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Volume 41 Issue 11
Nov.  2023
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LAI Liyong, XIA Tianshuang, YUE Xiaoqiang, XIN Hailiang. Mechanism of Artemisia annua L. in GIOP with kidney-yin deficiency based on network pharmacology[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(11): 672-679. doi: 10.12206/j.issn.2097-2024.202204115
Citation: LAI Liyong, XIA Tianshuang, YUE Xiaoqiang, XIN Hailiang. Mechanism of Artemisia annua L. in GIOP with kidney-yin deficiency based on network pharmacology[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(11): 672-679. doi: 10.12206/j.issn.2097-2024.202204115

Mechanism of Artemisia annua L. in GIOP with kidney-yin deficiency based on network pharmacology

doi: 10.12206/j.issn.2097-2024.202204115
  • Received Date: 2022-04-27
  • Rev Recd Date: 2022-11-01
  • Available Online: 2023-11-25
  • Publish Date: 2023-11-25
  •   Objective  To predict and preliminarily verify the potential targets and related signaling pathways of Artemisia annua L. in treating glucocorticoid-induced osteoporosis (GIOP) with kidney-yin deficiency by network pharmacology and in vitro experiments.   Methods  The pharmacological targets of Artemisia annua L. were obtained from TCMSP database and were converted to gene names through Uniprot database. The target genes of GIOP with kidney-yin deficiency were obtained from GeneCards database, OMIM database and Drugbank database, and the common target genes were obtained by cross analysis with drug target gene. Protein-protein interaction (PPI) network was constructed by String database, and visualization analysis and core targets screening were performed by Cytoscape 3.9.0. All common targets were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis through Metascape database. Finally, the prediction results were verified by in vitro experiments.   Results  Ninety-eight targets of Artemisia annua L. to GIOP with kidney-yin deficiency were screened, including 17 core genes. The results of GO and KEGG functional enrichment analysis indicated that Artemisia annua L. treating GIOP with kidney-yin deficiency was related to biological processes such as hormonal response, positive regulation of cell death and extracellular stimulation response, et al, as well as signaling pathways such as PI3K/AKT, AGE/RAGE, MAPK and IL-17 et al. The number of genes enriched in PI3K/AKT signaling pathway was the largest. In vitro experiment results showed that Artemisia annua L. promoted the proliferation of osteoblasts damaged by dexamethasone (DEX), increased alkaline phosphatase activity, activated PI3K/AKT pathway, and promoted the phosphorylation of AKT.   Conclusion  Artemisia annua L. treating GIOP with kidney-yin deficiency has the characteristics of multi-targets and multi-pathway, which could promote the proliferation and differentiation of osteoblasts through multiple pathways. The PI3K/AKT signaling pathway is an important pathway. Artemisia annua L. treating GIOP with kidney-yin deficiency might be related to its ability to promote the PI3K/AKT signaling pathway and promote the phosphorylation of AKT.
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Mechanism of Artemisia annua L. in GIOP with kidney-yin deficiency based on network pharmacology

doi: 10.12206/j.issn.2097-2024.202204115

Abstract:   Objective  To predict and preliminarily verify the potential targets and related signaling pathways of Artemisia annua L. in treating glucocorticoid-induced osteoporosis (GIOP) with kidney-yin deficiency by network pharmacology and in vitro experiments.   Methods  The pharmacological targets of Artemisia annua L. were obtained from TCMSP database and were converted to gene names through Uniprot database. The target genes of GIOP with kidney-yin deficiency were obtained from GeneCards database, OMIM database and Drugbank database, and the common target genes were obtained by cross analysis with drug target gene. Protein-protein interaction (PPI) network was constructed by String database, and visualization analysis and core targets screening were performed by Cytoscape 3.9.0. All common targets were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis through Metascape database. Finally, the prediction results were verified by in vitro experiments.   Results  Ninety-eight targets of Artemisia annua L. to GIOP with kidney-yin deficiency were screened, including 17 core genes. The results of GO and KEGG functional enrichment analysis indicated that Artemisia annua L. treating GIOP with kidney-yin deficiency was related to biological processes such as hormonal response, positive regulation of cell death and extracellular stimulation response, et al, as well as signaling pathways such as PI3K/AKT, AGE/RAGE, MAPK and IL-17 et al. The number of genes enriched in PI3K/AKT signaling pathway was the largest. In vitro experiment results showed that Artemisia annua L. promoted the proliferation of osteoblasts damaged by dexamethasone (DEX), increased alkaline phosphatase activity, activated PI3K/AKT pathway, and promoted the phosphorylation of AKT.   Conclusion  Artemisia annua L. treating GIOP with kidney-yin deficiency has the characteristics of multi-targets and multi-pathway, which could promote the proliferation and differentiation of osteoblasts through multiple pathways. The PI3K/AKT signaling pathway is an important pathway. Artemisia annua L. treating GIOP with kidney-yin deficiency might be related to its ability to promote the PI3K/AKT signaling pathway and promote the phosphorylation of AKT.

