Effect of Sodium Tanshinone Ⅱ A Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinase1/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective:To observe the effects of sodium tanshinone Ⅱ A sulfonate(STS)on angiotensin Ⅱ(Ang Ⅱ)-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase(P-ERK1/2).Methods:In the primary culture of neonatal rat myocardial cells.the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by[3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells.The expression of p-ERK1/2 was determined using Western blot and immunofluorescence Iabeling.Results:(1)The totaI protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ (1 μmol/L)for 24 h;STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein.(2)After pretreatment of myocardial cells with Ang Ⅱ(1 μ mol/L)for 5 min,the p-ERK1/2 protein expression was increased,with the most obvious effect shown at about 10 min;pretreatment of myocardial cells with STS at different doses(2,10,50 μ mol/L)for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner.(3)After the myocardial cells were stimulated by Ang Ⅱ(1 μ mol/L),the immunofluorescence of ERK1/2 rapidly appeared in the nucleus.The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS.Conclusion:STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ,and the mechanism may be associated with the inhibition of p-ERK1/2 expression.     

Inhibitory Effect of Sodium Ferulate on the Cardiomyocyte Hypertrophy Induced by AngiotensionⅡ

Objective:To investigate the effects of sodium ferulate(SF) on myocardial hypertrophy of rat and explore the protective mechanism. Methods:The myocardial hypertrophy was induced by 0.1 μmol·L~(-1) Ang II. The cytoactive was detected by MTT. The cultured cardiomyocytes from Sprague Dawley neonate rats were randomly divided into normal,model,L-arginine(L-arg 1 000 μmol·L~(-1)) group and SF(50,100,200 μmol·L~(-1)) group.To observe whether SF had nonspecific injurious effect on the cells,SF 200 μmol·L~(-1) was added into the normal cardiomyocytes and to determine whether the effect of SF on cardiomyocyte hypertrophy was associated with NO release,another two groups were established. NG-nitro-L-arginine-methyl ester(L-NAME) 1 500 μmol·L~(-1) combined with SF 200 μmol·L~(-1) or L-arg 1 000 μmol·L~(-1),respectively. Cardiomyocyte hypertrophy was confirmed by observing the histological changes and the measurements of cell diameter,protein content and ANF and β-MHC m RNA expression of the cells.The levels of NO,NOS and eNOS activity,the contents of cGMP and cAMP. The expression of eNOS were detected by Real time PCR and Western blotting. Results:(1) SF(50,100,200 μmol·L~(-1)) had no obvious side effect on cultured neonatal rat cardiomyocytes in vitro. In the group added 0.1 μmol·L~(-1) Ang Ⅱ alone,the cells displayed swollen,with undistinguishable border;the diameter and protein content of cardiomyocytes was increased remarkably,and the expression of ANF and β-MHC m RNA were up-regulated by Ang Ⅱ. SF and L-arg could ameliorate the cardiomyocyte hypertrophy which can be inhibit by L-NAME.(2) Compared with normal group,0.1 μmol · L~(-1) Ang II could decrease the NO content,NOS and eNOS activity in supernatant of cultured cardiomyocytes,decrease the content of cGMP and increase the content of cAMP incardiomyocytes,upregulation the expression of eNOS.SF-H and L-arg administrated could siginificantly ameliorate these changes. Conclusion:SF can inhibit cardiomyocyte hypertrophy induced Ang Ⅱ in rats. The probable mechanism involved to promote NO-cGMP signaling pathway and up-regulate the expression of eNOS.     

