自发性高血压大鼠肥厚左心室的蛋白质组学研究

目的 心肌肥厚是高血压患者心血管事件发生和死亡的主要危险因素,其机制仍未完全阐明.应用双向凝胶电泳/质谱的经典蛋白质组学方法,比较不同年龄自发性高血压大鼠(SHR)肥厚心肌的蛋白质表达谱的差异.方法 取4周、20周龄雄性SHR和WKY大鼠,观察血压和心肌肥厚情况;抽提其左心室组织蛋白,经二维凝胶电泳(2-DE)分离蛋白,银染后计算机图象分析寻找蛋白表达差异点,并以基质辅助激光解吸电离-飞行时间-飞行时间质谱测量法(MALDI-TOF-TOF MS)分析鉴定蛋白.结果 SHR在血压未升高时就已存在心肌肥厚.经二维胶图比较共找出27个差异蛋白点,质谱分析鉴定出20个差异蛋白,涉及能量代谢、线粒体氧化磷酸化和氧化应激反应等.其中13个蛋白的变化发生在4周龄SHR血压未升高时;另7个蛋白表达变化发生于20周龄血压持续升高状态下的SHR肥厚心肌中.结论 SHR肥厚心肌中脂肪酸氧化和葡萄糖有氧氧化中的酶下调,而糖酵解的关键酶明显上调,且这些变化大多发生在血压升高前;线粒体氧化磷酸化的多种酶和一些参与抗氧化作用的蛋白均发生了变化,提示在SHR的心肌肥厚过程中物质能量代谢和氧化应激也具有重要作用.     

自发性高血压大鼠肥厚左室心肌组织微小RNA-1与缝隙连接蛋白43的改变

目的观察自发性高血压大鼠(SHR)肥厚左室心肌组织微小RNA-1(miRNA-1)、缝隙连接蛋白43(Cx43)表达的变化及其关系,以探讨高血压性心肌肥厚发生室性心律失常(VA)的分子机制。方法 10只17周龄雄性SHR大鼠做为左室肥厚组(LVH组),10只8周龄雄性SHR大鼠做为对照组,通过病理学、心肌细胞横径的测量、实时荧光定量聚合酶链反应、免疫组织化学法及western blotting检测等方法 ,比较两组大鼠左室心肌组织病理学改变、miRNA-1及Cx43蛋白表达。结果①与对照组比较,LVH组的收缩压、舒张压升高,左室质量指数及心肌细胞横径均明显增大(P均0.05);miRNA-1表达水平明显升高,以及Cx43蛋白表达水平降低(0.27±0.10vs0.60±0.13,P0.05);②LVH组大鼠左室心肌组织miRNA-1与Cx43蛋白的表达水平呈显著负相关(r=-0.661,P0.05)。结论 miRNA-1可能通过抑制Cx43表达而参与高血压LVH发生VA。     

自发性高血压大鼠心肌fas基因表达与左室肥厚关系探讨

目的探讨自发性高血压大鼠(SHR)心肌fas表达及其与左室肥厚的关系. 方法自发性高血压大鼠(SHR)与正常血压大鼠各16只,尾套法测收缩压,逆转录-聚合酶链反应(RT-PCR)法测心肌fas mRNA表达.结果与WKY相比,SHR心肌fas表达、左室重量指数(LVMI)均显著升高,且二者呈正相关.结论自发性高血压大鼠早期心肌肥厚时心肌fas表达已经升高,可能参与后期心衰的发生.     

