基于血清代谢组学探讨青翘、老翘对急性肺损伤大鼠的作用机制

Investigation of Mechanism of Qingqiao and Laoqiao on Acute Lung Injury Rats Based on Serum Metabolomics

目的:通过研究青翘和老翘的化学成分差异,以及二者对急性肺损伤(ALI)大鼠血清代谢组学的影响,探讨青翘、老翘治疗ALI作用机制的异同。方法:24只SD雄性大鼠适应性喂养1周后,随机取6只大鼠作为空白组,其余18只大鼠采用气管滴注脂多糖(LPS)诱导ALI模型,造模成功后,随机分为模型组、青翘组(1.5 g·kg^(-1))与老翘组(1.5 g·kg^(-1)),每组6只。青翘组和老翘组每日灌胃相应剂量药液1次,空白组、模型组灌胃等体积生理盐水,连续给药3 d。通过苏木素-伊红(HE)染色、肺组织干湿质量比(W/D)值及大鼠肺泡灌洗液(BALF)中蛋白浓度综合评估大鼠肺组织的病理状况。采用酶联免疫吸附测定法(ELISA)检测BALF中白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)的表达水平。采用超高效液相色谱-四极杆-飞行时间串联质谱法(UPLC-Q-TOF-MS)鉴定并分析青翘、老翘的化学成分,并对各组大鼠血清进行代谢组学分析,结合多元统计分析,以变量重要性投影(VIP)值>1、t检验P<0.05及差异倍数(FC)≥1.5或FC≤0.5筛选青翘、老翘治疗ALI的差异代谢物。采用京都基因与基因组百科全书(KEGG)数据库结合MetaboAnalyst对筛选出的差异代谢物进行通路分析。结果:与空白组比较,模型组大鼠肺泡腔变大,肺泡破裂,肺间质出血,支气管渗出大量中性粒细胞及红细胞,BALF中蛋白浓度和肺组织W/D值显著升高(P<0.01);与模型组比较,青翘组、老翘组的大鼠肺支气管出血减轻,BALF中蛋白浓度和肺组织W/D值显著降低(P<0.01),肺部损伤明显缓解,以青翘组效果更为明显。与空白组比较,模型组BALF中炎症因子表达水平均显著升高(P<0.01),与模型组比较,青翘组、老翘组炎症因子表达水平均显著降低(P<0.01)。成分分析结果显示,青翘中共检测出63个化学成分,老翘中共检测出55个化学成分,其中二者共有成分47个,青翘独有成分16个,老翘独有成分8个。对大鼠血清代谢组学差异进行表征,青翘和老翘分别回调了19个和12个代谢产物。代谢通路富集分析显示,青翘通过影响亚油酸代谢、苯丙氨酸代谢、甘油磷脂代谢等6条关键代谢通路发挥治疗作用,老翘通过影响亚油酸代谢、花生四烯酸代谢、鞘磷脂代谢等6条关键代谢通路发挥治疗作用。结论:青翘、老翘对ALI均具有治疗作用,以青翘效果更优,二者均可通过调控氨基酸代谢与脂质代谢对ALI起到治疗作用,但二者影响的代谢通路有所差异。

Objective Serum metabolomics of acute lung injury(ALI)in rats was conducted using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS)to explore the similarities and differences in the mechanism of Qingqiao(harvested when the fruits of Forsythiae Fructus were initially ripe and still green in color)and Laoqiao(harvested when the fruits of Forsythiae Fructus were ripe)in the treatment of ALI.Method A total of 24 SD male rats were acclimatized and fed for 1 week,6 of them were randomly selected for the blank group and 18 for the experimental group.The ALI model was induced in the experimental group by tracheal intubation with lipopolysaccharide(LPS).After successfully constructing the ALI model,these rats was randomly divided into model group,Qingqiao group and Laoqiao group,with 6 rats in each group.The Qingqiao and Laoqiao groups were administered orally once a day at a dose of 1.5 g·kg^(-1),while the blank and model groups received an equivalent volume of saline for 3 consecutive days.The pathological conditions of rat lung tissues were comprehensively assessed by hematoxylin-eosin(HE)staining,wet-to-dry mass ratio(W/D)of lung tissues,and protein concentration in rat bronchoalveolar lavage fluid(BALF).The levels of interleukin(IL)-6,IL-1βand tumor necrosis factor(TNF)-αin BALF were quantified using enzyme-linked immunosorbent assay(ELISA).UPLC-Q-TOF-MS was used to identify and analyze the chemical compositions of Qingqiao and Laoqiao,and serum metabolomics of rats in each group was analyzed,combined with multivariate statistical analysis with variable importance in the projection(VIP)value>1,P<0.05 from t-test,and fold change(FC)≥1.5 or FC≤0.5 to screen the differential metabolites Qingqiao and Laoqiao for the treatment of ALI.The Kyoto Encyclopedia of Genes and Genomes(KEGG)database was used in combination with MetaboAnalyst for the metabolic pathway analysis of the screened differential metabolites.Result Compared with the blank group,rats in the model group exhibited enlarged alveolar lumen,ruptured alveoli,interstitial hemorrhage,bronchial exudation of a large number of neutrophils and erythrocytes,and a significant increase in the protein concentration in the BALF and the W/D value of the lung tissues(P<0.01).In contrast,compared with the model group,rats in the Qingqiao group and the Laoqiao group showed reduced bronchial hemorrhage in the lungs,and the protein concentration in the BALF and the W/D value of the lung tissues were significantly decreased(P<0.01),the lung injury was significantly alleviated,but more obvious in the Qingqiao group.Compared with the blank group,the expression levels of IL-6,IL-1βand TNF-αin the BALF of the model group were significantly higher(P<0.01).Additionally,compared with the model group,the expression levels of IL-6,IL-1βand TNF-αin the Qingqiao and Laoqiao groups were significantly lower(P<0.01).The chemical composition analysis of Qingqiao and Laoqiao revealed that 63 components were detected in Qingqiao and 55 components were detected in Laoqiao,with 47 common components,16 components unique to Qingqiao and 8 components unique to Laoqiao.Characterizing the differences in serum metabolomics in rats,19 and 12 metabolites were called back by Qingqiao and Laoqiao,respectively.The metabolic pathway enrichment analysis showed that Qingqiao exerted its therapeutic effects by affecting 6 key metabolic pathways,including linoleic acid metabolism,phenylalanine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,glycerophospholipid metabolism,α-linolenic acid metabolism,and arachidonic acid metabolism,and Laoqiao exerted therapeutic effects by affecting 6 key metabolic pathways,including linoleic acid metabolism,arachidonic acid metabolism,sphingolipid metabolism,phenylalanine metabolism,ascorbate and aldarate metabolism,and glycerophospholipid metabolism.Conclusion Qingqiao and Laoqiao have therapeutic effects on ALI,and Qingqiao is more effective.Both of them can play a therapeutic role in ALI by regulating amino acid metabolism and lipid metabolism,but the metabolic pathways affected by them are different.