Effects of Serum Containing Xinlikang (心力康) on Angiotensin Ⅱ Induced Hypertrophy in Cultured Neonatal Rat Cardiomyocytes

Objective: To evaluate the effects of Xinlikang (心力康,XLK) on angiotensinⅡ(AngⅡ) induced hypertrophic cultured neonatal rat's cardiomyocyte (CMC). Methods: Primary cultured neonatal rat's CMCs with the purity certified by immunohistochemical technique, were divided into three groups. Rats in the normal control group were untreated; those in the model group were established into hypertrophic models but underwent no treatment; and those in the XLK group were established to hypertrophic models and treated with XLK containing serum obtained from rats with aorta coarctation after 8 days of feeding with XLK. MTT and phase-contrast microscope were used to evaluate the effect of XLK on cell activity, pulsating rhythm and surface area; Atrial natriuretic peptide (ANP) expression was determined by radioimmunoassay; Protein content was determined by Bradford method; and DNA synthesis was detected by flow cytometric assay. Results: Immunohistochemistry results showed that more than 90% of the cells wereα-sarcometin actin stained positive cells. No significant effect of XLK on normal CMC was found. AngⅡcould significantly induce hypertrophy in CMCs, and XLK could significantly decrease the increased surface area and the accelerated pulsating rate in them. ANP expression was 780±38 Mg/L in the model group, and 430±23μg/L in the control group, and the elevated expression of ANP in model rats was significantly decreased in the XLK group;The DNA content in the G0/G1 and G2/M phases was significantly enhanced and at the same time it was accompanied with increase of total protein content in the model rats after being stimulated by AngⅡfor 24 h, showing that serum-containing XLK could also significantly suppress total protein synthesis (P<0. 05). Conclusion: XLK could improve AngⅡmediated pathological growth of CMCs without influencing the growth of normal CMCs, suggesting that XLK is probably an effective drug for treatment of myocardial hypertrophy and heart failure.     

EXPERIMENTAL WORK AND RESEARCH——Effects of Serum Containing Xinlikang （心力康） on Angiotensin Ⅱ Induced Hypertrophy in Cultured Neonatal Rat Cardiomyocytes

Objective: To evaluate the effects of Xinlikang (XLK) on angiotensin II (Ang II) induced hypertrophic cultured neonatal rat’s cardiomyocyte (CMC).Methods: Primary cultured neonatal rat’s CMCs with the purity certified by immunohistochemical technique, were divided into three groups. Rats in the normal control group were untreated;those in the model group were established into hypertrophic models but underwent no treatment;and those in the XLK group were established to hypertrophic models and treated with XLK containing serum obtained from rats with aorta coarctation after 8 days of feeding with XLK. MTT and phase-contrast microscope were used to evaluate the effect of XLK on cell activity, pulsating rhythm and surface area;Atrial natriuretic peptide (ANP) expression was determined by radioimmunoassay; Protein content was determined by Bradford method;and DNA synthesis was detected by flow cytometric assay.Results: Immunohistochemistry results showed that more than 90% of the cells were α-sarcometin actin stained positive cells. No significant effect of XLK on normal CMC was found. Ang II could significantly induce hypertrophy in CMCs, and XLK could significantly decrease the increased surface area and the accelerated pulsating rate in them. ANP expression was 780 ±38 μg/L in the model group, and 430 ±23 ώg/L in the control group, and the elevated expression of ANP in model rats was significantly decreased in the XLK group;The DNA content in the G₀/G₁ and G₂/M phases was significantly enhanced and at the same time i was accompanied with increase of total protein content in the model rats after being stimulated by Ang II for 24 h, showing that serum-containing XLK could also significantly suppress total protein synthesis (P < 0. 05).Conclusion: XLK could improve Ang II mediated pathological growth of CMCs without influencing the growth of normal CMCs, suggesting that XLK is probably an effective drug for treatment of myocardial hypertrophy and heart failure. Supported by Grant from Hubei Science and Technology Bureau Research Project (No. 2001AA309B)     

Effect of Atorvastatin on Expression of Peroxisome Proliferator-activated Receptor Beta/delta in AngiotensinⅡ-induced Hypertrophic Myocardial CellsIn Vitro

