Abstract
目的
基于网络药理学和分子对接技术探讨连花清瘟胶囊提高COVID-19临床治愈率的作用靶点、信号通路和生物学功能。
方法
利用TCMSP、SwissTarget Prediction、CooLGeN、GeneCards、DAVID等数据库，以连花清瘟胶囊中的连翘、金银花、炙麻黄等13味药来检索活性成分及其靶点蛋白，筛选与COVID-19及其临床表现(发热、咳嗽、乏力)共有的靶点蛋白及其信号通路与生物学功能，采用Gephi软件构建连花清瘟胶囊药味-活性成分-作用靶点-生物学功能网络图。
结果
连花清瘟胶囊中的连翘、金银花、炙麻黄等13味药中的MOL000522、MOL003283、MOL003365、MOL003006、MOL003014等160个活性成分通过MAPK1、IL6、HSP90AA1、TNF、CCL2等57个靶点蛋白、NOD-like receptor signaling pathway、Toll-like receptor signaling pathway等35条信号通路发挥治疗COVID-19或干预COVID-19病变过程的作用，影响COVID-19的临床表现。分子对接
结果
表明，MOL000522(来自连翘)、MOL001495(来自金银花)、MOL001494(来自金银花、炙麻黄)等83个化学成分与MAPK1、IL6、HSP90AA1等12个靶点蛋白的Total Score分值≥5.0，可形成稳定构象，具有较好结合活性。其中，Total Score值≥9.0的有：MOL000522(来自连翘)-MAPK1、MOL004989(来自甘草)-MAPK1、MOL003330(来自连翘)-MAPK1、MOL001495(来自金银花)-NLRP3、MOL001494(来自金银花、炙麻黄)-NLRP3、MOL004908(来自甘草、苦杏仁)-HSP90AA1、MOL004863(来自甘草)-HSP90AA1、MOL001749(来自板蓝根)-TLR9、MOL004806(来自甘草)-HSP90AA1、MOL001495(来自金银花)-AKT1。结论连花清瘟胶囊以多药味、多靶点、多信号通道和多生物学功能发挥提高COVID-19临床治愈率作用。
Keywords: 网络药理学, 分子对接, 连花清瘟胶囊, COVID-19, 临床治愈率
Abstract
Objective
To explore the potential targets, signal pathways and biological functions that mediate the effect of Lianhua Qingwen capsule in improving clinical cure rate of COVID-19 in light of network pharmacology and molecular docking technology.
Methods
TCMSP, Target, Prediction, CooLGeN, GeneCards, DAVID and other databases were searched for the active components and their target proteins from 13 herbs including Forsythia, Honeysuckle and roasted Ephedra used in Lianhua Qingwen capsule. The common target proteins, signal pathways and biological functions shared by these components and the clinical manifestations of COVID-19 (fever, cough, and fatigue) were identified to construct the network consisting of the component drugs in Lianhua Qingwen capsule, the active ingredients of, their targets of action, and the biological functions involved using Gephi software.
Results
A total 160 active components including MOL000522, and MOL003283, MOL003365, MOL003006, MOL003014 in 13 component drugs in Lianhua Qingwen capsule produced therapeutic effects against COVID-19 through 57 target proteins including MAPK1, IL6, HSP90AA1, TNF, and CCL2, involving 35 signaling pathways including NOD-like receptor signaling pathway and Toll-like receptor signaling pathway. The results of molecular docking showed that 83 chemical components had total scores no less than 5.0 for docking with 12 target proteins (including MAPK1, IL6, and HSP90AA1) with high binding activities to form stable conformations. The binding of MOL000522, MOL004989, and MOL003330 with MAPK1; MOL001495 and MOL001494 with NLRP3; MOL004908, MOL004863 and MOL004806 with HSP90AA1; MOL001749 with TLR9; and MOL001495 with AKT1 all had total scores exceeding 9.0.
Conclusion
Lianhua Qingwen capsule contains multiple effective ingredients to improve clinical cure rate of COVID-19, and its therapeutic effect is mediated by multiple protein targets, signal pathways and biological functions.
