腺苷A1受体与к阿片肽受体激活对异丙肾上腺素诱导的心肌细胞肥大的交互作用

目的观察腺苷A1受体与κ阿片肽受体(κ-OR)激活对异丙肾上腺素(Iso)诱导的心肌细胞肥大的交互作用及机制。方法体外培养大鼠乳鼠心肌细胞,以Iso 10μmol/L诱导心肌细胞肥大,观察腺苷A1受体激动剂R(-)-N6-(2-phenylIsopropyl)adenosine(R-PIA)1μmol/L和κ-OR激动剂U50,488H1μmol/L对其作用,进一步探讨腺苷A1受体拮抗剂8-cyclopentyl-1,3-dipropylxanthine(CPDPX)0.1μmol/L存在时κ-OR的激活对细胞肥大的影响和κ-OR拮抗剂nor-binaltorphimine(NOR-BNI)1μmol/L存在时腺苷A1受体的激活对心肌细胞肥大的影响。通过Lowry法测心肌细胞蛋白含量;消化分离法及计算机图像分析系统测细胞体积;以Fluo-3/AM为荧光探针,共聚焦显微镜下测量心肌细胞内[Ca2+]i变化;RT-PCR法检测心肌细胞心房钠尿肽(ANP)的mRNA表达。结果 10μmol/LIso可以诱导心肌细胞肥大,1μmol/L的R-PIA(腺苷A1受体激动剂)可以明显抑制Iso诱导的心肌细胞蛋白合成增加、体积增大、ANPmRNA表达增加、心肌细胞内[Ca2+]i荧光强度增大,该抑制作用可以被1μmol/Lκ-OR拮抗剂NOR-BNI部分阻断;κ-OR激动剂U50,488H1μmol/L可以明显抑制Iso诱导的心肌细胞蛋白合成增加、体积增大、ANPmRNA表达增加、心肌细胞内[Ca2+]i荧光强度增大,该抑制作用可以被腺苷A1受体拮抗剂CPDPX部分阻断。结论腺苷可通过激动A1受体、阿片肽可通过激动κ受体抑制Iso诱导的心肌肥大,二者可以通过影响心肌细胞内[Ca2+]i浓度的增大而交互抑制Iso诱导的心肌肥大。     

Apelin-13对H9c2心肌细胞增殖及ERK1/2信号通路活化的影响

目的分析Apelin-13对H9c2心肌细胞增殖及ERK1/2信号通路活化的影响。方法体外培养后的H9c2心肌细胞分为对照组、Apelin-13组、U0126(ERK1/2抑制剂)组和Apelin-13+U0126四组。对照组:只使用10%的FBS和DMSO处理;Apelin-13组:在对照组基础上分别加入浓度为0.0001,0.001,0.01和0.1μmol/L的Apelin-13(n=5);U0126干预组:在对照组基础上加入浓度为10μmol/L的U0126;Apelin-13+U0126组:在对照组基础上分别加入0.1μmol/L的Apelin-13和10μmol/L的U0126。各组依次恒温培养24 h后,采用MTT法测定H9c2心肌细胞在490 nm处各组的光密度值(OD值,n=5)并计算其细胞增值率,采用Western blot法测定各实验组ERK1/2信号通路磷酸化水平。结果与对照组比较,U0126干预组所测各细胞指标差异无统计学意义(P0.05)。与对照组比较,Apelin-13组的干预显著升高各组H9c2心肌细胞的OD值,促进了H9c2心肌细胞的增殖,升高了心肌细胞的p-ERK1/2表达量;并且0.001～0.1μmol/L Apelin-13四组间,细胞增值率及p-ERK1/2表达量有显著的统计学差异,随Apelin-13剂量的增加,各组的OD值、细胞增殖率及p-ERK1/2表达量亦显著升高(P0.05)。与Apelin-13组比较,Apelin-13+U0126组H9c2心肌细胞的OD值、细胞增值率及p-ERK1/2表达量显著降低(P0.05)。结论 Apelin-13促进了H9c2心肌细胞的增殖,并存在显著的剂量依赖性,Apelin-13通过ERK1/2信号通路调控了H9c2心肌细胞的细胞增殖。     

