the renin-angiotensin system, and its receptor, Mas, in rat, rabbit and human ovaries. We have also shown the involvement of Ang-(1-7) in the rabbit ovulatory process in vitro. In the present study, we observed that Ang-(1-7) stimulated the resumption of meiosis in oocytes of rat preovulatory follicles, reaching more than 30% of oocytes with germinal vesicle breakdown. The specific antagonist of the Mas receptor, A-779, inhibited the germinal vesicle breakdown induced by Ang-(1-7) and reduced the oocyte maturation stimulated by luteinizing hormone (LH). Immunohistochemistry showed that LH increased both Ang-(1-7) and angiotensin-converting enzyme 2 (ACE2) staining in preovulatory follicles. The effect of gonadotrophins on mRNA expression of Mas and ACE2 in ovaries of immature equine chorionic gonadotrophin-primed rats was analysed by real-time PCR after 6 h of human chorionic gonadotrophin (hCG) injection, which exhibits LH-like effects. After hCG treatment, ACE2 mRNA expression was higher in the ovaries of treated rats than in the ovaries of control rats, whereas Mas mRNA levels were unchanged. A-779 changed the steroidogenesis stimulated by LH. An increased testosterone concentration and decreased progesterone levels were measured in the follicle medium. In conclusion, our results suggest that LH upregulates the ACE2-Ang-(1-7)-Mas axis and that Ang-(1-7) promotes meiotic resumption, possibly as a gonadotrophin intermediate.     

ACE2, angiotensin-(1–7), and Mas: the other side of the coin

The renin–angiotensin system (RAS) has recently been extended by the addition of a novel axis consisting of the angiotensin-converting enzyme 2 (ACE2), the heptapeptide angiotensin (1–7) (Ang-(1–7)), and the G protein-coupled receptor Mas. ACE2 converts the vasoconstrictive and pro-oxidative peptide angiotensin II (Ang II) into Ang-(1–7) which exerts vasodilatory and antioxidative effects via its receptor Mas. Thereby, ACE2 regulates the local actions of the RAS in cardiovascular tissues and the ACE2/Ang-(1–7)/Mas axis exerts protective actions in hypertension, diabetes, and other cardiovascular disorders. Consequently, this novel RAS axis represents a promising therapeutic target for cardiovascular and metabolic diseases.     

ACE2内源性激动剂DIZE对糖尿病肾病大鼠的保护作用

目的:观察血管紧张素转换酶2(ACE2)内源性激动剂乙酰甘氨酸重氮氨苯脒(DIZE)对糖尿病肾病(DN)大鼠的保护作用。方法:30只Wistar大鼠随机分为正常对照组(NC组)、DN组和DIZE处理组(DIZE组)。DN组与DIZE组一次性腹腔注射链脲佐菌素(65 mg/kg)建立糖尿病模型,12周后糖尿病肾病大鼠模型建立后给予DIZE 15 mg·kg~(-1)·d~(-1)或等量生理盐水皮下注射4周处理。16周末称量体重和肾重,计算肾质量体质量比(KW/BW),收集血、尿标本,检测血糖(GLU)、24 h尿蛋白(24UP)及血清肌酐(SCr)等指标。通过PAS染色观察各组肾脏病理变化;ELISA法检测大鼠AngⅡ、Ang-(1-7)、TGF-β1及VCAM-1水平的变化;通过免疫组化观察collagenⅠ和FN蛋白表达的变化;利用实时荧光定量PCR(RT-qPCR)技术检测大鼠肾组织collagenⅠ和FN mRNA含量的变化;Western blot观察各组大鼠ACE2蛋白表达的变化。结果:DIZE显著提高了糖尿病大鼠ACE2的表达(P0.05),降低了糖尿病大鼠血浆AngⅡ含量(P0.05),提高了Ang-(1-7)的水平(P0.05)。与NC组大鼠相比,DN组与DIZE组大鼠的24UP、SCr和KW/BW明显升高(P0.05),collagenⅠ和FN mRNA水平及蛋白表达量增加,肾脏组织TGF-β1及VCAM-1明显上升(P0.05)。DIZE组与DN组大鼠相比,24UP和SCr水平降低(P0.05),GLU和KW/BW无明显差异,collagenⅠ和FN mRNA含量及蛋白表达量减少,肾脏组织TGF-β1及VCAM-1水平降低(P0.05)。结论:ACE2内源性激动剂DIZE显著提高了ACE2的活性,增加了Ang-(1-7)的含量,从而降低了肾脏纤维化及炎症水平,并对糖尿病肾病大鼠起到保护性作用。     

