平喘颗粒对慢性哮喘大鼠Notch通路相关因子的影响

摘要 目的：通过观测各组慢性哮喘大鼠Notch信号通路相关因子Notch2、Jagged1、HES-1的变化，以及肺组织气道重塑的改善状况，探讨平喘颗粒在哮喘的治疗方面所发挥的作用机制。方法：按随机法将60只大鼠均分至空白组、模型组、地塞米松组、平喘颗粒组中。除空白组外，剩余的3组大鼠经卵蛋白+氢氧化铝致敏，复制慢性哮喘大鼠模型，空白组用生理盐水代替致敏液。造模前各组予相应的药物灌胃处理。6周后造模完成，处死大鼠并取其肺组织进行HE染色，在光镜下观察病理变化。使用Real-time PCR法检测Notch2、Jagged1 mRNA，Western blot法检测HES-1蛋白的表达程度。结果：与空白组相比，其余各组肺组织均可观察到明显气道重塑改变，Notch2、Jagged1 mRNA及HES-1蛋白表达明显上升（P＜0.05）；与模型组相比，平喘颗粒和地塞米松可缓解气道重塑，各因子表达情况显著下降（P＜0.05），两组间比较则无明显差异（P＞0.05）。结论：Notch2、Jagged1、HES-1表达程度与气道重塑呈正相关，平喘颗粒通过降低Notch2、Jagged1、HES-1表达改善气道重塑，可能是其防治哮喘发生发展的机制。     

Hesperetin-7,3'-<Emphasis Type=Italic>O</Emphasis>-dimethylether selectively inhibits phosphodiesterase 4 and effectively suppresses ovalbumin-induced airway hyperresponsiveness with a high therapeutic ratio

Background  

Hesperetin was reported to selectively inhibit phosphodiesterase 4 (PDE4). While hesperetin-7,3'-O-dimethylether (HDME) is a synthetic liposoluble hesperetin. Therefore, we were interested in investigating its selectivity on PDE4 and binding ability on high-affinity rolipram-binding sites (HARBs) in vitro, and its effects on ovalbumin-induced airway hyperresponsiveness in vivo, and clarifying its potential for treating asthma and chronic obstructive pulmonary disease (COPD).     

Effect of atrial natriuretic peptide on bronchial tone in anesthetized rabbits

The effect of atrial natriuretic peptide (ANP) on histamine-induced bronchoconstriction was studied in vivo (in normoxic and in hypoxic rabbits) and in vitro. Thirty-two anesthetized rabbits, spontaneously breathing room air or 10% O₂, received infusions of ANP (20, 40, or 80 ng/min/kg normoxia; 20 ng/min/kg hypoxia) or the vehicle for 100 min. After 75 min of ANP infusion, bronchoconstriction was induced inhaling histamine; respiratory resistance (Rrs) was measured prior to and until 20 min posthistamine. The results show that the histamine-induced increase in Rrs was significantly reduced by ANP 80 ng/kg/min in normoxia, and by ANP 20 ng/kg/min in hypoxia. In vitro, ANP had no effect on tracheal and bronchial smooth muscle precontracted with histamine or acetylcholine. These results show that ANP can decrease a histamine-induced bronchoconstriction in vivo but not in vitro, suggesting an indirect mechanism of action.     

Intranasal mite allergen induces allergic asthma-like responses in NC/Nga mice

Airway responses induced by intranasal administration of mite allergen without adjuvant were studied in NC/Nga mice. A crude extract of Dermatophagoides farinae (Df) was administered for 5 consecutive days and a single intranasal challenge booster dose was given 1 week after the last sensitization. 24 h after the single challenge, the airway hyperresponsiveness (AHR) was measured and the bronchoalveolar lavage fluid (BALF) was analyzed for numbers of eosinophils and neutrophils, and both cytokine and chemokine levels. There were marked increases in number of eosinophils in the BALF, AHR, Th2 cytokines (IL-5 and IL-13), and chemokine (eotaxin-1 and eotaxin-2) levels in the BALF following Df exposure. C57BL/6N, A/J, BALB/c, and CBA/JN mouse strains were also exposed to Df crude extract, but all of the measured responses were strongest in NC/Nga mice. Furthermore, Df-exposed NC/Nga mice showed the goblet cell hyperplasia, pulmonary eosinophilic inflammation, and increases in both total serum IgE and Df-specific IgG1. After intranasal exposure of NC/Nga mice to crude extract of Dermatophagoides pteronyssinus, the BALF eosinophilia and AHR were similar to responses induced by Df. None of the study parameters were increased in response to intranasal exposure to ovalbumin. These data demonstrated that NC/Nga mice developed allergic asthma-like responses after intranasal exposure to mite allergens.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.     

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.     

A distinct subset of natural killer T cells produces IL-17, contributing to airway infiltration of neutrophils but not to airway hyperreactivity

Activated natural killer T (NKT) cells produce a broad range of cytokines, including IL-4 and IFN-γ, that determine immunomodulatory functions in various animal models. In this report, we show that a well-known proinflammatory cytokine, IL-17 is also produced by a distinct population of NKT cells upon TCR stimulation. Administration of α-galactosylceramide (α-GalCer), a strong agonist of NKT cells, induces rapid IL-17 production by a small population of NKT cells, mostly belonging to a population different from that of IL-4- and IFN-γ-producing NKT cells. IL-17-producing NKT cells showed unresponsiveness after stimulation of α-GalCer as conventional NKT cells. During airway inflammation induced by pulmonary activation of NKT cells with α-GalCer, IL-17 contributes to the infiltration of neutrophils into the airway but has no effect on airway hyperreactivity (AHR). These results indicate that TCR stimulation induces IL-17 expression by a novel population of NKT cells and may help to explain diverse NKT cell functions.     

