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Volume 40 Issue 3
Jul.  2022
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YU Dan, CHI Xiaowei. Mechanism of Psoralea corylifolia Linn on liver injury based on bioinformatics[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(3): 254-258. doi: 10.12206/j.issn.1006-0111.202103054
Citation: YU Dan, CHI Xiaowei. Mechanism of Psoralea corylifolia Linn on liver injury based on bioinformatics[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(3): 254-258. doi: 10.12206/j.issn.1006-0111.202103054

Mechanism of Psoralea corylifolia Linn on liver injury based on bioinformatics

doi: 10.12206/j.issn.1006-0111.202103054
  • Received Date: 2021-03-22
  • Rev Recd Date: 2021-06-04
  • Available Online: 2023-11-06
  • Publish Date: 2022-05-25
  •   Objective  To explore the mechanism of Psoralea corylifolia Linn (PCL) on liver injury by establishing the biological function and pathway network of PCL components, targets and protein interactions based on bioinformatics.   Methods  The components of PCL and potential liver-injury related targets were collected from TCMIP database. The targets of PCL were predicted by the reverse pharmacophore matching method. Cytoscape software was applied for the construction of active components-targets network map. Protein-protein interaction network was constructed by STRING database. Gene ontology functional enrichment analysis and KEGG pathway enrichment analysis were conducted to predict the liver injury mechanism of PCL.   Results  22 components were identified from PCL with the corresponding 31 potential liver injury targets, mainly on serum albumin (ALB), glutathione S-transferase P (GSTP1), transthyretin (TTR) and peroxisome proliferator activated receptor gamma (PPARG) by PPI network analysis. The chemical carcinogenesis, adenosine 5 '- monophosphate activated protein kinase (AMPK) signal, PPAR signal, liver enzyme P450 and its harmful substance metabolism, glutathione metabolism and other signaling pathways were selected by KEGG analysis.   Conclusion  The active components of PCL may target on ALB, GSTP1, TTR and PPARG to regulate AMPK and PPAR signaling pathways, leading to liver injury.
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Mechanism of Psoralea corylifolia Linn on liver injury based on bioinformatics

doi: 10.12206/j.issn.1006-0111.202103054

Abstract:   Objective  To explore the mechanism of Psoralea corylifolia Linn (PCL) on liver injury by establishing the biological function and pathway network of PCL components, targets and protein interactions based on bioinformatics.   Methods  The components of PCL and potential liver-injury related targets were collected from TCMIP database. The targets of PCL were predicted by the reverse pharmacophore matching method. Cytoscape software was applied for the construction of active components-targets network map. Protein-protein interaction network was constructed by STRING database. Gene ontology functional enrichment analysis and KEGG pathway enrichment analysis were conducted to predict the liver injury mechanism of PCL.   Results  22 components were identified from PCL with the corresponding 31 potential liver injury targets, mainly on serum albumin (ALB), glutathione S-transferase P (GSTP1), transthyretin (TTR) and peroxisome proliferator activated receptor gamma (PPARG) by PPI network analysis. The chemical carcinogenesis, adenosine 5 '- monophosphate activated protein kinase (AMPK) signal, PPAR signal, liver enzyme P450 and its harmful substance metabolism, glutathione metabolism and other signaling pathways were selected by KEGG analysis.   Conclusion  The active components of PCL may target on ALB, GSTP1, TTR and PPARG to regulate AMPK and PPAR signaling pathways, leading to liver injury.

YU Dan, CHI Xiaowei. Mechanism of Psoralea corylifolia Linn on liver injury based on bioinformatics[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(3): 254-258. doi: 10.12206/j.issn.1006-0111.202103054
Citation: YU Dan, CHI Xiaowei. Mechanism of Psoralea corylifolia Linn on liver injury based on bioinformatics[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(3): 254-258. doi: 10.12206/j.issn.1006-0111.202103054
  • 补骨脂(Psoralea corylifolia Linn)为豆科植物,以干燥成熟果实入药,性温,味辛、苦,归肾、脾经。有温肾助阳,纳气平喘,温脾止泻等功效,主治骨质疏松症、骨软化症、关节痛、哮喘、白癜风、银屑病、遗尿尿频、肾虚作喘等疾病,在中医临床治疗中被广泛应用于脾肾阳虚的治疗,但长期或者超量使用补骨脂及其制剂可导致肝损伤,临床表现主要为皮肤巩膜黄染、尿黄、乏力和食欲不振,同时伴有丙氨酸转移酶(ALT)、总胆红素(T-Bil)和直接胆红素(D-Bil)等生化指标不同程度升高,肝脏肿大或肝脏弥漫性病变。实验研究也表明,长期或大剂量服用补骨脂可引起大鼠不同程度的肝损伤。然而,补骨脂肝损伤成分和潜在机制至今尚未得到阐明。在本研究中,我们通过生物信息学工具评价补骨脂诱导肝损伤的潜在作用机制,为补骨脂肝损伤的临床和基础研究提供参考依据。