Keywords: network pharmacology, molecular docking, Lianhua Qingwen capsule, coronavirus disease 2019, clinical cure rate
中华人民共和国国家卫生健康委办公厅和中华人民共和国国家中医药管理局办公室发布的《新型冠状病毒肺炎诊疗方案》(试行第七版)(国卫办医函〔2020〕184号)[1]明确新型冠状肺炎(COVID-19)临床主要表现为发热、干咳和乏力。在中医治疗中，明确COVID-19属于中医“疫”病范畴，病因为感受“疫戾”之气，各地可根据病情、当地气候特点以及不同体质情况进行辩证论治，并推荐连花清瘟胶囊用于医学观察期的治疗。
Hu等[2]的最新研究论文阐述了连花清瘟胶囊用于临床治疗COVID-19的疗效，即经过连花清瘟治疗组治疗14 d(4粒/次，3次/d)后，连花清瘟胶囊治疗组的恢复率达91.5%，明显高于对照组(82.4%)，且连花清瘟胶囊治疗组对于发热、乏力、咳嗽等症状的治愈时间中位数明显缩短。该文认为，从安全性和有效性角度来看，可以考虑使用连花清瘟胶囊改善COVID-19的临床症状(发热、干咳和乏力)。
以系统生物学理论为基础的网络药理学，通过对生物系统的网络分析来选取特定信号节点，构建活性成分-蛋白靶点-信号通路之间的复杂网络来探讨药物的作用机制。现有网络药理学研究多关注药物作用于靶点蛋白用于疾病的治疗[3-4]，对于疾病与其临床症状共有靶点蛋白间的网络药理学研究少有报道。COVID-19的临床主要表现为发热、干咳和乏力。涉及发热、干咳和乏力等临床症状的靶点蛋白较多，就COVID-19而言，这些临床症状似应与COVID-19间存在共有的靶点蛋白。连花清瘟胶囊改善COVID-19的临床症状提高临床治愈率似应是作用于COVID-19及其临床症状(发热、干咳和乏力)共有靶点蛋白的结果。为此，为更全面地研究连花清瘟胶囊提高COVID-19临床治愈率的机制和物质基础，本文以“fever ”“、coughing ”“、fatigue ”等临床表现及“2019-nCoV”、“COVID-19”为疾病关键词，筛选出“fever ”、“coughing ”、“fatigue ”、“2019-nCoV”、“COVID-19”中的共同靶点蛋白。同时选取连花清瘟胶囊中连翘、金银花等13味中药，对涉及到的活性成分进行筛选，通过网络药理学方法研究连花清瘟胶囊提高COVID-19临床治愈率的潜在作用靶点和信号通路及生物学过程，并采用分子对接技术研究核心活性成分与主要通路中的靶点蛋白的对接，探讨连花清瘟胶囊提高COVID-19临床治愈率的机制和物质基础。
1. 资料和方法
1.1. 数据资料来源
1.1.1. 药材来源
连花清瘟胶囊(国药准字Z20040063)由石家庄以岭药业股份有限公司生产。成分为：连翘、金银花、炙麻黄、炒苦杏仁、石膏、板蓝根、绵马贯众、鱼腥草、广藿香、大黄、红景天、薄荷脑、甘草。功能主治为清瘟解毒、宣肺泄热。用于治疗流行性感冒属热毒袭肺证，症见：发热或高热，恶寒，肌肉酸痛，鼻塞流涕，咳嗽，头痛，咽干咽痛，舌偏红，苔黄或黄腻等。
1.1.2. 活性成分来源
通过中药系统药理学数据库与分析平台(TCMSP，Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform，<a href="https://tcmspw.com/tcmsp.php" target="_blank">https://tcmspw.com/tcmsp.php</a>)及有关文献[<xref ref-type="bibr" rid="b5">5</xref>]，分别以连花清瘟胶囊中的13味药来检索活性成分，其中炙麻黄、炒苦杏仁、绵马贯众分别按麻黄、苦杏仁、贯众进行检索。选择符合口服生物利用度(oral bioavailability, OB)≥ 30%、类药性(drug like, DL)≥0.18并合并各药味中相同的活性成分，绘制结构式并以MDL SDfile(*.sdf)格式保存(检索日期：2020年10月28日)。将这些活性成分的MDL SDfile(*.sdf)格式结构式导入SwissTargetPrediction数据库(<a href="http://www.swisstargetprediction.ch/" target="_blank">http://www.swisstargetprediction.ch/</a>)，检索符合Probability≥0.1的活性成分靶点蛋白(检索日期：2020年11月2日)。
1.1.3. 候选靶标来源
利用CooLGeN (<a href="http://ci.smu.edu.cn/CooLGeN/Home.php" target="_blank">http://ci.smu.edu.cn/CooLGeN/Home.php</a>)、GeneCards(<a href="https://www.genecards.org/" target="_blank">https://www.genecards.org/</a>)、TTD (<a href="http://bidd.nus.edu.sg/group/cjttd/" target="_blank">http://bidd.nus.edu.sg/group/cjttd/</a>)数据库，以“fever ”、“coughing ”、“fatigue ”、“2019-nCoV”、“COVID-19”为关键词，收集临床表现及疾病靶点蛋白(选择标示符为All human genes)，筛选临床表现与疾病共有靶点蛋白，再进一步选择与药材中活性成分相匹配的靶点蛋白。
1.2. 靶点通路及生物学功能注释分析
将靶点蛋白复制至DAVID数据库(<a href="https://david.ncifcrf.gov/tools.jsp" target="_blank">https://david.ncifcrf.gov/tools.jsp</a>)的列表中，选择标示符为official_ gene_symbol，物种注释为Homo sapiens，进行靶点蛋白的GOTERM_BP_DIRECT、GOTERM_CC_DIRECT、GOTERM_MF_DIRECT富集分析和Kyoto Encyclopedia of Genes and Genomes(KEGG)_PATHWAY通路注释分析。
1.3. 活性成分-蛋白靶点-生物学功能网络分析和构建
根据预测结果，使用Gephi0.9.2软件将筛选出来的活性成分、蛋白靶点和生物学功能构建活性成分-蛋白靶点-生物学功能网络。
1.4. 分子对接分析
本文以SYBYL-X 2.1.1对接软件对靶点蛋白和化学成分进行分子对接分析，获得Total Score值。
2. 结果
2.1. 连花清瘟胶囊各药味中活性成分
通过TCMSP及有关文献[5]共检索到连花清瘟胶囊各药味中符合OB≥30%、DL≥0.18的活性成分(合并各药味中相同的活性成分)210个。
2.2. 靶点的预测
将“2.1”中符合OB≥30%、DL≥0.18的活性成分以MDL SDfile(*.sdf)格式导入SwissTargetPrediction数据库，获得蛋白靶点信息24215条，涉及1320个化学靶点蛋白。筛选符合Probability≥0.1的活性成分靶点蛋白，获得蛋白靶点信息12 274条，涉及1159个化学靶点蛋白。
利用CooLGeN、GeneCards、TTD数据库，以“2019-nCoV”、“COVID-19”为关键词，共收集疾病靶点蛋白(All human genes)261个(检索日期：2020年10月28日)。以“fever ”、“coughing ”、“fatigue ”为关键词，分别收集疾病临床表现靶点蛋白7237、4157、7127个。“fever ”“、coughing ”“、fatigue ”与COVID-19的共有靶点蛋白分别为154、176、106个。筛选各药材中与“fever ”“、coughing ”“、fatigue ”与COVID-19的共有靶点蛋白相同的靶点蛋白及对应的化学成分。并进一步确定药材、临床表现及COVID-19中共同的靶点蛋白共计57个(图 1)。
1.