丹参酮ⅡA磺酸钠对血管紧张素Ⅱ诱导的大鼠心肌细胞外信号调节激酶1/2的作用

目的研究丹参酮ⅡA磺酸钠(STS)对血管紧张素Ⅱ(AngⅡ)诱导的心肌肥大反应中细胞外信号调节激酶1/2(ERK1/2)是否有抑制作用。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;用免疫荧光标记法和Western-blot测定磷酸化ERK1/2蛋白(p-ERK1/2)表达。结果1)AngⅡ(1μmol/L)处理24h,心肌细胞[3H]-亮氨酸掺入率、蛋白含量明显增加,STS能明显抑制AngⅡ介导心肌细胞[3H]-亮氨酸掺入率、蛋白含量的增加;2)AngⅡ刺激心肌细胞可见胞核内出现磷酸化ERK1/2荧光染色,丹参酮ⅡA可阻断AngⅡ引起的ERK1/2活化、入核过程。3)用AngⅡ(1μmol/L)处理心肌细胞5min,磷酸化ERK1/2蛋白(p-ERK1/2)表达即开始增加,10min左右时最明显。4)STS剂量依赖性抑制AngⅡ诱导的心肌细胞磷酸化ERK1/2蛋白表达。结论STS可以抑制AngⅡ诱导的心肌肥厚,其机制与抑制磷酸化ERK1/2表达有关。     

腺苷A_1受体激动抑制心肌细胞肥大

目的利用体外培养的乳鼠心肌细胞,观测腺苷 A_1受体激动剂,R(-)-N6-(2-phenylisopropyl)adenosine(R-PIA)对异丙肾上腺素(Iso)诱导的心肌细胞肥大的抑制作用,并且探讨其作用机制特别是与 CaMK Ⅱ表达的关系及对心肌细胞[Ca~(2+)]i 瞬间变化的影响。方法新生大鼠心肌细胞分组给药,以10μmol/L 的异丙肾上腺素(β肾上腺素激动剂,β-AR)诱导心肌肥大,观察1 μmol/L 的 R-PIA 的作用以及钙调素激酶Ⅱ特异性抑制剂 KN93(0.2 μmol/L)防止腺苷 A_1受体的激活对心肌肥厚的作用。[~3H]亮氨酸 leucine标记法测定心肌细胞蛋白合成;Western blot 法测细胞核内 CaMK ⅡδB的表达水平;Till 图像测定系统,以 Fura-2做荧光标志,观察心肌细胞[Ca~(2+)]i 瞬间变化。结果 1 μmol/L 的 R-PIA 可以明显抑制 Iso 诱导的蛋白合成增加[(974.8±58.6)vs(1220.8±240.5)计数/(min·孔),P<0.01],抑制[Ca~(2+)]i 瞬变增加[(189.9±10.7)vs(...     

U50488对大鼠离体心心律及心肌细胞内游离钙的影响

观察选择性Kappa(κ)阿片受体激动剂U50488对大鼠离体心心律及心肌细胞内游离钙([Ca2+]i)的作用.结果显示:U50488可诱发心律失常并增加静息心肌细胞[Ca2+]i,U50488诱发心律失常及增加心肌细胞[Ca2+]i的作用可被选择性κ阿片受体拮抗剂Nor-binaltorphimine(Nor-BNI)及选择性磷酯酶C抑制剂U73122、新霉素及链霉素所阻断.表明心脏κ阿片受体激活所致的心律失常是由于磷酸肌醇信号传导通路激活引起心肌细胞[Ca2+]i增多所致.     

