川芎嗪抑制大鼠气道平滑肌细胞增殖和诱导凋亡的研究

目的研究川芎嗪对大鼠气道平滑肌细胞(ASMCs)增殖和凋亡的影响。方法采用改良组织块和胰酶消化联合培养原代ASMCs,以血小板源生长因子(PDGF)诱导ASMCs增殖建立模型。采用MTT法检测不同浓度川芎嗪作用不同时间对ASMCs增殖的抑制,Hoechst33342染色检测细胞凋亡,Western blot检测ERK1/2蛋白表达情况。结果 MTT检测川芎嗪处理(6,12,24,36和48 h)后,与对照组比较,12.5μmol/L组、25μmol/L组、50μmol/L组和100μmol/L组的细胞平均抑制率均显著增加(P均<0.05)。Hoechst 33342染色观察到50μmol/L组、100μmol/L组和200μmol/L组川芎嗪中强荧光细胞比例随川芎嗪剂量增大而增多,细胞核内多个不均一蓝染现象。川芎嗪(200μmol/L组)和PDGF(20 pg/L)共同处理后,随作用时间增加,ERK1/2蛋白表达水平显著降低。结论川芎嗪对ASMCs增殖有抑制作用,同时高浓度的川芎嗪可促进ASMCs凋亡,可能与下调ERK1/2的表达有关。     

Esculetin regulates the phenotype switching of airway smooth muscle cells

Airway remodeling is one important feature of childhood asthma, which is one of the most common chronic childhood diseases. Phenotype switching of airway smooth muscle cells (ASMCs), defined as a reversible switching between contractile and proliferative phenotypes, plays an important role in the process of airway remodeling. Esculetin has shown antiinflammatory action in animal models of asthma; however, the effects of esculetin on ASMC phenotype switching have not been investigated. In the present study, platelet‐derived growth factor (PDGF) was used to induce the phenotype modulation of ASMCs. The results demonstrated that esculetin pretreatment mitigated the PDGF‐caused inhibitory effects on expressions of contractile phenotype protein markers, including calponin and SM22α. Esculetin also inhibited PDGF‐induced migration and proliferation of ASMCs. Besides, the PDGF‐induced expressions of extracellular matrix components, collagen I and fibronectin, were attenuated by esculetin pretreatment. Furthermore, PDGF‐caused activation of PI3K/Akt pathway in ASMCs was inhibited by esculetin. These findings suggest that esculetin might exert its inhibitory effect on PDGF‐induced ASMC phenotype switching through inhibition of PI3K/Akt pathway.     

针刺对哮喘大鼠气道重建模型气道平滑肌细胞T型钙通道蛋白表达的影响

目的:观察针刺抗哮喘气道重建效应,并探讨气道平滑肌细胞T型钙通道在哮喘气道重建效应中的作用机制。方法:将32只大鼠随机分为正常组、模型组、针刺组、假针刺组,每组8只。后3组均造模:大鼠一次性腹腔注射卵蛋白10mg和氢氧化铝200mg的生理盐水混悬液1mL以及1mL灭活百日咳菌苗处于致敏状态14d。模型组第15天起连续14d每天超声雾化吸入1%卵蛋白诱发哮喘30min。针刺组大鼠造模后亦连续14d每天超声雾化吸入1%卵蛋白前针刺"大椎""风门""肺俞",每次30min,每2天1次。假针刺组与针刺组取穴及疗程相同,造模后每天超声雾化吸入1%卵蛋白前针刺穴位皮肤1mm,不留针。正常组不予干预处理。检测各组大鼠气道阻力评价呼吸功能,采用肺组织病理学检测评价气道重建情况,并运用免疫组化技术检测大鼠气道平滑肌T型钙通道3个亚单位Cav3.1、Cav3.2、Cav3.3蛋白表达情况。结果:①模型组气道阻力高于正常组与针刺组(均P<0.05),而针刺组气道阻力低于假针刺组(P<0.05)。②模型组气道壁厚度与气道基底膜周径比值(Awt/Pbm)高于正常组及针刺组(均P<0.05),而针刺组Awt/Pbm比值小于假针刺组(P<0.05)。模型组气道外径与气道内径比值(Po/Pi)高于正常组和针刺组(均P<0.05),针刺组Po/Pi比值小于假针刺组(P<0.05)。③模型组气道平滑肌细胞Cav3.1、Cav3.2平均光密度均高于正常组、针刺组(均P<0.05),模型组气道平滑肌细胞Cav3.3平均光密度高于正常组(P<0.05),低于假针刺组(P<0.05)。结论:针刺能抑制哮喘气道重建,降低气道阻力,抑制气道平滑肌增生,其机制可能与抑制气道平滑肌细胞T型钙通道蛋白,尤其是Cav3.1蛋白表达水平相关。     

