黄芪甲苷治疗PM2.5致肺损伤的转录网络整合研究及实验验证

Integrated Transcriptional Networks and Experiment of Astragaloside IV on PM2.5-Induced Lung Injury

目的挖掘黄芪甲苷介导铁死亡治疗PM2.5致肺损伤的调控网络,并进行实验验证。方法通过数据库、R语言和Perl软件筛选出黄芪甲苷介导铁死亡调控PM2.5致肺损伤的关键基因,构建lncRNA-miRNA-mRNA转录网络进行富集分析。体内实验分为药效和机制两部分验证,药效部分中,35只雄性无特定病原体(Specific pathogen free,SPF)级SD大鼠,随机分为5组。采用喉镜下气管滴注PM2.5诱导肺损伤模型。通过肺组织苏木精-伊红染色(Hematoxylin-eosin,HE)、湿/干重比、支气管肺泡灌洗液中肿瘤坏死因子α(Tumour necrosis factor-α,TNF-α)和白介素1β(Interleukin,IL-1β)浓度评估肺损伤程度;通过透射电镜和蛋白免疫印迹法检测铁死亡水平。机制部分中,28只雄性SPF级SD大鼠,随机分为4组。RSL3在PM2.5造模前1 h腹腔注射,其余组的干预及检测指标同药效部分。结果挖掘出黄芪甲苷介导铁死亡治疗PM2.5致肺损伤的16个关键lncRNA、9个关键miRNA和6个关键mRNA组成的lncRNA-miRNA-mRNA转录网络。体内实验证实,与空白组相比,PM2.5组大鼠肺损伤评分明显增高(P<0.0001),伴肺水肿(P<0.01);BALF中TNF-α、IL-1β浓度升高(P<0.0001);透射电镜下可见到线粒体典型的铁死亡改变;肺组织Nrf2、GPX4和HO-1下调(P<0.0001),p-p38 MAPK和p-ERK上调(P<0.01)。与PM2.5组相比,黄芪甲苷高剂量组中上述趋势显著逆转,肺损伤评分降低(P<0.01),肺水肿减轻(P<0.05),TNF-α、IL-1β浓度降低(P<0.01),线粒体铁死亡改善,肺组织Nrf2、GPX4、HO-1升高(P<0.0001),p-p38 MAPK和p-ERK降低(P<0.05)。而铁死亡激动剂RSL3的干预明显消除了黄芪甲苷的治疗作用。结论黄芪甲苷对PM2.5致肺损伤具有保护作用,其机制可能是抑制MAPK信号通路活化,从而减轻铁死亡,这与构建的转录网络整合预测结果类似。

Objective We performed data mining on the regulatory network of Astragaloside IV on PM2.5-induced lung injury,and verified the results of the experiment in vivo.Methods Crucial targets of Astragaloside IV on PM2.5-induced lung injury by regulating ferroptosis were filtered out using the database,Perl language,and R software.Then,the lncRNA-miRNA-mRNA transcriptional networks were conducted and the GO and KEGG enrichment analyses were performed.This system is experimentally validated in two parts,pharmacodynamic validation and mechanism validation.In pharmacodynamic validation,35 SPF-class SD rats were randomly divided into 5 groups.Rats were exposed to PM2.5 via intratracheal instillation.Rats were sacrificed 24 h after the last PM2.5 injection.The degree of lung injury were assessed by hematoxylin-eosin(HE),lung wet/dry(W/D)weight ratio,and the TNF-αand IL-1βin the bronchoalveolar lavage.The ferroptosis was observed by transmission electron microscope and Western blot analysis.In mechanism validation,28 SPF-class SD rats were randomly divided into 4 groups.RSL3 were administered intraperitoneally 1h prior to PM2.5 adiministration.Intervention in other groups and detection of indicators were the same as mechanism validation.Results 16 lncRNAs,9 miRNAs,and 6 mRNAs were screened to build the lncRNA-miRNA-mRNA transcriptional network of Astragaloside IV on PM2.5-induced lung injury by regulating ferroptosis.The lung injury score of rats in the PM2.5 group was higher than that in the control group(P<0.0002),accompanied by pulmonary edema(P<0.01),increasing levels of TNF-αand IL-1β(P<0.0001),and classic changes of ferroptosis in mitochondrial ultrastructure by TEM.The levels of Nrf2,GPX4,and HO-1 in the PM2.5 group decreased(P<0.0001),while the levels of p-p38 MAPK and p-ERK increased(P<0.01)compared with the control group.The above indicators were reversed in high-dose Astragaloside IV compared with the PM2.5 group,accompanied by decreased lung injury score(P<0.01)and pulmonary edema(P<0.05),down-regulated TNF-αand IL-βin BALF(P<0.01),improved ferroptosis in mitochondrial ultrastructure,up-regulated levels of Nrf2,GPX4 and HO-1(P<0.0001),and decreased levels of p-p38 MAPK and p-ERK(P<0.05).However,the effects of Astragaloside IV were abolished by RSL3,a ferroptosis activator.Conclusion Astragaloside IV has a protective role against PM2.5-induced lung injury.The mechanism may be decreasing ferroptosis by inhibiting the activation of the MAPK signaling pathway,which is similar to our integrated transcriptional networks results.