钙神经素信号途径在前列腺素F2α诱导乳鼠心肌细胞肥大中的作用

目的:探讨Ca2 /钙调蛋白(CAM)依赖的钙神经素(CAN)信号途径在前列腺索F2α(PGF2α)诱导心肌细胞肥大中的作用.方法:用PGF2α(1 μmol/L,0.1μmol/L,0.01 μmol/L,0.001μmol/L)刺激Wistar乳鼠心肌细胞,并用CaN特异性抑制剂环胞素A(CsA)加以干预.以心肌细胞总蛋白含量和心肌细胞面积作为反映心肌肥大的指标,用免疫印迹(Western-blot)测定心肌细胞内CaN-α蛋白表达,用Fura 2/AM为荧光指示剂测定心肌细胞内游离钙浓度([Ca2 ]i).结果:除0.001μmol/L浓度的PGF2α外,1.0μmol/L,0.1μmol/L,0.01μmol/L的PGF2α均可明显提高心肌细胞内蛋白质浓度,而其中0.1μmol/L PGF2α作用最显著,增加了(67.78±11.19)%,并且使乳鼠心肌细胞面积增加了(68.94±1.52)%.0.1μmol/L PGF2α刺激的心肌细胞CaN-α蛋白表达增加和[Ca2 ]i提高,并且加入CsA可阻断0.1μmol/L PGF2α诱导的乳鼠心肌细胞肥大效应.结论:PGF2α可能通过Ca2 /CaM-CaN信号途径刺激乳鼠心肌细胞发生肥大.     

丹参酮ⅡA磺酸钠对血管紧张素Ⅱ诱导的大鼠心肌细胞外信号调节激酶1/2的作用

目的 研究丹参酮ⅡA磺酸钠(STS)对血管紧张素Ⅱ(Ang Ⅱ)诱导的心肌肥大反应中细胞外信号调节激酶1/2(ERK1/2)是否有抑制作用.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]-亮氨酸掺人法测定蛋白合成速率作为心肌肥大指标;用免疫荧光标记法和Western-blot测定磷酸化ERK1/2蛋白(p-ERK1/2)表达.结果 1)Ang Ⅱ(1 μmol/L)处理24 h,心肌细胞[3H]-亮氨酸掺入率、蛋白含量明显增加,STS能明显抑制Ang Ⅱ介导心肌细胞[3H]-亮氨酸掺人率、蛋白含量的增加;2)Ang Ⅱ刺激心肌细胞可见胞核内出现磷酸化ERK1/2荧光染色,丹参酮ⅡA可阻断AngⅡ引起的ERK1/2活化、入核过程.3)用Ang Ⅱ(1 μmol/L)处理心肌细胞5 min,磷酸化ERK1/2蛋白(p-ERK1/2)表达即开始增加,10 min左右时最明显.4)STS剂量依赖性抑制Ang Ⅱ诱导的心肌细胞磷酸化ERK1/2蛋白表达.结论 STS可以抑制Ang Ⅱ诱导的心肌肥厚,其机制与抑制磷酸化ERK1/2表达有关.     

姜黄素对血管紧张素Ⅱ诱导的乳鼠心肌细胞细胞外信号调节激酶表达的影响

目的从细胞外信号调节激酶1/2(ERK1/2)激活角度研究姜黄素(curcumin)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的心肌肥大反应的作用。方法体外培养新生大鼠心肌细胞,分为AngⅡ组(10-6mol/L AngⅡ)、姜黄素组(10-6mol/L AngⅡ+2.5×10-8g/L姜黄素)及对照组(正常培养的心肌细胞)。3组分别培养24h,收集心肌细胞,[3H]-亮氨酸掺入法测定心肌细胞蛋白合成速率,免疫印迹法测定ERK1/2表达。应用SPSS11.0软件进行统计学分析。结果①3组间心肌细胞蛋白合成速率比较有统计学意义(F=20.68,P0.01),AngⅡ(10-6mol/L)处理24h,心肌细胞[3H]-亮氨酸掺入率明显增加,姜黄素能明显抑制AngⅡ介导心肌细胞[3H]-亮氨酸掺入率的增加;②AngⅡ(10-6mol/L)处理心肌细胞10min,磷酸化ERK1/2蛋白(p-ERK1/2)表达明显增加,预先以姜黄素2.5×10-8g/L处理心肌细胞30min,发现姜黄素可明显抑制AngⅡ诱导的心肌细胞磷酸化ERK1/2蛋白表达。结论姜黄素可抑制AngⅡ诱导的心肌细胞ERK1/2的表达,这可能是姜黄素治疗心肌肥厚的重要机制之一。     