Relaxant effect of flavonoid naringenin on contractile activity of rat colonic smooth muscle

Ethnopharmacological relevance

Disturbed gastrointestinal (GI) motility can be associated with smooth muscle abnormalities and dysfunction. Exploring innovative approaches that can modulate the disturbed colonic motility are of great importance for clinical therapeutics. Naringenin, a flavonoid presented in many traditional Chinese herbal medicines, has been shown to have a relaxant effect on different smooth muscles. The aim of the present study was to investigate the effect of naringenin on regulation of GI motility.

Material and methods

Mechanical recording was used to investigate the effect of naringenin on isolated rat colonic smooth muscle spontaneous contractions. Whole cell patch clamp, intracellular [Ca²⁺] concentration ([Ca²⁺]_i) and membrane potential measurements were examined on primary cultures of colonic smooth muscle cells (SMCs). A neostigmine-stimulated rat model was utilized to investigate the effect of naringenin in vivo.

Results

Naringenin induced a concentration-dependent inhibition (1–1000 μM) on rat colonic spontaneous contraction, which was reversible after wash out. The external Ca²⁺ influx induced contraction and [Ca²⁺]_i increase were inhibited by naringenin (100 μM). In rat colonic SMCs, naringenin-induced membrane potential hyperpolarization was sensitive to TEA and selective large-conductance calcium-activated K⁺ (BK_Ca) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, naringenin stimulated an iberiotoxin-sensitive BK_Ca current, which was insensitive to changes in the [Ca²⁺]_i concentration. Furthermore, naringenin significantly suppressed neostigmine-enhanced rat colon transit in vivo.

Conclusion

Our results for the first time demonstrated the relaxant effect of flavonoid naringenin on colon smooth muscle both in vitro and in vivo. The relaxant effect of naringenin was attributed to direct activation of BK_Ca channels, which subsequently hyperpolarized the colonic SMCs and decreased Ca²⁺ influx through VDCC. Naringenin might be of therapeutic value in the treatment of GI motility disorders.     

阳和平喘颗粒对气道平滑肌细胞增殖的影响

目的:研究阳和平喘颗粒在体外对支气管哮喘大鼠气道平滑肌增殖及PI3K/PKB通路相关因子的影响。方法:将阳和平喘颗粒含药血清及PI3K/PKB通路阻断剂分别组合作用于体外培养的支气管哮喘大鼠气道平滑肌细胞,噻唑蓝比色法(MTT)检测大鼠气道平滑肌细胞的增值情况,实时聚合酶链反应(Realtime-PCR)检测阳和平喘颗粒对磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(PKB)、肾上腺素受体(AR)、内皮型一氧化氮合酶(e NOS)、增殖细胞核抗原(PCNA)mRNA表达水平的影响,蛋白印迹法(Western-Blot)检测阳和平喘颗粒对PI3K、PKB、AR、eNOS、PCNA蛋白表达水平的影响,酶联免疫吸附测定(ELISA)阳和平喘颗粒对磷脂酰肌醇二磷酸(PIP2)和磷脂酰肌醇三磷酸(PIP3)表达水平的影响。结果:50%阳和平喘颗粒(18.45g/kg)含药血清对支气管哮喘大鼠气道平滑肌细胞的增殖具有明显的抑制作用,阳和平喘颗粒含药血清及PI3K/PKB通路阻断剂能显著抑制支气管哮喘大鼠细胞内PI3K、PKB、PIP2、PIP3和PCNA的表达,促进eNOS和AR的生成。结论:阳和平喘颗粒具有在体外通过PI3K/PKB通路治疗支气管哮喘大鼠气道重塑的作用。     

针刺治疗腓肠肌损伤的临床观察

目的：观察针刺治疗腓肠肌损伤的临床疗效。方法：运用针灸方法治疗腓肠肌损伤58例，取穴头针足运感区、肌肉疼痛起止点。结果：58例患者一般5-10天治愈，治愈50例，有效8例，总有效率迭100％。结论：针刺治疗腓肠肌损伤，有显著的治疗效果，值得临床进一步推广应用。     

