BDNF经TrkB-Ca^(2+)信号通路对哮喘小鼠气道高反应的调控研究

Regulation of BDNF on airway hyperresponsiveness via TrkB-Ca^(2+)signaling pathway in asthmatic mice

为探讨脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)经酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)-Ca^(2+)信号影响哮喘小鼠气道高反应的分子机制,应用卵白蛋白(ovalbumin,OVA)建立支气管哮喘小鼠模型,进行肺泡灌洗液(broncho alveolar lavage fluid,BALF)细胞计数及分类计数;H-E染色观察肺组织炎性改变并测定气道壁厚度;ELISA检测BALF、血清、肺组织中BDNF水平;免疫组化、Western blotting和qRT-PCR检测肺组织中BDNF、TrkB蛋白和mRNA的表达变化;瞬时转染技术将BDNF小干扰RNA(si-BDNF)和蛋白激酶C(protein kinase C,PKC)小干扰RNA(si-PKC)转染入被分离的哮喘模型组小鼠肺动脉平滑肌细胞内沉默BDNF和PKC的表达,比色法测定肺组织细胞中Ca^(2+)水平变化。结果显示,与对照组比较,哮喘模型组小鼠BALF中细胞总数、嗜酸性粒细胞、淋巴细胞和中性粒细胞显著增加(P<0.05),单核细胞显著减少(P<0.05);肺组织中有大量炎性细胞浸润,且气道壁变厚(P<0.05);BALF、血清和肺组织中BDNF水平均显著升高(P<0.01);肺组织中BDNF、TrkB蛋白和mRNA水平均显著升高(P<0.001);肺组织细胞中Ca^(2+)水平上调(P<0.01),抑制BDNF和PKC后肺组织细胞中Ca^(2+)水平下调(P<0.05)。该研究提示,BDNF可能通过激活下游PKC信号通路使肺组织Ca^(2+)水平增加,血管收缩,进而参与哮喘气道高反应的形成。

This study aims to explore the molecular mechanism of brain-derived neurotrophic factor(BDNF)on airway hyperresponsiveness in asthmatic mice through tyrosine kinase receptor B(TrkB)-Ca^(2+)signaling.The asthmatic mouse model was established by ovalbumin(OVA).Broncho alveolar lavage fluid(BALF)was obtained to perform cell count and differential count.H-E staining was used to observe the inflammatory changes in lung tissue and measure airway wall thickness.ELISA was used to detect the levels of BDNF in BALF,serum,and lung tissue.The expressions of BDNF and TrkB protein and mRNA in lung tissue were detected by immunohistochemistry,Western blotting,and qRT-PCR.BDNF small interfering RNA(si-BDNF)and protein kinase C(PKC)small interfering RNA(si-PKC)were transfected into pulmonary artery smooth muscle cells isolated from mice of the asthma model group to knock down BDNF and PKC expression and the levels of Ca^(2+)in lung tissue cells were measured by colorimetry.The results showed that compared to those of the control group,the total number of cells,eosinophils,lymphocytes,and neutrophils in BALF of the asthma model group were significantly increased(P<0.05),while the number of monocytes was significantly decreased(P<0.05).There were more inflammatory cells infiltrated in the lung tissue of the asthma model group and the airway wall became thicker(P<0.05).The levels of BDNF in BALF,serum,and lung tissue were significantly increased(P<0.01).The levels of BDNF,TrkB protein,and mRNA were significantly increased(P<0.001).The level of Ca^(2+)in lung tissue cells was up-regulated(P<0.01),but down-regulated after inhibition of BDNF and PKC(P<0.05).In conclusion,BDNF may increase the level of Ca^(2+)and vasoconstriction by activating the downstream PKC signaling pathway,resulting in airway hyperresponsiveness in asthma.