结缔组织生长因子诱导大鼠心肌细胞肥大的作用及其与细胞外信号调节激酶1/2的关系

目的 观察结缔组织生长因子(CTGF)诱导大鼠心肌细胞肥大的作用,探讨其作用与细胞外信号调节激酶1/2(ERK1/2)的关系.方法 以培养的新生Sprague-Dawley(SD)大鼠心肌细胞为实验模型,用图象分析法测定心肌细胞表面积,用[3H]-亮氨酸掺入法测定心肌细胞蛋白合成速率,用考马斯亮兰法测定心肌细胞蛋白含量,用蛋白免疫印迹法(Western blot)测定心肌细胞总ERK1/2(t-ERK1/2)与磷酸化ERK1/2(p-ERK1/2)的蛋白表达水平.结果 (1)随着CTGF浓度的增加,心肌细胞表面积呈剂量依赖性增加,其中10、25、50、100 μg/L的CTGF组心肌细胞表面积分别为(929.9±132.2)、(1411.3±129.2)、(1732.0±153.0)、(2040.6±205.4)μm2,均明显高于对照组心肌细胞表面积[(606.3±72.7)μm2,P均＜0.01];100 μmol/L的PD98059明显减少CTGF诱导的心肌肥大(P＜0.01).(2)随着CTGF浓度的增加,心肌细胞蛋白合成速率与蛋白含量呈剂量依赖性增加,CTGF组心肌细胞[3H]-亮氨酸掺入率明显高于对照组(P＜0.01);ERK1/2抑制剂PD98059明显减少CTGF诱导的心肌细胞[3H]-亮氨酸掺入率及蛋白含量(P＜0.01).(3)随着CTGF浓度的增加,心肌细胞p-ERK1/2表达呈剂量依赖性增高,5、10、25、50、100 μg/L的CTGF组的心肌细胞p-ERK1/2表达明显高于对照组,而t-ERK1/2在各组表达差异不明显.结论 CTGF可诱导心肌细胞肥大,该作用可能是通过ERK1/2的磷酸化来实现的.     

黄芩素联合U0126调控人膀胱癌T24细胞凋亡的分子机制

目的探讨黄芩素联合U0126促进人膀胱癌T24细胞系凋亡的分子机制。方法将膀胱癌T24细胞株随机分为对照组,黄芩素组(加入20μmol/L黄芩素培养24 h),U0126组(加入20μmol/L U0126培养24 h),黄芩素联合U0126组(加入20μmol/L黄芩素和20μmol/L U0126共同培养24 h)。CCK8检测各组细胞增殖;流式细胞仪检测T24细胞周期和凋亡;Real Time PCR和Western印迹检测细胞外调节蛋白激酶(ERK)1/2、B淋巴细胞瘤-2基因(Bcl)-2相关X蛋白(Bax)、Bcl-2和P38 mRNA和蛋白的表达。结果黄芩素或U0126及黄芩素联合U0126作用可显著抑制T24细胞增殖,G0～G1期细胞增多,S期细胞减少,诱导细胞早期凋亡和晚期凋亡,Bax表达增加,Bcl-2表达减少,ERK1/2和P38 mRNA水平显著降低,ERK1/2和P38磷酸化水平显著降低,与对照组比较差异有统计学意义(P0. 05);而黄芩素联合U0126作用对细胞的改变更加显著(P0. 01)。结论黄芩素联合U0126均可通过调控ERK1/2和P38信号通路,抑制人膀胱癌细胞增殖,改变细胞周期,进而诱导肿瘤细胞凋亡。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.