    • TCMIP (http://lsp.nwu.edu.cn/, Version 2.3);Pharm Mapper (http://59.78.96.61/pharmmapper/);Uniprot数据库中UniprotKB检索功能(http://www.uniprot.org/);String数据库(https://string-db.org/, Version 4.5.0);Disgenet(https://www.disgenet.org/);DAVIA基因富集分析网站 (http://david.ncifcrf.gov/summary.jsp);Cytoscape软件(Version 2.7.1);Chembiodraw Ultra软件 (Version14.0)

    • 通过TCMIP数据库获取补骨脂所有化学成分,再结合文献挖掘与整理,筛选出补骨脂的主要成分,并对成分进行预测。

      登陆Pharm Mapper服务器,上传上述补骨脂成分,获得虚拟筛选结果。利用UniProt数据库中UniProtKB搜索功能,输入蛋白名称,限定物种为人,预测补骨脂成分的作用靶点。

      在Disgenet数据库中搜索药物性肝损伤相关的基因,去除重复基因和假阳性基因,与上述Pharm Mapper 服务器中返回的靶点相匹配,得到补骨脂成分诱导肝损伤的潜在作用靶点。

    • 将补骨脂诱导肝损伤的作用靶点导入String数据库,利用Multiple proteins工具,选择“Homo Sapiens”为生物体,获取蛋白相互作用。将补骨脂成分和作用靶点信息以源节点和节点直接导入Cytoscape3.7.1,构建药材-成分-靶标-疾病网络。借助Network Analyzer工具对网络进行拓扑参数分析,并筛选出补骨脂诱导肝损伤的关键靶蛋白。

    • 用DAVID 6.7 (http://david.ncifcrf.gov/summary.jsp) 对补骨脂药物性肝损伤PPI网络中的节点蛋白进行GO注释和KEGG通路分析,并采用R软件对结果进行图形化表示。

    • 从TCMIP数据库中共筛选出的32个相关化合物,其中补骨脂素、补骨脂酚、补骨脂查尔酮、异补骨脂素和甲氧补骨脂素等22个符合Lipinski5规则,而补骨脂素、异补骨脂素和补骨脂酚为补骨脂主要成分。因此,上述22个化合物全部纳入进一步研究,化合物名称及详细信息见表1

      化合物相对分子
      质量
      AlogP氢键供
      体数
      氢键受
      体数
      Pub
      ChemID
      双羟异补骨脂定368.302.0375316096
      补骨脂酚338.402.8255320772
      补骨脂定336.304.7255281806
      异新补骨脂查尔酮298.293.0255318608
      异补骨脂二氢黄酮324.404.124193679
      补骨脂异黄酮醛282.252.52544257227
      槐属香豆雌烷 A334.304.01514630492
      补骨脂呋喃查尔酮340.403.5356476086
      新补骨脂查尔酮298.293.0255320052
      补骨脂二氢黄酮甲醚338.404.41410337211
      补骨脂香豆雌烷B352.303.1265321820
      补骨脂乙素324.405.1345281255
      新补骨脂异黄酮322.404.4245320053
      补骨脂查尔酮324.405.1346450879
      补骨脂色烯素322.404.5245321800
      异补骨脂定336.304.11512304285
      甲氧补骨脂素216.191.9044114
      补骨脂呋喃香豆精186.162.3033083848
      异补骨脂素186.162.00310658
      补骨脂素186.162.3036199
      补骨脂香豆雌烷A353.303.1265321811
      补骨脂定-2',3'-环氧化物352.303.32644257529
    • 将补骨脂22个成分输入Pharm Mapper中,得到所有潜在蛋白质靶点,经UniProt转化为基因ID,并与Disgenet数据库中8类药物性肝损伤挖掘的疾病基因ID根据匹配度由高到低进行排序,去除重复后,匹配共得到31个补骨脂潜在的肝损伤作用靶点,见表2