活性成分与COVID-19及其临床表现(发热、咳嗽、乏力)的靶点蛋白
Target proteins of active ingredients in Lianhua Qingwen capsule, COVID-19 and its clinical manifestations (fever, cough, and fatigue).
活性成分与COVID-19及其临床表现(发热、咳嗽、乏力)的共有靶点蛋白共计57个：ABL1、ACE、ACE2、AGTR1、AKT1、ALK、AR、BTK、CALCA、CASP1、CASP3、CCL2、CCR5、CTSL、CXCL8、DHODH、DPP4、EGFR、FLT3、G6PD、HPSE、HSP90AA1、HSPA5、IDH1、IL2、IL6、JAK1、JAK2、LNPEP、MAPK1、MCL1、MGMT、MPO、MTOR、NFKB1、NLRP3、NR3C1、P2RX7、PARP1、PIK3CG、PLAT、PLAUR、PLG、PPARA、REN、ROS1、TK1、TLR4、TLR7、TLR9、TNF、TRPV1、TRPV4、TTR、VCP、VEGFA、XPO1，涉及11味药、160个活性成分(注：本文活性成分均以Mol ID表示，具体成分名称可检索TCMSP数据库)。药材、化学成分及靶点蛋白信息(以MAPK1、NLRP3、HSP90AA1、TLR9、AKT为代表)：
AKT1靶点蛋白：MOL000006(金银花、连翘、炙麻黄)，MOL000098(甘草、广藿香、金银花、连翘、鱼腥草、炙麻黄)，MOL000239、MOL000354、MOL004828、MOL004863、MOL004866、MOL004891、MOL004904、MOL004949、MOL004961(甘草)，MOL000422(甘草、金银花、连翘、绵马贯众、鱼腥草、炙麻黄)，MOL001495、MOL003044、MOL003117(金银花)，MOL001735(板蓝根)，MOL002235(大黄)，MOL002823、MOL002881、MOL005842(炙麻黄);
HSP90AA1靶点蛋白：MOL000500、MOL004806、MOL004838、MOL004849、MOL004855、MOL004856、MOL004857、MOL004863、MOL004864、MOL004883、MOL004884、MOL004904、MOL004905、MOL004910、MOL004911、MOL004935、MOL004945、MOL004949、MOL004959、MOL004980、MOL004988、MOL004989、MOL005001、MOL005008、(甘草)，MOL001040(绵马贯众)，MOL002311、MOL004908(甘草、苦杏仁)，MOL003283、MOL003370(连翘)，MOL005016(甘草、广藿香);
MAPK1靶点蛋白：MOL000522、MOL003283、MOL003330、MOL003370(连翘)，MOL001728、MOL001774、MOL001803(板蓝根)，MOL004806、MOL004808、MOL004838、MOL004891、MOL004905、MOL004959、MOL004988、MOL004989 (甘草)，MOL004908(甘草、苦杏仁)，MOL005911(广藿香);
NLRP3靶点蛋白：MOL001495(金银花)，MOL001494(金银花、炙麻黄);
TLR9靶点蛋白：MOL000392、MOL000417、MOL002844、MOL004815、MOL004833、MOL004879、MOL004885、MOL004891、MOL004907、MOL004957、MOL004990、MOL005016 (甘草)，MOL001728、MOL001749(板蓝根)，MOL004908(甘草、苦杏仁)。
2.3. 靶点信号通路与Gene Ontology(GO)分析
将连花清瘟胶囊中各药味活性成分与COVID-19及其临床表现(发热、咳嗽、乏力)共有的57个靶点蛋白导入DAVID数据库，进行靶点蛋白的GOTERM_BP_DIRECT、GOTERM_CC_DIRECT、GOTERM_MF_DIRECT富集分析和KEGG_PATHWAY通路注释分析。信号通路主要涉及NOD-like receptor signaling pathway、Toll-like receptor signaling pathway、Renin-angiotensin system等35条信号通路。其中Benjamini correction < 0.01的信号通路共有10条(图 2)。
2.
连花清瘟胶囊中活性成分潜在靶点的KEGG信号通路富集分析
Enrichment analysis of KEGG signal pathway of potential targets of the active components in Lianhua Qingwen capsule.
连花清瘟胶囊中的160个活性成分通过MAPK1，IL6，HSP90AA1，TNF，CCL2，NFKB1，CASP1，NLRP3等靶点蛋白参与COVID-19及其临床表现(发热、咳嗽、乏力)的生物过程、细胞组成和分子功能。生物过程主要与regulation of apoptosis、regulation of programmed cell death等有关，细胞组成主要与cell surface、extracellular space等有关，分子功能主要与protein tyrosine kinase activity、ATP binding等有关(图 3、4)。
3.
连花清瘟胶囊中活性成分潜在靶点与COVID-19及其临床表现(发热、咳嗽、乏力)相关的GOTERM_BP_FAT富集分析
GOTERM_BP_FAT enrichment analysis of the potential targets of active components in Lianhua Qingwen capsule and the biological functions related to COVID-19 and its clinical manifestations (fever, cough, and fatigue) (Benjamini correction ≤0.0001).