Cistus incanus and Cistus monspeliensis inhibit the contractile response in isolated rat smooth muscle

The lyophilized aqueous extracts from Cistus incanus L. (CI) and Cistus monspeliensis L. (CM) collected in Sicily were studied in order to evaluate their myorelaxant activity by using isolated smooth muscle of rat ileum and rat aorta. Both CI and CM extracts concentration-dependently inhibited the contractile response to acetylcholine (ACh), phenylephrine (PE) and to 100 mM KCl. The concentration-contraction curves to ACh in ileum and to PE in aorta, were displaced to the right by Cistus extracts in a non-competitive manner, with a depression of the maximum contractile response. The EC50 (microg/ml) of CM and CI were: ileum/KCl, CM 457+/-99, CI 681+/-80; ileum/ACh 100 microM, CM 297+/-66, CI 335+/-41; aorta/KCl, CM 360+/-21, CI 843+/-36; and aorta/PE 10 microM, CM 287+/-33, CI 451+/-58. The two extracts resulted almost equi-active in ileum, whereas CM was more active than CI in aorta. These data indicate that Cistus extracts act as spasmolytic on intestinal and vascular smooth muscle. The antagonism they exert on ACh-, PE- and KCl-evoked contractions seems to be functional, because it is not specifically directed toward any particular receptor; furthermore, a calcium-antagonist activity seems unlikely, since the extracts are capable of completely block the contractile response to agonists.     

Anti-proliferative effect of Euphorbia stenoclada in human airway smooth muscle cells in culture

The ethanolic extract of a Malagasy species Euphorbia stenoclada (ES) (Euphorbiaceae), traditionally used as a herbal remedy against asthma and acute bronchitis, was tested to evaluate possible anti-proliferative activity on human airway smooth muscle cells (HASMC). The ES ethanolic extract totally abolished the interleukin-1beta (IL-1beta) induced proliferation of HASMC (IC(50)=0.73+/-0.08 microg/mL). No cytotoxic effect was observed up to 20 microg/mL. A bioassay-guided fractionation of the ethanolic extract was performed by reversed-phase (RP) flash chromatography, giving five fractions (FA to FE) where fraction FE was the only active one (IC(50)=0.38+/-0.02 microg/mL). The purification of this bioactive fraction FE was carried out by RP-HPLC affording six sub-fractions 1-6, and only sub-fraction 5 kept the anti-proliferative activity. Its major constituent was identified as quercetin (IC(50)=0.49+/-0.12 microg/mL) by means of HPLC/UV/MS and co-elution with the authentic standard. Quercitrin was also identified in the fraction FE but was inactive. A structure-activity relationship with flavonols determined that methylation reduced the anti-proliferative activity whereas glycosylation abolished it. The present study shows that the anti-proliferative properties of Euphorbia stenoclada are mediated through the presence of quercetin that may explain the traditional use of this plant as a remedy against asthma.     

Parthenolide inhibits the contractile responses of rat stomach fundus to fenfluramine and dextroamphetamine but not serotonin

The isolated rat stomach fundus preparation, a sensitive bioassay to evaluate serotonin-(5-HT) like activity, was used as a model to study the effects of parthenolide (PAR), a component of Tanacetum parthenium (feverfew), on 5-HT storage, release and stimulation of the 5-HT_2B receptor. Cumulative-concentration response curves to 5-HT and the indirect-acting serotonergics fenfluramine (F) and dextroamphetamine (DA) on fundus were obtained in the presence and absence of 1 × 10⁻⁶ to 1 × 10⁻⁵ PAR. 5-HT release elicited by F and DA was indirectly assessed by comparing the contraction elicited by these compounds on tissues from reserpine-treated, l-p-chlorophenylalanine (1-PCPA)-treated and untreated rats. The observed order of agonist potencies on intact fundus was: 5-HT > DA > F and the order of intrinsic activity was: 5-HT > DA > F. PAR did not show agonist effects nor antagonism toward 5-HT on rat fundus at all concentrations used. However, PAR antagonized non-competitively the effects of F and DA. Contractile responses to 5-HT were not significantly different on mucosa-denuded fundus and tissue strips from untreated, 1-PCPA- and reserpine-treated rats. PAR appears to inhibit 5-HT release mediated responses by the indirect-acting 5-HT agonists on fundal tissue.     

