荞麦花叶中芦丁对血管紧张素Ⅱ诱导的新生大鼠心肌细胞肥大的体外抑制作用

目的 观察荞麦花叶中芦丁(RBFL)对新生大鼠心肌细胞肥大的作用,并探讨其对蛋白激酶C(PKC)信号途径的影响.方法 选用培养的新生大鼠心肌细胞为研究对象,以10-7mol·L-1血管紧张素Ⅱ(AngⅡ)刺激建立体外心肌细胞肥大模型,并用不同浓度的RBFL作用于细胞,测定心肌细胞表面积,3H-亮氨酸掺入法测定心肌细胞蛋白质的合成速率,免疫细胞化学法测定心肌细胞PKC蛋白的表达,同位素底物掺入法检测PKC活性.结果 10-7mol·L-1AngⅡ能显著增加心肌细胞表面积和蛋白质合成速率、PKC蛋白的表达及其活性;1～10 mg·L-1RBFL能剂量依赖性地抑制AngⅡ诱导的心肌细胞表面积和蛋白质合成速率的增加、PKC蛋白的表达及其活性,并具有统计学意义.结论 RBFL能明显抑制培养的新生大鼠心肌细胞的肥大,此作用可能与其对心肌细胞中PKC信号途径的调控有关.     

碘化N-正丁基氟哌啶醇通过蛋白激酶C途径保护缺氧复氧心肌细胞

目的研究碘化N-正丁基氟哌啶醇(F2)对缺氧复氧(H/R)所致心肌细胞损伤的作用及蛋白激酶C(PKC)活性的影响。方法用1～3d的Sprague-Dawley(SD)新生乳鼠进行心肌细胞原代培养,取生长4～5d的心肌细胞制作H/R模型。采用非放射性同位素法测定PKC活性、蛋白免疫印迹法检测PKC蛋白转位;双抗体夹心光化学测定法和酶联免疫吸附试验(ELISA)方法分别检测培养心肌细胞上清液中肌钙蛋白(cTnI)浓度和肿瘤坏死因子-α(TNF-α)分泌观察心肌细胞损伤状况。结果心肌细胞缺氧复氧时,PKC总活性明显升高,PKCα和βⅡ均发生转位。F2和cPKC特异性抑制剂G6976可减少cTnI泄漏和TNF-α的分泌,减轻心肌细胞H/R所致的损伤;PKC激动剂thymeleatoxin(TXA)拮抗F2的心肌保护作用。结论 F2对培养心肌细胞H/R损伤具有保护作用,可能与其抑制钙依赖PKCα转位有关。     

糖基化终产物对人外周血单个核细胞蛋白激酶C的影响

目的探讨糖基化终产物(AGEs)对人外周血单个核细胞(PBMC)蛋白激酶C(PKC)活性的影响以及氨基胍(AG)是否影响AGEs的作用.方法分离人PBMC,加入AGEs不同浓度及不同时间共同孵育.用[γ-32P]ATP掺入外源性底物的方法测定PBMCPKC活性.结果(1)PBMC在AGEs刺激下,PKC活性渐升高,15min时达高峰.(2)与对照组相比,随AGEs浓度增加,PKC活性显著升高(P＜0.05～0.01).(3)PKC活性随着刺激物AGEs孵育时间或葡萄糖修饰浓度的增加而逐步升高(P＜0.05～0.01).(4)经AG处理的AGEs增强细胞PKC活性的作用较对照AGEs组明显减弱(P＜0.05).结论AGEs随浓度或孵育时间增加可增强PBMC的PKC总活性.经AG处理的AGEs增强细胞PKC活性的作用明显减弱.     

白花前胡甲素对Ang Ⅱ诱导的心肌细胞肥大及核转录因子c-Jun蛋白表达的影响

目的:探讨白花前胡甲素(Pd-Ia)对AngⅡ诱导的心肌细胞肥大及核转录因子c-Jun蛋白表达水平的影响。方法:体外原代培养乳鼠心肌细胞;免疫细胞化学检测核转录因子c-Jun蛋白表达情况;[3H]亮氨酸掺入法及[3H]胸腺嘧啶核苷掺入法检测细胞内蛋白、DNA合成。结果:AngⅡ(10-6 mol·L-1)刺激心肌细胞2 h后,细胞核内c-Jun蛋白表达显著增强,为对照组的2．8倍(P<0．01);24 h后,细胞内DNA、蛋白合成显著增加(P<0．05)。白花前胡甲素对此有显著抑制作用(P< 0．05)。结论:白花前胡甲素能够拮抗AngⅡ诱导的心肌细胞肥大,其机制可能与抑制AngⅡ诱导的心肌细胞c-Jun蛋白表达上调有关。     

