基于网络药理学及实验验证的“麻黄-葶苈子”药对治疗支气管哮喘的作用研究Mechanism of "Ephedrae Herba-Descurainiae Semen Lepidii Semen" combination in treatment of bronchial asthma based on network pharmacology and experimental verification
张贝贝;曾梦楠;张钦钦;王茹;贾菊芳;郭彭莉;刘萌;冯卫生;郑晓珂;
ZHANG Bei-bei;ZENG Meng-nan;ZHANG Qin-qin;WANG Ru;JIA Ju-fang;GUO Peng-li;LIU Meng;FENG Wei-sheng;ZHENG Xiao-ke;School of Pharmacy,Henan University of Chinese Medicine;Engineering and Technology Center for Chinese Medicine Development of Henan Province;Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province and Education Ministry of P.R.China,Henan University of Chinese Medicine;
摘要(Abstract):
网络药理学结合体内实验探究“麻黄-葶苈子”药对治疗哮喘的作用机制,为临床用药提供理论依据。首先,利用网络药理学预测“麻黄-葶苈子”治疗哮喘的潜在靶点,构建“中药-活性成分-靶点-通路-疾病”的关系网络,并对潜在靶点进行Gene Oncology (GO)功能分析和Kyoto Encyclopedia of Genes and Genomes (KEGG)信号通路富集分析,分子对接模拟关键候选活性成分与核心基因的结合活性。然后,通过腹腔注射卵白蛋白(ovalbumin, OVA)致敏液和雾化激发构建OVA哮喘大鼠模型,分为正常组、模型组、地塞米松组(dexamethasone, DEX, 0.075 mg·kg(-1))和“麻黄-葶苈子”药对组(MT,配伍比例1∶1.5),苏木素-伊红(HE)、马松(Masson)和过碘酸雪夫(PAS)染色观察“麻黄-葶苈子”对哮喘大鼠肺脏和气管病理变化及杯状细胞增生情况的影响,酶联免疫吸附(ELISA)法检测大鼠血清中转化生长因子β1(TGF-β1)、白细胞介素6(IL6)和白细胞介素10(IL10)水平,进而通过实时荧光定量PCR(qRT-PCR)和Western blot法检测网络药理学预测的核心基因丝裂原活化蛋白激酶8(MAPK8)、cyclin D1(CCND1)、表皮生长因子受体(EGFR)、IL6、磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(Akt)的mRNA和蛋白表达水平。网络药理学分析表明“麻黄-葶苈子”治疗哮喘的潜在核心基因为MAPK8、CCND1、IL6和EGFR,并且可能与PI3K/Akt信号通路相关,“麻黄-葶苈子”药对中活性成分槲皮素和β-谷甾醇作用于哮喘的靶点较多,且分子对接结果显示槲皮素和β-谷甾醇与MAPK、PI3K和Akt具有较好的结合活性;体内实验结果表明“麻黄-葶苈子”可有效改善OVA哮喘大鼠症状,改善肺部组织病理变化,减少杯状细胞产生,减轻哮喘大鼠炎症反应,抑制MAPK8、CCND1、EGFR和IL6的表达,调节PI3K/Akt信号通路。因此,推测“麻黄-葶苈子”可能通过调节PI3K/Akt信号通路改善大鼠哮喘症状,抑制哮喘大鼠炎症反应,槲皮素和β-谷甾醇可能是“麻黄-葶苈子”治疗哮喘的候选活性成分。
This study aims to investigate mechanism of "Ephedrae Herba-Descurainiae Semen Lepidii Semen" combination(MT) in the treatment of bronchial asthma based on network pharmacology and in vivo experiment, which is expected to lay a theoretical basis for clinical application of the combination. First, the potential targets of MT in the treatment of bronchial asthma were predicted based on network pharmacology, and the "Chinese medicine-active component-target-pathway-disease" network was constructed, followed by Gene Oncology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the potential targets. Molecular docking was used to determine the binding activity of key candidate active components to hub genes. Ovalbumin(OVA, intraperitoneal injection for sensitization and nebulization for excitation) was used to induce bronchial asthma in rats. Rats were classified into control group(CON), model group(M), dexamethasone group(DEX, 0.075 mg·kg(-1))和“麻黄-葶苈子”药对组(MT,配伍比例1∶1.5),苏木素-伊红(HE)、马松(Masson)和过碘酸雪夫(PAS)染色观察“麻黄-葶苈子”对哮喘大鼠肺脏和气管病理变化及杯状细胞增生情况的影响,酶联免疫吸附(ELISA)法检测大鼠血清中转化生长因子β1(TGF-β1)、白细胞介素6(IL6)和白细胞介素10(IL10)水平,进而通过实时荧光定量PCR(qRT-PCR)和Western blot法检测网络药理学预测的核心基因丝裂原活化蛋白激酶8(MAPK8)、cyclin D1(CCND1)、表皮生长因子受体(EGFR)、IL6、磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(Akt)的mRNA和蛋白表达水平。网络药理学分析表明“麻黄-葶苈子”治疗哮喘的潜在核心基因为MAPK8、CCND1、IL6和EGFR,并且可能与PI3K/Akt信号通路相关,“麻黄-葶苈子”药对中活性成分槲皮素和β-谷甾醇作用于哮喘的靶点较多,且分子对接结果显示槲皮素和β-谷甾醇与MAPK、PI3K和Akt具有较好的结合活性;体内实验结果表明“麻黄-葶苈子”可有效改善OVA哮喘大鼠症状,改善肺部组织病理变化,减少杯状细胞产生,减轻哮喘大鼠炎症反应,抑制MAPK8、CCND1、EGFR和IL6的表达,调节PI3K/Akt信号通路。因此,推测“麻黄-葶苈子”可能通过调节PI3K/Akt信号通路改善大鼠哮喘症状,抑制哮喘大鼠炎症反应,槲皮素和β-谷甾醇可能是“麻黄-葶苈子”治疗哮喘的候选活性成分。
