Ethanol Inhibits Contractility of Esophageal Smooth Muscle Strips

Acute ethanol (EtOH) in vivo decreases both the pressure of the lower esophageal sphincter (LES) and the amplitude of contractions of the smooth muscle of the lower esophageal body (LEB) in both man and cat. However, the mechanism of this inhibitory effect of EtOH is unclear. This inhibitory effect could be caused by a direct effect of EtOH on the esophagus or be secondary to known inhibitory effects of EtOH on the central nervous system. To this end, we evaluated the in vitro effect of EtOH on contractility of smooth muscle strips from both LES and LEB. Circular muscle strips from LES and LEB were isolated from cats. Changes in resting tension of LES strips and changes in stimulant-induced tension of LES or LEB strips were measured in the presence of up to five concentrations of EtOH (12.5–100 mM). Stimulants included electric field stimulation (EFS) and carbachol. EtOH at 75 mM significantly decreased resting LES tension. EtOH also decreased maximal contractile responses to carbachol in both LES and LEB and increased the EC₅₀ of carbachol for LES, but not LEB. EtOH also modulated EFS-induced esophageal contractility; EtOH potentiated EFS-induced "on-response relaxation" in LES and decreased EFS-induced "off-response contractions" in LEB. EtOH-induced inhibition of esophageal contractility seemed to be reversible. EtOH did not result in muscle fatigue. Thus, EtOH can directly inhibit contractility of the esophagus, and does so reversibly and at pharmacologically relevant concentrations.     

Differences in the glutathione system of cultured aortic smooth muscle cells from young and aged rats

We studied the relation between the glutathione (GSH) system and cell proliferation in a model of smooth muscle cells (SMC) derived from the thoracic aorta of 4–6-week-old (young) and 15-month-old (aged) rats. SMC from aged rats showed greater levels of total non-protein thiol compounds (T-SH), increased glutathione transferase (GST) and increased glutathione reductase (GSSG-Red) activities compared with cells from young rats. These changes were associated with an increased proliferation rate of SMC from aged rats. To evaluate the role of GSH on cell proliferation better, a specific inhibitor of gamma-glutamyl-cystein synthetase, -buthionine-SR-sulphoximine (BSO) was used. BSO showed a dose-dependent inhibition of cell growth, with an IC₅₀ of 10⁻⁴ M, after 48–72 h of incubation. Removal of BSO restored cell growth, further suggesting a link between GSH levels and vascular cell proliferation. The inhibitory effect of BSO was about two times greater on SMC from young than on SMC from aged rats. BSO showed 56% inhibition on the proliferation of SMC from young rats and 32% inhibition on SMC from aged rats (10⁻⁴ M, 72 h of incubation). A parallel reduction of GSH levels of 38% and 19% for SMC from young and aged rats, respectively, was observed, suggesting that age-related factors may influence the involvement of GSH system in cell proliferation.     

Revised morphology and hemodynamics of the anorectal vascular plexus: impact on the course of hemorrhoidal disease

Purpose  The pathogenesis of hemorrhoidal disease is based mainly on the vascular hyperplasia theory. The aim of this study was to reassess the morphology and the functional mechanisms of the anorectal vascular plexus with regard to hemorrhoidal disease. Materials and methods  The anorectal vascular plexus was investigated in 17 anorectal and five hemorrhoidectomy specimens by means of conventional histology and immunohistochemistry. Vascular corrosion casts from two fresh rectal specimens were used for scanning electron microscopy. Transperineal color Doppler ultrasound (CDUS) with spectral wave analysis (SWA) was performed in 38 patients with hemorrhoidal disease and 20 healthy volunteers. Results  The anorectal vascular plexus was characterized by a network of submucosal vessels exhibiting multiple thickened venous vessels separated by distinct sphincter-like constrictions. CDUS and SWA showed significant flow differences in peak velocities (6.8 ± 1.3 cm/s vs. 10.7 ± 1.5 cm/s; P = 0.026) and acceleration velocities (51 ± 4 ms vs. 94 ± 11 ms; P = 0.001) of afferent vessels between the control group and patients with hemorrhoidal disease. Conclusions  Coordinated filling and drainage of the anorectal vascular plexus is regulated by intrinsic vascular sphincter mechanisms. Both morphological and functional failure of this vascular system may contribute to the development of hemorrhoidal disease.     

