虫草素对小鼠慢性哮喘模型支气管平滑肌收缩的作用及机制研究

目的 评价虫草素及虫草素与糖皮质激素联合应用对慢性哮喘模型小鼠气道平滑肌收缩的调节作用及可能机制.方法 将32只C57/BL6小鼠随机分成对照组和慢性哮喘模型组,利用肌动描记仪比较p38蛋白激酶抑制剂SB239063(10-6M)、虫草素(10-6M)以及糖皮质激素地塞米松(10-6M)单独或联合孵育前、后由乙酰胆碱(acetylcholine,Ach)诱导的支气管收缩反应,同时应用蛋白印迹检测支气管组织中p38蛋白激酶和热休克蛋白27(heat shock proteins 27,HSP27)的磷酸化状态.结果 模型组小鼠的支气管收缩较对照组加重,有统计学意义.对照组和模型组p38蛋白激酶抑制剂SB239063孵育1h后对Ach介导的收缩反应较空白对照组下降(P＜0.05),地塞米松单独和地塞米松联合虫草素在模型组小鼠Ach诱导支气管平滑肌收缩力的作用均较对照组降低(P＜0.05);地塞米松单独和地塞米松联合虫草素孵育后,对照组和模型组小鼠支气管组织p38蛋白激酶和HSP27磷酸化水平较Ach单纯作用组降低,而且在模型组,使用虫草素干预后p38蛋白激酶和HSP27磷酸化水平较Ach单纯作用组下降(P值均＜0.05).结论 Ach诱导的离体支气管收缩反应具有良好的可重复性,卵清白蛋白吸入可导致Ach诱导的支气管收缩反应加重,糖皮质激素联合虫草素治疗较糖皮质激素单独治疗更有效抑制Ach诱导的气道平滑肌收缩,此种潜在的协同作用可能通过更大程度地抑制p38蛋白激酶信号通路为机制.     

不同剂量虫草活力素对大鼠支气管哮喘模型慢性气道炎症的作用机制研究

目的 对不同剂量虫草活力素(简称虫草)在大鼠支气管哮喘(简称哮喘)模型慢性气道炎症中的抑制作用及相关机制进行初步探讨.方法 Wister大鼠,随机分为对照组、单纯模型组、虫草20 mg/kg、50 mg/kg组、布地奈德(BUD)组及BUD联合虫草素干预组.卵蛋白致敏建立大鼠哮喘模型后给予药物干预8周,对各组支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)进行细胞分类计数及观察肺组织的病理变化,同时应用双抗体夹心ABC-ELISA法测定血清和BALF的哮喘相关细胞因子水平.结果 与单纯模型组比较,虫草两种剂量治疗组均降低慢性哮喘大鼠BALF的细胞总数和嗜酸粒细胞数,差异有统计学意义(P<0.05),并呈剂量相关性;HE染色显示大剂量虫草、BUD治疗组及联合用药组较单纯模型组炎症细胞浸润、平滑肌肥厚及黏膜肺组织水肿等炎症表现明显减轻;较单纯模型组,虫草治疗组血清及BALF的白介素4(IL-4)、IL-14水平下降,干扰素γ升高,差异有统计学意义(P<0.05),并呈剂量相关性.结果 一定剂量的虫草可通过上调T辅助细胞1(Th1)相关细胞因子,下调Th2相关细胞因子,减少嗜酸粒细胞等炎症细胞渗出,抑制哮喘的慢性气道炎症,并与糖皮质激素具有协同作用.     

