摘要
目的 探讨阿纳其根醇提取物(EEAP)对咳嗽变异性哮喘(CVA)大鼠气道炎症的影响及与肠道菌群的相关性.方法 建立CVA大鼠模型40只,随机分为CVA模型组、EEAP低剂量组(160 mg/kg)、EEAP中剂量组(320 mg/kg)、EEAP高剂量组(640 mg/kg)、阳性药组(醋酸泼尼松,2.50 mg/kg),每组8只;另外,取未造模的8只SD大鼠作为对照组.根据分组连续干预30 d后,收集各组大鼠肺泡灌洗液、肺组织及粪便.通过ELISA法检测肺泡灌洗液中白介素4(IL-4)、IL-13、IL-17和干扰素-γ(IFN-γ)水平,采用高通量测序检测大鼠肠道菌群,使用Western印迹法检测大鼠肺组织中缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)表达.结果 各组大鼠肺泡灌洗液中IL-4、IL-13、IL-17和IFN-γ水平差异均有统计学意义(F=63.832、11.869、11.266、4.028,P均＜0.05).与模型组相比,EEAP中剂量、高剂量组和阳性药组大鼠肺泡灌洗液中IL-4、IL-13、IL-17水平明显降低,EEAP高剂量组和阳性药组大鼠肺泡灌洗液IFN-γ水平明显升高,差异均有统计学意义(P均＜0.05).各组大鼠肺组织HIF-1α和VEGF蛋白表达差异均有统计学意义(F=55.898、26.835,P均＜0.05).与模型组相比,EEAP中剂量、高剂量组及阳性药组大鼠肺组织HIF-1α和VEGF蛋白表达明显降低,差异均有统计学意义(P均＜0.05).Spearman相关性分析结果显示,Prevotellaceae_NK3B31_group 与 IL-4、IL-13、IL-17 水平成负相关(r=-0.605、-0.460、-0.522,P 均＜0.05),与 IFN-γ 成正相关(r=0.397,P=0.025).结论 EEAP可抑制CVA大鼠支气管炎症因子IL-4、IL-13、IL-17的表达,并与调控肠道的差异菌群呈现相关性,初步提示EEAP可参与肠-肺轴改善CVA.
Abstract
Objective To investigate the effect of ethanol extract of Anacyclus pyrethrum root(EEAP)on airway inflammation in rats with cough variant asthma(CVA)and its correlation with intestinal flora.Methods Forty rats with CVA were established and randomly divided into CVA model group,EEAP low-dose group(160 mg/kg),EEAP medium-dose group(320 mg/kg),EEAP high-dose group(640 mg/kg),and positive drug group(prednisone acetate,2.50 mg/kg),with 8 rats in each group;SD rats without modeling were taken as control group.After continuous intervention for 30 days according to the grouping,bronchoalveolar lavage fluid,lung tissue and feces of rats in each group were collected.The levels of interleukin-4(IL-4),IL-13,IL-17 and interferon-γ(IFN-γ)in bronchoalveolar lavage fluid were detected by ELISA;the intestinal flora of rats was detected by high-throughput sequencing,and the expressions of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)in lung tissue of rats were detected by Western blotting.Results There were significant differences in IL-4,IL-13,IL-17 and IFN-γ levels in alveolar lavage fluid of rats in each groups(F=63.832,11.869,11.266,4.028;all P＜0.05).Compared with the model group,the levels of IL-4,IL-13,and IL-17 in bronchoalveolar lavage fluid of rats in the medium-dose,high-dose,and positive drug groups of EEAP were significantly decreased,while the level of IFN-γ in bronchoalveolar lavage fluid of rats in the high-dose and positive drug groups of EEAP was significantly increased(all P＜0.05).There were significant differences in HIF-1α and VEGF protein expression in lung tissues of rats in each groups(F=55.898,26.835;all P＜0.05).Compared with the model group,the expressions of HIF-1α and VEGF proteins in lung tissue of rats in the medium-dose,high-dose,and positive drug groups of EEAP were significantly decreased(all P＜0.05).Spearman correlation analysis showed that Prevotellaceae_NK3B31_group were negatively correlated with IL-4,IL-13,and IL-17 levels(r=-0.605,-0.460,-0.522;all P＜0.05),and positively correlated with IFN-γ(r=0.397,P=0.025).Conclusion EEAP could inhibit the expression of bronchial inflammatory factors IL-4,IL-13,and IL-17 in CVA rats,and was correlated with the differential flora that regulate the intestine,which preliminarily suggests that EEAP might be participate in the intestinal-lung axis to improve CVA.
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