自发性高血压大鼠心肌fas基因表达与左室肥厚关系探讨

目的 探讨自发性高血压大鼠（SHR）心肌fas表达及其与左室肥厚的关系。方法 自发性高血压大鼠（SHR）与正常血压大鼠各16号，尾套法测收缩压，逆转录-聚合酶边反应（RT-PCR）法测心肌fas mRNA表达。结果 与WKY相比，SHR心肌fas表达、左室重量指数(LVMI)均显著升高，且二者呈正相关。结论 自发性高血压大鼠早期心肌肥厚时心肌fas表达已经升高，可能参与后期心衰的发生。     

Apelin-13在自发性高血压大鼠心肌组织的表达及其与心肌肥厚和心功能的关系

目的观察Apelin-13在自发性高血压大鼠(SHR)心肌组织的表达改变,探讨其与心肌肥厚和心功能的关系。方法选取清洁级4周龄和20周龄雄性自发性高血压大鼠和WKY(Wistar-Kyoto)大鼠,按周龄及种属分为4组,每组8只。分别测定无创尾动脉血压及心脏质量指数;超声心动图和血流动力学系统评估心室重构和心功能;HE染色评价心肌细胞及排列情况。Western blot法检测心肌组织Apelin-13、APJ的蛋白表达。结果 1Apelin-13、APJ在SHR心肌组织中呈低表达(P0.05),20周龄SHR较4周龄SHR更明显(P0.05)。2 SHR的收缩压(SBP)、左心室舒张期末压(LVEDP)、心脏质量指数(HW/BW)、心室质量指数(LVW/BW)、舒张期室间隔厚度(IVSD)和左心室舒张期末后壁厚度(LVWPd)明显升高(P0.05),左心室舒张期末内径(LVEDd)、左心室射血分数(EF)、左心室短轴缩短率(FS)和左心室压力最大下降速率(-dp/dtmax)明显降低(P0.05);心肌细胞明显肥大、排列紊乱。20周龄SHR与4周龄SHR相比,上述指标改变更加明显(P0.05)。3心肌组织Apelin-13与IVSD、HW/BW、LVW/BW以及LVEDd、LVEDP呈负相关(P0.05),与EF、FS和-dp/dtmax呈正相关(P0.05)。结论 Apelin-13在SHR心肌组织中呈低表达,其与心肌肥厚指标呈负相关,与心功能呈正相关,提示其可能影响高血压的左心室心肌肥厚和心功能。     

自发性高血压大鼠肥厚左室心肌组织微小RNA-1与内向整流钾通道2.1表达的改变及其意义

目的观察自发性高血压大鼠(SHR)肥厚的左室心肌组织微小RNA-1、内向整流钾通道2.1(Kir2.1)表达的变化及其关系,以探讨高血压左心室肥厚(LVH)发生室性心律失常的分子机制。方法取10只17周龄雄性SHR为LVH组,10只8周龄雄性SHR为阳性对照组,10只17周龄雄性WKY大鼠作为空白对照组,通过HE染色、心肌细胞横径测量、实时荧光定量聚合酶链反应(qRT-PCR)、免疫组织化学法及Western blot检测等方法,检测大鼠左室心肌组织病理学改变、微小RNA-1表达、Kir2.1蛋白表达水平的改变。结果①与空白对照组比较,LVH组和阳性对照组的收缩压、舒张压明显升高(分别P<0.01,P<0.05);②与两对照组相比,LVH组的左室质量指数及心肌细胞横径均明显增大(均P<0.05),左室心肌细胞明显肥大,心肌间质增多,伴随着微小RNA-1表达水平明显升高,Kir2.1蛋白表达水平显著降低(P<0.05);③LVH组大鼠左室心肌组织微小RNA-1与Kir2.1蛋白的表达水平呈负相关(r=-0.720,P<0.05)。结论SHR肥厚左室心肌组织微小RNA-1表达上调,并伴随Kir2.1表达下调。     

当归挥发油对自发性高血压大鼠ACE2基因表达的影响

目的探讨当归挥发油对自发性高血压大鼠(SHR)心肌血肌紧张素转换酶(ACE)2基因表达的影响及作用机制。方法将40只8周龄雄性SHR随机分为模型组,当归挥发油组(低、高)和卡托普利组,并以同周龄的Wistar大鼠作为正常对照组。分别灌胃4 w后,取大鼠心肌组织,HE染色观察心肌组织形态学的变化实时荧光定量PCR法检测ACE2 mRNA表达量,Western印迹检测ACE2蛋白的表达水平。结果当归可降低SHR血压;HE染色显示,与模型组相比较,当归挥发油各组病变分别有不同程度的减轻;与模型组相比,当归高、低剂量组ACE2 mRNA及蛋白表达水平均明显升高(P<0.01)。结论当归挥发油对SHR血压有调节作用,可能通过ACE2基因发挥降压作用。     