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

Summary： The effects of cyclosporine A （CsA） on Angiontensin Ⅱ （Ang Ⅱ ）-induced protein contents, c los protein levels and cytosolic Ca^2＋ level （[Ca^2＋]i） in cultured eardiomyocytes of neonatal rats were observed. Total protein contents were determined by Bradford method. The expression of c-fos protein was detected by Western blot. （[Ca^2＋]i） labeled with fluorescent probe Fluo-3/AM was measured under a laser scanning confoeal microscope. The results revealed that as compared with control, the total protein contents were increased in cardiomyocytes treated with Ang Ⅱ （10-1 mol/ L）, which could be inhibited by CsA in a dose-dependent manner. It was found that Ang Ⅱ could increase the c-los protein expression, which could be inhibited by CsA in a dose-dependent manner. Ang Ⅱ induced the [Ca^2＋]i elevation in cardiomyocytes. CsA did not influence the resting intracellular Ca^2＋ , but inhibited significantly the Ang Ⅱ-induced [Ca^2＋]i elevation. It was concluded that CsA can suppress the Ang Ⅱ-induced c-fos protein expression and [Ca^2＋]i elevation in single cardiomyocyte, which might play a role in the prevention of Ang Ⅱ-induced cardiomyocyte hypertrophy by CsA.     

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.     

Nardosinone protects H9c2 cardiac cells from angiotensin II-induced hypertrophy

Pathological cardiac hypertrophy induced by angiotensin Ⅱ （Ang Ⅱ ） can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots ofNardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on Ang Ⅱ-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosi- none （25, 50, 100, and 200μmol/L） or Ang Ⅱ （1 μmol/L）. Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by Ang Ⅱ. The mRNA expression of ANP, BNP and 13-MHC was obviously elevated in Ang Ⅱ-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang Ⅱ-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.     

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)     

阿托伐他汀对心肌肥大的抑制作用及FoxO1表达的影响

目的探讨阿托伐他汀(atorvastatin)对血管紧张素Ⅱ(AngⅡ)诱导的肥大心肌细胞的抑制作用及对转录因子FoxO1表达的影响。方法将H9C2细胞分为3组,对照组:未给予任何干预;心肌肥大组:给予AngⅡ(10-7 mol/L)刺激细胞;阿托伐他汀组:预先给予atorvastatin(10-5 mol/L),30min后AngⅡ(10-7 mol/L)刺激细胞。western-blotand real time PCR检测H9C2细胞FoxO1蛋白及mRNA含量,脑钠肽(brain natriuretic pepide,BNP)作为判断心肌肥大的指标。结果 AngⅡ诱导心肌细胞肥大后FoxO1表达较正常心肌细胞明显降低(P<0.05),而给予阿托伐他汀干预的肥大心肌细胞其FoxO1表达较肥大心肌明显升高(P<0.05)。结论阿托伐他汀可能通过上调FoxO1表达,从而抑制心肌肥大。     

Effects of cyclosporine A on angiotensin II-induced cardiomyocyte hypertrophy in neonatal rats

Summary 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 pay a role in the prevention of Ang II-induced cardiomyocyte hypertrophy by CsA. Han Zhaomin, female, born in 1974, Pharmacist     

冬虫夏草提取液对肾小管上皮细胞Klotho表达和凋亡的影响

目的：研究冬虫夏草(cordyceps sinensis, CS)提取液及氯沙坦(losartan, Los) 对血管紧张素II (Ang II) 刺激后的大鼠肾小管上皮细胞 NRK-52E中Klotho（Kl）,P53,P21表达和凋亡的影响,探讨CS对Ang II诱导肾小管上皮细胞凋亡影响的机制。方法：CS（0, 5,10,20,40,80 mg/L）单独或与Ang II (1×10-8 mol/L) 共同培养24 h。确定CS的最佳干预浓度后设立对照组,Ang II (1×10-8 mol/L) 组,Ang II (1×10-8 mol/L) +CS (40 mg/L) 组,Ang II (1×10-8 mol/L) + Los (1×10-5 mol/L) 组和Ang II (1×10-8 mol/L) +CS (40 mg/L) + Los (1×10-5 mol/L) 组,培养24 h后进行实验。四甲基偶氮唑盐（MTT）比色法检测细胞的增殖;分别用RT-PCR和Western 印迹法检测Kl,P53和P21 mRNA及蛋白的表达;分光光度法检测caspase-3的活性;Annexin V-FITC双染法及流式细胞仪检测细胞凋亡率。结果：一定浓度范围的CS单独作用可促进NRK-52E细胞增殖,还能拮抗Ang II对其增殖的抑制(P＜0.01或P＜0.05);Ang II可下调Kl mRNA及蛋白表达,上调P53和P21 mRNA及蛋白表达,增加caspase-3活性和细胞凋亡率 (均P＜0.01);加入CS或/和Los后,Kl mRNA及蛋白表达明显上调,P53和P21 mRNA及蛋白表达下调,caspase-3活性和细胞凋亡率明显降低(均P＜0.05),各药物干预组间的作用差异无统计学意义(P＞0.05)。结论：CS可逆转Ang II诱导的Kl低表达,并抑制P53及P21的表达,从而抑制Ang II诱导的肾小管上皮细胞凋亡,可能是其对高血压肾损害起保护作用的机制之一。     