DIZE对糖尿病心肌病大鼠心功能的保护作用

目的:观察研究血管紧张素转换酶2(ACE2)激动剂重氮氨苯脒乙酰甘氨酸盐(DIZE)对糖尿病心肌病(DCM)大鼠心功能的保护作用。方法:30只健康雄性Wistar大鼠随机分为正常对照组、DCM组和DIZE处理组(DIZE组),每组10只。DCM组与DIZE组一次性腹腔注射链脲佐菌素65 mg/kg,建立糖尿病模型,12周后经超声检查选出符合糖尿病心肌病的大鼠模型,分别给予DIZE 15 mg·kg~(-1)·d~(-1)或等体积生理盐水,皮下泵入给药4周处理。第16周末行超声检测心功能后麻醉处死大鼠。通过HE染色和Masson染色观察大鼠心肌组织形态学变化,Wsetern blot、ELISA和免疫组化等方法观察ACE2、血管紧张素(Ang)Ⅱ、Ang-(1-7)、白细胞介素(IL)-1、IL-6和结缔组织生长因子(CTGF)等指标的变化。结果:DIZE显著提高了糖尿病大鼠心肌中ACE2的表达,DIZE组较DCM组心肌组织中胶原含量明显降低,IL-1和IL-6蛋白表达水平明显下降,心功能得到明显改善(P0.05)。结论:ACE2内源性激动剂DIZE显著提高了ACE2的水平,降低了炎症水平,从而对DCM大鼠心功能起到保护作用。     

Angiotensin-(1-7) attenuated long-term hypoxia-stimulated cardiomyocyte apoptosis by inhibiting HIF-1α nuclear translocation via Mas receptor regulation

Extreme hypoxia often leads to myocardial apoptosis and causes heart failure. Angiotensin-(1-7)Ang-(1-7) is well known for its cardio-protective effects. However, the effects of Ang-(1-7) on long-term hypoxia (LTH)-induced apoptosis remain unknown. In this study, we found that Ang-(1-7) reduced myocardial apoptosis caused by hypoxia through the Mas receptor. Activation of the Ang-(1-7)/Mas axis down-regulated the hypoxia pro-apoptotic signaling cascade by decreasing the protein levels of hypoxia-inducible factor 1α (HIF-1α) and insulin-like growth factor binding protein-3 (IGFBP3). Moreover, the Ang-(1-7)/Mas axis further inhibited HIF-1α nuclear translocation. On the other hand, Ang-(1-7) activated the IGF1R/PI3K/Akt signaling pathways, which mediate cell survival. However, the above effects were abolished by A779 treatment or silencing of Mas expression. Taken together, our findings indicate that the Ang-(1-7)/Mas axis protects cardiomyocytes from LTH-stimulated apoptosis. The protective effect of Ang-(1-7) is associated with the inhibition of HIF-1α nuclear translocation and the induction of IGF1R and Akt phosphorylation.     