4.
连花清瘟胶囊中活性成分潜在靶点与COVID-19及其临床表现(发热、咳嗽、乏力)相关的富集分析
GOTERM_CC_FAT (A) and GOTERM_MF_FAT (B) enrichment analysis of the potential target of active components in Lianhua Qingwen capsule and the biological function related to COVID-19 and its clinical manifestations (fever, cough, fatigue) (Benjamini correction ≤0.01).
2.4. 活性成分-靶点蛋白-信号通路网络的构建
使用Gephi 0.9.2软件构建化学成分-靶点蛋白-信号通路网络。连花清瘟胶囊中的160个活性成分通过57个靶点蛋白、35条信号通路发挥治疗COVID-19或干预COVID-19病变过程的作用，网络关系复杂、蛋白靶点与信号通路多(图 5)。
5.
连花清瘟胶囊中药味-部分活性成分-靶点蛋白-作用通路网络图
Network diagram of medicinal ingredients (
)-active component (
)-target protein (
)
-action pathway (
) in Lianhua
Qingwen capsule.
2.5. 部分活性成分与靶点蛋白分子对接结果分析
鉴于NOD-like receptor signaling pathway、Tolllike receptor signaling pathway的Benjamini correction较小，为5.8E-5、1.5E-6。为此，以NOD-like receptor signaling pathway、Toll-like receptor signaling pathway涉及的靶点蛋白MAPK1(PDB ID:6SLG)、IL6(PDB ID:1ALU)、HSP90AA1(PDB ID:3O0I)、TNF(PDB ID: 6M95)、CCL2(PDB ID:5J5Y)、NFKB1(PDB ID: 1LE9)、CASP1(PDB ID:1RWV)、NLRP3(PDB ID: 3VWE)、PIK3CG(PDB ID: 2A4Z)、AKT1(PDB ID: 4GAH)、TLR4(PDB ID:3FXI)、TLR7(PDB ID:5T1S)、TLR9(PDB ID:5WYX)为例，对靶点蛋白和化学成分进行分子对接结果分析，验证连花清瘟胶囊化学成分与靶点蛋白的作用。配体与受体间低能量的稳定构象预示着二者间有较大的作用可能性。一般以结合能≤−5 kJ/mol或Total Score值≥5.0作为筛选标准。本文以SYBYL-X 2.1.1对接软件进行分子对接，获得Total Score值(表 1)。分子对接结果表明，MOL000522、MOL001495、MOL001494等83个化学成分与MAPK1、IL6、HSP90AA1等12个靶点蛋白的Total Score值≥5.0。Total Score值≥9.0的代表性核心活性成分与靶点蛋白分子对接模式(图 6)。
1.
NOD-like and Toll-like receptor signaling pathway各化学成分与靶点蛋白作用的Total Score值
Total score values of each chemical component interacting with target protein in NOD like and total like receptor signaling pathway
| No. | Medicinal materials | Chemical composition | Target protein | Total Score |
| MOL001728 | 4.9750 | |||
| Isatidis radix | MOL001774 | 4.7116 | ||
| MOL001803 | 8.7373 | |||
| MOL004806 | 8.2133 | |||
| MOL004808 | 6.5849 | |||
| MOL004838 | 5.7862 | |||
| Glycyrrhizae radix et rhizoma | MOL004891 | 6.0748 | ||
| MOL004905 | 4.2735 | |||
| 1 | MOL004959 | MAPK1 | 7.1327 | |
| MOL004988 | 5.8852 | |||
| MOL004989 | 9.2094 | |||
| Glycyrrhizae radix et rhizoma、Armeniacae semen amarum | MOL004908 | 6.7727 | ||
| Pogostemonis herba | MOL005911 | 5.7615 | ||
| MOL000522 | 10.231 | |||
| Forsythiae fructus | MOL003283 | 5.6543 | ||
| MOL003330 | 9.0596 | |||
| MOL003370 | 5.8832 | |||
| Isatidis radix | MOL001774 | 2.7712 | ||
| 2 | Armeniacae semen amarum | MOL002211 | IL6 | 7.2272 |
| Forsythiae fructus | MOL003365 | 5.8637 | ||
| MOL000500 | 7.0339 | |||
| MOL004806 | 9.0092 | |||
| MOL004838 | 5.4535 | |||
| MOL004849 | 8.8892 | |||
| MOL004855 | 8.9802 | |||
| MOL004856 | 6.7809 | |||
| MOL004857 | 7.8582 | |||
| MOL004863 | 9.4006 | |||
| MOL004864 | 8.6671 | |||
| MOL004883 | 8.3102 | |||
| MOL004884 | 7.3729 | |||
| MOL004904 | 5.8715 | |||
| Glycyrrhizae radix et rhizoma | MOL004905 | 1.7412 | ||
| MOL004910 | 7.6057 | |||
| MOL004911 | 7.1548 | |||
| 3 | MOL004935 | HSP90AA1 | 8.6197 | |
| MOL004945 | 7.7389 | |||
| MOL004949 | 7.6895 | |||
| MOL004959 | 7.5331 | |||
| MOL004980 | 7.0161 | |||
| MOL004988 | 6.5771 | |||
| MOL004989 | 7.9668 | |||
| MOL005001 | 8.1583 | |||
| MOL005016 | 7.0515 | |||
| MOL005008 | 5.7821 | |||
| Glycyrrhizae radix et rhizoma、Armeniacae semen amarum | MOL004908 | 9.5050 | ||
| MOL002311 | 6.2047 | |||
| Pogostemonis herba | MOL005916 | 6.7172 | ||
| Forsythiae fructus | MOL003283 | 6.3775 | ||
| MOL003370 | 6.1435 | |||
| Ryopteridis crassirhizomatis rhizoma | MOL001040 | 6.2675 | ||
| Isatidis radix | MOL001790 | 8.6973 | ||
| MOL004848 | 7.6923 | |||
| MOL004905 | -13.0491 | |||
| 4 | Glycyrrhizae radix et rhizoma | MOL004935 | TNF | 8.3922 |