Objective To explore the effect of atorvastatin on cardiac hypertrophy and to determine the potential mechanism involved. Methods Anin vitro cardiomyocyte hypertrophy from neonatal rats was induced with angiotensinⅡ (AngⅡ) stimulation. Before AngⅡ stimulation, the cultured rat cardiac myocytes were pretreated with atorvastatin at different concentrations (0.1, 1, and 10μmol/L). The following parameters were evaluated: the myocyte surface area,3H-leucine incorporation into myocytes, mRNA expressions of atrial natriuretic peptide, brain natriuretic peptide, matrix metalloproteinase 9, matrix metalloproteinase 2, and interleukin-1β, mRNA and protein expressions of theδ/β peroxisome proliferator-activated receptor (PPAR) subtypes. Results It was shown that atorvastatin could ameliorate AngⅡ-induced neonatal cardiomyocyte hypertrophy in the area of cardiomyocytes,3H-leucine incorporation, and the expression of atrial natriuretic peptide and brain natriuretic peptide markedly. Meanwhile, atorvastatin also inhibited the augmented mRNA level of several cytokines in hypertrophic myocytes. Furthermore, the down-regulated expression of PPAR-δ/β at both the mRNA and protein levels in hypertrophic myocytes could be significantly reversed by atorvastatin treatment. Conclusions Atorvastatin could improve AngⅡ-induced cardiac hypertrophy and inhibit the expression of cytokines. Such effect might be partly achieved through activation of the PPAR-δ/β pathway.     

Construction of shRNA Targeted to the Rat Angiotensin Ⅱ Type 1 Receptors and Its RNAi in Cytoplasma

Though remarkable progress has been madeinhypertension mechanisms in the last few decades ,the target of treating essential hypertension is stillfocusing pri marily on the rennin-angiotensin-aldo-sterone system(RAAS) .RAAS promotes vascularsmooth muscle constriction, remodels the struc-ture and function of the vascular system,enhancessodiumreabsorption in the kidney by sti mulatingaldosterone secretion. Therefore , RAAS is a cen-tral regulator of hypertension and plays a key roleinthe path…     

Effects and Mechanism of Weinaokang(维脑康) on Reperfusioninduced Vascular Injury to Cerebral Microvessels after Global Cerebral Ischemia

<正>Objective:To study the effects of the Weinaokang(维脑康,WNK),the active compounds extracted from Ginkgo,Ginseng,and saffron,on ischemia/reperfusion(I/R)-induced vascular injury to cerebral microvessels after global cerebral ischemia.Methods:Male C57BL/6J mice were randomly divided into 5 groups(10 animals/group):the sham group(0.5%CMC-Na,20 mL/kg),the I/R model group(0.5%CMC-Na, 20 mL/kg),the I/R+Crocin control group(20 mg/kg),the I/R+high dose WNK group(20 mg/kg),and the I/R+low dose WNK group(10 mg/kg).Bilateral common carotid artery occlusion(BCCAO,20 min) in mice, followed by 24 h reperfusion,was built.The generation of nitric oxide(NO),the activity of nitric oxide synthase (NOS),the phosphorylation of extracellular signal-regulated kinase 1/2(ERK1/2),and the expression of matrix metalloproteinases-9(MMP-9) and G protein-coupled receptor kinase 2(GRK2) in cortical microvascular homogenates were evaluated.The ultrastructural morphology of cortical microvascular endothelial cells (CMEC) was observed.Results:The transient global cerebral ischemia(20 min),followed by 24 h of reperfusion, significantly promoted the generation of NO and the activity of NOS.The reperfusion led to serious edema with mitochondrial injuries in the cortical CMEC,as well as enhanced membrane GRK2 expression and reduced cytosol GRK2 expression.Furthermore,enhanced phosphorylation of ERK1/2 and decreased expression of MMP-9 were detected in cortical micovessels after l/R(20 min/24 h).As well as the positive control Crocin(20 mg/kg, 21 days),pre-treatment with WNK(20,10 mg/kg,21 days) markedly inhibited nitrative injury and modulated the ultrastructure of CMEC.Furthermore,WNK inhibited GRK2 translocation from cytosol to the membrane(at 20 mg/kg) and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels.Conclusion:WNK and its active compounds(Crocin) are effective to suppress l/R-induced vascular injury to cerebral microvessels after global cerebral ischemia with the target on GRK2 pathways.     