桑杞清眩颗粒干预自发性高血压大鼠心肌细胞凋亡的实验研究

目的观察桑杞清眩颗粒对高血压心肌重构的抑制作用及其可能机制。方法选8只4周龄雄性WKY大鼠和16只4周龄雄性自发性高血压(SHR)大鼠,WKY大鼠作为对照组(WKY),SHR大鼠随机分为模型组(SHR组)和给药组(SQ+SHR组),每组8只。每组在治疗16周后处死大鼠,尾套法测量SHR尾动脉收缩压、舒张压、平均压,计算心脏重量指数;用脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色来检测凋亡心肌细胞;用蛋白免疫印迹法检测大鼠心肌组织中B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化型半胱天冬酶-3(cleaved Caspase-3)。结果与WKY组相比,桑杞清眩颗粒显著抑制SHR大鼠收缩压的升高、心脏重量指数的增加、抑制高血压引起的大鼠心肌细胞凋亡、Bcl-2的降低及Bax和cleaved Caspase-3的增加。结论桑杞清眩颗粒可以抑制SHR大鼠血压升高,减轻心肌肥厚,其作用机制可能与抑制自发性高血压大鼠心肌细胞凋亡有关。     

自发性高血压大鼠心肌增殖细胞核抗原(PCNA)的表达以及厄贝沙坦和咪哒普利的影响

目的探讨自发性高血压大鼠(SHR)左心室肌增殖细胞核抗原(PCNA)的表达以及咪哒普利、厄贝沙坦的影响. 方法选用13周龄的SHR30只,雌性9只,雄性21只,体重228.5±39 g,随机分为三组,使得每组雌性3只,雄性7只,分别设为SHR组,厄贝沙坦组,咪哒普利组;另选同源同系Wistar-Kyoto 大鼠(WKY大鼠)10只,雌性5只,雄性5只,体重206.1 g±49.1 g,作为正常对照组(WKY组).实验期14周.观察指标血压、左室重量/体重(LVW/BW)、左室厚度/体重、心肌PCNA蛋白水平.结果 SHR组血压、LVW/BW、左室厚度/体重均增高,心肌PCNA蛋白的表达明显增加; 咪哒普利组、厄贝沙坦组血压、LVW/BW、左室厚度/体重、心肌增殖核抗原(PCNA)蛋白的表达均比SHR组低.结论 26周龄SHR左心室肌PCNA蛋白的表达明显升高,咪哒普利、厄贝沙坦不仅可以良好地控制血压,而且可以抑制自发性高血压大鼠左心室重塑,并可以降低SHR左心室肌PCNA蛋白的表达,二者对SHR左室肥厚的抑制效应可能与降低PCNA蛋白的表达有关系.     

黄素腺嘌呤二核苷酸通过激活SCAD抑制大鼠病理性心肌肥厚和心肌纤维化

目的探究黄素腺嘌呤二核苷酸(FAD)在自发性高血压大鼠病理性心肌肥厚和心肌纤维化中的作用及防治机制。方法选取12周龄的自发性高血压大鼠(SHR)及周龄匹配的Wistar大鼠。经尾静脉注射FAD(每天1μmol/kg)治疗10周后,采用无创血压测量仪检测大鼠的血压及心率;超声心动图和组织学方法观察病理性心肌肥厚和心肌纤维化程度;Western blot方法检测短链酰基辅酶A脱氢酶(SCAD)、CollagenⅠ、CollagenⅢ、α-SMA的蛋白表达水平;免疫荧光单标法进一步验证SCAD的蛋白表达水平;荧光定量PCR检测SCAD、ANF、脑利钠肽(BNP)以及CollagenⅠ、CollagenⅢ、α-SMA的mRNA表达水平;检测SCAD酶活性、ATP、游离脂肪酸、BNP及活性氧含量。结果自发性高血压大鼠经FAD治疗后,收缩压和心率均明显降低;病理性心肌肥厚和心肌纤维化程度得到明显改善;心肌组织中SCAD的mRNA、蛋白表达、酶活性及ATP含量均显著增高,游离脂肪酸和活性氧水平明显降低(P0.05)。结论 FAD可能通过激活SCAD,改善心肌能量代谢,减少氧化应激,从而抑制自发性高血压大鼠病理性心肌肥厚和心肌纤维化。     