人主动脉硫酸乙酰肝素蛋白聚糖对胎儿主动脉平滑肌细胞c-fos和c-myc基因表达的影响

本室以往的研究发现硫酸乙酰肝素蛋白聚糖对培养的胎儿主动脉平滑肌细胞的增殖有明显的抑制作用。本实验采用Ｎｏｒｔｈｅｒｎｂｌｏｔ的方法观察硫酸乙酰肝素蛋白聚糖对胎儿主动脉平滑肌细胞的ｃ－ｆｏｓ，ｃ－ｍｙｃ原癌基因的表达的影响，发现前者能抑制后者的表达。因此硫酸乙酰肝素蛋白聚糖抑制平滑肌细胞增殖的途径之一可能为抑制原癌基因的表达。     

Electromechanical effects of leukotriene D4 on ferret tracheal muscle and its muscarinic responsiveness

We investigated the possible electrophysiological processes by which leukotriene D₄ (LTD₄) affects airway smooth muscle and its responsiveness to acetylcholine (ACh). For study in vitro, preparations of ferret tracheal muscle (dissected free of overlying mucosal and submucosal layers) were used. These preparations were arranged so that force transducers and glass intracellular microelectrodes (having tip resistances of 35–60 megohm) could be used to measure isometric force generation and cell membrane potential (Em) simultaneously from muscle stimulated by LTD₄. At rest, the muscle was electrically and mechanically quiescent and had an Em of −59±0.2 mV (mean±SEM). We found that ferret tracheal muscle cells were relatively sensitive to LTD₄, and that both the resulting depolarization (beginning at 10⁻¹⁰ M LTD₄) and force generation (produced by higher concentrations) progressed in a concentration-dependent manner. Depolarization by 10⁻⁹ M LTD₄ elicited electrical oscillations. These oscillations were accompanied by phasic contractile activity at 5 × 10⁻⁹ M LTD₄. Verapamil abolished these oscillations and diminished force substantially. We also found that ACh depolarized and contracted the muscle in a concentration-dependent manner. It caused electrical oscillations at ≥ 10⁻⁶ M. Diltiazem abolished these oscillations and markedly diminished force generation without affecting Em. Preexposure of airway muscle preparations for 20 min to a concentration (10⁻¹⁰ M) of LTD₄ that, by itself, did not produce significant force, substantially augmented the voltage-tension relationship of the muscle upon ACh stimulation. We conclude that there is an electrical basis for the slow, prolonged force generation of airway muscle caused by LTD₄, and that LTD₄ potentiates the electromechanical responsiveness of the airway muscle to muscarinic stimulation.     

花刺参粘多糖对大鼠血管平滑肌细胞粘附分子表达的影响

目的观察花刺参粘多糖对肿瘤坏死因子α诱导的大鼠血管平滑肌细胞血管细胞粘附分子1和细胞间粘附分子1表达的影响,以探讨花刺参粘多糖抗动脉粥样硬化的可能机制。方法组织贴块法体外原代培养大鼠血管平滑肌细胞,采用流式细胞术、逆转录聚合酶链反应和细胞粘附实验方法,观察花刺参粘多糖对肿瘤坏死因子α诱导的平滑肌细胞血管细胞粘附分子1、细胞间粘附分子1蛋白及mRNA水平表达及单核细胞与平滑肌细胞粘附的影响。结果肿瘤坏死因子α刺激后平滑肌细胞血管细胞粘附分子1和细胞间粘附分子1表达增强,花刺参粘多糖(1.5~6.0 g/L)可从蛋白和mRNA水平浓度依赖性地抑制其表达,流式细胞分析示血管细胞粘附分子1的荧光强度由176.4±3.5减少到80.7±1.9(P<0.01),细胞间粘附分子1的荧光强度由92.2±2.9减少到58.3±2.1(P<0.01);逆转录聚合酶链反应结果发现血管细胞粘附分子1的相对光密度值由0.61±0.03减少到0.41±0.04(P<0.01),细胞间粘附分子1的相对光密度值由0.41±0.01减少到0.30±0.03(P<0.05);粘附实验发现花刺参粘多糖可减少单核细胞与平滑肌细胞粘附,光密度值由0.467±0.062减少到0.256±0.029(P<0.05)。结论花刺参粘多糖可抑制肿瘤坏死因子α诱导的平滑肌细胞血管细胞粘附分子1和细胞间粘附分子1的表达,减少单核细胞与平滑肌细胞的粘附和聚集,这可能对减轻血管壁的炎症反应、延缓动脉粥样硬化发生发展起一定作用。     

丹参对家兔实验性动脉粥样硬化预防作用的研究

丹参预防组主动脉斑块面积占主动脉总面积的百分比为（１９．８５±１１．７９％），明显小于动脉粥样硬化（ＡＳ）模型组（３７．７９±１０．８０％），Ｐ＜０．０５。丹参有显著保护超氧化物歧化酶（ＳＯＤ）活性和清除脂质过氧化物中间产物丙二醛（ＭＤＡ）的作用。丹参能明显抑制在高脂条件下３Ｈ－ＴｄＲ掺入培养的平滑肌（ＳＭＣ），抑制ＳＭＣ、ＤＮＡ合成。本研究初步探讨了丹参抑制家兔实验性ＡＳ病变发生发展的作用机制。     