| MOL004959 | 5.2719 | |||
| MOL004989 | 8.7198 | |||
| MOL005013 | -12.0236 | |||
| Forsythiae fructus | MOL003365 | 5.5913 | ||
| 5 | Forsythiae fructus | MOL000791 | CCL2 | 6.5440 |
| 6 | Glycyrrhizae radix et rhizoma 、Armeniacae semen amarum | MOL002311 | NFKB1 | 3.3351 |
| Glycyrrhizae radix et rhizoma | MOL005000 | 3.8014 | ||
| MOL002844 | 5.1758 | |||
| Glycyrrhizae radix et rhizoma | MOL005013 | 2.5680 | ||
| 7 | Lonicerae japonicae flos | MOL003006 MOL003014 | CASP1 | 5.2431 5.6239 |
| Forsythiae fructus | MOL003330 | 3.3774 | ||
| Lonicerae japonicae flos、Ephedrae | MOL001494 | NLRP3 | 9.8683 | |
| 8 | herba praeparata cum melle | |||
| Lonicerae japonicae flos | MOL001495 | 10.1359 | ||
| MOL001803 | 6.2639 | |||
| Isatidis radix | MOL001814 | 6.6419 | ||
| Rhei radix et rhizome | MOL002235 | 7.3826 | ||
| Glycyrrhizae radix et rhizoma、Pogostemonis herba、japonicae flos、Forsythiae fructus、Houttuyniae herba、Ephedrae herba | MOL000098 | 6.0542 | ||
| praeparata cum melle | MOL000239 | 6.1381 | ||
| MOL000354 | 7.2169 | |||
| MOL000500 | 5.8799 | |||
| MOL002565 | 3.9326 | |||
| MOL002844 | 4.9174 | |||
| MOL004810 | 6.4536 | |||
| MOL004866 | 8.1841 | |||
| Glycyrrhizae radix et rhizoma | MOL004891 | 3.8563 | ||
| MOL004904 | 7.6479 | |||
| 9 | MOL004905 | PIK3CG | -4.5592 | |
| MOL004941 | 4.8181 | |||
| MOL004961 | 6.2743 | |||
| MOL004966 | 5.1168 | |||
| MOL004974 | 6.9209 | |||
| MOL004989 | 8.4427 | |||
| MOL005007 | 6.3041 | |||
| Pogostemonis herba | MOL005911 | 7.0538 | ||
| Lonicerae japonicae flos | MOL003095 | 7.6467 | ||
| Armeniacae semen amarum | MOL012922 | 6.5879 | ||
| MOL000173 | 5.3401 | |||
| MOL003290 | 7.2718 | |||
| Forsythiae fructus | MOL003308 | 7.3495 | ||
| MOL003330 | 7.7492 | |||
| Ryopteridis crassirhizomatis rhizoma | MOL002605 | 0.8139 | ||
| MOL002823 | 6.0577 | |||
| Ephedrae herba praeparata cum melle | MOL005842 | 5.6310 | ||
| Isatidis radix | MOL001735 | 5.9142 | ||
| Rhei radix et rhizome | MOL002235 | 6.8423 | ||
| Glycyrrhizae radix et rhizoma、Pogostemonis herba、 japonicae flos、Forsythiae fructus、Houttuyniae herba、Ephedrae herba | MOL000098 | 5.8223 | ||
| 10 | praeparata cum melle | MOL000422 | AKT1 | 5.4487 |
| Glycyrrhizae radix et rhizoma、Lonicerae japonicae flos、Forsythiae fructus、ryopteridis crassirhizomatis rhizoma、Houttuyniae herba、Ephedrae herba praeparata cum melle | ||||
| MOL000239 | 5.8410 | |||
| MOL000354 | 6.6103 | |||
| MOL004828 | 5.9371 | |||
| MOL004863 | 8.0458 | |||
| Glycyrrhizae radix et rhizoma | MOL004866 | 7.7877 | ||
| MOL004891 | 4.2970 | |||
| MOL004904 | 4.6532 | |||
| MOL004949 | 7.8296 | |||
| MOL004961 | 6.2984 | |||
|
Lonicerae japonicae flos、Forsythiae fructus、Ephedrae herba praeparata
cum melle |
MOL000006 | AKT1 | 6.0612 | |
| MOL001495 | 9.7707 | |||
| Lonicerae japonicae flos | MOL003044 | 6.1451 | ||
| MOL003117 | 5.1368 | |||
| MOL002823 | 5.2484 | |||
| Ephedrae herba praeparata cum melle | MOL002881 | 7.1609 | ||
| MOL005842 | 6.3957 | |||
| 11 | Ryopteridis crassirhizomatis rhizoma | MOL002605 | TLR4 | 4.8851 |
| 12 | Glycyrrhizae radix et rhizoma | MOL004988 | TLR7 | 7.2271 |
| Isatidis radix | MOL001728 | 6.1353 | ||
| MOL001749 | 10.2153 | |||
| Glycyrrhizae radix et rhizoma、Armeniacae semen amarum | MOL004908 | 4.3256 | ||
| MOL000392 | 6.7941 | |||
| MOL000417 | 7.4214 | |||
| MOL002844 | 6.8041 | |||
| MOL004815 | 8.9438 | |||
| 13 | MOL004833 | TLR9 | 5.7856 | |
| Glycyrrhizae radix et rhizoma | MOL004879 | 8.0629 | ||
| MOL004885 | 5.5260 | |||
| MOL004891 | 5.4322 | |||
| MOL004907 | 6.7363 | |||
| MOL004957 | 8.3794 | |||
| MOL004990 | 8.3002 | |||
| MOL005016 | 7.7733 |
6.
