哮喘模型大鼠气道迷走中枢去抑制的机制及胞外核苷酸代谢和调节异常在其中的作用

Asthma is a chronic inflammatory airway disease with augmented tonic and reflex airway vagal activity. In its lethal attack, airway vagal tone is exaggerated and persistent even without consistent severe airway inflammation; inhalation of glucocorticoids and application of β2-adrenergic agonists are poorly effective whereas assistant anti-cholinergic agents are satisfactory in its control. The central mechanisms of exaggerated airway vagal tone in lethal asthma attack are unknown. In our preliminary study, microglia were found to be activated in the sensory relay center [the nucleus tractus solitarius (NTS)] and airway vagal efferent centers. In this proposal, we hypothesize that microglia in airway vagal centers are alternatively and predominantly polarized to their M2 type releasing brain-derived neurotrophic factor (BDNF), which, via activation of its TrkB receptors in airway vagal preganglionic neurons (AVPNs), causes up-regulation of Na+-K+-2Cl- co-transporter 1 (NKCC1) and down-regulation of K+-Cl- co-transporter 2 (KCC2) in these neurons, leading to weakened or reversed response of them to inhibitory neurotransmitters, and consequently enhances airway vagal tone. Since ATP plays an important role in the activation of microglia, we further hypothesize that the polarization of microglia in the air vagal centers of asthmatics is mediated by down-regulation of ectonucleotidases CD39 and CD73, and upregulation and activation of purinergic P2X4 receptors (P2X4R). ELISA, imunohistochemical staining, western-blot, RT-PCR, patch-clamp, shRNA interference and pulmonary function analysis are to be used to test these hypotheses. Execution of this project is significant for revealing the central mechanisms of enhanced airway vagal tone in lethal asthma attack and for discovery of new anti-asthma therapy.

哮喘是慢性气道炎性疾病,伴紧张和反射性气道迷走神经活动增强。严重发作时,气道迷走神经活动呈与炎症程度无关的持续紧张,但中枢机制不明;吸入糖皮质激素结合β2肾上腺素受体激动剂疗效很差,但抗胆碱药能有效控制。在哮喘大鼠气道迷走中枢,我们前期发现小胶质细胞(MG)被激活,在本项目进一步假设这种MG激活以M2型为主,且其分泌的BDNF通过激活神经元上的TrkB受体,上调1型Na+-K+-2Cl-同向转运体,下调2型K+-Cl-同向转运体,使神经元对抑制性神经递质的反应减弱甚至反转为兴奋,发生去抑制。ATP是重要的MG激活物质。我们假设哮喘时MG激活由外核苷酸酶CD39和CD73表达下调、嘌呤能P2X4型受体上调并激活所介导。拟用ELISA,免疫组化,western-blot,RT-PCR,膜片钳,shRNA干扰和肺功能分析等方法予以验证,以期揭示严重哮喘时气道迷走神经紧张性增强的机制并发现新疗法。

在新生大鼠延髓脑片，本项目用膜片钳电生理方法证明：胞外ATP通过P2X受体易化AVPN的兴奋性突触传入，并对神经元有直接兴奋性作用，而其代谢产物腺苷则通过A1型受体呈现基本相反的作用。 .在卵清蛋白敏化的成年SD大鼠哮喘模型，本项目发现：①气道迷走中枢小胶质细胞（MG）激活，同时BDNF在MG和气道迷走节前神经元（AVPN）表达均增加；②气道迷走中枢1型Na+-K+-2Cl-同向转运体(NKCC1)表达增加，2型K+-Cl-同向转运体(KCC2)表达下降，NKCC1与KCC2的比值升高。③小脑延髓池注射抑制性神经递质-氨基丁酸在对照和哮喘模型大鼠均可增强气道迷走张力，但在哮喘模型大鼠的作用更强。小脑延髓池预先注射NKCC1抑制剂布美它尼（bumetanide）在正常动物可翻转、在哮喘模型鼠可抑制-氨基丁酸的作用，证明在正常动物，神经元Cl-稳态在其对抑制性递质的反应中起到决定作用，而这一作用在哮喘模型大鼠则因NKCC1/KCC2的表达变化而发生改变，提示NKCC1抑制剂可通过恢复AVPN的Cl-稳态降低哮喘时的气道迷走高张性；④腹腔或侧脑室慢性注射MG激活抑制剂米诺环素可逆转卵清蛋白敏化引起的中枢神经化学、肺功能和肺泡灌洗液内卵清蛋白特异性IgE浓度的变化，提示MG激活抑制剂可通过其中枢性非抗生素作用改善抑制肺部炎症，改善肺功能；⑤哮喘模型大鼠气道迷走中枢CD73的表达和活性都显著下降，脑脊液ATP浓度升高；⑥在哮喘模型大鼠气道迷走中枢，P2X受体中只有P2X4亚型表达升高，同时腺苷A1受体表达下降。但免疫荧光研究发现P2X4受体在脑干仅表达在神经元，而不表达在非神经细胞，提示哮喘时中枢胶质细胞的激活与胞外ATP无关；⑦哮喘模型大鼠气道迷走神经（喉返神经）的紧张性和节律性放电活动均增强，这种变化可被小脑延髓池急性注射P2X4受体拮抗剂所拮抗。P2X4受体拮抗剂在对照大鼠也可显著降低甚至消除气道迷走神经（喉返神经）的传出放电活动，提示P2X4受体在气道迷走神经的生理性活动及其与哮喘有关的增强中均起到很重要的作用，P2X4受体拮抗剂可作为潜在的中枢性平喘药物。