Effect of Angiotensin Ⅱ on Cord Blood CD~(34+) Cells Expansion in vitro

In the development of the study on hematopoi etic stem cells, various hematopoietic cell growthfactors have received wide and profound research.Recently, some researchers wondered whether thebiological factors out of hematopoietic system hadsimilar effects on blood cells production and matu ration. AngiotensinⅡ is one of them. AngiotensinⅡ is recognized as a regulator of body water, elec trolyte and blood pressure equilibrium. But recentclinical experiments and laboratory studies…     

Effect of Electroacupuncture Stimulation on mRNA Expression of Angiotensinogen,Angiotensin Ⅱ Type 1 Receptor,Endothelin-1,and Endothelin A Receptor in Spontaneously Hypertensive Rat Aorta

Objective: To observe the effect of electroacupuncture(EA) stimulation on the expressions of angiotensinogen(AGT), angiotensin Ⅱ type 1 receptor(AT1R), endothelin-1(ET1), and endothelin A receptor(ETAR) m RNA in spontaneously hypertensive rat(SHR) aorta. Methods: Eighteen male SHRs were randomly divided into three groups, an SHR group, an SHR Baihui(DU 20) and Zusanli(ST 36) acupoint(SHR-AP) group, and an SHR non-acupoint(SHR-NAP) group, with 6 rats in each group. Six Wistar rats were used as a control. Rats in the SHR-AP group were stimulated by DU 20 and ST 36 acupoints, both of which were connected with EA. EA was handled one time every Monday, Wednesday and Friday, for total 24 times(8 weeks). SHRNAP rats were acupointed at a 15°angle flat into 0.5 cm to two points, which were 1 and 2 cm from rail tip separately. EA parameters were the same as the SHR-AP rats. SHR control rats and Wistar rats were fixed without EA. Real-time quantitative polymerase chain reaction(PCR) was used to measure AGT, AT1 R, ET1, and ETAR m RNA expression in rat aorta. Results: EA stimulation significantly reduced rat aorta vascular AGT, ET1, ETAR and AT1 R m RNA expressions in the SHR-AP and SHR-NAP groups(P0.01). Among these four genes, AT1 R m RNA expression was significantly lower in the SHR-AP than in the SHR-NAP group(P0.01). Conclusion: EA could reduce the AT1 R m RNA expression in SHR-AP rat aorta, indicating a potential mechanism for the hypotensive effects of EA.     

In Silico Screening of Potential Chinese Herbal Medicine Against COVID-19 by Targeting SARS-CoV-2 3CLpro and Angiotensin Converting Enzyme Ⅱ Using Molecular Docking

Objective: To seek potential Chinese herbal medicine(CHM) for the treatment of coronavirus disease 2019(COVID-19) through the molecular docking of the medicine with SARS-CoV-2 3 CL hydrolytic enzyme and the angiotensin converting enzyme Ⅱ(ACE2) as receptors, using computer virtual screening technique, so as to provide a basis for combination forecasting. Methods: The molecular docking of CHM with the SARS-Cov-2 3 CL hydrolase and the ACE2 converting enzyme, which were taken as the targets, was achieved by the Autodock Vina software. The CHM monomers acting on 3 CLpro and ACE2 receptors were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, the active ingredients were selected, and the key CHMs and compounds were speculated. Based on the perspective of network pharmacology, the chemical-target network was constructed, and the functional enrichment analysis of gene ontology and the pathway enrichment analysis of Kyoto encyclopedia of genes and genomes were carried out by DAVID to speculate about the mechanism of action of the core drug pairs. Results: There are6 small molecule compounds that have the optimal binding energy with the two target proteins. Among 238 potential anti-COVID-19 herbs screened in total, 16 kinds of CHM containing the most active ingredients, and5 candidate anti-COVID-19 herbs that had been used in high frequency, as well as a core drug pair, namely,Forsythiae Fructus-Lonicerae Japonicae Flos were selected. Conclusion: The core drug pair of Forsythiae Fructus-Lonicerae Japonicae Flos containing multiple components and targets is easy to combine with 3 CLpro and ACE2, and exerts an anti-COVID-19 pneumonia effect through multi-component and multi-target, and plays the role of anti-COVID-19 pneumonia in multi-pathway.     

HO-1在AMPK抑制心肌肥大中的作用

目的】 研究AMP激活的蛋白激酶 (AMPK)及血红素氧合酶HO-1激活对心肌肥大的影响,以及HO-1在AMPK抑制心肌肥大中的作用?【方法】 以培养的新生大鼠心肌细胞为研究对象,采用蛋白免疫印迹杂交方法检测5-氨基-4甲酰胺咪唑核糖核苷酸(AICAR)对AMPK的磷酸化程度及对HO-1的影响,并测定心肌细胞 [3H]-亮氨酸掺入率及心肌细胞表面积,观察AMPK及HO-1心肌细胞肥大的影响?【结果】 AICAR对AMPK及HO-1均有激活作用,AICAR能阻断AngⅡ引起的心肌肥大效应;阻断HO-1的激活则能减弱AICAR的抑制作用?【结论】 AMPK具有抑制心肌肥大的作用,AMPK能激活HO-1,HO-1在AMPK抗心肌肥大的机制中起重要作用     