Altered expression of BK channel beta1 subunit in vascular tissues from spontaneously hypertensive rats

Correlation of blood pressure (BP) with expression levels of large-conductance, voltage- and Ca2+-activated K+ (BK) channel beta1 subunit in vascular tissues from spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and Sprague-Dawley rats (SD) at different ages was investigated. Systolic BP and BK beta1 expression in mesenteric arteries at either mRNA or protein levels were not different among 4-week-old SHR, WKY, and SD. With hypertension developed at 7 weeks and reached plateau at 12 weeks, expression levels of BK beta1 mRNA in mesenteric arteries and aortae from SHR during this period of time were significantly higher than in age-matched normotensive WKY. The BK beta1 protein expression was significantly higher in mesenteric arteries from 12-week-old but not 7-week-old SHR when compared with age-matched WKY and SD. The BK beta1 protein levels in aortae were not different among 7-week-old SHR, WKY, and SD but were significantly lower in 12-week-old WKY than in age-matched SHR and SD. Captopril treatment normalized BP of 12-week-old SHR. This treatment downregulated BK beta1 protein in mesenteric arteries but upregulated it in aortae. No significant difference in BK alpha subunit expression was detected in mesenteric arteries from three strains of rats as well as the captopril-treated SHR. It appears that expression patterns of BK beta1 in vascular tissues vary depending on tissue types, animal age, and animal strains. Expression of BK beta1 in mesenteric arteries is closely correlated with BP in SHR. Increased BK beta1 expression in mesenteric arteries may represent a compensatory reaction to limit the development of hypertension.     

NEPHRIN EXPRESSION IN THE POST-NATAL DEVELOPING KIDNEY IN NORMOTENSIVE AND HYPERTENSIVE RATS

Nephrin is a slit diaphragm protein and its expression in the developing kidney is largely unknown. In this study, we explored the expression of nephrin in the developmental kidney in spontaneously hypertensive (SHR) and in Wistar-Kyoto (WKY) rats at different time points, from day 5 after birth to adulthood. Real time RT-PCR, in situ hybridization and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin in the kidney. SHR had hypertension at week 10 and albuminuria at week 20. Nephrin expression in both SHR and WKY increased from day 5 to adulthood. Furthermore, both gene and protein expression of nephrin were significantly lower in SHR after birth when compared to WKY at the same age. These findings suggest that both in normotensive and hypertensive rats, nephrin expression increased from birth to the adult age and that down-regulation of nephrin in SHR evident from the early developmental kidney to adulthood may contribute to the development of albuminuria in adult SHR.     

Nephrin expression in the post-natal developing kidney in normotensive and hypertensive rats

Nephrin is a slit diaphragm protein and its expression in the developing kidney is largely unknown. In this study, we explored the expression of nephrin in the developmental kidney in spontaneously hypertensive (SHR) and in Wistar-Kyoto (WKY) rats at different time points, from day 5 after birth to adulthood. Real time RT-PCR, in situ hybridization and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin in the kidney. SHR had hypertension at week 10 and albuminuria at week 20. Nephrin expression in both SHR and WKY increased from day 5 to adulthood. Furthermore, both gene and protein expression of nephrin were significantly lower in SHR after birth when compared to WKY at the same age. These findings suggest that both in normotensive and hypertensive rats, nephrin expression increased from birth to the adult age and that down-regulation of nephrin in SHR evident from the early developmental kidney to adulthood may contribute to the development of albuminuria in adult SHR.     