一氧化氮诱导体外人气管平滑肌细胞凋亡

目的 观察一氧化氮（NO）是否在体外诱导人气管平滑肌细胞（airway smooth muscle,ASMCs）凋亡。方法 ASMCs用SNAP或SNP或8－溴－环磷酸鸟苷（8－Br-cGMP）处理。通过四甲基偶氮唑盐（dimethythiazol-zay17-2,5diphenylte-trazdium,MTT）法在6、，12，18，24，36h检测SNAP或SNP或8－Br-cGMP对细胞生长的作用。电镜、琼脂糖电泳、原位末端标记和免疫组化链霉菌抗生素蛋白－过氧化酶（streptavidin-peroxidase，SP）法技术用于检测凋亡。结果 ①SNAP组和SNP组ASMc存活的数量降低，而对照组和cGMP组无变化（P＜0．01）。②SNAP组和SNP组电镜显示核皱缩，染色体浓聚和凋亡小体形成。③用SNAP或SNP处理，ASMCs的凋亡指数显著升高（P＜0．01），但对照组成8－Br-CGMP组没有不同。④在SNAP或SNP组，琼脂糖电泳代表了核苷酸DNA长度整倍数的特征性的DNA梯状条带（大约180－200bp）。⑤SNAP或SNP在体外以时间和剂量表达显著高于对照组或8－Br-cGMP组，但Bcl－2基因表达是低于对照组或8－Br-cGMP组。结论 ①NO在体外以时间和剂量依赖的方式诱导人ASMCs凋亡。②NO这些作用通过的通路不涉及到鸟苷酸环化酶和cGMP依赖的蛋白激酶。③对人ASMCs的这种负性调节机制也许与治疗哮喘的气道重建有关。     

茶多酚对大鼠血管平滑肌细胞增殖的影响

目的　探讨茶多酚对离体大鼠胸主动脉血管平滑肌细胞增殖的影响。方法　体外培养大鼠主动脉血管平滑肌细胞 ,应用不同剂量的茶多酚作用 2 4 h后 ,采用 MTS/ PES法确定血管平滑肌细胞的增殖状态。应用流式细胞术测定细胞周期。结果　与对照组相比 ,茶多酚对血管平滑肌细胞增殖具有明显的抑制作用 (P<0 .0 0 1)。流式细胞术检测结果表明 ,茶多酚可以使血管平滑肌细胞大部分处于 G0 / G1 期 ,与对照组相比有明显差异 (P<0 .0 5 ) ,并且可以诱导其凋亡。结论　茶多酚可明显抑制离体大鼠血管平滑肌细胞的增殖。     

Tetramethylpyrazine inhibits angiotensin II-increased NAD(P)H oxidase activity and subsequent proliferation in rat aortic smooth muscle cells

Tetramethylpyrazine (TMP) is the major component extracted from the Chinese herb, Chuanxiong, which is widely used in China for the treatment of cardiovascular problems. The aims of this study were to examine whether TMP may alter angiotenisn II (Ang II)-induced proliferation and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with TMP and then stimulated with Ang II, [3H]-thymidine incorporation and the ET-1 expression was examined. Ang II increased DNA synthesis which was inhibited by TMP (1-100 microM). TMP inhibited the Ang II-induced ET-1 mRNA levels and ET-1 secretion. TMP also inhibited Ang II-increased NAD(P)H oxidase activity, intracellular reactive oxygen species (ROS) levels, and the ERK phosphorylation. Furthermore, TMP and antioxidants such as Trolox and diphenylene iodonium decreased Ang II-induced ERK phosphorylation, and activator protein-1 reporter activity. In summary, we demonstrate for the first time that TMP inhibits Ang II-induced proliferation and ET-1, partially by interfering with the ERK pathway via attenuation of Ang II-increased NAD(P)H oxidase and ROS generation. Thus, this study delivers important new insight in the molecular pathways that may contribute to the proposed beneficial effects of TMP in cardiovascular disease.     

针灸及推拿治疗肌紧张性发音障碍临床观察

目的 观察针灸及推拿治疗肌紧张性发音障碍的临床疗效.方法 将35例肌紧张性发音障碍患者随机分成治疗组和声休组.治疗组18例,选择针刺疗法治疗,并配合颈周穴位推拿,共治疗3周.声休组17例,患者休息,无特殊治疗.观察3周后2组声道不适指数(VTD)评分、嗓音障碍指数(VHI)评分和嗓音主观评分(GRABS)等变化.结果 ...     