代表性核心活性成分与靶点蛋白分子对接模式
Molecular docking mode of representative core active components and target proteins (Total Score ≥9.0).
3. 讨论
通过网络药理学研究连花清瘟胶囊与COVID-19的临床表现(“fever ”“、coughing ”“、fatigue ”)关系结果可知，在涉及到的35条信号通路中，Benjamini correction最小的2个信号通路为NOD-like receptor signaling pathway、Toll-like receptor signaling pathway，Benjamini correction分别为5.8E-5、1.5E-6。NOD受体可识别进入细胞内的病原微生物及其产物，快速启动信号传递，激活天然免疫效应机制; Toll受体可识别细胞外病原体，并将信号传递至细胞内激发机体天然免疫系统。二种模式识别受体可独立进行自我辨识，同时又相互联系协调，在启动天然免疫反应中共同发挥重要作用，可激活免疫系统，破坏免疫耐受状态，纠正自身免疫性疾病[6-7]。连花清瘟胶囊提高COVID-19的临床治愈率可能主要与提高患者自身免疫能力、抑制炎症反应有关。为此，以NOD-like receptor signaling pathway、Toll-like receptor signaling pathway涉及的靶点蛋白及其化学成分进行了分子对接结果分析，验证连花清瘟胶囊化学成分与靶点蛋白的作用。MOL000522、MOL001495、MOL001494等83个化学成分与MAPK1、IL6、HSP90AA1等12个靶点蛋白的Total Score值≥5.0，Total Score值≥9.0涉及到的主要靶点蛋白有MAPK1、NLRP3、HSP90AA1、TLR9、AKT1。
MAPK可调节细胞增殖、分化、死亡、应激反应和凋亡等关键细胞活动[8]，在细胞生命中起着重要作用[9]。MAPK1是多种生化信号的整合点，参与细胞增殖分化、转录调控和细胞的有丝分裂自噬等多种生物学过程，在治疗各种炎症性疾病中发挥重要作用，也是急性肺损伤(ALI)炎症反应和LPS诱导的细胞损伤的重要调节因子[10-14]。由本文网络药理学和分子对接结果可知，来自连翘、甘草、板蓝根、苦杏仁、广藿香的14个化学成分与MAPK1靶点蛋白的分子对接结果Total Score值>5.0，其中MOL000522(连翘)、MOL004989(甘草)、MOL003330(连翘)与MAPK1靶点蛋白的分子对接结果Total Score值>9.0。连花清瘟胶囊作用于MAPK1靶点蛋白，可能通过细胞凋亡调控、细胞程序性死亡的调控等生物学途径，提高患者自身免疫能力、抑制炎症反应发挥提高COVID-19临床治愈率作用。
NLRP3炎症小体作为机体固有免疫及应激系统的重要防御成分，不但可以识别细菌、病毒等病原体引发机体发生固有免疫应答，还负责炎症反应的激活[15-17]，参与了多种疾病发生和进展[18]，在先天免疫中起着重要作用[19]。近年来，愈来愈多的研究表明NLRP3炎性体在肺部感染性疾病中参与并发挥多种作用[20]。如在各种类型ALI中介导了炎症介质的生成和炎症细胞的浸润，增加肺泡上皮细胞的通透性，促进肺水肿的形成[21]。抑制或缺失NLRP3可特异性减轻辐射和脂多糖治疗引起的小鼠肺部炎症[22]。中药成分也可通过抑制NLRP3炎症小体活化，继而减轻脂多糖诱导的ALI[23-25]。由本文网络药理学和分子对接结果可知，MOL001494(金银花、炙麻黄)、MOL001495(金银花)与NLRP3靶点蛋白的分子对接结果Total Score值>9.0。连花清瘟胶囊作用于NLRP3靶点蛋白，可能通过细胞凋亡调控、细胞程序性死亡的调控等生物学途径，提高机体固有免疫及应激功能，激活并参与炎症反应，在提高COVID-19临床治愈率中发挥重要作用。
作为鸟流感病毒结合受体的细胞表面蛋白HSP90AA1在感染早期通过HSP90AA1-AKT-MTOR途径诱导自噬。一旦病毒识别，HSP90AA1和AKTMTOR通路的直接连接会触发自噬，这是控制感染的关键步骤[26]。由本文网络药理学和分子对接结果可知，来自连翘、甘草、苦杏仁、广藿香、绵马贯众的30个化学成分与HSP90AA1靶点蛋白的分子对接结果Total Score值>5.0，其中MOL004908(甘草、苦杏仁)、MOL004863、MOL004806(甘草)与HSP90AA1靶点蛋白的分子对接结果Total Score值>9.0。连花清瘟胶囊作用于HSP90AA1靶点蛋白，可能通过对有机物的反应、细胞生物合成过程的正调控等生物学途径，触发自噬控制病毒感染，在提高COVID-19临床治愈率中发挥重要作用。
TLR9可激活固有免疫系统，参与传染病检测[27-28]。TLR9和磷脂酰肌醇-3-激酶γ(PI3Kγ)是免疫应答中非常重要的效应因子。免疫应答需要PI3Kγ，其中TLR9是相关的触发因子[29-30]。在稳态条件下，不适当的TLR9反应可导致严重的自体炎症性疾病，阻断TLR9炎症通路被激活，控制疾病进展和炎症并发症[31]。在肺缺血再灌注过程中释放的线粒体DNA(mtDNA)会触发TLR9依赖性网络的形成，并导致肺损伤[32]。由本文网络药理学和分子对接结果可知，来自板蓝根、甘草、苦杏仁的14个化学成分与TLR9靶点蛋白的分子对接结果Total Score值>5.0，其中MOL001749(板蓝根)与TLR9靶点蛋白的分子对接结果Total Score值>9.0。连花清瘟胶囊作用于TLR9靶点蛋白，可能通过对伤害的反应、细胞因子产生的调节、细胞生物合成过程的正调控等生物学途径，激活固有免疫系统，在预防疾病进展和炎症并发症的发展中起到作用[31]，提高COVID-19临床治愈率。