This study aims to investigate mechanism of "Ephedrae Herba-Descurainiae Semen Lepidii Semen" combination(MT) in the treatment of bronchial asthma based on network pharmacology and in vivo experiment, which is expected to lay a theoretical basis for clinical application of the combination. First, the potential targets of MT in the treatment of bronchial asthma were predicted based on network pharmacology, and the "Chinese medicine-active component-target-pathway-disease" network was constructed, followed by Gene Oncology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the potential targets. Molecular docking was used to determine the binding activity of key candidate active components to hub genes. Ovalbumin(OVA, intraperitoneal injection for sensitization and nebulization for excitation) was used to induce bronchial asthma in rats. Rats were classified into control group(CON), model group(M), dexamethasone group(DEX, 0.075 mg·kg(-1)), and MT(1∶1.5) group. Hematoxylin and eosin(HE), Masson, and periodic acid-Schiff(PAS) staining were performed to observe the effect of MT on pathological changes of lungs and trachea and goblet cell proliferation in asthma rats. The levels of transforming growth factor(TGF)-β1, interleukin(IL)6, and IL10 in rat serum were detected by enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein levels of mitogen-activated protein kinase 8(MAPK8), cyclin D1(CCND1), IL6, epidermal growth factor receptor(EGFR), phosphatidylinositol 3-kinase(PI3 K), and protein kinase B(Akt) by qRT-PCR and Western blot. Network pharmacology predicted that MAPK8, CCND1, IL6, and EGFR were the potential targets of MT in the treatment of asthma, which may be related to PI3 K/Akt signaling pathway. Quercetin and β-sitosterol in MT acted on a lot of targets related to asthma, and molecular docking results showed that quercetin and β-sitosterol had strong binding activity to MAPK, PI3 K, and Akt. In vivo experiment showed that MT could effectively alleviate the symptoms of OVA-induced asthma rats, improve the pathological changes of lung tissue, reduce the production of goblet cells, inhibit the inflammatory response of asthma rats, suppress the expression of MAPK8, CCND1, IL6, and EGFR, and regulate the PI3 K/Akt signaling pathway. Therefore, MT may relieve the symptoms and inhibit inflammation of asthma rats by regulating the PI3 K/Akt signaling pathway, and quercetin and β-sitosterol are the candidate active components.