不同剂量虫草活力素对大鼠支气管哮喘模型慢性气道炎症的作用机制研究

目的对不同剂量虫草活力素（简称虫草）在大鼠支气管哮喘（简称哮喘）模型慢性气道炎症中的抑制作用及相关机制进行初步探讨。方法Wister大鼠,随机分为对照组、单纯模型组、虫草20mg／kg、50mg／kg组、布地奈德（BUD）组及BUD联合虫草素干预组。卵蛋白致敏建立大鼠哮喘模型后给予药物干预8周,对各组支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）进行细胞分类计数及观察肺组织的病理变化,同时应用双抗体夹心ABC—ELISA法测定血清和BALF的哮喘相关细胞因子水平。结果与单纯模型组比较,虫草两种剂量治疗组均降低慢性哮喘大鼠BALF的细胞总数和嗜酸粒细胞数,差异有统计学意义（P〈0．05）,并呈剂量相关性;HE染色显示大剂量虫草、BUD治疗组及联合用药组较单纯模型组炎症细胞浸润、平滑肌肥厚及黏膜肺组织水肿等炎症表现明显减轻;较单纯模型组,虫草治疗组血清及BALF的白介素4（IL-4）、IL-14水平下降,干扰素γ升高,差异有统计学意义（Pd0．05）,并呈剂量相关性。结论 一定剂量的虫草可通过上调T辅助细胞1（Th1）相关细胞因子,下调Th2相关细胞因子,减少嗜酸粒细胞等炎症细胞渗出,抑制哮喘的慢性气道炎症,并与糖皮质激素具有协同作用。     

虫草活力素对大鼠慢性支气管哮喘模型气道重塑作用初探

目的对虫草活力素（简称虫草）在大鼠支气管哮喘（简称哮喘）模型气道重塑中的作用及相关机制进行初步探讨。方法 50只Wister大鼠[6～8周龄,（200±20）g],随机数字表法分为阴性对照组（A）、慢性哮喘单纯模型组（B）（卵清白蛋白系统致敏和反复激发）、慢性哮喘＋虫草50mg/kg治疗8周组（C）、慢性哮喘＋布地奈德（BUD）8周治疗组（D）及慢性哮喘＋BUD联合虫草8周治疗组（E）。肺组织切片HE染色观察病理变化并检测气道管壁厚度,采用免疫组织化学法检测气道转化生长因子β1（TGF-β1）表达水平,应用逆转录PCR法检测肺组织A2a腺苷受体（A2aAR）mRNA表达。数据统计采用ANOVA检验进行组间分析,Mann-Whitney检验进行组内两两比较,Spearman等级相关分析。结果①HE染色显示所有药物干预组较单纯模型组炎症细胞浸润、平滑肌肥厚及黏膜肺组织水肿等炎症表现明显减轻,以D和E组最为明显;②5组气道管壁厚度依次分别为（9.89±2.09）、（21.72±3.16）、（14.94±1.96）、（12.29±2.75）和（12.25±2.32）μm2/μm,F=31.37,P〈0.0001,所有药物干预组气道壁厚度均高于对照组（C组、D组和E组与A组比较后的P值分别为0.0001、0.0524、0.0524）,且低于单纯模型组（C组、D组和E组与B组比较后的P值均〈0.0001）,差异有统计学意义,C组气道壁较D组和E组增厚,差异有统计学意义（P值分别为0.0232和0.0147）;③5组TGF-β1表达强阳性率分别为（2.08±1.63）%、（29.37±5.08）%、（12.30±3.26）%、（10.78±3.35）%及（7.43±2.84）%,F=86.45,P〈0.0001,所有药物干预组TGF-β1蛋白表达均高于对照组（C组、D组和E组与A组比较后的P值均〈0.0001）,且低于单纯模型组（C组、D组和E组与B组比较后的P值均〈0.0001）,差异有统计学意义;E组较C组的蛋白表达有统计学意义下降（P=0.0052）;④5组肺组织A2aARmRNA表达相对强度依次为（0.35±0.06）、（0.27±0.10）、（0.52±0.07）、（0.24±0.05）及（0.47±0.08）,F=26.46,P〈0.0001,C组和E组A2aARmRNA表达高于A组（P值分别为0.0001及0.0007）、B组（P值分别为0.0002及0.0003）和D组（P值均〈0.0001）,差异有统计学意义,D组表达低于A组,差异有统计学意义（P=0.0003）;⑤所有大鼠支气管壁厚度与气道TGF-β1蛋白表达存在正相关（r=0.80,P〈0.01）,而C组大鼠肺组织A2aARmRNA表达和TGF-β1蛋白表达呈负相关（r=-0.68,P=0.03）。结论虫草对哮喘模型的气道重塑具有一定的改善作用,其机制可能通过增加慢性哮喘模型大鼠肺组织A2aARmRNA转录,抑制气道TGF-β1的生成,从而减轻气道重塑,且虫草与糖皮质激素联合应用对于降低TGF-β1的表达具有潜在协同作用。     

