基于UPLC-Q-TOF/MS探讨感咳方药效物质基础及作用机制的研究

Study on therapeutic material basis and mechanism of Ganke Formula based on UPLC-Q-TOF/MS

目的 探讨感咳方的药效物质基础及其止咳作用的相关机制。方法 用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS),分析鉴定感咳方入血成分。将30只大鼠随机分为对照组、模型组和低、中、高剂量实验组,每组6只。用烟熏法+冷刺激建立急性支气管炎大鼠模型。低、中、高剂量实验组分别按1.86、4.66、9.32 g·kg^(-1)(生药/体质量)的剂量灌胃给予感咳方,对照组和模型组均灌胃给予等体积0.9%NaCl,每天给药1次,连续7 d。用酶联免疫吸附试验法检测大鼠肺组织中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)含量,用蛋白质印迹法检测大鼠肺组织中磷脂酰肌醇3-激酶(PI3K)和磷酸化蛋白激酶B(p-Akt)蛋白的表达水平。结果 UPLC-Q-TOF/MS分析鉴定得到26个入血成分。对照组、模型组和中、高剂量实验组的IL-1β含量分别为(57.80±7.67)、(186.48±8.50)、(166.05±9.90)和(143.19±6.31)pg·mL^(-1),TNF-α含量分别为(47.14±8.55)、(316.22±9.49)、(68.93±7.94)和(65.93±7.10)pg·mL^(-1),PI3K蛋白相对表达水平分别为0.38±0.05、0.97±0.10、0.68±0.15和0.56±0.10,p-Akt蛋白相对表达水平分别为0.34±0.14、0.93±0.05、0.63±0.16和0.49±0.14。模型组的上述指标与对照组和高剂量实验组比较,在统计学上差异均有统计学意义(P<0.01,P<0.05)。结论 感咳方可通过调控PI3K/Akt信号通路,干预炎症和免疫调节因子表达,改善气道炎症,从而发挥止咳作用。

Objective To explore the therapeutic material basis and antitussive mechanism of Ganke Formula.Methods Ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF/MS) technology was used to analyze and identify the components of Ganke Formula in serum.30 rats were randomly divided into control group,model group,experimental-L group,experimental-M group and experimental-H group,6 rats per group.Acute bronchitis rat model was established by smoke inhalation and cold stimulus.The experimental-L,-M,-H groups were respectively given 1.86,4.66,9.32 g·kg^(-1)(crude drug/weight) dose of Ganke Formula per day by intragastric administration for 7 days.The control group and model group were given the equal amount of normal saline.The contents of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in rat lung tissue were determined by enzyme-linked immunosorbent assay.The expressions of phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (p-Akt) in lung were detected by Western blotting.Results Twenty-six absorbed components have been identified by UPLC-Q-TOF/MS.The contents of IL-1β in control group,model group,experimental-M,-H group were (57.80±7.67),(186.48±8.50),(166.05±9.90) and (143.19±6.31)pg·m L^(-1);TNF-α contents were (47.14±8.55),(316.22±9.49),(68.93±7.94) and (65.93±7.10)pg·m L^(-1);the relative expression levels of PI3K in lung tissues were 0.38±0.05,0.97±0.10,0.68±0.15 and0.56±0.10;the relative expression levels of p-Akt were 0.34±0.14,0.93±0.05,0.63±0.16 and 0.49±0.14,respectively.Compared with the model group,the above indicators in the experimental-H group and control group were statistically different (P<0.01 or P<0.05).Conclusion Ganke Formula can intervene in the expression of inflammatory and immune regulatory factors by regulating PI3K/Akt signaling pathway,improve airway inflammation,and thus exert cough relieving effects.