Lentivirus-mediated overexpression of angiotensin-(1-7) attenuated ischaemia-induced cardiac pathophysiology

Myocardial infarction (MI) results in cell death, development of interstitial fibrosis, ventricular wall thinning and ultimately, heart failure. Angiotensin-(1-7) [Ang-(1-7)] has been shown to provide cardioprotective effects. We hypothesize that lentivirus-mediated overexpression of Ang-(1-7) would protect the myocardium from ischaemic injury. A single bolus of 3.5 × 10(8) transducing units of lenti-Ang-(1-7) was injected into the left ventricle of 5-day-old male Sprague-Dawley rats. At 6 weeks of age, MI was induced by ligation of the left anterior descending coronary artery. Four weeks after the MI, echocardiography and haemodynamic parameters were measured to assess cardiac function. Postmyocardial infarction, rats showed significant decreases in fractional shortening and dP/dt (rate of rise of left ventricular pressure), increases in left ventricular end-diastolic pressure, and ventricular hypertrophy. Also, considerable upregulation of cardiac angiotensin-converting enzyme (ACE) mRNA was observed in these rats. Lentivirus-mediated cardiac overexpression of Ang-(1-7) not only prevented all these MI-induced impairments but also resulted in decreased myocardial wall thinning and an increased cardiac gene expression of ACE2 and bradykinin B2 receptor (BKR2). Furthermore, in vitro experiments using rat neonatal cardiac myocytes demonstrated protective effects of Ang-(1-7) against hypoxia-induced cell death. This beneficial effect was associated with decreased expression of inflammatory cytokines (tumour necrosis factor-α and interleukin-6) and increased gene expression of ACE2, BKR2 and interleukin-10. Our findings indicate that overexpression of Ang-(1-7) improves cardiac function and attenuates left ventricular remodelling post-MI. The protective effects of Ang-(1-7) appear to be mediated, at least in part, through modulation of the cardiac renin-angiotensin system and cytokine production.     

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.     

血管紧张素-(1-7)/Mas受体轴通过调控NF-κB通路保护心肌细胞对抗高糖诱导的损伤

目的:探讨血管紧张素-(1-7)[Ang-(1-7)]/Mas受体轴能否通过抑制核因子-κB(NF-κB)通路对抗高糖(HG)引起的心肌细胞损伤。方法:应用细胞计数检测试剂盒(CCK-8)检测心肌细胞存活率;双氯荧光素(DCFH-DA)染色荧光显微镜照相法检测胞内活性氧(ROS)水平;Hoechst 33258核染色荧光显微镜照相测定凋亡细胞的形态及数量的变化;JC-1染色法测定线粒体膜电位(MMP);应用免疫蛋白印迹法测定NF-κB p65和cleaved caspase-3蛋白的表达水平。结果:应用35 mmol/L葡萄糖分别处理H9c2心肌细胞30、60、90、120和150 min均能明显增加磷酸化(p)NF-κB p65的水平,其中60 min时,表达水平增加最明显;1μmol/L Ang-(1-7)与HG共同处理心肌细胞60 min能抑制HG对p-NF-κB p65表达的上调作用;0.1~30μmol/L的Ang-(1-7)与HG分别共处理心肌细胞24 h均能抑制HG的细胞毒性,使细胞存活率增多;另一方面,1μmol/L Ang-(1-7)能抑制HG引起的细胞凋亡、氧化应激、线粒体损伤等,使凋亡细胞数量、cleaved caspase-3表达、ROS生成水平及MMP丢失减少;但是10μmol/L Ang-(1-7)/Mas受体拮抗剂A-779能明显阻断上述的Ang-(1-7)的心肌细胞保护作用;与Ang-(1-7)的作用相似,100μmol/L PDTC(NF-κB抑制剂)与HG共处理心肌细胞24 h也能显著抑制上述的HG损伤作用。结论:Ang-(1-7)/Mas受体轴可通过抑制NF-κB通路对抗HG诱导的心肌细胞损伤。     