关键词(KeyWords):
麻黄;葶苈子;网络药理学;支气管哮喘
Ephedrae Herba;Descurainiae Semen Lepidii Semen;network pharmacology;bronchial asthma
基金项目(Foundation): 国家重点研发计划项目(2019YFC1708800);; 河南省高层次人才特殊支持计划“中原千人计划”-中原领军人才项目(ZYQR201810080)
作者(Authors):
张贝贝;曾梦楠;张钦钦;王茹;贾菊芳;郭彭莉;刘萌;冯卫生;郑晓珂;
ZHANG Bei-bei;ZENG Meng-nan;ZHANG Qin-qin;WANG Ru;JIA Ju-fang;GUO Peng-li;LIU Meng;FENG Wei-sheng;ZHENG Xiao-ke;School of Pharmacy,Henan University of Chinese Medicine;Engineering and Technology Center for Chinese Medicine Development of Henan Province;Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province and Education Ministry of P.R.China,Henan University of Chinese Medicine;
DOI: 10.19540/j.cnki.cjcmm.20220211.403
参考文献(References):
- [1] 李竹英,孙丽丽,王婷,等.中药对哮喘气道重塑相关蛋白作用的实验研究进展[J].中国实验方剂学杂志,2021,27(3):208.
- [2] 屠新敏,赵欢欢,杨江,等.支气管哮喘中医证候及其生物标志物的研究进展 [J].时珍国医国药,2020,31(5):1212.
- [3] 江泽强,贾珊珊,孟翔鹤,等.基于网络药理学探讨乌梅治疗哮喘的作用机制 [J].中华中医药学刊,2020,39(2):81.
- [4] 朱慧志,张玉,胡国俊,等.基于中医传承辅助系统分析名老中医胡国俊治疗支气管哮喘用药规律 [J].中国实验方剂学杂志,2015,21(5):210.
- [5] 蒋建明.葶苈定喘汤加减治疗急慢性支气管炎39例临床观察 [J].长春中医药大学学报,2011,27(4):612.
- [6] 李新,杨玉芬,朱燕.葶苈子为主药治疗小儿痰多咳喘30例 [J].中国基层医药,2001,2(2):182.
- [7] 屈悦,屈文萍,李瑶,等.基于网络药理学的麻黄-甘草药对治疗小儿哮喘作用机制 [J].辽宁中医杂志,2020,47(8):22.
- [8] 李亚格,克迎迎,王梦梦,等.基于典型热症动物模型物质能量代谢的葶苈子及其性味拆分组分药性归属探讨 [J].中草药,2020,51(13):3465.
- [9] 曹雅雯,汤岐梅,侯雅竹,等.葶苈子治疗心力衰竭的药理研究进展 [J].中西医结合心脑血管病杂志,2019,17(20):3123.
- [10] 史佩玉,樫林,陈国铭,等.基于网络药理学的葶苈子潜在作用机制研究 [J].中国药房,2019,30(20):2823.
- [11] 刘庆林.葶苈子药对的临床应用 [J].光明中医,2006,21(12):58.
- [12] 安祯祥,张安邦,何远利,等.赤芍治疗肝纤维化网络药理学机制探讨 [J].亚太传统医药,2021,17(2):132.
- [13] 赵子樟,李佳晌,宿树兰,等.基于网络药理学及细胞实验的乳香-没药功效成分抗炎机制研究 [J].中国中药杂志,2021,46(21):5674.
- [14] 王小兰,赫金丽,张国顺,等.桑白皮水煎液及化学拆分组分止咳祛痰平喘作用研究 [J].世界科学技术(中医药现代化),2014,16(9):1951.
- [15] 王盟,帕力姑·买买提力,李慧,等.钩藤碱固体脂质纳米粒对哮喘小鼠miR-155/p38MAPK轴的影响 [J].天津医药,2021,49(11):1154.
- [16] 庞亚荣,席建宏,王志旺,等.磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路参与哮喘气道炎症反应的研究现状 [J].中国临床药理学杂志,2021,37(14):1897.
- [17] 曲一帆,徐凤英,王玉珍,等.基于网络药理学和分子对接技术探索黄酮类化合物治疗新型冠状病毒肺炎(COVID-19)的作用机制 [J].包头医学院学报,2020,36(3):74.
- [18] HADJIGOL S,NETTO K G,MALTBY S,et al.Lipopolysaccharide induces steroid-resistant exacerbations in a mouse model of allergic airway disease collectively through IL-13 and pulmonary macrophage activation [J].Clin Exp Allergy,2020,50(1):82.
- [19] 王瀚,马紫童,唐秀凤,等.基于网络药理学探寻淫羊藿治疗哮喘的关键靶标及作用机制 [J].世界中医药,2020,16(5):742.