虫草活力素对大鼠慢性支气管哮喘模型气道重塑作用初探

目的 对虫草活力素(简称虫草)在大鼠支气管哮喘(简称哮喘)模型气道重塑中的作用及相关机制进行初步探讨.方法 50只Wister大鼠[6～8周龄,(200±20)g],随机数字表法分为阴性对照组(A)、慢性哮喘单纯模型组(B)(卵清白蛋白系统致敏和反复激发)、慢性哮喘+虫草50 mg/kg治疗8周组(C)、慢性哮喘+布地...     

支气管哮喘气道重塑平滑肌增生机制

支气管哮喘是严重影响人类正常生活的慢性疾病之一,尤其是在气道重塑发生之后.由于气道重塑的不可逆,使支气管哮喘的治疗陷入瓶颈状态.气道重塑最主要的病理学改变为气道平滑肌细胞增殖导致的平滑肌层增厚,其原因是机体慢性炎症和长期免疫应答,那么参与炎症反应和免疫应答的细胞因子以及平滑肌细胞的来源就成为研究气道重塑的重要途径.在此...     

支气管哮喘气道重塑平滑肌增生机制

支气管哮喘是严重影响人类正常生活的慢性疾病之一,尤其是在气道重塑发生之后。由于气道重塑的不可逆,使支气管哮喘的治疗陷入瓶颈状态。气道重塑最主要的病理学改变为气道平滑肌细胞增殖导致的平滑肌层增厚,其原因是机体慢性炎症和长期免疫应答,那么参与炎症反应和免疫应答的细胞因子以及平滑肌细胞的来源就成为研究气道重塑的重要途径。在此,对近几年的研究作一综述。     

蛋白激酶C在支气管哮喘模型大鼠气道平滑肌细胞增殖中的信号转导机制研究

目的　探讨蛋白激酶C(PKC)信号转导途径在支气管哮喘 (简称哮喘 )大鼠气道平滑肌细胞 (ASMC)增殖中的作用。方法　 (1) 4 8只Wistar大鼠分为哮喘组 (A组 )及对照组 (B组 ) ,根据激发时间 (2、4、8周 )又分别分为A1、A2 、A3 组和B1、B2 、B3 组 ,其中A、B组大鼠各 12只 ,A2 、A3 、B2 及B3 组各 6只。用流式细胞术、四甲基偶氮唑盐 (MTT)法、增殖细胞核抗原 (PCNA)染色等方法观察每组ASMC增殖 ;(2 )用PKC激活剂 12 肉蔻酰 13 乙酸佛波酯 (PMA)及抑制剂Ro 31 82 2 0分别干预A1、B1组ASMC ,观察ASMC增殖的变化 ;(3)用逆转录 聚合酶链测定 (RT PCR)和免疫细胞化学法检测A1、A2 、A3 组和B1组ASMCPKC α的表达。结果　 (1)A组ASMCS期比例、吸光度 (A)值、PCNA表达增高 ,与B组比较差异有显著性 (P <0 0 1)。 (2 )A1组ASMCS期比例、A值、PCNA阳性表达率在干预前分别为 (19± 3) %、0 4 5 9± 0 0 36、(80± 10 ) % ;10nmol/LPMA处理后分别为 (2 7± 4 ) %、0 5 99± 0 0 78、(95± 9) % ;5 0nmol/LPMA处理后为 (14± 3) %、0 346± 0 0 38、(5 3± 8) % ;Ro 31 82 2 0处理后为 (14± 3) %、0 343± 0 0 4 8、(4 9± 8) %。各干预剂处理后与处理前比较差异均有显著性 (P <0 0 1) ;5 0nmol/LPMA处     

