基于抗炎和促红细胞生成作用的九味补血口服液改善血虚证小鼠造血功能的机制研究

Mechanism Study of Jiuwei Buxue Oral Liquid on Improving Hematopoietic Function in Blood Deficiency Mice Based on Anti-inflammation and Erythropoiesis Effects

关键词:  九味补血口服液  血虚证  网络药理学  炎症  促红细胞生成  缺氧诱导因子-1

Key words:  Jiuwei Buxue Oral Liquid  syndrome of blood deficiency  network pharmacology  inflammation  erythropoiesis  Hypoxia-inducible factor-1 (HIF-1)

摘要:

为探讨九味补血口服液改善血虚证小鼠造血功能的作用及机制，采用超高效液相色谱串联四级杆飞行时间质谱(UPLC-Q-TOF-MS/MS)技术鉴定九味补血口服液中的化学成分;利用蛋白互作和网络分析手段筛选九味补血口服液治疗贫血的关键网络靶点，并对关键网络靶点进行基因本体(GO)分析、京都基因与基因组百科全书(KEGG)通路富集分析;同时采用分子对接技术将潜在活性成分与关键网络靶点对接，评估活性分子与靶点的结合能力。在动物实验中，采用环磷酰胺建立小鼠血虚模型，设立对照组和模型组，复方阿胶浆口服液组(30 mL/kg)及九味补血口服液高、中、低剂量组(10.0、5.0、2.5 mL/kg)，每天灌胃给药1次，连续32 d。检测全血中红细胞(RBC)、血红蛋白(HGB)和红细胞压积(HCT)水平，并利用qRT-PCR法和免疫印迹法对关键靶点及通路在血虚证小鼠肾脏的表达进行验证。结果显示，在91种鉴定的九味补血口服液化学成分中，满足口服生物利用度(OB)≥30%和药物相似性(DL)≥0.18的条件，且具有相应靶点的潜在活性成分共计15个。分析九味补血口服液成分-血虚疾病靶点网络，获得九味补血口服液治疗贫血的关键靶点15个，主要富集于核因子-κB (NF-κB)信号通路(NF-κB signaling pathway)、代谢通路(Metabolic pathway)、缺氧诱导因子-1(HIF-1)信号通路(HIF-1 signaling pathway)、Toll样受体信号(Toll-like receptor signaling pathway)通路等。分子对接结果显示，甘草苷、橙皮素与Toll样受体4(TLR4)具有强烈的结合能力。动物实验结果显示，与对照组相比，血虚证小鼠RBC、HGB和HCT水平明显降低，TLR4/NF-κB p65介导的炎性信号增强，缺氧诱导因子-1α (HIF-1α)/促红细胞生成素(Erythropoietin，EPO)介导的促红细胞生成信号受抑制(P<0.05或P<0.01)。与模型组相比，九味补血口服液各剂量组可显著升高血虚证小鼠RBC、HGB和HCT水平，不同程度地逆转TLR4、NF-κB p65变化，并上调HIF-1α、EPO mRNA或蛋白的表达水平(P<0.05或P<0.01)。因此，九味补血口服液可以改善血虚证小鼠的贫血状态，其机制可能与促进肾脏中HIF-1α/EPO介导的促红细胞生成作用、减少TLR4/NF-κB p65介导的肾脏炎症反应相关。

Abstract:

This study aimed to investigate the effect and mechanism of Jiuwei Buxue Oral Liquid on improving hematopoietic function in blood deficiency mice.The chemical constituents in Jiuwei Buxue Oral Liquid were identified by Ultra-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS/MS).The key network targets of Jiuwei Buxue Oral Liquid in the treatment of anemia were screened by protein interaction and network analysis,and the key network targets were analyzed by Gene Ontology (GO) analysis and KEGG pathway enrichment analysis.At the same time,molecular docking technology was used to dock potential active components with key network targets to evaluate the binding ability of active molecules to targets.In the animal experiment,the blood deficiency model of mice was established by cyclophosphamide,and the control group and model group,Fufang E Jiao Jiang Oral Liquid group (30 mL/kg) and Jiuwei Buxue Oral Liquid high,medium and low dose groups (10.0,5.0,2.5 mL/kg) were established.The mice were intra-gastrically administered once a day for 32 consecutive days.The levels of Red Blood Cell (RBC),Hemoglobin (HGB) and Hematocrit (HCT) in whole blood were detected,and the expression of key targets and pathways in the kidney of mice with blood deficiency syndrome was verified by qRT-PCR and Western blotting.The results showed that among the 91 identified chemical components of Jiuwei Buxue Oral Liquid,a total of 15 potential active components that met the conditions of oral bioavailability (OB)≥30% and drug similarity (DL)≥0.18 and had corresponding targets were obtained.The component-blood deficiency disease target network of Jiuwei Buxue Oral Liquid was analyzed,and 15 key targets of Jiuwei Buxue Oral Liquid in the treatment of anemia were obtained,which were mainly enriched in Nuclear Factor-κB (NF-κB) signaling pathway,Metabolic pathway,Hypoxia-inducible factor-1(HIF-1) signaling pathway,Toll-like receptor signaling pathway and other pathways.Molecular docking results showed that Liquiritin and Hesperetin had strong binding ability with Toll-like receptor 4 (TLR4).The results of animal experiments showed that compared with the control group,the levels of RBC,HGB and HCT in blood deficiency mice were significantly decreased,and the inflammatory signal mediated by TLR4/NF-κB p65 was enhanced.And the signal of Hypoxia-inducible factor-1α (HIF-1α)/Erythropoietin (EPO) mediated erythropoiesis was inhibited (P<0.05 or P<0.01).Compared with model group,the levels of RBC,HGB and HCT in mice with blood deficiency syndrome were significantly increased in each dose group of Jiuwei Buxue Oral Liquid.The changes of TLR4 and NF-κB p65 were reversed to varying degrees,and the expression levels of HIF-1α and EPO mRNA or protein were up-regulated (P<0.05 or P<0.01).Therefore,Jiuwei Buxue Oral Liquid can improve the anemia status of mice with blood deficiency syndrome,and its mechanism may be related to the promotion of HIF-1α/EPO-mediated erythropoiesis in the kidney and the reduction of TLR4/NF-κB p65-mediated renal inflammatory response.