Differential expression of D2-like dopamine receptors in the kidney of the spontaneously hypertensive rat

OBJECTIVE: To compare the expression and cellular distribution of D(2)-like dopamine receptors in the kidney of the spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat. DESIGN: Renal D(2)-like receptor protein expression and distribution has not been studied in the SHR. Since changes in D(2)-like receptor expression and/or distribution may contribute to the dysregulation of renal dopamine and D(1A) receptor function, we examined the expression of the three subtypes of D(2)-like receptors (D(2), D(3) and D(4)) in SHR and WKY rat kidneys. METHODS: Western blot analysis and confocal immunocytochemistry with specific polyclonal antipeptide antibodies directed against the receptor subtypes, were used to assess protein expression. RESULTS: There were no differences in protein expression and cellular immunolocalization of the D(2) receptor subtypes between SHR and WKY rats. Expression of the 50 kDa D(3) receptor was reduced in the cortex of the SHR; no differences in D(3) receptor levels were seen in the inner medulla of SHR and WKY rats. The D(4) receptor polypeptides were overexpressed in the cortex of SHR, while in the inner medulla no difference in expression of the D(4) receptor proteins was observed between SHR and WKY rats. Immunocytochemistry also showed increased immunostaining of D(4) receptors in tubular structures in the cortex, but diminished staining in the SHR inner medulla. CONCLUSION: The observed differences in expression and distribution of D(3) and D(4) dopamine receptors between cortex and inner medulla of the kidneys of SHR and WKY rats may contribute to the aberrant state of dopaminergic-mediated natriuresis in SHR.     

硫化氢对自发性高血压大鼠血管炎症反应的调节作用

目的 探讨新型气体信号分子硫化氢(H2S)对自发性高血压大鼠(SHR)血管炎症反应的调节作用.方法 4周龄Wistar-Kyoto(WKY)雄性大鼠和SHR雄性大鼠分为4组:WKY大鼠对照组、SHR对照组、SHR+硫氢化钠(NaHS,H2S供体)组及SHR+炔丙基甘氨酸(PPG,H2S生成关键酶抑制剂)组,饲养至9周.尾容积法测清醒时大鼠血压,免疫组织化学方法检测主动脉内皮细胞膜细胞间黏附分子-1(ICAM-1)、核转录因子-κB p65(NF-κB p65)和核转录因子抑制因子-α(IκB-α)的蛋白表达,原位杂交分析检测主动脉内皮细胞ICAM-1 mRNA的表达.结果 SHR对照组血压显著高于WKY对照组(P<0.05),主动脉内皮细胞ICAM-1、ICAM-1 mRNA、胞核NF-κB p65明显高(P均<0.01),胞浆IκB-α蛋白表达明显低(P<0.01).SHR+NaHS组血压显著低于SHR对照组,主动脉内皮细胞ICAM-1、ICAM-1 mRNA、胞核NF-κB p65蛋白表达明显低(P<0.05),胞浆IκB-α蛋白表达明显增高(P<0.05),SHR+PPG组主动脉内皮细胞ICAM-1、ICAM-1mRNA、胞核NF-κB p65蛋白表达更高(P<0.05),胞浆IκB-α蛋白表达显著低(P<0.05).结论 H2S可通过抑制SHR血管炎症发挥抗高血压效应.H2S的抗血管炎症效应可能通过上调IκB-α的表达,降低NF-κB p65的表达,抑制ICAM-1 mRNA的转录和蛋白表达实现.     