气道平滑肌细胞表现型在慢性哮喘中的作用

气道的重塑和重建在哮喘发病及气道高反应性形成中的作用已越来越为人们所认识。平滑肌细胞向合成表现型转变，主要表现为肌细胞的增生和肥厚，伴有上皮下纤维化，基膜增厚，气道狭窄。多种介质对细胞表现型改变有调节作用，基因表达参与了气道的重塑和重建。     

气道平滑肌异常在支气管哮喘病理生理中的作用

支气管哮喘（简称哮喘）的发病过程中,气道平滑肌细胞在哮喘相关的炎症介质的作用下,出现肥大、增殖活性增强、凋亡减少、迁移、表型转变及动力学异常等改变。此种气道平滑肌的异常最终导致了气道阻力增加和气道高反应性。因此,深入了解导致气道平滑肌异常的表现及机制,寻找更加合理有效的治疗靶点,将是未来哮喘治疗的新方向。     

Mfge8 suppresses airway hyperresponsiveness in asthma by regulating smooth muscle contraction

Airway obstruction is a hallmark of allergic asthma and is caused primarily by airway smooth muscle (ASM) hypercontractility. Airway inflammation leads to the release of cytokines that enhance ASM contraction by increasing ras homolog gene family, member A (RhoA) activity. The protective mechanisms that prevent or attenuate the increase in RhoA activity have not been well studied. Here, we report that mice lacking the gene that encodes the protein Milk Fat Globule-EGF factor 8 (Mfge8^−/−) develop exaggerated airway hyperresponsiveness in experimental models of asthma. Mfge8^−/− ASM had enhanced contraction after treatment with IL-13, IL-17A, or TNF-α. Recombinant Mfge8 reduced contraction in murine and human ASM treated with IL-13. Mfge8 inhibited IL-13–induced NF-κB activation and induction of RhoA. Mfge8 also inhibited rapid activation of RhoA, an effect that was eliminated by an inactivating point mutation in the RGD integrin-binding site in recombinant Mfge8. Human subjects with asthma had decreased Mfge8 expression in airway biopsies compared with healthy controls. These data indicate that Mfge8 binding to integrin receptors on ASM opposes the effect of allergic inflammation on RhoA activity and identify a pathway for specific inhibition of ASM hypercontractility in asthma.     

Inhaled corticosteroid prevents the thickening of airway smooth muscle in murine model of chronic asthma

Airway smooth muscle growth contributes to the mechanism of airway hyperresponsiveness (AHR) in asthma. Although current steroid use demonstrates anti-inflammatory activity, there is little reported on the action of corticosteroid on smooth muscle of the asthmatic airway. The present study investigated the effect of inhaled corticosteroid on the thickening of airway smooth muscle in bronchial asthma. We developed a mouse model of airway remodeling including smooth muscle thickening in which ovalbumin (OVA)-sensitized female BALB/c-mice were repeatedly exposed to intranasal OVA administration twice a week for 3 months. Mice were treated intranasally with fluticasone during the OVA challenge. Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation compared with control mice. In addition, the mice chronically exposed to OVA developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Intranasal administration of fluticasone inhibited the development of eosinophilic inflammation, and importantly, thickening of the smooth muscle layer. Moreover, intranasal fluticasone treatment reduced the transforming growth factor (TGF)-beta 1 level in bronchoalveolar lavage fluid and regulated active TGF-beta 1 signaling with a reduction in the expression of phospho-Smad2/3 and the concomitant up-regulation of Smad7 in lung tissue sections. These results suggest that intranasal administration of fluticasone can modulate the remodeling of airway smooth muscle via regulation of TGF-beta 1 production and active TGF-beta 1 signaling.     

Molecular mechanisms underlying airway smooth muscle contraction and proliferation: implications for asthma

Airway smooth muscle (ASM) plays a key role in bronchomotor tone, as well as in structural remodeling of the bronchial wall. Therefore, ASM contraction and proliferation significantly participate in the development and progression of asthma. Many contractile agonists also behave as mitogenic stimuli, thus contributing to frame a hyperresponsive and hyperplastic ASM phenotype. In this review, the molecular mechanisms and signaling pathways involved in excitation-contraction coupling and ASM cell growth will be outlined. Indeed, the recent advances in understanding the basic aspects of ASM biology are disclosing important cellular targets, currently explored for the implementation of new, more effective anti-asthma therapies.     