Ang-(1-7)与Mas结合促进NIT细胞分泌胰岛素

目的体外研究Ang-(1-7)对NIT细胞胰岛素分泌的影响及其潜在机制。方法将NIT细胞在不同浓度Ang-(1-7)(10-8～10-3mol/L)培养24 h,ELISA法检测NIT细胞对葡萄糖刺激的胰岛素分泌功能。用RT-PCR法,从NIT细胞中扩增Mas、GLUT-2和TGF-β1基因的全长cDNA序列。将NIT细胞在10-5mol/L Ang-(1-7)条件下培养48 h,QRT-PCR方法检测NIT细胞Mas、GLUT-2及TGF-β1的mRNA表达,Western印迹方法测定NIT细胞Mas、GLUT-2及TGF-β1的蛋白表达。结果 NIT细胞系随着细胞外Ang-(1-7)浓度(10-8～10-3mol/L)的增加胰岛素分泌增加,10-5mol/L Ang-(1-7)组胰岛素分泌量为(8.86±0.53)mIU/L,显著高于对照组(8.06±0.39)mIU/L(P<0.05)。与对照组相比,经10-5mol/L Ang-(1-7)预处理的NIT细胞Mas及GLUT-2的mRNA和蛋白表达上调(P<0.05);相反,经10-5mol/L Ang-(1-7)预处理的NIT细胞TGF-β1的mRNA和蛋白表达水平下降(P<0.05)。结论Ang-(1-7)与Mas结合能够促进NIT细胞分泌胰岛素,这可能与提高NIT细胞摄取葡萄糖的能力、抑制纤维化进程等相关。     

缺氧诱导因子-1α抑制剂对大鼠局部脑缺血再灌注损伤的影响

目的研究大鼠局部脑缺血再灌注损伤时,缺氧诱导因子-1α(HIF-1α)抑制剂2-甲氧雌二醇(2ME2)对损伤脑组织的影响。方法雄性SD大鼠60只随机分为假手术组、大脑中动脉阻塞再灌注组(MCAO组)、假性治疗组(DMSO组)、2ME2治疗组(2ME2组)。治疗组在术后1h腹腔注射相应剂量药物。观察各组大鼠神经行为学缺陷;再灌注7d,TTC染色观察脑梗死体积变化;再灌注24h,Western blotting检测HIF-1α及其下游基因的表达变化;制备脑组织切片分别作Nissl染色、免疫组织化学染色及原位缺口末端标记(TUNEL)。结果2ME2组神经功能较MCAO及DMSO组有明显改善(P<0.05),同时,2ME2组梗死面积明显减小(P<0.05)。Western blotting结果显示,HIF-1α的表达经2ME2治疗后降低,其下游基因VEGF、BNIP3及Caspase-3的表达有同样的变化。Nissl染色可见2ME2治疗组皮质神经元结构较清晰,胞体肿胀、核固缩、核溶解程度较模型组及假性治疗组明显减轻,淡染区域减小;免疫组织化学法观察到2ME2组HIF-1α、Caspase-3、BNIP3、VEGF及TUNEL标记的阳性细胞数明显减少(P<0.05)。结论2ME2可能通过降低HIF-1α水平并下调其下游的BNIP3和VEGF的表达,降低血脑屏障的通透性并减少凋亡因子Caspase-3的作用,发挥神经元的保护作用。     

Angiotensin-(1-7) induces ovulation and steroidogenesis in perfused rabbit ovaries

A local renin-angiotensin system has been described in several organs, including the ovary; however, data indicating a role for angiotensin II in the induction of ovulation are controversial. We have previously shown the presence of a novel peptide, angiotensin-(1-7) [Ang-(1-7)], in the rat ovary and its effect on steroidogenesis. The objective of the present study was to determine whether Ang-(1-7) plays a role in ovulation. We first determined the presence and distribution of Ang-(1-7) and the receptor Mas in rabbit ovaries by immunohistochemistry. Angiotensin-(1-7) and Mas immunoreactivity were observed in interstitial cells and oocytes of immature ovaries. Immunoreactivity for Ang-(1-7) and Mas was also observed in theca and granulosa cells of preovulatory follicles in ovaries of gonadotrophin-stimulated rabbits. To verify the effect of Ang-(1-7) in ovulation and steroidogenesis, we used isolated ovaries from immature rabbits pretreated with equine chorionic gonadotrophin (50 i.u., 48 h before the experiment) and then perfused in vitro. The ovulatory efficiency was determined by the number of oocytes compared with the number of preovulatory follicles present in the ovary. Angiotensin-(1-7) stimulated oestradiol production and enhanced ovulatory efficiency, which was blocked by the specific Ang-(1-7) antagonist, A-779. Ovulation induced by human chorionic gonadotrophin was also antagonized by A-779. These results show, for the first time, the involvement of a novel regulatory peptide system, Ang-(1-7) and Mas, in the ovulatory process. More importantly, because A-779 antagonized hCG-induced ovulation, it may be inferred that Ang-(1-7) plays an important role in ovulation, possibly as a mediator of gonadotrophin action.     

Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7) or an angiotensin II receptor antagonist

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome. Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin (Ang) II have been implicated in the pathogenesis of ARDS. A counter-regulatory enzyme of ACE, ie ACE2 that degrades Ang II to Ang-(1-7), offers a promising novel treatment modality for this syndrome. As the involvement of ACE and ACE2 in ARDS is still unclear, this study investigated the role of these two enzymes in an animal model of ARDS. ARDS was induced in rats by intratracheal administration of LPS followed by mechanical ventilation. During ventilation, animals were treated with saline (placebo), losartan (Ang II receptor antagonist), or with a protease-resistant, cyclic form of Ang-(1-7) [cAng-(1-7)]. In bronchoalveolar lavage fluid (BALF) of ventilated LPS-exposed animals, ACE activity was enhanced, whereas ACE2 activity was reduced. This was matched by enhanced BALF levels of Ang II and reduced levels of Ang-(1-7). Therapeutic intervention with cAng-(1-7) attenuated the inflammatory mediator response, markedly decreased lung injury scores, and improved lung function, as evidenced by increased oxygenation. These data indicate that ARDS develops, in part, due to reduced pulmonary levels of Ang-(1-7) and that repletion of this peptide halts the development of ARDS.     

Mas基因沉默对血管紧张素-(1-7)拮抗血管紧张素Ⅱ诱导的大鼠肾间质成纤维细胞活化的影响

 目的： 探讨Mas基因沉默后对血管紧张素-(1-7) ［Ang-(1-7)］拮抗血管紧张素Ⅱ（AngⅡ）诱导的大鼠肾间质成纤维细胞活化的影响。方法：（1）有效Mas siRNA的筛选：设计合成3对不同序列针对Mas基因的siRNA,瞬时转染大鼠肾间质成纤维细胞（NRK-49F）,实验分5组：Mas siRNA序列1、Mas siRNA序列2、Mas siRNA序列3、阴性siRNA序列及正常对照组。转染后48 h,分别用半定量RT-PCR及Western blotting检测Mas mRNA和蛋白的表达水平。（2）研究有效Mas siRNA对Ang-(1-7)拮抗AngⅡ的影响：实验分6组：正常对照组、AngⅡ组、Ang-(1-7)组、AngⅡ+Ang-(1-7)组、阴性siRNA对照组和Mas siRNA转染组。分别培养72 h后,细胞免疫化学法检测细胞活化标志物α-平滑肌肌动蛋白（α-SMA）的表达,ELISA法检测上清液中细胞外基质成分Ⅰ型胶原（ColⅠ）的含量。结果：（1）与组相比,Mas siRNA各组Mas基因的表达均下降,其中以Mas siRNA序列2最明显（P<0.05）。 （2）有效Mas siRNA转染组在加AngⅡ+Ang-(1-7)干预72 h后,较非转染组和阴性siRNA转染组α-SMA及ColⅠ表达均增加,差异有统计学意义（P<0.05）。结论：Mas siRNA能有效抑制Mas的表达,使Ang-(1-7)拮抗AngⅡ诱导的大鼠肾间质成纤维细胞活化作用明显减弱。     