慢病毒介导ACE2过表达对血管紧张素Ⅱ诱导的大鼠肝细胞的白蛋白表达及迁移能力的影响

目的探讨血管紧张素转换酶2（Angiotensin-converting enzyme 2, ACE2）过表达对血管紧张素Ⅱ（Angiotensin II, Ang
Ⅱ）诱导的肝细胞白蛋白表达下调及细胞迁移性增强的抑制作用。方法常规培养大鼠肝细胞系BRL-3A细胞株,予以Ang II
（10-7 mol/L）进行刺激,观察不同处理时间（24、48 h）组肝细胞内白蛋白、波形蛋白（vimentin）表达及细胞迁移性的改变;通过构
建lentiACE2慢病毒载体,建立稳定过表达ACE2的细胞系,并分别用AngⅡ、A779进行干预,观察肝细胞内相应蛋白表达及其
迁移性的改变。采用细胞免疫荧光、Western blot检测细胞中蛋白水平变化;Transwell迁移实验观察不同处理组细胞迁移能力
的改变。结果AngⅡ处理组肝细胞内vimentin表达显著增加、albumin表达减低,细胞迁移能力显著增强,并具有时间依赖效
应。ACE2 过表达大鼠肝细胞albumin 表达明显升高,vimentin 蛋白表达下降,该作用被MAS受体抑制剂A779 阻断。同时
ACE2过表达显著抑制AngⅡ的作用,vimentin合成显著下降,albumin表达水平显著升高,细胞迁移能力受到抑制。结论ACE2
过表达可抑制AngⅡ诱导的肝细胞albumin表达下调及迁移能力增强。
     

NAD对AngⅡ诱导的心肌成纤维细胞Ⅰ型胶原mRNA表达的影响

目的   探讨烟酰胺腺嘌呤二核苷酸(NAD) 对血管紧张素II（AngII)诱导的大鼠心肌成纤维细胞I型胶原mRNA表达的作用。方法   提取新生Wistar大乳鼠原代心肌成纤维细胞,传代培养,采用2~4代细胞。实验分为空白对照组,AngII (0.01、 0.1、 1μmol／L)组,NAD(250μmol／L)组, AngII(1μmol／L)+ NAD (250μmol／L )组,FAM组,Sirt1-siRNA+AngII(1μmol／L)组,Sirt1-siRNA 组,Sirt1-siRNA+ NAD（250μmol／L）组,Sirt1-siRNA+Ang II(1mol／L)+NAD（250μmol／L）组。刺激24h后,提取总RNA, Real time RT-PCR法分别检测SIRT1和I型胶原mRNA的表达。结果   心肌成纤维细胞I型胶原mRNA的表达随着AngⅡ浓度升高明显增加(P＜0.05),呈浓度依赖性。与空白对照组比较,NAD（250μmol／L）组SIRT1 mRNA表达增高(P＜0.05)。与AngⅡ（1μmol／L）组比较,AngII (1μmol／L)+ NAD(250μmol／L )组I型胶原mRNA的表达明显减少(P＜0.01)。相对于FAM组,Sirt1-siRNA转染后各组SIRT1 mRNA的表达减少(P＜0.05),而心肌成纤维细胞I型胶原mRNA表达增多(P＜0.05)。结论   NAD可以抑制心肌成纤维细胞 I型胶原mRNA的表达,SIRT1影响I型胶原mRNA的表达,它们对Ang II诱导的心肌纤维化有保护作用。     