Aldehyde dehydrogenase 3A1 protects airway epithelial cells from cigarette smoke-induced DNA damage and cytotoxicity

Aldehyde dehydrogenase 3A1 (ALDH3A1), an ALDH superfamily member, catalyzes the oxidation of reactive aldehydes, highly toxic components of cigarette smoke (CS). Even so, the role of ALDH3A1 in CS-induced cytotoxicity and DNA damage has not been examined. Among all of the ALDH superfamily members, ALDH3A1 mRNA levels showed the greatest induction in response to CS extract (CSE) exposure of primary human bronchial epithelial cells (HBECs). ALDH3A1 protein accumulation was accompanied by increased ALDH enzymatic activity in CSE-exposed immortalized HBECs. The effects of overexpression or suppression of ALDH3A1 on CSE-induced cytotoxicity and DNA damage (γH2AX) were evaluated in cultured immortalized HBECs. Enforced expression of ALDH3A1 attenuated cytotoxicity and downregulated γH2AX. SiRNA-mediated suppression of ALDH3A1 blocked ALDH enzymatic activity and augmented cytotoxicity in CSE-exposed cells. Our results suggest that the availability of ALDH3A1 is important for cell survival against CSE in HBECs.     

Phosphodiesterase 4 inhibitors modulate beta2-adrenoceptor agonist-induced human airway hyperresponsiveness

Chronic exposure of human isolated bronchi to beta2-adrenergic agonists, especially fenoterol, potentiates smooth muscle contraction in response to endothelin-1 (ET-1), a peptide implicated in chronic inflammatory airway diseases. 5'-Cyclic adenosine monophosphate (cAMP) pathways are involved in fenoterol-induced hyperresponsiveness. The present study investigated whether chronic elevation of intracellular cAMP by other pathways than beta2-adrenoceptor stimulation provokes bronchial hyperresponsiveness. Samples from eighteen human bronchi were sensitized to ET-1 by prolonged incubation with 0.1 microM fenoterol (15 h, 21 degrees C), or, under similar conditions, were incubated with a selective type-3 phosphodiesterase inhibitor (1 microM siguazodan), two selective type-4 phosphodiesterase inhibitors (0.1 microM rolipram and 0.1 microM cilomilast), a combination of fenoterol and rolipram (0.1 microM each) or of fenoterol and cilomilast (0.1 microM each). Rolipram and cilomilast, but not siguazodan, induced hyperresponsiveness (p < 0.01 and p < 0.05 vs. paired controls, respectively) similar to the fenoterol effect. Fenoterol-induced bronchial hyperresponsiveness was significantly enhanced by coincubation with cilomilast (p < 0.05 vs. fenoterol alone) but not with rolipram. Our results suggest that prolonged activation of intracellular cAMP through phosphodiesterase 4 inhibition induces hyperresponsiveness to ET-1 in human isolated bronchi. However, differences in subcellular localization of phosphodiesterase 4 may provoke divergent responsiveness patterns when human bronchi are continuously exposed to selective phosphodiesterase inhibitors with or without beta2-adrenergic agonists.     

Mitochondrial ROS and NLRP3 inflammasome in acute ozone-induced murine model of airway inflammation and bronchial hyperresponsiveness

Oxidative stress is a key mechanism underlying ozone-induced lung injury. Mitochondria can release mitochondrial reactive oxidative species (mtROS), which may lead to the activation of NLRP3 inflammasome. The goal of this study was to examine the roles of mtROS and NLRP3 inflammasome in acute ozone-induced airway inflammation and bronchial hyperresponsiveness (BHR). C57/BL6 mice (n?=?8/group) were intraperitoneally treated with vehicle (phosphate buffered saline, PBS) or mitoTEMPO (mtROS inhibitor, 20?mg/kg), or orally treated with VX-765 (caspse-1 inhibitor, 100?mg/kg) 1?h before the ozone exposure (2.5?ppm, 3?h). Compared to the PBS-treated ozone-exposed mice, mitoTEMPO reduced the level of total malondialdehyde in bronchoalveolar lavage (BAL) fluid and increased the expression of mitochondrial complexes II and IV in the lung 24?h after single ozone exposure. VX-765 inhibited ozone-induced BHR, BAL total cells including neutrophils and eosinophils, and BAL inflammatory cytokines including IL-1α, IL-1β, KC, and IL-6. Both mitoTEMPO and VX-765 reduced ozone-induced mtROS and inhibited capase-1 activity in lung tissue whilst VX-765 further inhibited DRP1 and MFF expression, increased MFN2 expression, and down-regulated caspase-1 expression in the lung tissue. These results indicate that acute ozone exposure induces mitochondrial dysfunction and NLRP3 inflammasome activation, while the latter has a critical role in the pathogenesis of ozone-induced airway inflammation and BHR.