      序号Uniprot ID基因靶点蛋白靶点
      1P28161GSTM2glutathione S-transferase Mu 2
      2O75469NR1I2nuclear receptor subfamily 1
      group I member 2
      3P02768ALBserum albumin
      4P11712CYP2C9cytochrome P450 2C9
      5P09211GSTP1glutathione S-transferase P
      6P04179SOD2superoxide dismutase [Mn], mitochondrial
      7Q96RI1NR1H4bile acid receptor
      8P05089ARG1arginase-1
      9P00374DHFRdihydrofolate reductase
      10P02774GCvitamin D-binding protein
      11P00390GSRglutathione reductase, mitochondrial
      12P09601HMOX1heme oxygenase 1
      13P05019IGF1insulin-like growth factor I
      14P80188LCN2neutrophil gelatinase-associated lipocalin
      15P17931LGALS3galectin-3
      16P08253MMP272 000 type IV collagenase
      17P00491PNPpurine nucleoside phosphorylase
      18Q08257CRYZquinone oxidoreductase
      19Q07869PPARAperoxisome proliferator-activated receptor alpha
      20Q00796SORDsorbitol dehydrogenase
      21P49888SULT1E1sulfotransferase 1E1
      22O00204SULT2B1sulfotransferase 2B1
      23P36897TGFBR1TGF-beta receptor type-1
      24P02766TTRtransthyretin
      25Q14994NR1I3nuclear receptor subfamily 1
      group I member 3
      26Q13133NR1H3oxysterols receptor LXR-alpha
      27P37231PPARGperoxisome proliferator-activated receptor gamma
      28O60760HPGDShematopoietic prostaglandin D synthase
      29P04035HMGCR3-hydroxy-3-methylglutaryl-coenzyme A reductase
      30P06702S100A9protein S100-A9
      31P09488GSTM1glutathione S-transferase Mu 1
    • 采用Cytoscape软件的Merge功能构建补骨脂成分-靶点-信号通路-肝损伤网络模型,如图1所示(绿色椭圆代表靶点,黄色椭圆代表成分,粉红色菱形代表疾病,紫色椭圆表示补骨脂),节点代表活性或靶点,边代表成分、作用靶点和疾病间的相互关联,其中共有60个节点,327个边。节点的大小与度的大小呈正比关系,节点越大表示该节点的度越大。依据拓扑分析,13个成分的度值大于10,分别为补骨脂素、补骨脂定、补骨脂酚、异新补骨脂查尔酮、异补骨脂黄酮、补骨脂异黄酮醛、异补骨脂定、双羟异补骨脂定、新补骨脂查尔酮、补骨脂二氢黄酮甲醚、补骨脂乙素、补骨脂查尔酮、新补骨脂异黄酮;具有较高度值的靶点蛋白有血清白蛋白(ALB)、谷胱甘肽S-转移酶P(GSTP1)、运甲状腺素蛋白(TTR)和过氧化物酶体增殖物激活受体γ(PPARG)。

      采用DAVID在线富集平台进行生物学功能分析,获得103个GO注释(黄色表示生物学过程、绿色表示细胞组成、蓝色表示分子功能)。GO分析表明,靶蛋白大多富集于RNA聚合酶Ⅱ启动子启动转录、类固醇激素介导的信号通路、信号转录、DNA模板转录正调控、RNA聚合酶Ⅱ启动子转录的正调控、氧化还原反应、固有免疫应答、细胞对脂多糖(LPS)的反应、细胞氧化解毒等生物学过程,见图2。此外,KEGG富集分析表明,5条通路(P<0.05)受补骨脂的影响,包括化学物致癌、AMPK信号通路、PPAR信号通路、肝药酶P450(CYP)有害物代谢和谷胱甘肽代谢通路等,结果见图3