The mechanism of ergonovine-induced airway smooth muscle contraction

There have been three articles in the clinical literature concerning ergonovine maleate-induced bronchospasm. The effect of this alkaloid on isolated canine tracheobronchial smooth muscle was analyzed to investigate the mechanism of ergonovine-induced airway smooth muscle contraction. Both ergonovine and serotonin (5-hydroxytryptamine, 5HT) contracted canine tracheal smooth muscle with EC50 1.4 X 10(-8) M and 5.1 X 10(-7) M, respectively. The maximal contractile response observed with ergonovine was approximately 30% less than that observed with 5HT. In diverse blockers such as methysergide, atropine, prazosin, propranolol, pyrilamine and cimetidine, only methysergide competitively blocked both ergonovine and 5HT responses with a similar calculated dissociation constant (pKB); 8.3 +/- 0.2 against ergonovine and 8.5 +/- 0.2 against 5HT (n = 6, p = 0.9). The relative affinity and efficacy of ergonovine and 5HT were determined by use of a partial irreversible antagonist, phenoxybenzamine. The calculated affinity of ergonovine was about 16 times higher than that of 5HT. The relative efficacy of EC100 for ergonovine was 0.2 but at EC10 it was 41.9 (5HT efficacy = 1). Ergonovine 10(-9) M or 10(-8) M shifted the 5HT dose-response curve to the right without reducing the maximal response, but the shift was nonparallel. Ergonovine and 5HT also contracted canine bronchial smooth muscle in a dose dependent manner with EC50 6.4 X 10(-8) M and 1.8 X 10(-7) M, respectively. The dose-responses curve of these two agonists were competitively blocked by methysergide 10(-6) M. These data indicate that ergonovine directly contracts canine tracheobronchial smooth muscle as a result of its combination with 5HT receptors. This effect may result in the precipitation of an asthmatic attack in susceptible individuals.     

气道平滑肌肥大细胞浸润与哮喘的关系

肥大细胞与气道平滑肌相互作用在哮喘气道高反应的产生中起重要作用。肥大细胞产生多种脂类介质、趋化性细胞因子、细胞因子和酶类，它们与气道平滑肌细胞相互作用产生对收缩性刺激的高反应性和增生性，并且激活气道平滑肌细胞产生干细胞因子和其它趋化性细胞因子、细胞因子及生长因子，它们补充、分化和保持肥大细胞。哮喘气道的肥大细胞浸润是T细胞依赖的，来自T细胞及其它来源的Th2型细胞因子对循环及组织中肥大细胞前体的扩展起作用。     

Phenotype modulation of airway smooth muscle in asthma

The biological responses of airway smooth muscle (ASM) are diverse, in part due to ASM phenotype plasticity. ASM phenotype plasticity refers to the ability of ASM cells to change the degree of a variety of functions, including contractility, proliferation, migration and secretion of inflammatory mediators. This plasticity occurs due to intrinsic or acquired abnormalities in ASM cells, and these abnormalities or predisposition of the ASM cell may alter the ASM response and in some cases recapitulate disease hallmarks of asthma.These phenotypic changes are ultimately determined by multiple stimuli and occur due to alterations in the intricate balance or reversible state that maintains ASM cells in either a contractile or synthetic state, through processes termed maturation or modulation, respectively. To elucidate the role of ASM phenotype in disease states, numerous in vitro studies have suggested a phenotypic switch in ASM primary cell cultures as an explanation for the plethora of responses mediated by ASM cells. Moreover, there is overwhelming evidence suggesting that the immunomodulatory response of ASM is due to the acquisition of a synthetic phenotype; however, whether this degree of plasticity is present in vivo as opposed to cell culture-based models remains speculative. Nonetheless, this review will give an overall scope of ASM phenotypic markers, triggers of ASM phenotype modulation and novel therapeutic approaches to control ASM phenotype plasticity.