LAI Liyong, XIA Tianshuang, YUE Xiaoqiang, XIN Hailiang. Mechanism of Artemisia annua L. in GIOP with kidney-yin deficiency based on network pharmacology[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(11): 672-679. doi: 10.12206/j.issn.2097-2024.202204115
Citation: LAI Liyong, XIA Tianshuang, YUE Xiaoqiang, XIN Hailiang. Mechanism of Artemisia annua L. in GIOP with kidney-yin deficiency based on network pharmacology[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(11): 672-679. doi: 10.12206/j.issn.2097-2024.202204115
  • 作者通过网络药理学和体外实验,对青蒿治疗肾阴虚型糖皮质激素性骨质疏松(GIOP)的潜在作用靶点及相关信号通路进行预测和初步验证。研究结果表明, 青蒿治疗肾阴虚型GIOP具有多靶点-多通路的特点,可以通过多条途径促进成骨细胞的增殖、分化,其中PI3K/AKT信号通路是一条重要的通路,青蒿治疗肾阴虚型骨质疏松可能与其能够促进PI3K/AKT信号通路,促进AKT的磷酸化有关。

    • 骨质疏松症(osteoporosis, OP)是一种以骨密度(bone mineral density, BMD) 降低、骨量丢失、骨微结构破坏为主要特点的全身代谢性骨病,以骨脆性增加、易发生骨折为主要特征[1]。随着老龄化的加剧,OP的发生率越来越高,在我国50岁以上的人群中,OP的发生率为19.2%,其中女性为32.1%,男性为6.0%[2]。OP可分为原发性OP和继发性OP,在继发性OP中,糖皮质激素性OP(glucocorticoid-induced osteoporosis, GIOP)的发生率排在第一位[3],目前骨质疏松症(OP)是一种以骨密度(BMD) 降低、骨量丢失、骨微结构破坏为主要特点的全身代谢性骨病,以骨脆性增加、易发生骨折为主要特征[1]。随着老龄化的加剧,OP的发生率越来越高,在我国50岁以上的人群中,OP的发生率为19.2%,其中女性为32.1%,男性为6.0%[2]。OP可分为原发性OP和继发性OP,在继发性OP中,GIOP的发生率排在第一位[3],GIOP治疗上西医使用的药物有钙制剂、活性维生素 D、双磷酸盐类以及雌激素等,这些药物不仅忽略了糖皮质激素这个罪魁祸首,还有可能产生一系列的副作用[4],寻找安全有效且有针对性的药物显得尤为重要。

      中医认为糖皮质激素是“纯阳”之品[5],使用过量易伤阴津;肾主骨,肾虚是导致骨质疏松的关键病机,因此,肾阴虚证是GIOP的一个主要证型,临床研究也证明,肾阴虚证是GIOP患者的一个易患证型[6]。青蒿为菊科蒿属植物黄花蒿植物Artemisia annua L.的干燥地上部分,分布遍及全国,味苦、辛,性寒,归肝、胆经,具有清虚热、除骨蒸的作用[7],对于过量使用糖皮质激素造成的阴虚火旺、烦躁不安,有一定的疗效,临床上也常用青蒿加减处方治疗糖皮质激素使用过度,造成阴虚火旺、虚火上炎的病症[8-9],同时,体内实验发现,青蒿在多种骨丢失模型动物上,可以促进骨组织的再生,提高骨密度[10],青蒿在治疗骨质疏松方面具有广阔的应用前景。然而,目前关于青蒿治疗OP,特别是在GIOP方面的研究尚有限,为此,本研究应用网络药理学和体外实验,结合中医基础理论知识,研究青蒿治疗肾阴虚型GIOP的关键靶点和信号通路,以期明确中药青蒿抗肾阴虚型GIOP的作用及机制,为骨质疏松的临床治疗提供新思路和方法。

