青蒿素基于PI3K/Akt信号通路减轻卵清蛋白哮喘大鼠的气道炎症和气道重塑的分析

Attenuation of Airway Inflammation and Airway Remodeling in Ovalbumin Asthmatic Rats by Artemisinin through PI3K/Akt Signaling Pathway

目的:探究青蒿素通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路减轻哮喘气道炎症和气道重塑的分子作用机制。方法:50只雄性SD大鼠随机分为空白组、模型组、青蒿素低、中、高剂量组,每组各10只。建立卵清蛋白(OVA)诱导大鼠哮喘模型,造模成功后,空白组、模型组分别每天给予尾静脉注射1.0 mL·kg^(-1)生理盐水,青蒿素低、中、高剂量组每天给予尾静脉注射青蒿素(12.5、25、50 mg·kg^(-1)),持续7 d。采用氯化乙酰胆碱法测定气道阻力;流式细胞术测定各组肺泡灌洗液中细胞数量、种类变化;苏木素-伊红(HE)染色法测定气道内皮组织形态变化;CytoTox 96法测定细胞凋亡情况;酶联免疫吸附测定法(ELISA)测定NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体、白细胞介素(IL)-1β和IL-10含量表达;蛋白免疫印迹法(Western blot)检测各组肺泡支气管组织磷酸化(p)-PI3K/p-Akt水平。结果:与空白组比较,模型组细胞总数、巨噬细胞总数、嗜酸性粒细胞总数、淋巴细胞总数、中性粒细胞总数明显提高(P<0.05);HE染色显示,大鼠气道黏膜水肿明显,炎症细胞大量浸润(P<0.05);细胞凋亡率明显升高(P<0.05);炎性小体NLRP3、IL-1β、IL-10含量明显上升(P<0.05);p-PI3K/p-Akt明显增高(P<0.05)。与模型组比较,青蒿素低、中、高浓度干预后,细胞总数、巨噬细胞总数、嗜酸性粒细胞总数、淋巴细胞总数、中性粒细胞总数明显下降(P<0.05);HE染色显示大鼠气道黏膜水肿程度减轻,炎症细胞浸润面积大幅减少(P<0.05);细胞凋亡率明显降低(P<0.05);炎性小体NLRP3、IL-1β、IL-10含量明显下降(P<0.05);p-PI3K/p-Akt水平显著降低(P<0.05)。结论:青蒿素可显著抑制NLRP3炎症小体激活并减少细胞焦亡,降低炎性细胞表达,可减轻哮喘大鼠的气道炎症表现及气道重塑,可能与p-PI3K/p-Akt调控有关,可为青蒿素治疗支气管哮喘提供实验依据。

Objective:To investigate the molecular mechanism of action of artemisinin in attenuating asthmatic airway inflammation and airway remodeling through the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.Method:Fifty male SD rats were randomly divided into blank group,model group,and low-dose,medium-dose,and high-dose groups of artemisinin,with 10 rats in each group.The ovalbumin(OVA)-induced asthma model of the rats was established,and after successful modeling,the blank group and model group received tail vein injection of 1.0 mL·kg^(-1)normal saline,while the low-dose,medium dose,and high-dose groups of artemisinin received tail vein injection of 12.5,25,and 50 mg·kg^(-1)artemisinin daily for seven days.Airway resistance was measured by the acetylcholine chloride method.Cell number and species changes in the alveolar lavage fluid of each group were determined by flow cytometry.Morphological changes in airway endothelial tissue were determined by the hematoxylin-eosin(HE)staining method.Apoptosis was determined by CytoTox 96 method,and enzyme-linked immunosorbent assay(ELISA)method was used to determine the NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome,interleukin-1β(IL-1β),and interleukin-10(IL-10)expression.Western blot method was used to detect the(p)-PI3K/p-Akt level in the alveolar bronchial tissue of each group.Result:Compared with the blank group,the total number of cells,total number of macrophages,total number of eosinophils,total number of lymphocytes,and total number of neutrophils were significantly higher in the model group(P<0.05).HE staining showed that the airway mucosa of the rats had obvious edema,and a large number of inflammatory cells were infiltrated(P<0.05).The rate of apoptosis was significantly higher(P<0.05),and the levels of the inflammasome NLRP3,IL-1β,and IL-10 increased significantly(P<0.05).p-PI3K/p-Akt level increased significantly(P<0.05).Compared with the model group,the total number of cells,total number of macrophages,total number of eosinophils,total number of lymphocytes,and total number of neutrophils were significantly decreased after the intervention of artemisinin at low,medium,and high concentrations(P<0.05).HE staining showed that the degree of edema of the airway mucosa of the rats was reduced,and the area of the inflammatory cell infiltration was drastically reduced(P<0.05).The apoptosis rate was significantly reduced(P<0.05),and the levels of the inflammasome NLRP3,IL-1β,and IL-10 decreased significantly(P<0.05).p-PI3K/p-Akt level decreased significantly(P<0.05).Conclusion:Artemisinin significantly inhibits NLRP3 inflammasome activation,reduces cellular pyroptosis and inflammatory cell expression,and attenuates airway inflammatory manifestations and airway remodeling in asthmatic rats,which may be related to the regulation of p-PI3K/p-Akt,and the results may provide laboratory insights and basis for the treatment of bronchial asthma with artemisinin.