乌贼墨对H22癌细胞TPK、PKC及PKA活性的影响

利用γ-^23P－ATP作为反应启动剂掺入外源性底物的方法测定TPK、PKC及PKA活性,研究了乌贼墨对H22癌细胞TPK、PKC及PKA活性的影响。结果表现：癌细胞质膜、胞浆及线粒体TPK活性均显著升高而以质膜TPK活性升高最为显著;胞浆、线粒体及胞核PKC活性均显著升高而以胞核PKC升高最为明显;胞浆内PKA活性升高。给予乌贼墨作用后,癌细胞质膜、胞冻及线粒体TPK活性均显著下降;线粒体及胞核PKC活性均显著下降,胞浆PKA活性继续明显升高。结果提示：乌贼墨能明显的逆转癌细胞异常改变的TPK、PKC和PKA活性,引起Ras－MAPK信号传导系统的正调节作用减弱和负调节作用增强,结果抑制或阻断了Ras－MAPK信号传导系统的信号及联传导,使癌细胞由去分化性增殖向分化增殖转变,产生抗癌促分化作用。     

转化生长因子β1在大鼠心肌细胞肥大中的作用

目的探讨转化生长因子β1(transforminggrowth factorβ1，TGF-β1)在诱导大鼠心肌细胞肥大中的作用。方法培养新生大鼠的心肌细胞，检测[^3H]-Leu的掺入量和胚胎抗原心房利钠因子(atrial natriuretic factor，ANF)的表达。结果不同浓度的转化生长因子β1均能明显增加心肌细胞[^2H]-Leu的掺入量和ANF的表达。结论转化生长因子β1能诱导大鼠的心肌细胞肥大。     

尿促皮素诱导乳大鼠心肌细胞肥大的作用及信号传导机制

目的探讨尿促皮素(urocortin)诱导大鼠心肌细胞肥大的作用及其信号传导机制。方法实验分8组,正常对照组、尿促皮素0.1μmol.L-1组、星形孢菌素(Sta)1μmo.lL-1、H890.1μmol.L-1和维拉帕米(Ver)1μmol.L-1组及尿促皮素分别加Sta,H89和Ver组。采用体外培养的乳大鼠心肌细胞,应用尿促皮素0.1μmol.L-1诱导心肌肥大,观察Sta1μmol.L-1,H890.1μmo.lL-1和Ver1μmol.L-1的作用,进一步探讨尿促皮素0.1μmol.L-1诱导心肌肥厚的作用机制。用消化分离法及计算机图像分析系统检测心肌细胞直径;[3H]亮氨酸掺入法测定心肌细胞蛋白质的合成;用Lowry法检测心肌细胞蛋白质含量;用Western蛋白印迹法测定心房钠尿肽(ANP)表达;采用Till阳离子测定系统,以Fura-2/AM为荧光探针,观察心肌细胞[Ca2+]i瞬间变化。结果尿促皮素使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别增加30.9%,36.3%,35.5%和34.7%;尿促皮素+Sta组使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别降低了16.5%,22.1%,18.1%和21.3%;尿促皮素+H89组使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别降低了16.6%,21.5%,19.5%和20.6%;尿促皮素+Ver组使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别降低了17.1%,20.9%,17.9%及19.9%;尿促皮素能够使心肌细胞[Ca2+]i瞬间变化水平增高,Sta,H89和Ver能够降低尿促皮素引起的心肌细胞[Ca2+]i瞬间变化升高。结论尿促皮素可能通过蛋白激酶C和蛋白激酶A信号途径影响L-型Ca2+通道,进而影响细胞[Ca2+]i瞬间变化水平,诱导乳大鼠心肌细胞肥大。     