AKT1是调节细胞存活的信号通路中的一个中心节点。AKT1调控的多种途径在细胞内通过100多种细胞底物的磷酸化进行沟通[33]。如肺纤维化和高碳酸血症。特发性肺纤维化是一种进行性间质性肺炎，以成纤维细胞聚集、胶原沉积和细胞外基质重塑为特征。AKT1通过诱导巨噬细胞产生IL-13来调节肺纤维化，表明靶向AKT1可能同时阻断特发性肺纤维化的纤维化过程[34]。高碳酸血症是严重急性和慢性肺部疾病患者死亡的危险因素。靶向AKT1或升高CO2信号的下游通路可以增强巨噬细胞抗病毒宿主的防御能力，并改善晚期肺部疾病高碳酸血症患者的临床预后[35]。由本文网络药理学和分子对接结果可知，来自甘草、金银花、连翘、绵马贯众、鱼腥草、炙麻黄、广藿香、金银花、板蓝根、大黄的18个化学成分与AKT1靶点蛋白的分子对接结果Total Score值>5.0，其中MOL001495(金银花)与AKT1靶点蛋白的分子对接结果Total Score值>9.0。连花清瘟胶囊作用于AKT1靶点蛋白，可能通过细胞凋亡调控、细胞程序性死亡的调控、细胞死亡调控、多细胞生物过程的正调控等生物学途径，在提高COVID-19临床治愈率中发挥重要作用。
COVID-19属中医肺络病，其临床病理特征是免疫系统功能障碍和炎症反应引起的深气道、肺泡的损害，还可见重度肺充血。连花清温胶囊以络病理论为指导，对许多病毒如sars-CoV、mers-CoV和炎症反应有抑制作用，已纳入国家的COVID-19诊断和治疗计划，其理论组方特色和临床基础研究已得到广泛认可，是治疗呼吸系统疾病的代表性药物[36]。最新研究表明[36]，连花清瘟胶囊可抑制体外培养的COVID-19活性，显著缓解COVID-19患者的发热、咳嗽、乏力等症状，为连花清瘟胶囊治疗COVID-19提供了理论和临床依据。
Biography
鄢海燕，教授，E-mail: yhy0801@126.com
Funding Statement
2018年度安徽省省级质量工程项目(2018jyxm1273)
Contributor Information
鄢 海燕 (Haiyan YAN), Email: yhy0801@126.com.
邹 纯才 (Chuncai ZOU), Email: zouchc@163.com.
References
- 1.国家卫生委员会和国家中医药管理局.《新型冠状病毒肺炎诊疗方案》(试行第七版)[EB/OL]. 2020年3月4日, <a href="http://www.gov.cn/zhengce/zhengceku/2020-03/04/content_5486705.htm" target="_blank">http://www.gov.cn/ zhengce/zhengceku/2020-03/04/content_5486705.htm</a>.
- 2.Hu K, Guan WJ, Bi Y, et al. Efficacy and safety of Lianhuaqingwen capsules, a repurposed Chinese herb, in patients with coronavirus disease 2019: a multicenter, prospective, randomized controlled trial. Phytomedicine. 2020;2020:153242. doi: 10.1016/j.phymed.2020.153242. [Hu K, Guan WJ, Bi Y, et al. Efficacy and safety of Lianhuaqingwen capsules, a repurposed Chinese herb, in patients with coronavirus disease 2019: a multicenter, prospective, randomized controlled trial [J]. Phytomedicine, 2020, 2020: 153242.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.魏 亚男, 朱 博冉, 姬 梦姣, et al. 基于网络药理学探究补阳还五汤防治动脉粥样硬化的作用机制. 中国药理学通报. 2020;36(12):1750–5. doi: 10.3969/j.issn.1001-1978.2020.12.021. [魏亚男, 朱博冉, 姬梦姣, 等.基于网络药理学探究补阳还五汤防治动脉粥样硬化的作用机制[J].中国药理学通报, 2020, 36(12): 1750-5.] [DOI] [Google Scholar]
- 4.刘 华熙, 吕 智豪, 田 春阳, et al. 肾病Ⅲ号方治疗慢性肾脏病蛋白尿的机制:基于网络药理学方法. http://www.j-smu.com:81/CN/10.12122/j.issn.1673-4254.2019.02.16. 南方医科大学学报. 2019;39(2):227–34. doi: 10.12122/j.issn.1673-4254.2019.02.16. [刘华熙, 吕智豪, 田春阳, 等.肾病Ⅲ号方治疗慢性肾脏病蛋白尿的机制:基于网络药理学方法[J].南方医科大学学报, 2019, 39(2): 227-34.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.王 雪晶, 谢 雪, 罗 鑫, et al. 大株红景天化学成分研究(I) 中草药. 2015;46(23):3471–4. [王雪晶, 谢雪, 罗鑫, 等.大株红景天化学成分研究(I)[J].中草药, 2015, 46(23): 3471-4.] [Google Scholar]
- 6.张维康. NLRP3炎症复合体在呼吸机相关性肺损伤中的作用机制研究[D].南宁: 广西医科大学, 2016.