一氧乙酰旋覆花内酯对血管紧张素Ⅱ诱导的肥大心肌细胞的保护性作用

目的 探讨ABL在心肌细胞肥大中的作用及其机制。方法 体外培养H9C2心肌细胞,血管紧张素Ⅱ（AngⅡ）构建心肌细胞肥大模型;模型组给予1μmol/L AngⅡ刺激24h、ABL处理组给予AngⅡ刺激,同时给予5μmol/L和10μmol/L ABL处理24h。采用心肌细胞肌动蛋白α-actinin染色评价心肌细胞大小,采用RT-PCR检测肥厚相关基因心房利钠肽（ANP）、脑钠肽（BNP）、肌球蛋白重链β（β-MHC）的表达;蛋白印迹（Western blot）法检测信号通路蛋白的表达。结果 与模型组比较,5μmol/L和10μmol/L ABL处理组心肌细胞面积明显降低（P<0.05）;ANP、BNP、β-MHC肥厚相关基因表达降低（P<0.05）,且其抗心肌细胞肥大呈现剂量依赖性（P<0.05）。Western blot法检测结果显示ABL处理组AMPKa磷酸化水平增高（P<0.05）,而Akt、mTOR、GSK3β的磷酸化明显抑制（P<0.05）;AMPKa拮抗剂Compound C可阻断ABL的心肌保护作用。结论 ABL可通过激活AMPKa信号通路,调节Akt/Mtor/GSK3β对AngⅡ诱导的心肌细胞肥大发挥保护性作用。     

Effects of Cyclosporine A on Angiotensin Ⅱ-induced Cardiomyocyte Hypertrophy in Neonatal Rats

The effects of cyclosporine A (CsA) on Angiontensin Ⅱ (Ang Ⅱ )-induced protein contents, c-fos protein levels and cytosolic Ca2+ level ([Ca2+ ]i) in cultured cardiomyocytes of neonatal rats were observed. Total protein contents were determined by Bradford method. The expression of c-fos protein was detected by Western blot. [Ca2+ ]i labeled with fluorescent probe Fluo-3/AM was measured under a laser scanning confocal microscope. The results revealed that as compared with control, the total protein contents were increased in cardiomyocytes treated with Ang Ⅱ (10-7 mol/L), which could be inhibited by CsA in a dose-dependent manner. It was found that Ang Ⅱ could increase the c-fos protein expression, which could be inhibited by CsA in a dose-dependent manner.Ang Ⅱ induced the [Ca2+ ]i elevation in cardiomyocytes. CsA did not influence the resting intracellular Ca2+ , but inhibited significantly the Ang Ⅱ-induced [Ca2+ ]i elevation. It was concluded that CsA can suppress the Ang Ⅱ-induced c-fos protein expression and [Ca2+ ]i elevation in single cardiomyocyte, which might play a role in the prevention of Ang Ⅱ -induced cardiomyocyte hypertrophy by CsA.     

血管紧张素Ⅱ诱导心肌细胞肥大后缝隙连接蛋白Cx43表达的变化

 [目的]研究血管紧张素Ⅱ诱导心肌细胞肥大后连接蛋白43（Cx43）表达的变化及与细胞周期分布的关系。[方法]分离培养大鼠心肌细胞,用血管紧张素Ⅱ诱导心肌肥大,72h后用RT-PCR和免疫荧光方法观察心肌细胞Cx43基因和蛋白表达,用流式细胞仪测定法观察心肌细胞周期分布变化以及Cx43表达量的关系。[结果]血管紧张素Ⅱ处理后的心肌细胞表现细胞肥大且细胞活力增强,S期、G2-M期细胞百分比增加,细胞内G2-M（二倍体）DNA含量降低,Cx43蛋白表达低于对照组,Cx43mRNA表达水平也较对照组明显下调,Cx43蛋白表达下调与细胞周期分布的改变相关。[结论]血管紧张素Ⅱ诱导心肌细胞肥大后Cx43基因表达明显下调,导致Cx43蛋白表达亦出现下调,这一改变可能与心肌肥大过程中的细胞周期变化有关,提示血管紧张素Ⅱ可能通过调控Cx43基因的表达而参与缝隙连接重构过程,而Cx43表达的变化可能与心肌肥大的机制有关。     