虎杖苷对缺血缺氧下心肌细胞蛋白激酶C的影响

目的探讨虎杖苷对缺血缺氧作用下心肌细胞蛋白激酶C(PKC)活性的影响及其抗休克的机制。方法取大鼠乳鼠心室肌培养心肌细胞,用Koyama方法复制心肌细胞缺血缺氧模型。用γ-闪烁计数仪测定PKC的活性。结果正常状态下心肌细胞胞质PKC经虎杖苷(PD)刺激后活性下降(vs正常组P<0.05),但胞膜的PKC活性上升(vs正常组P<0.05)。缺血缺氧后胞质和胞膜PKC的活性均下降(vs正常组P<0.05),再经PD处理后胞质胞膜的PKC活性均上升(vs缺血缺氧组P<0.05,vs正常组P>0.05)。结论PD对缺血缺氧作用下的心肌有保护作用,可能是通过调节PKC的活性来发挥作用的,这可能是PD抗休克的分子机制之一。     

丙泊酚对血管紧张肽Ⅱ诱导心肌细胞肥大及ROS/JNK1/2通路的影响

目的 探讨丙泊酚对血管紧张肽Ⅱ(angiotensin Ⅱ, Ang Ⅱ)诱导的心肌细胞肥大及活性氧(ROS)/ 活化c-Jun氨基末端激酶(c-Jun N-teminal kinase1/2, JNK1/2)通路的影响. 方法 培养1～2 d龄乳鼠心肌细胞,相差显微镜观察心肌细胞直径、[³H] 亮氨酸掺入法测定蛋白合成速率作为心肌肥大指标;细胞内ROS水平用ROS敏感的荧光探针2,7 dichlorofluorescein diacetate(DCFH-DA)反映. 比色法测定NADPH氧化酶(NADPH oxidase, Nox)活性. 免疫印记法测定JNK1/2磷酸化水平. 结果 Ang Ⅱ能显著提高心肌细胞直径、蛋白合成速率、Nox活性及ROS水平,并明显促进JNK1/2磷酸化,以上效应被不同浓度丙泊酚所抑制. 结论丙泊酚可抑制Ang Ⅱ诱导的乳鼠心肌细胞肥大,其机制可能与下调ROS/JNK1/2通路有关.     

内源性中叶素可减轻血管紧张素Ⅱ诱导的乳鼠心肌细胞肥大

目的研究内源性中叶素(IMD)与心肌细胞肥大间的相互作用关系。方法血管紧张素Ⅱ(AngⅡ)孵育乳鼠心肌细胞构建心肌肥大模型。应用Western blot及放射免疫法测定心肌细胞IMD产生和分泌,实时定量PCR(Real-timePCR)方法检测心肌细胞IMD及其受体系统降钙素受体样受体/受体活性修饰蛋白(CRLR/RAMPs)基因表达的变化。并以[3H]-亮氨酸([3H]-Leu)摄入及脑钠素(BNP)基因表达作为心肌细胞肥大的指标。结果 AngⅡ孵育下调心肌细胞IMD生成、表达,并影响其受体系统的基因表达。反过来,利用IMD抗体及其受体阻断剂阻断内源性IMD的生物学效应可增强AngⅡ诱导的心肌细胞肥大反应。结论内源性IMD及其受体系统参与了心肌肥大的发生、发展,对其生成、表达的干预有可能成为今后防治心肌细胞肥大的新途径。     

槲皮素对HL—60细胞中蛋白激酶C和酪氨酸蛋白激酶活性的影响

目的：研究槲皮素（Ｑｕｅ）对ＨＬ－６０细胞中的细胞溶质和胞膜蛋白激酶Ｃ（ＰＫＣ）酪氨酸蛋白激酶（ＴＰＫ）活性的影响。方法：活细胞数的计数用苔盼兰拒染法，用组蛋白ⅢＳ，（γ＾３２Ｐ）ＡＴＰ与ＰＫＣ酶液一起保温测定ＰＫＣ活性，用聚谷氨酸．酪氨酸（４：１）多肽），（γ＾３２Ｐ）ＡＴＰ与ＴＰＫ酶液一起保温测定ＴＰＫ活性，结果：Ｑｕｅ对ＨＬ－６０细胞的增殖有抑制作用，呈剂量依赖关系，处理４８小时后，其ＩＣ５     