    • 在GeneCards数据库(https://www.Genecards.org)、OMIM数据库(https://omim.org)和Drugbank数据库(https://go.drugbank.com)中,以“glucocorticoid induced osteoporosis”作为关键词进行检索,获得GIOP的靶点基因,去除两个数据库的重复结果,获得GIOP疾病靶点基因。

    • 根据全国中西医结合虚证与老年病研究专业委员会[11]1986年对虚证辩证标准的修订,阴虚证诊断标准:主证:①五心烦热(palms and soles);②咽燥口干(dry throat and thirst );③舌红或少苔、无苔(red tongue or less fur or without fur);④脉细数(thin and rapid pulse)。次证:①午后升火(tidal fever);②便结而尿短赤(constipation and scanty dark urine);③盗汗(night sweating)。诊断条件:具备主证3项,次证1项。在GeneCards数据库中,对各症状关键词进行检索,筛选出4个主证检索结果中至少出现3次的靶点,与次证检索结果交叉分析后得到阴虚靶点。肾虚证诊断标准为:①腰脊酸痛(soreness and weakness of waist);②胫酸膝软或足跟痛(soreness and weakness of knees or heel pain);③耳鸣或耳聋(tinnitus or deaf);④发脱或齿摇(alopecia or toothmobilit)y;⑤尿后有余沥或失禁(urinary incontinence or poor urination);⑥性功能减退、不育、不孕(sexual dysfunction and infertility or sterility)。诊断条件:具备3项。在GeneCards数据库中,对各症状关键词进行检索,筛选出至少出现3次的靶点为肾虚靶点。将阴虚靶点与肾虚靶点交叉分析后获得肾阴虚靶点。将GIOP疾病靶点与肾阴虚靶点交叉分析后获得肾阴虚型GIOP靶点。

    • 在中药系统药理学数据库与分析平台(TCMSP:http://tcmspw.com/tcmsp.php)上,以Herb:青蒿、OB≥30%、DL≥0.18为条件进行检索,确定青蒿的活性成分。

    • 利用TCMSP的靶点预测模型,进一步预测青蒿活性成分的相关靶点,通过UniProt数据库(http://www.Uniprot.org/)对预测靶点添加基因名。最后利用微生信在线软件(http://www.bioinformatics.com.cn/)将肾阴虚型GIOP靶点基因与青蒿靶点基因进行交集分析,并绘制出韦恩图。

    • 将“青蒿-肾阴虚型GIOP”共同靶点基因数据导入String在线软件(https://string-db.org /),选择种属为“Homo sapiens”,设置最小互作得分为0.4,构建出PPI网络,将数据导入Cytoscape软件中进行可视化分析,并利用CytoNCA插件进行拓扑分析,以度中心度(DC)和介度中心度(BC)均大于上四分位数的靶点作为核心靶点,进一步分析。

    • 为进一步阐述青蒿对肾阴虚型GIOP的作用机制,将“青蒿-肾阴虚型GIOP”共同靶点基因数据导入metascape数据库(https://metascape.org/gp/index.html)进行GO功能富集分析和KEGG功能富集分析,并通过微生信在线软件绘制相关柱状图和气泡图。

    • 青蒿购于河北安国市场,经海军军医大学药学系生药学教研室辛海量副教授鉴定,密封存放于干燥阴凉处。称取青蒿粉末1g,加入60%乙醇50 ml,室温冷浸7 d,过滤得滤液,55 ℃旋蒸浓缩后,55 ℃烘干备用[12-13]

      其他试剂及生产厂家:地塞米松(大连美仑);胎牛血清(Gibco,美国);DMEM培养基等细胞培养试剂(天津灏洋);碱性磷酸酶(ALP)试剂盒(南京建成);BCA蛋白检测试剂盒、MTT(上海碧云天生物技术有限公司);PI3K、AKT、p-AKT、GADPH抗体(CST公司)。