    • 在临床上,补骨脂被广泛应用于治疗骨质疏松、银屑病等多种疾病,其所导致的药物性肝损伤已引起人们关注,但对于其肝损伤成分及分子机制的研究尚不深入。为了探究补骨脂所致肝损伤的作用机制,本研究从补骨脂的成分出发,借助生物信息学技术构建了补骨脂药材-成分-靶点-信号通路-肝损伤网络,分析补骨脂中成分与靶点的作用关系,为其导致肝损伤作用机制研究提供了参考。

      通过数据库检索,我们发现补骨脂主要含有补骨脂素、异补骨脂素、补骨脂酚、补骨脂定、新补骨脂异黄酮和补骨脂二氢黄酮甲醚等22个成分[1-5] ,其中补骨脂素、异补骨脂素和补骨脂酚等为主要成分[5-6]。补骨脂中个别成分已被发现具有导致肝损伤的作用,如研究发现补骨脂素与补骨脂酚可通过降低BSEP蛋白的表达,升高NTCP和CYP7A1蛋白的表达,使得进入肝细胞的胆汁酸增多,进而肝细胞中胆汁酸的含量异常升高,最终导致胆汁淤积性肝损伤[7-9];此外,补骨脂素通过上调细胞周期蛋白E1和p27蛋白水平并下调细胞周期蛋白D1的水平,使肝细胞周期阻滞进而导致肝再生和代偿能力下降而诱发小鼠轻度肝损伤和L02肝细胞损伤;而异补骨脂素通过抑制MRP2和MRP3导致HepG2的细胞肝损伤[10-11]。补骨脂酚在体外的生物利用度远低于补骨脂素和异补骨脂素,但在体内补骨脂酚的CYP450代谢作用较强[12],补骨脂酚在体内复杂环境中的吸收和代谢可能是导致其在体内外肝损伤差异的主要原因。

      通过GO分析整合预测,得到补骨脂导致肝损伤生物功能包括氧化还原反应、固有免疫应答、细胞对LPS的反应、细胞氧化解毒等。目前已知,氧化还原反应与免疫应答在药物性肝损伤发病机制中起关键作用。有研究发现,可溶性T细胞免疫球蛋白结构域和含黏蛋白结构域分子-3(sTIM-3)通过促进自噬和调节单核/巨噬细胞功能,减轻炎症反应和肝损伤[13]。氧化应激介导的脂质过氧化可导致胶原合成增加,氧化还原稳态失衡会干扰线粒体功能障碍进而导致药物引起的肝损伤。ROS可影响过氧化物酶体增殖物激活的受体调节脂肪酸氧化以及丝裂原活化蛋白激酶(MAPK)和相关的应激敏感激酶活力而调节肝细胞凋亡[14]。LPS参与了肝损伤中炎症或免疫破坏,可通过激活TLR-2和TLR-4途径促进炎症活动,并且LPS诱导的短暂缺血破坏了呼吸链复合物的活性,增强了ROS,从而进一步增强肠道高通透性,并能显著诱导肝损伤[15-16]

      KEGG富集分析结果表明,补骨脂所致肝损伤可能涉及AMPK、PPAR、CYP等多条信号通路。AMPK是NF-κB的上游蛋白,被认为是维持ADP/AMP/ATP水平的关键细胞代谢传感器,AMPK信号通路的激活可抑制非典型NF-κB途径,从而保护肝脏免受损伤[17-18]。PPAR信号通路在肝损伤和肝纤维化中起着关键作用,在活化的HSCs中,PPAR-γ活性降低,从而导致HSCs从维甲酸储存状态向产生ECM的肌成纤维细胞转化,可促进CCl4诱导的肝损伤和纤维化,然而,抑制PPARG可阻止Fsp27α mRNAs的表达,进而减少乙醇诱导小鼠线粒体活性氧生成所导致的肝损伤[19-20]。肝脏CYP酶在药物代谢和清除中发挥关键作用,例如CYP2E1可增加活性氧的数量,进而导致氧化应激肝损伤[21-22]。基于上述结果和理论基础,有必要开展补骨脂AMPK、PPAR等信号通路的作用研究,从而为其肝损伤机制提供理论依据。

      综上所述,本研究通过生物信息学技术构建补骨脂药材-成分-靶点-信号通路-肝损伤网络对补骨脂成分所致肝损伤作用机制进行分析预测,研究结果充分表明了补骨脂多成分-多靶点-多通路的肝损伤作用特点,为进一步开展补骨脂致肝损伤作用机制的研究提供了新思路。

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