Effect of Shengmai injection (生脉注射液) on vascular endothelial and heart functions in patients with coronary heart disease complicated with diabetes mellitus

Objective： To study the effect of Shengmai injection （生脉注射液, SMI） on vascular endothelial and heart functions in coronary heart disease patients complicated with diabetes mellitus （CHD-DM）. Methods： One hundred and twenty patients with CHD-DM, their diagnosis confirmed by coronary arteriography, were equally randomized into a control group treated with conventional treatment and a treated group treated with conventional treatment plus SMI. The changes in blood levels of nitric oxide （NO）, endothelin-1 （ET-1） and angiotensin Ⅱ （Ang Ⅱ）, as well as endothelium-dependent vascular dilating function and heart function in the patients were observed before treatment and after the 3-week treatment. Results： After being treated with SMI for 3 weeks, in the treated group, blood level of NO was raised significantly from 69.8±33.1 μmol/L to 120.1±50.8μmol/L, and ET-1 was lowered from 70.1±32.1 ng/L to 46.2±21.3 ng/L, respectively （P〈0.01）; that of Ang Ⅱ was lowered from 81.3±24.3 ng/L to 50.2±27.3 ng/L （P〈0.01）; brachial arterial post-congestion blood flow increasing rate was raised from 389.4±26.3% to 459.3±27.8% （P〈0.01）; and the improvement in heart function as seen through the ejection fraction （EF） was increased from 44±5% to 68±6% （P〈0.01）, all the changes being more significant than those in the control group （all P〈0.01）. Conclusion： SMI can improve not only the endothelial function in CHD-DM patients, but also heart contraction significantly.     

Effect of angiotensin Ⅱ and angiotensin Ⅱ type 1 receptor antagonist on the proliferation,contraction and collagen synthesis in rat hepatic stellate cells

Background Angiotensin Ⅱ （Ang Ⅱ） is a very important vasoactive peptide that acts upon hepatic stellate cells （HSCs）, which are major effector cells in hepatic cirrhosis and portal hypertension. The present study was aimed to investigate the effects of Ang Ⅱ and angiotensin Ⅱ type 1 receptor antagonist （AT1RA） on the proliferation, contraction and collagen synthesis in HSCs.
Methods HSC-T6 rat hepatic stellate cell line was studied. The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by ^3H-thymidine incorporation. The effects of angiotensin Ⅱ and AT1RA on HSCs contraction were studied by analysis of the contraction of the collagen lattice. Cell culture media were analyzed by RT-PCR to detect secretion of collagen Ⅰ （Col Ⅰ）, collagen Ⅲ （Col Ⅲ） and transforming growth factor β1 （TGF-β1） by enzyme linked immunosorbent assay. HSC was harvested to measure collagen Ⅰ, collagen Ⅲ and tissue inhibitor of metalloproteinase-1 （TIMP-1） mRNA expression.
Results Ang Ⅱ （（1×10^-10-1×10^-4）mol/L）stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells. AT1RA inhibited angiotensin Ⅱ induced proliferation of HSCs. A linear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin Ⅱ （1×10^-9-1×10^-5 mol/L） and with time over 48 hours. AT1RA blocks angiotensin Ⅱ induced contraction of collagen lattice. Col Ⅰ, Col Ⅲ and TGF-β1 levels of the Ang Ⅱ group were higher than those of control group and this increase was downregulated by AT1RA. The mRNA expressions of Col Ⅰ, Col Ⅲ and TIMP-1 were higher in HSCs from the Ang Ⅱ group than the control group and downregulated by AT1RA.
Conclusions Angiotensin Ⅱ increased DNA synthesis and proliferation of HSCs in a dose-dependent manner, stimulated the contraction of HSCs dose- and time-dependently. Angiotensin also promoted excretion of Col I, Col Ⅲ and TGF-β1 lev