    • 以二次消化法从新生大鼠颅骨盖中分离获得原代成骨细胞[14]

    • 以含10%胎牛血清和1%青/链霉素的DMEM培养基培养成骨细胞,置于37 ℃、5%CO2培养箱中培养,待细胞铺满80%~90%时,用0.25%胰蛋白酶消化传代培养。

    • 取3-8代的成骨细胞计算其数目,配制成细胞浓度为1×104 个/ml细胞悬液接种于96孔板。24 h后分别更换为含药培养液(DEX: 10 μmol/L;AE: 100、50、 25 μg/ml)。给药48 h后采用MTT法检测成骨细胞的增殖情况。

      取3-8代的成骨细胞计算其数目,配制成细胞浓度为5×104 个/ml细胞悬液接种于24孔板。24 h后分别更换为含药培养液(给药浓度同上)。培养过程中每3 d更换1次含药培养液。第8d裂解细胞,收集细胞裂解液,于4 ℃、13800×g 离心5 min。用对硝基苯磷酸二钠法测定细胞ALP活性。

    • 将3-8代的成骨细胞裂解,提取细胞总蛋白,根据BCA试剂盒进行蛋白定量。采用Western-blot技术对PI3K、AKT和p-AKT水平进行检测。

    • 实验结果以“均值±标准差”($ \bar x$±s)表示。采用SPSS 22.0软件进行数据分析,选用单因素方差分析(One-Way ANOVA)进行组间变量的比较分析。

    • 通过Genecards数据库共检索到GIOP相关靶点基因1741个,通过OMIM数据库共检索到GIOP相关靶点基因311个,通过Drugbank数据库共检索到GIOP相关靶点基因30个,将3个数据库检索结果合并后去除重复基因后,最终获得2039个。通过Genecards数据库检索获得阴虚靶点基因876个,肾虚靶点基因4603个,交叉分析后获得肾阴虚靶点基因836个。将肾阴虚靶点基因和GIOP靶点基因交叉分析后获得522个肾阴虚型GIOP靶点基因(图1)。

    • 通过TCMSP平台共检索到青蒿中OB≥30%,DL≥0.18的活性成分22个,见表1

      分子名称 生物利用度(%) 药物相似度(DL)
      新西兰牡荆苷_qt 59.85 0.21
      醋酸酯 58.02 0.52
      脱氧青蒿素 54.47 0.26
      青蒿烯 54.36 0.31
      万寿菊素 53.11 0.34
      牡荆黄素_qt 52.18 0.21
      泽兰黄醇素 50.8 0.41
      双氢青蒿素 50.75 0.3
      青蒿素 49.88 0.31
      异鼠李素 49.6 0.31
      青蒿亭 49.55 0.48
      茵陈黄酮 48.96 0.41
      槲皮素 46. 43 0.28
      维采宁-2_qt 45.84 0.21
      豆甾醇 43.83 0.76
      线蓟素 43. 46 0.34
      六氟磷酸钠 42.6 0.37
      山奈酚 41.88 0.24
      谷甾醇 36.91 0.75
      木犀草素 36.16 0.25
      怪柳黄素 32.86 0.31
      玄参黄酮 30.35 0.3
    • 通过TCMSP和UniProt数据库获得214个青蒿活性成分靶点基因。将其与肾阴虚型GIOP疾病靶点基因交叉分析(图1),“青蒿-肾阴虚型GIOP”交集靶点基因共有98个。

    • 运用String在线软件构建出“青蒿-肾阴虚型GIOP”共同靶点PPI网络(图2),共有98个节点,1935条相互关系,见图2。利用CytoNCA插件计算网络节点的DC,BC上四分位数为59、101.49248,故以DC≥59、BC≥101.49248为筛选条件,筛选青蒿治疗肾阴虚型GIOP的核心靶点,共得到17个核心靶点,如表2