- [20] 付智慧,赵灵灵,周霖,等.基于网络药理学探讨大承气汤治疗脓毒症的作用机制及关键靶点通路验证[J].中国中药杂志,2021,46(20):5351.
- [21] 曹礼慧,高珊珊,袁中文.枸杞子-菊花药对治疗高血压作用机制的网络药理学研究[J].今日药学,2021,31(9):669.
- [22] CHEN X,ZHOU S,LI H.Evodiamine alleviates severe pneumonia induced by methicillin-susceptible Staphylococcus aureus following cytomegalovirus reactivation through suppressing NF-κB and MAPKs [J].Int J Mol Med,2018,42(1):3247.
- [23] 郑文江,谢富淇,黄慧婷,等.基于网络药理学的射干-麻黄药对治疗支气管哮喘的分子机制探讨 [J].中国实验方剂学杂志,2019,25(19):182.
- [24] 曾幸坤,郭红荣,俞小卫,等.维生素D对原代培养哮喘大鼠气道平滑肌细胞增殖的影响 [J].南京医科大学学报,2013,33(8):1070.
- [25] 卓荦,史家欣,李小民.PI3K/AKT/mTOR信号通路对脓毒症中细胞自噬的调控研究进展 [J].重庆医学,2019,48(11):1927.
- [26] 黄家喜,鲍翠玉,李晶.PI3K/Akt通路在糖尿病心肌病中的研究进展 [J].中国药理学通报,2019,35(9):1202.
- [27] JOO J D,KIM M,D′AGATI V D,et al.Ischemic preconditioning provides both acute and delayed protection against renal ischemia and reperfusion injury in mice [J].J Am Soc Nephrol,2006,17(11):3115.
- [28] 许照,王展,周开放,等.麻黄-杏仁对药对组胺大鼠气道上皮细胞损伤模型EGFR与PI3K表达的影响 [J].中药材,2018,41(9):2206.
- [29] 骆明旭,罗丹,赵万红.槲皮素药理作用研究进展 [J].中国民族民间医药,2014,23(17):12.
- [30] 许成辰,徐婷贞,张敏,等.槲皮素对支气管哮喘模型小鼠NLRP3/Caspase-1炎症小体的抑制作用研究 [J].中国新药与临床药理,2019,30(8):899.
- [31] KOC K,GEYIKOGLU F,CAKMAK O,et al.The targets of beta-sitosterol as a novel therapeutic against cardio-renal complications in acute renal ischemia/reperfusion damage [J].Naunyn Schmiedebergs Arch Pharmacol,2020,394(3):469.
- [32] MAHAJAN S G,MEHTA A A.Suppression of ovalbumin-induced Th2-driven airway inflammation by beta-sitosterol in a guinea pig model of asthma [J].Eur J Pharmacol,2011,650(1):458.
- [33] 陈欧,李国勇,刘爱红,等.网络药理学预测麻黄治疗哮喘的抗炎作用机制 [J].山东大学学报(医学版),2019,57(1):55.
- 张贝贝
- 曾梦楠
- 张钦钦
- 王茹
- 贾菊芳
- 郭彭莉
- 刘萌
- 冯卫生
- 郑晓珂
ZHANG Bei-bei- ZENG Meng-nan
- ZHANG Qin-qin
- WANG Ru
- JIA Ju-fang
- GUO Peng-li
- LIU Meng
- FENG Wei-sheng
- ZHENG Xiao-ke
- School of Pharmacy
- Henan University of Chinese Medicine
- Engineering and Technology Center for Chinese Medicine Development of Henan Province
- Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province and Education Ministry of P.R.China
- Henan University of Chinese Medicine
- 张贝贝
- 曾梦楠
- 张钦钦
- 王茹
- 贾菊芳
- 郭彭莉
- 刘萌
- 冯卫生
- 郑晓珂
ZHANG Bei-bei- ZENG Meng-nan
- ZHANG Qin-qin
- WANG Ru
- JIA Ju-fang
- GUO Peng-li
- LIU Meng
- FENG Wei-sheng
- ZHENG Xiao-ke
- School of Pharmacy
- Henan University of Chinese Medicine
- Engineering and Technology Center for Chinese Medicine Development of Henan Province
- Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province and Education Ministry of P.R.China
- Henan University of Chinese Medicine