Inhibition of hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by a mTOR siRNA-loaded cyclodextrin nanovector

The proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH), an intractable disease, for which pharmacotherapy is limited and only slight improvement in survival outcomes have achieved over the past few decades. RNA interference provides a highly promising strategy to the treatment of this chronic lung disease, while efficient delivery of small interfering RNA (siRNA) remains a key challenge for the development of clinically acceptable siRNA therapeutics. With the aim to construct useful nanomedicines, the mammalian target of rapamycin (mTOR) siRNA was loaded into hybrid nanoparticles based on low molecular weight (Mw) polyethylenimine (PEI) and a pH-responsive cyclodextrin material (Ac-aCD) or poly(lactic-co-glycolic acid) (PLGA). This hybrid nanoplatform gave rise to desirable siRNA loading, and the payload release could be modulated by the hydrolysis characteristics of carrier materials. Fluorescence observation and flow cytometry quantification suggested that both Ac-aCD and PLGA nanovectors (NVs) may enter PASMCs under either normoxia or hypoxia conditions as well as in the presence of serum, with uptake and transfection efficiency significantly higher than those of cationic vectors such as PEI with Mw of 25 kDa (PEI25k) and Lipofectamine 2000 (Lipo 2k). Hybrid Ac-aCD or PLGA NV containing siRNA remarkably inhibited proliferation and activated apoptosis of hypoxic PASMCs, largely resulting from effective suppression of mTOR signaling as evidenced by significantly lowered expression of mTOR mRNA and phosphorylated protein. Moreover, these hybrid nanomedicines were more effective than commonly used cationic vectors like PEI25k and Lipo 2k, with respect to cell growth inhibition, apoptosis activation, and expression attenuation of mTOR mRNA and protein. Therefore, mTOR siRNA nanomedicines based on hybrid Ac-aCD or PLGA NV may be promising therapeutics for diseases related to hypoxic abnormal growth of PASMCs.     

钙化血管平滑肌细胞肾上腺髓质素及其受体系统的基因表达上调

探讨钙化的血管平滑肌细胞肾上腺髓质素生成和肾上腺髓质素受体系统一降钙素受体样受体和受体活性修饰蛋白基因表达的改变及其病理意义。采用β-甘油磷酸盐诱导培养的大鼠血管平滑肌细胞钙化;放射免疫法测定血管平滑肌细胞分泌的肾上腺髓质素含量;半定量逆转录聚合酶链反应测定细胞肾上腺髓质素、降钙素受体样受体和受体活性修饰蛋白的mRNA水平;原子吸收分光光度计测定细胞钙含量;碱性磷酸酶试剂盒测定血管平滑肌细胞碱性磷酸酶活性;β液体闪烁计数仪测定45Ca2+放射活性。结果发现,与非钙化血管平滑肌细胞比较,钙化血管平滑肌细胞内钙含量、45Ca2+摄入及碱性磷酸酶活性分别增加118％、174％和7倍(P<0.01);钙化细胞肾上腺髓质素分泌量增高99％(P<0.01),肾上腺髓质素、降钙素受体样受体、受体活性修饰蛋白2和3的mRNA水平分别增加78％、93.7％、91.8％和109.5％(P均<0.01)。肾上腺髓质素与降钙素受体样受体、受体活性修饰蛋白2和3的mRNA水平呈正相关,其相关系数分别为0.83、0.92和0.93(P均<0.01)。结果提示,血管平滑肌细胞肾上腺髓质素 旁/自分泌功能改变可能参与血管钙化的调节过程。     

血小板源生长因子对大鼠血管平滑肌细胞表达血管细胞粘附分子1的影响

目的本实验研究血小板源生长因子对血管平滑肌细胞表达血管细胞粘附分子1的影响以及对血管平滑肌细胞与单核细胞粘附的作用。方法利用基因芯片和RT-PCR技术检测鼠主动脉血管平滑肌细胞经血小板源生长因子处理0min、20min、6h后血管平滑肌细胞表达血管细胞粘附分子1的mRNA表达变化;细胞粘附实验观察血管平滑肌细胞经血小板源生长因子分别处理0min、20min、6h后与单核细胞粘附情况。结果血小板源生长因子对鼠血管平滑肌细胞表达血管细胞粘附分子1的表达有显著的诱导作用,其诱导作用在20min即已出现(P<0.05),6h时明显增强(P<0.01)。细胞粘附实验显示随着血小板源生长因子的作用时间的延长,血管平滑肌细胞与单核细胞的粘附率增高,血小板源生长因子处理20min和6h后粘附率是对照组的1.92倍和3.04倍(P<0.05)。结论血小板源生长因子明显诱导血管平滑肌细胞中血管平滑肌细胞表达血管细胞粘附分子1的表达,促进血管平滑肌细胞与单核细胞的粘附,从而参与了损伤早期,白细胞向内膜下迁移的炎症反应。