Expression of the Na+/H+ exchanger regulatory protein family in genetically hypertensive rats

OBJECTIVE: To examine a possible involvement of a regulatory protein of Na+/H+ exchanger (NHE) in the increased renal NHE activity in spontaneously hypertensive rats (SHR), we investigated mRNA expression of inhibitory members of the NHE regulatory protein family, NHERF1 and NHERF2, in the kidney. DESIGN: Prehypertensive 4-week-old and hypertensive 11-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were used to determine the changes in NHE activity and NHERF family expression in the kidney. Dahl salt sensitive (DS) and resistant rats were also used to examine whether these changes are specific for SHR. METHODS: mRNA expression in the kidney was quantified by RNase protection assay. The NHE activity in primary cultured proximal tubular cells was measured as Na-dependent pHi recovery rate by the NH4Cl prepulse technique with 2'7'-bis-(2-carboxyethyl)-5.6-carboxyfluorescein (BCECF). RESULTS: NHERF1 mRNA expression was significantly decreased in both prehypertensive and hypertensive SHR in comparison with age-matched WKY rats, whereas NHERF2 mRNA expression was significantly increased in SHR only in the hypertensive period. Antihypertensive treatment did not abolish these changes seen in control SHR. On the other hand, hypertensive DS rats fed a high-salt diet showed significant decreases in NHE activity and NHE3 mRNA expression compared with normotensive DS rats fed a low-salt diet, without significant changes in NHERF1 and NHERF2 mRNA expression. CONCLUSION: These results suggest that decreased expression of NHERF1 may be related to the enhanced NHE activity in SHR and that these changes are likely to be genetically determined, whereas the increased NHERF2 expression may be induced as a compensatory mechanism.     

Decreased expression of Na+-H+ exchanger isoforms 1 and 3 in denervated spontaneously hypertensive rat kidney

To determine whether the sympathetic nerve plays a role in the regulation of Na⁺-H⁺ exchange (NHE) in the kidney of spontaneously hypertensive rats (SHR), we investigated the expression of NHE and NHE regulatory protein family (NHERF) in the denervated kidneys compared with intact kidneys. Twelve-week-old male SHR and age-matched Wistar Kyoto (WKY) rats were used. SHR were randomly assigned to the renal denervated (RDNX, n = 8) or Sham (n = 8) groups. The protein and mRNA expression of NHE1, NHE3, NHERF1 and NHERF2 were assessed in the kidney of the groups. Following the renal denervation, immunohistochemistry and western blot analysis showed that NHE1 and NHE3 protein were signi?cantly decreased in the kidney compared with Sham group. NHERF1 protein expression was signi?cantly increased in RDNX compared with Sham group, whereas NHERF2 protein expression was signi?cantly decreased after renal denervation. Similar results were observed at the mRNA level of NHE1, NHE3, NHERF1 and NHERF2 expression. The present ?ndings suggest that the renal sympathetic nervous system plays a role in the regulation of NHE1 and NHE3 in the kidney of SHR, and NHERF1 may be involved in the expression of NHE3 in the kidney of SHR.     

Altered gene expression of prostacyclin synthase and prostacyclin receptor in the thoracic aorta of spontaneously hypertensive rats.

OBJECTIVE: The aim of this study was to evaluate the possible role of prostacyclin (PGI2) in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). METHODS: Measurement of mRNA and protein levels of PGH synthase (PGHS)-1, PGI2 synthase and the PGI2 receptor, in the thoracic aorta was performed in SHR aged 5, 10, 20, and 40 weeks old and in age-matched normotensive Wistar-Kyoto (WKY) rats with a competitive polymerase chain reaction method and immunoblotting. Aortic production of 6-keto-PGF1 alpha, the main metabolite of PGI2, was also measured. RESULTS: Compared with age-matched WKY rats, PGHS-1 mRNA and protein levels in the thoracic aorta of SHR increased with age, reaching three- and twofold higher than WKY rats at 40 weeks old, respectively. PGI2 synthase mRNA and protein levels in SHR were significantly higher than in WKY rats at 20 and 40 weeks old. In contrast, PGI2 receptor mRNA levels in SHR were consistently lower than in WKY rats at all ages. CONCLUSIONS: These results provide evidence that hypertension elicits alterations in levels of arachidonic acid metabolites, including PGH2 and PGI2. They also suggest that the decreased expression of PGI2 receptor mRNA in prehypertensive SHR could be one of the causes of hypertension in SHR.