木犀草素对气道平滑肌细胞PI3 K/AKT通路的影响

目的观察木犀草素对小鼠气道平滑肌细胞增殖的影响及其与PI3 K/AKT通路的关系。方法分离并培养小鼠气道平滑肌细胞,α-actin免疫细胞化学染色鉴定,细胞分4组:对照组、PDGF组、PDGF+LUT组、LUT组,MTT法检测细胞增殖,Western Blot方法观察p-AKT表达的变化。结果 MTT检测发现,PDGF刺激气道平滑肌细胞后A490值较对照组明显增加(P<0.01),PDGF+LUT组A490值较PDGF组明显降低(P<0.05),LUT组A490值较对照组无明显变化;Western Blot发现,对照组p-AKT极低,PDGF组明显升高,PDGF+LUT组p-AKT较PDGF组降低。结论木犀草素能够通过抑制AKT的磷酸化水平,限制体外培养的气道平滑肌细胞的PI3 K/AKT通路激活,从而发挥抑制小鼠气道平滑肌增殖的作用。     

黄连水煎剂对未孕大鼠离体子宫平滑肌运动的影响

目的 :探讨黄连水煎剂对未孕大鼠离体子宫平滑肌运动的影响。方法 :利用BL 3 10智能型生物信号显示与处理系统记录平滑肌条的等长收缩活动。结果 :黄连水煎剂能明显延长收缩波持续时间 ,增加收缩波面积且有显著正相剂量效应关系 ,还可增加收缩波的振幅但无剂量效应关系 ,黄连水煎剂的这作用可被异搏定 (L型电压依赖性Ca2 通道阻断剂 )所阻断。结论 :黄连水煎剂在体外可增强子宫平滑肌的收缩 ,该作用是通过L型钙通道而发挥的。     

小檗碱对大鼠胃窦平滑肌细胞收缩作用的影响(英文)

目的 :观察小檗碱对大鼠胃窦平滑肌细胞的收缩作用。方法 :采用Bitar法分离制备大鼠胃窦平滑肌细胞悬液 ;将 3种不同浓度的小檗碱加入细胞悬液中 ,用测微尺观察细胞长度的变化。结果 :与对照组相比 ,终浓度为 0 .5 g·L-1 ,1 g·L-1 的小檗碱可显著促进大鼠胃窦平滑肌细胞收缩 (P <0 .0 5 ) ,并呈剂量 效应关系。结论 :小檗碱对大鼠胃窦平滑肌细胞有直接收缩作用。     

针刺悬钟穴治疗突发性项肌痉挛案

马某某,女,18岁,2004年10月9日就诊,门诊号:00132732.主诉:无明显诱因项肌痉挛作痛2小时.查:神倦、痛苦貌,头部强直性后仰,时向左偏、时向右偏,体温、心率、呼吸、血压等均无异常,唯项部双侧斜方肌僵硬触痛.否认落枕史、外伤史,否认近期上呼吸道感染史,否认既往有类似发病史和器质性疾病史.     

丹皮酚对脂多糖诱导损伤的与平滑肌细胞共培养的大鼠血管内皮细胞黏附功能的影响

目的：观察脂多糖（LPS）诱导的与平滑肌细胞（SMC）共培养的内皮细胞（VEC）与大鼠单核细胞黏附功能的改变及丹皮酚（paeonal,Pae）的干预作用。方法：组织块预消化贴壁法原代培养大鼠血管内皮细胞和大鼠血管平滑肌细胞;采用Transwell小室建立VEC-SMC共培养模型;采用LPS诱导VEC的损伤;MTT法和LDH法检测VEC活力;ELISA法检测VEC分泌IL-1β和TNF-α的水平;免疫细胞化学法检测ICAM-1表达水平;孟加拉玫瑰红染色法检测VEC与单核细胞的黏附功能。结果：LPS诱导VEC损伤的质量浓度为100 μg·L^-1,时间为7 h;Pae（15,30,60 μmol·L^-1）对以上细胞模型作用24 h可有效抑制LPS诱导的VEC损伤,显著提高LPS导致的VEC存活率的下降;降低损伤VEC分泌IL-1β和TNF-α的水平,同时减少ICAM-1的表达;从而抑制LPS诱导的VEC与单核细胞的黏附。结论：Pae通过抑制IL-1β和TNF-α表达而保护VEC免受LPS的损伤,可以通过降低ICAM-1的表达水平达到抑制VEC与单核细胞的黏附的作用。