Recent advances in the angiotensin-converting enzyme 2-angiotensin(1-7)-Mas axis

In the past few years, the classical concept of the renin-angiotensin system (RAS) has experienced substantial conceptual changes. The identification of: the renin/prorenin receptor; the angiotensin-converting enzyme homologue, ACE2, as an angiotensin peptide-processing enzyme and a virus receptor for severe acute respiratory syndrome, the Mas as a receptor for angiotensin (1-7) [Ang(1-7)], and the possibility of signaling through ACE have contributed to switch our understanding of the RAS from the classical limited-proteolysis linear cascade to a cascade with multiple mediators, multiple receptors and multifunctional enzymes. With regard to Ang(1-7), the identification of ACE2 and of Mas as a receptor implicated in its actions contributed to decisively establish this heptapeptide as a biologically active member of the RAS cascade. In this review, we will focus on the recent findings related to the ACE2-Ang(1-7)-Mas axis and, in particular, on its putative role as an ACE-Ang II-AT(1) receptor counter-regulatory axis within the RAS.     

The role of angiotensin-(1–7) receptor Mas in spermatogenesis in mice and rats

Evidence regarding the components of the renin–angiotensin (Ang) system suggests that this system plays an important role in male reproduction. However, there are few data available in the literature on the effects of Ang-(1–7) on the male reproductive system. The present study investigated the effects of the genetic deletion and chronic blockage of Ang-(1–7) receptor Mas on spermatogenesis and male fertility. The localization of Mas in mouse and rat testes was determined by binding assays and immunofluorescence, whereas the testis structure and spermatogenic process were morphologically and stereologically analysed by light microscopy. Ang-(1–7) binding and immunofluorescence revealed the presence of Mas in the testes of mice and rats. Although the total numbers of Sertoli and Leydig cells per testis and Leydig cell size were similar in both wild-type and Mas -deficient mice, Mas ^−/– animals exhibited a significant reduction in testis weight and a greater volume of apoptotic cells, giant cells and vacuoles in the seminiferous epithelium. In both mice and rats, an increased number of apoptotic cells were found during meiosis. Due to disturbed spermatogenesis, daily sperm production was markedly reduced in Mas ^−/– mice. Moreover, chronic infusion of A-779 [an Ang-(1–7) antagonist] in rats significantly increased the total number of apoptotic cells and primary spermatocytes in particular stages of spermatogenesis. Taken together, these findings strongly suggest that Ang-(1–7) receptor Mas plays an important role in the regulation of spermatogenesis.     

rhAng-1通过PKCα/MLC通路影响局灶性脑缺血再灌注大鼠血脑屏障通透性

目的研究重组人血管生成素1(rhAng-1)对局灶性脑缺血再灌注大鼠血脑屏障内皮细胞骨架的影响。方法大鼠随机分8组:(1)假手术组;(2)缺血组;(3-5)生理盐水+缺血再灌注12h/48h/7d组;(6-8)Ang-1+缺血再灌注12h/48h/7d组。用伊文思兰渗透性实验检测血脑屏障通透性;Westernblot法检测微血管内皮细胞PKCα和p-MLC的表达。结果 Ang-1可明显降低局灶性脑缺血再灌注大鼠血脑屏障通透性,可使脑微血管内皮细胞PKCα和p-MLC表达水平减少。结论 Ang-1降低血脑屏障通透性与降低PKCα/MLC通路表达相关。     

Inhibition of endothelin-1 by the competitive ETA receptor antagonist Ro 61-1790 reduces lesion volume after cold injury in the rat