红景天苷通过调节miR-30d-5p表达减弱大鼠心肌细胞肥大的机制研究

[目的]研究红景天苷(salidroside,SAL)调控微小RNA(microRNA,miR)改善大鼠心肌肥大的机制。[方法]采用苯肾上腺素(phenylephrine,PE)刺激新生大鼠心肌细胞,建立心肌细胞肥大模型,以miR基因芯片筛选出3组细胞(正常对照组、模型组和SAL组)中有显著差异的miR,利用生物信息学方法和双荧光素酶报告基因确定靶基因。以Real-time PCR与Western blot分别检测靶基因mRNA及蛋白表达。在分别以SAL、候选miR模拟物和抑制物干预后,检测心肌细胞的表面积,分析靶基因和靶蛋白的表达。[结果]基因芯片结果表明,模型组与正常对照组有14种miR存在显著差异,SAL组与模型组有10种miR存在显著差异。模型组miR-30d-5p表达水平显著低于正常对照组和SAL组(P＜0.05),而SAL组miR-30d-5p表达量与正常对照组无统计学差异(P＞0.05)。生物信息学方法和双荧光素酶报告基因测定确定葡萄糖调节蛋白78(glucose regulated protein78,GRP78)是miR-30d-5p的靶基因。SAL能够上调PE刺激诱导的肥大心肌细胞的miR-30d-5p水平,抑制心肌细胞表面积增大;miR-30d-5p模拟物能抑制心肌细胞GRP78蛋白表达,而miR-30d-5p抑制物的作用结果相反。[结论]miR-30d-5p具有抑制心肌肥大的作用,SAL可能通过增加心肌细胞中miR-30d-5p的表达,抑制GRP78 mRNA及蛋白表达,从而改善心肌肥大。     

机械刺激诱导心肌细胞肥大的分子机制

机械负荷过度将导致病理性心肌肥大并严重危害人类健康。机械刺激作用于心肌细胞后,可通过细胞表面的机械感受器（如整合素）或分泌细胞因子（血管紧张素Ⅱ及内皮素等）,激活一系列信号通路,主要的有丝裂原激活蛋白激酶通路、詹纳斯激酶／信号转导蛋白转录激活因子通路及由钙离子介导的信号通路。心肌细胞将机械信号转化为化学信号,调节肥大相关基因的表达,致使心肌细胞发生肥大。     

非对称二甲基精氨酸诱导大鼠心肌细胞肥大的作用

目的：探讨非对称二甲基精氨酸（ADMA）诱导心肌细胞肥大的作用机制。方法：原代取材新生SD大鼠的心肌细胞进行细胞培养。原代培养的第4天，用不同浓度的ADMA（4～16μmol／L）和L-精氨酸（L-arg）（0．8～3．2mmol/L）处理心肌细胞，24h后测定细胞上清液中一氧化氮（NO）的含量、一氧化氮合酶（NOS）的活性，细胞内氧活性物质（ROS）的水平、RNA含量、总蛋白量以及心肌细胞表面积。结果：①4μmol／L，8μmol／L，16μmol／L的ADMA分别能剂量依赖性使细胞内NO的含量减少，NOS的活性降低（P〈0．05）；②16μmol／L的ADMA能使心肌细胞内ROS的水平升高、细胞内RNA含量和总蛋白含量增加以及心肌细胞表面积增加（P〈0．05）；⑧0．8mmol／L，1．6mmol／L，3．2mmol/L的L-arg对ADMA诱导的心肌细胞内ROS水平的升高、细胞内RNA含量和总蛋白含量的增加以及心肌细胞表面积的增加产生剂量依赖性的抑制作用（P〈0．05）。结论：ADMA能够诱导心肌细胞肥大，NO的前体L-arg能够剂量依赖性地抑制ADMA诱导的心肌细胞肥大。     

不同剂量维生素E对去甲肾上腺素诱导的心肌细胞肥大的影响

目的：观察不同剂量维生素E对去甲肾上腺素(NE)诱导心肌细胞肥大的影响。方法：培养乳鼠心肌细胞暴露于NE(20μmol／L)和维生素E(5μmol／L，50μmol／L，500μmol／L)36h，检测心肌细胞^3H-亮氨酸掺入率和蛋白质含量的变化。结果：NE可导致心肌细胞^3H-亮氨酸掺入率和蛋白质含量明显增高，50μmol／L，500μmol／L的维生素E干预可抑制NE的致心肌细胞肥大的作用。结论：NE的促心肌细胞肥大作用可能与活性氧生成增加有关，维生素E以剂量依赖方式抑制NE的促肥大作用。