丝裂素活化蛋白激酶反义寡核苷酸对血管紧张素Ⅱ诱导的大鼠心肌？…

AIM: To investigate the inhibitory effect of down-regulating mitogen activated protein kinase (MAPK) on c-myc gene expression and further on cardiac fibroblast proliferation. METHODS: Cultured neonatal rat cardiac fibroblasts was pretreated with a phosphorothioate-protected 17-mer antisense MAPK oligodeoxynucleotide (ODN) directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection. A 17-mer sense and mismatch sequence MAPK ODN were used as controls. After liposomal transfecting, cells were exposed to angiotensin II (Ang II) 10 nmol.L-1 for 5 min and then harvested in lysis buffer. MAPK activity was measured by Western blot and P-81 phosphocellulose filter paper method by using [gamma-32P]ATP and myelin basic protein as substrates. c-myc mRNA expression stimulated by Ang II for 30 min was measured by Northern blot. DNA synthesis and collagen protein synthesis induced by Ang II for 24 h were measured by [3H]thymidine incorporation and [3H]Proline incorporation, respectively. RESULTS: Antisense ODN 0.2 mumol.L-1 reduced Ang II-induced MAPK activities by 72%, MAPK protein expression by 80%, and suppressed c-myc mRNA expression by 97%, respectively. [3H]thymidine incorporation and [3H]proline incorporation in Ang II-induced cardiac fibroblast were inhibited by 59% and 58%, respectively. CONCLUSION: A 17-mer MAPK antisense oligonucleotide directed againsts the initiation of translation sites of MAPK could specifically inhibit Ang II-stimulated cultured neonatal rat cardiac fibroblast proliferation through down-regulating MAPK activity and further depleting c-myc mRNA expression.     

Effect of PKC-zeta mediating Ang II-stimulated activation of CCDPK on rat cardiac fibroblast proliferation

AIM: To investigate whether the effect of angiotensin (Ang) II or epidermal growth factor (EGF) on cardiac fibroblast proliferation involved in activation of extracellular signal-regulated kinase (ERK) 1/2 or Ca(2+)-calmodulin dependent protein kinase(CCDPK) mediated by protein kinase C (PKC)-zeta. METHODS: Relative activity of CCDPK was measured by Western blotting. DNA synthesis was assayed by [3H]thymidine incorporation. RESULTS: PDBU caused no decrease in Ang II- and 10% FCS-stimulated CCDPK activity and DNA synthesis. In contrary, 65% or 75% EGF- or tetradecanoylphorbol acetate (TDPA, formally called PMA)--stimulated CCDPK activity and 38% or 42% [3H]thymidine incorporation treated by PDBU were inhibited, respectively. Meanwhile 70% and 72% CCDPK activities induced by Ang II and EGF were inhibited by PD 98059, respectively. CONCLUSION: PKC-zeta mediated Ang II-induced activation of CCDPK and cardiac fibroblast proliferation.     

缓激肽B2受体拮抗剂艾替班特降低卡托普和对培养新生大鼠心肌细胞 …

AIM: To study whether endogenous kinins are negative modulators in the growth of cardiomyocytes and their possible cellular and molecular mechanisms. METHODS: Cultured neonatal rat cardiomyocytes were used. Intracellular RNA and protein synthesis were measured by [3H]uridine incorporation and [3H]leucine incorporation, respectively. The expression level of proto-oncogene c-myc, c-fos mRNA was observed by Northern blotting. RESULTS: Exposure of cardiomyocytes to captopril (Cap, 100 mumol.L-1) for 48 h inhibited the rates of [3H]Urd and [3H]Leu incorporations by 25% and 26%, respectively, and for 2 h inhibited c-myc, c-fos mRNA expression by 75% and 55%, respectively. Treatment of angiotensin II (Ang II, 1 mumol.L-1) for 48 h significantly increased the rates of [3H]Urd and [3H]Leu incorporations and for 1 h induced c-myc, c-fos mRNA overexpression, which were reduced by pretreatment with Cap (100 mumol.L-1). Icatibant acetate (Hoe 140, a specific antagonist of bradykinin B2 receptor) 0.1-10 mumol.L-1 blocked the effects of Cap in a concentration-dependent manner. CONCLUSION: Endogenous kinins exhibited a negative modulatory effect on growth of cardiomyoctes via BK B2 receptor.     