The aim of the present study was to investigate whether endothelin-1 (ET-1) in cerebral arteries is inhibited by the new, non-peptidergic ET(A) receptor antagonist Ro 61-1790 and, if it is, whether that inhibition reduces the lesion volume induced by cold injury in the parietal cortex. In vitro experiments were performed by measuring the isometric contractions of the rat middle cerebral and basilar arteries. A cold lesion was induced in vivo by the application of a pre-cooled (-78 degrees C) copper cylinder (diameter 3 mm) to the intact dura of rats for 6 s. After 24 h, lesion volume was determined by the triphenyltetrazolium method. In vitro, ET-1 (10(-12) - 3x10(-7) M) caused a dose-dependent contraction under resting conditions in the middle cerebral and basilar arteries of control rats. Ro 61-1790 (3x10(-9) M, 10(-7) M) shifted the concentration-effect curves for ET-1 in a parallel fashion (Emax unaltered). Post-treatment with Ro 61-1790 (10(-7)-10(-5) M) also inhibited the prior contraction elicited by ET-1 (3x10(-9) M) significantly. In vitro ET-1 application 3 h after the intracerebroventricular in vivo administration of Ro 61-1790 showed that the antagonist had reached the arteries and was bound to their ET(A) receptors. Intracerebroventricular pre-treatment of Ro 61-1790 reduced significantly the lesion volume by 23% after the injury. We conclude that ET-1 is involved in the development of secondary brain damage and that intracerebroventricular treatment with Ro 61-1790 reduces the size of the brain lesion caused by cold injury.     

Netrin-1 attenuates ischemic stroke-induced apoptosis

In the present study we tested the protective effects of netrin-1 in stroke and investigated the potential underlying mechanisms. When we performed middle cerebral artery occlusion (MCAO) on adult mice, up-regulation of the receptor uncoordinated gene 5H2 (UNC5H2) but not its ligand netrin-1 was detected with RT-PCR and immunohistochemistry. Injection of netrin-1, 1 day after MCAO, significantly reduced infarct volume at 3 days after MCAO as revealed by functional magnetic resonance imaging. The ischemic cortex was preserved when netrin-1 was continuously administered. Fluoro-Jade and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling staining showed that netrin-1 reduced the number of dying neurons and apoptotic cells after MCAO. Ischemia-induced p53 expression was attenuated by netrin-1. We also tested the ability of netrin-1 to attract intrinsic neuronal stem cells to the infarct area. Both DCC and UNC5H2 were expressed in neurosphere culture and netrin-1 attracted stem cells in an in vitro transwell assay. However, in vivo netrin-1 administration did not enhance the MCAO-induced stem cell migration toward the infarct area. Our study shows that UNC5H2 expression was elevated after MCAO and administration of netrin-1 protected infarct tissue from p53-mediated apoptosis. These data indicate that the p53/dependent receptor pathway is involved in ischemic stroke pathology and suggest possible new stroke therapies.     

Engeletin protects against cerebral ischemia/reperfusion injury by modulating the VEGF/vasohibin and Ang-1/Tie-2 pathways

Engeletin is a natural derivative of Smilax glabra rhizomilax that exhibits anti-inflammatory activity and suppresses lipid peroxidation. In the present study, we sought to elucidate the mechanistic basis for the neuroprotective and pro-angiogenic activity of engeltin in a human umbilical vein endothelial cells (HUVECs) oxygen-glucose deprivation and reoxygenation (OGD/R) model system and a middle cerebral artery occlusion (MCAO) rat model of cerebral ischemia and reperfusion injury. These analyses revealed that engeletin (10, 20, or 40 mg/kg) was able to reduce the infarct volume, increase cerebral blood flow, improve neurological function, and bolster the expression of vascular endothelial growth factor (VEGF), vasohibin-2 (Vash-2), angiopoietin-1 (Ang-1), phosphorylated human angiopoietin receptor tyrosine kinase 2 (p-Tie2), and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in MCAO rats. Similarly, engeletin (100, 200, or 400 nM) markedly enhanced the migration, tube formation, and VEGF expression of HUVECs in an OGD/R model system, while the VEGF receptor (R) inhibitor axitinib reversed the observed changes in HUVEC tube formation activity and Vash-2, VEGF, and CD31 expression. These data suggested that engeletin exhibited significant neuroprotective effects against cerebral ischemia and reperfusion injury in rats, and improved cerebrovascular angiogenesis by modulating the VEGF/vasohibin and Ang-1/Tie-2 pathways.