调补肺肾法调控脂筏区“开关”治疗哮喘的协同增效机制研究

Bronchial asthma is a disease of airway hyperreactivity. The imbalance of β2 adrenergic receptor(β2AR)/cAMP/protein kinase A(KPA) signal pathway which present in the lipid rafts zone of airway epithelial cell plays a key role in the pathogenesis of bronchial asthma patient, which is mediated by β2AR. Inhalation β2AR-agonist combined with low-dose corticosteroids is a main way in clinical treatment. But long-term use of β2AR-agonist can lead to β2AR phosphorylation and then enter the lipid raft zone, which can affect the activity of β2AR and contribute to resistant. During the clinic, we find the Chinese Medicine of "nourishing lung and kidney" can reduce the dosage and frequency of the mentionedinhalation medicine, therefore we hypothesized that Chinese Medicine of "nourishing lung and kidney" may enhance the function of β2AR-agonist by regulating the switch of lipid rafts, reducing the degree of phosphorylation of β2AR and preventing it from entering the lipid raft. This study intends to carry out related animal experiment to elaborate the synergistic mechanism of the Chinese Medicine of "nourishing lung and kidney" during the treatment of asthma by using Chinese Medicine of "nourishing lung and kidney" combine with different dose of western-drug to control and from airway function, histomorphology and signal pathway imbalance. And clear the molecular mechanism and function of Chinese Medicine which is by enhancing the transmission of β2AR signal pathway, from the cytological level. This study wil contribute to clear the importance of ChineseMedicine of "nourishing lung and kidney" during the treatment of asthma, rich the scientific connotation of the "Aioi from Jinshui" theory, and we can furtherenhance their clinical value.

支气管哮喘患者存在于气道上皮细胞脂筏区中的β2肾上腺素受体（β2AR）/cAMP/蛋白激酶A（PKA）信号通路失衡在其发病机制中起关键作用。临床主要通过吸入β2AR激动剂联合糖皮质激素治疗，但长期应用可导致β2AR磷酸化而进入脂筏区内部，影响其活性，进而产生耐药。我们发现调补肺肾中药在哮喘治疗中可以减少吸入β2AR激动剂联合低剂量糖皮质激素用量及频次，因此提出假说调补肺肾中药通过调控脂筏区开关，降低β2AR磷酸化程度，抑制其进入脂筏区部，从而增强β2AR激动剂的治疗作用。本研究拟开展动物实验，通过调补肺肾中药联合不同剂量西药组干预，从气道功能、组织形态及信号通路失衡等方面，阐述调补肺肾法在治疗哮喘中的协同增效机制，并从细胞学水平明确其增强β2AR信号通路转导的分子作用机理。本研究将有助于明确调补肺肾法在哮喘治疗中的重要作用，丰富中医学“金水相生”的科学内涵，提升其临床应用价值。

支气管哮喘患者气道上皮细胞脂筏区中的β2肾上腺素受体（β2AR）/cAMP/蛋白激酶A（PKA）信号通路的失衡是其发病机制的关键。目前临床治疗主要为吸入β2AR激动剂联合糖皮质激素。但长期应用可导致β2AR磷酸化而进入脂筏区内部，影响其活性，进而产生耐药。课题组前期研究发现“调补肺肾”中药在哮喘治疗中可以减少吸入β2AR激动剂联合低剂量糖皮质激素用量及频次，因此提出假说“调补肺肾”中药可通过调控脂筏区开关，降低β2AR磷酸化程度，抑制其进入脂筏区内部，从而增强β2AR激动剂的治疗作用。 .研究一，建立小鼠“支气管哮喘”模型，通过调补肺肾中药联合不同剂量西药组干预，从气道功能、组织形态及信号通路失衡等方面，阐述“调补肺肾法”在治疗哮喘中的协同增效机制。采用Western Blot及RT-PCR法检测小鼠气管、肺组织中β2AR、磷酸化β2AR、cAMP、PKA以及其mRNA的表达。研究结果显示，“调补肺肾法”中药制剂联合高剂量福莫特罗组结局指标优于空白组、模型组、单纯西药组与中药制剂联合低剂量福莫特罗组，结果有统计学意义（P<0.05）；“调补肺肾法”中药制剂联合低剂量福莫特罗组结局指标优于空白组、模型组、单纯西药组，结果有统计学意义（P<0.05）。从体外研究角度证实“调补肺肾法”中药制剂可以通过降低β2AR磷酸化程度，抑制β2AR进入脂筏区内部，启动脂筏区“开关”，与β2AR激动剂联用治疗哮喘起协同增效的作用。.研究二以β2-CHO细胞为研究对象，采用双荧光素酶报告基因活性检测等检测手段，揭示含“调补肺肾”中药小鼠血清对协同增强β2AR信号通路转导的分子作用机理。实验结果显示，与空白组相比，给药组与阳性对照组的β2受体均未被激活。依据实验结果，我们推测出“补肺颗粒”协同增效的分子机制：“调补肺肾”中药可与胆固醇竞争性结合在细胞膜脂筏区，降低细胞膜胆固醇密度，增加细胞膜内侧的流动性，改变其中信号分子蛋白的构象，削弱脂筏对β2AR信号通路信号分子的限制，使得Gas蛋白从脂筏区中游离出来，增加β2AR和Gas的偶联，使得β2AR由非活化状态变成了活化状态。