      靶点信息简称度中心度
      (DC)
      介度中心度
      (BC)
      RAC-α丝氨酸/苏氨酸
      蛋白激酶
      AKT184495.8247
      肿瘤坏死因子TNF83336.63745
      白细胞介素-6IL681281.4354
      细胞肿瘤抗原p53TP5379273.47903
      血管内皮生长因子AVEGFA78200.2001
      白细胞介素-1βIL1B76199.86818
      基质金属蛋白酶-9MMP973146.52397
      转录因子AP-1JUN71167.16982
      前列腺素G/H合成酶-2PTGS271140.30101
      半胱氨酸蛋白酶-3CASP371120.33017
      表皮生长因子受体EGFR68294.5016
      表皮生长因子EGF67107.6285
      白细胞介素-8CXCL867127.67476
      雌激素受体ESR165251.7144
      C-C基序趋化因子配体CCL265101.49248
      过氧化物酶体增殖物
      激活受体γ
      PPARG63118.61439
      血红素氧合酶-1HMOX159156.89531
    • GO富集分析获得5612个细胞生物学过程,其中BP分析获得4649个条目,MF分析获得585个条目,CC分析获得378个条目,根据富集的基因数进行降序排列,分别选取前10个条目建立BP、MF、CC三合一柱状图(图3)。生物学过程(BP)主要涉及激素应答、性腺发育、细胞死亡的正向调节等,细胞成分(CC)包括细胞外基质、转录调节复合物、膜筏等,分子功能(MF)包括信号受体调节活性、蛋白结构域特异性结合、激酶结合等。

      KEGG富集分析获得225个信号过程,根据富集的基因数进行降序排列,选取前20个条目建立气泡图(图4),KEGG富集分析主要涉及的信号通路有PI3K/AKT信号通路,IL-17信号通路,AGE/RAGE信号通路,MAPK信号通路等,说明青蒿治疗肾阴虚型GIOP可能是从多条信号通路联合发挥作用的,其中PI3K/AKT信号通路上富集的共同靶点最多。

    • DEX损伤成骨细胞后,其增殖能力及ALP活性显著降低。药物治疗后,青蒿提取物可显著促进DEX损伤的成骨细胞的增殖,提高ALP活性,促进DEX损伤的成骨细胞的增殖(图5)。

    • 药物处理DEX损伤的成骨细胞48 h后,Western blot结果显示,与空白组比较,模型组p-AKT/AKT比值、PI3K蛋白表达水平明显下降,给予青蒿提取物治疗后,p-AKT/AKT比值、PI3K蛋白表达水平明显提高(图6)。

    • 研究发现,PI3K/AKT通路与氧化应激有密切的联系,一方面,过量的活性氧(ROS)可抑制AKT的磷酸化[15],另一方面,磷酸化的AKT也可以通过Nrf2/HO-1通路抑制氧化应激[16]。在OP的治疗药物探索过程中,抗氧化是一条重要的线索,如啤酒花中的黄腐酚,杜仲中的绿原酸,黄连中的小檗碱,其抗骨质疏松的机制均与抗氧化有关[17-19],现代药理研究已经证明青蒿具有抗氧化,抗炎,调节免疫等作用[20-21],但青蒿中化学成分众多,虽然目前多以倍半萜类成分青蒿素含量作为其质量评价标准[22]。然而,除了倍半萜类成分外,青蒿中还存在黄酮类成分、有机酸类成分等等[23],青蒿的抗肾阴虚型GIOP的机制可能与抗氧化有关。研究发现,倍半萜类成分、黄酮类成分、有机酸类成分均存在一定的抗氧化效果[24-27],青蒿的抗肾阴虚型GIOP的机制可能与抗氧化有关。因此,研究青蒿是否在抗OP方面存在作用,青蒿中哪种单体成分在其抗OP的过程中起主要作用值得进一步探究。

      本研究以网络药理学为基础,初步筛选出了青蒿抗肾阴虚型GIOP的98个共同靶点,通过拓扑分析,筛选出了17个核心靶点。GO分析和KEGG富集分析发现这些共同靶点参与了多个生物学过程,且参与PI3K/AKT,IL-17,AGE/AGE,MAPK等多个信号通路,其中PI3K/AKT信号通路上富集的共同靶点最多。研究表明,PI3K/AKT信号通路可通过影响成骨细胞的增殖和分化,抑制OP的发生[28]。本研究体外实验亦证实青蒿可以促进DEX损伤的成骨细胞的增殖和分化,激活PI3K/AKT信号通路,促进AKT的磷酸化,验证了网络药理学的预测结果,为后续青蒿抗GIOP研究提供了科学依据。

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