丹参酮IIA磺酸钠对血管紧张素II诱导的心肌肥大及p-ERK表达的影响

目的:观察丹参酮IIA磺酸钠(STS)对血管紧张素II(Ang II)诱导的心肌肥大及p-ERK表达的影响。方法:培养新生大鼠心肌细胞,考马斯亮蓝法测定心肌细胞蛋白含量、[3H]-亮氨酸掺入法测定蛋白合成速率作为心肌肥大指标;用West-ern-blot测定p-ERK表达。结果:STS能显著降低Ang II诱导的心肌细胞总蛋白含量、蛋白合成速率上升,同时对p-ERK表达具有剂量、时间依赖性抑制作用。结论:STS可以抑制Ang II诱导的心肌肥大,机制与抑制p-ERK表达有关。     

C-type natriuretic peptide inhibits rat mesangial cell proliferation by a phosphorylation-dependent mechanism

We studied the effects of C-type natriuretic peptide (CNP) on rat cultured mesangial cell proliferation. (1) Exposure to CNP (10 nM–1 μM for 72 h) inhibited [³H]thymidine incorporation into mesangial cells in a concentration-dependent manner. Atrial natriuretic peptide (1 nM–1 μM), a peptide related to CNP, also decreased [³H]thymidine incorporation into these cells in a concentration-dependent manner. (2) Both CNP (10 nM-1 μM) and atrial natriuretic peptide (10 nM-1 μM) also decreased mesangial cell number. (3) The cyclic GMP analog, 8-bromo-cyclic GMP (100 μM and 1 mM), mimicked the inhibitory effects of CNP and atrial natriuretic peptide on [³H]thymidine incorporation into mesangial cells, whereas inhibitors of protein kinase C, protein kinase A, and protein kinase G reduced the effect of both natriuretic peptides. Moreover, the phoshpatase inhibitor, calyculin A, increased [³H]thymidine incorporation into mesangial cells. (4) CNP and atrial natriuretic peptide decreased interleukin-1-, interleukin-6-, platelet derived growth factor-, angiotensin II-induced [³H]thymidine incorporation into mesangial cells. These results suggest that CNP exerts inhibitory effects on mesangial cell proliferation and that this effects depend on protein phosphorylation pathways. Received: 17 March 1997 / Accepted: 29 August 1997     

Inhibition of human lymphocyte transformation by two aryloxyalkylamidines

The incorporation of [3H]thymidine into human lymphocytes stimulated by phytohaemagglutinin (PHA) was inhibited by anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and xylamidine. These amidines antagonize 5-HT, but 5-HT did not alter [3H]thymidine incorporation. 501C inhibited PHA-induced lymphocyte transformation as observed by [3H]thymidine incorporation, [3H]uridine incorporation, [3H]leucine incorporation, DNA content, potassium content, and histological examination. 501C also inhibited increased [3H]thymidine incorporation in human mixed lymphocyte cultures. The IC50 of 501C for inhibition of these processes lay between 4 and 8 microM. When added late in culture (after 6-8 h) 501C was less effective. Possible mechanisms by which 501C inhibits transformation are discussed.     

Regulation of cyclic AMP level and synthesis of DNA, RNA and protein by quercetin in Ehrlich ascites tumor cells.

Quercetin (3.3, 4′, 5.7-pentahydroxy flavone) at the concentration of 10^?4 M as well as 10^?2 M theophylline and 10^?3 M dibutyryl cyclic AMP caused at least 85 per cent inhibition of [³H]thymidine incorporation in Ehrlich ascites tumor cells. At the same concentrations, these drugs decreased [³H]uridine and [³H]-L-leucine incorporation by 50–60 per cent and 35–45 per cent respectively. Ouabain (10^?3 M), the specific inhibitor of Na⁺-K⁺ pump system, did not alter the incorporation of [³H]thymidine and [³]uridine, but decreased the incorporation of [³H]-L-leucine in these cells. Treatment of Ehrlich ascites tumor cells with the polyanion dextran sulfate did not change the inhibitory effect of quercetin, theophylline and dibutyrlyl cyclic AMP on [³H]thymidine incorporation. On the other hand, this polyanion decreased the inhibitory effect of these drugs on incorporation of [³H]uridine and abolished completely their effect on incorporation of [³H]-L-leucine.