全基因转录组测序分析双氢青蒿素改善脂多糖诱导小鼠急性肺损伤机制

Mechanisms of amelioration of lipopolysaccharide-induced acute lung injury in mice by dihydroartemisinin using whole-genome sequencing

目的基于全基因转录组测序技术分析双氢青蒿素(DHA)改善脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的作用及机制。方法ip给予LPS 10 mg·kg-1诱导ALI小鼠模型。模型成功后,ip给予DHA 20 mg·kg-1(模型+DHA组),同时设正常对照组,每组10只。给药24 h后,取肺测定小鼠肺湿/干重比(W/D);制备并计数肺泡灌洗液(BALF)中炎症细胞数;苏木精-伊红染色法观察小鼠肺组织病理改变;酶联免疫吸附法(ELISA)检测BALF中白细胞介素1β(IL-1β),IL-6和肿瘤坏死因子α(TNF-α)分泌水平,实时荧光定量PCR(RT-qPCR)检测肺组织中IL-1β,IL-6和TNF-αmRNA表达水平。全基因转录组测序,结合生物信息分析筛选小鼠肺组织差异表达基因,进行GO和KEGG通路富集分析,并进行RT-qPCR验证。结果与模型组相比,模型+DHA组小鼠肺W/D值显著降低(P<0.01),病理损伤显著减轻,BALF中各类炎症细胞显著降低(P<0.01);肺组织中IL-1β,IL-6和TNF-αmRNA表达均显著降低(P<0.05)。全基因转录组测序及生物信息分析发现,ALI模型小鼠肺组织内免疫相关的胎盘特异蛋白8(Plac8)、Toll样受体7基因(TLR7)显著上调(P<0.01),给予DHA干预后则均显著下调(P<0.05,P<0.01)。RT-qPCR验证结果显示,模型+DHA组ALI小鼠肺组织中Plac8和TLR7基因表达变化趋势与转录组测序结果一致。GO和KEGG通路富集分析显示,Plac8和TLR7基因的作用主要与细胞因子生成调控、T/B细胞活化及其信号转导、趋化因子信号转导、NF-κB信号转导等相关,即与机体对外界应激而产生的炎症反应和免疫应答相关。结论DHA可减轻LPS诱导的ALI小鼠肺水肿,改善肺部炎症,减轻ALI,其作用机制可能与其降低TLR7和Plac8 mRNA表达、负调控其参与的信号转导通路,进而调控炎症相关细胞因子分泌和免疫细胞活化等免疫反应有关。

OBJECTIVE To investigate the effect and mechanism of dihydroartemisinin(DHA)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice using whole-genome sequencing.METHODS An ALI mouse model was established via intraperitoneal injection of 10 mg·kg-1 lipopolysaccharide.The mice were divided into normal control group(n=10),model group(n=10)and model+DHA group(n=10).The mice in the model+DHA group were injected intraperitoneally with 20 mg·kg-1 DHA,while those in the normal control group and LPS group were injected intraperitoneally with solvent of DHA,saline containing 1%Tween 80 and 10%Macrogol 400.The mice were executed 24 h after drug administration.The wet and dry weight ratio(W/D)of lung tissue was calculated.Hematoxylin-eosin(HE)staining was used to observe histopathological damage in the lung.Classified counts of inflamma⁃tory cells in alveolar lavage fluid were performed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-1β(IL-1β),IL-6,and tumor necrosis factor-α(TNF-α)in alveolar lavage fluid.Real-time quantitative PCR(RT-qPCR)was used to detect mRNA levels of placenta-specific 8(Plac8),Toll-like receptor 7(TLR7),IL-1β,IL-6 and TNF-αin lung tissue.The whole gene transcriptome was sequenced by RNA transcriptome sequencing(RNA-seq)using the Illumina HiSeq high-throughput sequencing platform before the function and signal pathway of differentially expressed gene mRNA between the groups were enriched and analyzed using GO and KEGG enrichment analysis methods.RESULTS Compared with the model group,the lung W/D values of mice,the pathological damage,inflammatory cells in alveolar lavage fluid,expression levels of IL-1β,IL-6 and TNF-αin alveolar lavage fluid(P<0.01,P<0.01,P<0.01),and the mRNA expression levels of IL-1β,IL-6 and TNF-αwere significantly reduced in lung tissues in the model+DHA group(P<0.01,P<0.05,P<0.05).Whole gene transcriptome sequencing revealed that immune-related Plac8 and TLR7 genes were significantly upregu⁃lated(P<0.01)in mouse lung tissue of the model group but significantly downregulated(P<0.05)in mouse lung tissue of the model+DHA group.The results of RT-qPCR of Plac8 and TLR7 verified the results of whole gene transcriptome sequencing.GO and KEGG analysis showed that Plac8 and TLR7 were mainly related to the regulation of cytokine production,T/B cell activation and signal transduction,chemo⁃kine signal transduction and NF-κB signal transduction.CONCLUSION DHA might reduce LPS-induced lung damage and ameliorate the inflammatory condition in lungs of ALI mice.The mechanism of action may be that DHA negatively regulates the signaling pathways involved in TLR7 and Plac8 by decreasing the expressions of TLR7 and Plac8 mRNA before regulating a series of immune responses such as secretion of inflammation-related cytokines and activation of immune cells,thereby reducing inflam⁃matory damage in lungs.