- 7.章 旭之, 马 毅. NOD样受体在免疫耐受中的作用. 中国免疫学杂志. 2017;33(6):930–3. doi: 10.3969/j.issn.1000-484X.2017.06.026. [章旭之, 马毅. NOD样受体在免疫耐受中的作用[J].中国免疫学杂志, 2017, 33(6): 930-3.] [DOI] [Google Scholar]
- 8.Kaur P, Garg M, Hombach-Barrigah A, et al. MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex. Sci Rep. 2017;7(1):10202. doi: 10.1038/s41598-017-09725-w. [Kaur P, Garg M, Hombach-Barrigah A, et al. MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex[J]. Sci Rep, 2017, 7(1): 10202.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Huang C, Liu LY, Li ZF, et al. Effects of small interfering RNAs targeting MAPK1 on gene expression profile in HeLa cells as revealed by microarray analysis. Cell Biol Int. 2008;32(9):1081–90. doi: 10.1016/j.cellbi.2008.04.019. [Huang C, Liu LY, Li ZF, et al. Effects of small interfering RNAs targeting MAPK1 on gene expression profile in HeLa cells as revealed by microarray analysis[J]. Cell Biol Int, 2008, 32(9): 1081-90.] [DOI] [PubMed] [Google Scholar]
- 10.Cao SL, Han XG, Ding C, et al. Molecular cloning of the duck mitogen-activated protein kinase 1 (MAPK1) gene and the development of a quantitative real-time PCR assay to detect its expression. Poult Sci. 2014;93(9):2158–67. doi: 10.3382/ps.2013-03796. [Cao SL, Han XG, Ding C, et al. Molecular cloning of the duck mitogen-activated protein kinase 1 (MAPK1) gene and the development of a quantitative real-time PCR assay to detect its expression[J]. Poult Sci, 2014, 93(9): 2158-67.] [DOI] [PubMed] [Google Scholar]
- 11.Zhu Siliang, Song Wenke, Sun Yanqi, et al. MiR-342 attenuates lipopolysaccharide-induced acute lung injury via inhibiting MAPK1 expression. Clinical and experimental pharmacology & physiology. 2020;47:1448–54. doi: 10.1111/1440-1681.13315. [Zhu Siliang, Song Wenke, Sun Yanqi, et al. MiR-342 attenuates lipopolysaccharide-induced acute lung injury via inhibiting MAPK1 expression[J]. Clinical and experimental pharmacology & physiology, 2020, 47: 1448-54.] [DOI] [PubMed] [Google Scholar]
- 12.Di Paola R, Crisafulli C, Mazzon E, et al. Effect of PD98059, a selective MAPK3/MAPK1 inhibitor, on acute lung injury in mice. Int J Immunopathol Pharmacol. 2009;22(4):937–50. doi: 10.1177/039463200902200409. [Di Paola R, Crisafulli C, Mazzon E, et al. Effect of PD98059, a selective MAPK3/MAPK1 inhibitor, on acute lung injury in mice[J]. Int J Immunopathol Pharmacol, 2009, 22(4): 937-50.] [DOI] [PubMed] [Google Scholar]
- 13.Jing Zhao, Li Li, Ling Peng. MAPK1 up-regulates the expression of MALAT1 to promote the proliferation of cardiomyocytes through PI3K/AKT signaling pathway. International J Clinical and Experimental Pathology. 2015;8(12):15947–53. [Jing Zhao, Li Li, Ling Peng. MAPK1 up-regulates the expression of MALAT1 to promote the proliferation of cardiomyocytes through PI3K/AKT signaling pathway[J]. International J Clinical and Experimental Pathology, 2015, 8(12): 15947-53.] [PMC free article] [PubMed] [Google Scholar]
- 14.Hirota Y, Yamashita S, Kurihara Y, et al. Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways. Autophagy. 2015;11(2):332–43. doi: 10.1080/15548627.2015.1023047. [Hirota Y, Yamashita S, Kurihara Y, et al. Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways[J]. Autophagy, 2015, 11(2): 332-43.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Hoque R, Sohail M, Malik A, et al. TLR9 and the NLRP3 inflammasome link acinar cell death with inflammation in acute pancreatitis. Gastroenterology. 2011;141(1):358–69. doi: 10.1053/j.gastro.2011.03.041. [Hoque R, Sohail M, Malik A, et al. TLR9 and the NLRP3 inflammasome link acinar cell death with inflammation in acute pancreatitis[J]. Gastroenterology, 2011, 141(1): 358-69.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Qu C, Bonar SL, Hickman-Brecks CL, et al. NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms. FASEB J. 2015;29(4):1269–79. doi: 10.1096/fj.14-264804. [Qu C, Bonar SL, Hickman-Brecks CL, et al. NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms[J]. FASEB J, 2015, 29(4): 1269-79.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Peeters PM, Perkins TN, Wouters EFM, et al. Silica induces NLRP3 inflammasome activation in human lung epithelial cells. Part Fibre Toxicol. 2013;10(1):3. doi: 10.1186/1743-8977-10-3. [Peeters PM, Perkins TN, Wouters EFM, et al. Silica induces NLRP3 inflammasome activation in human lung epithelial cells[J]. Part Fibre Toxicol, 2013, 10(1): 3.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.王 金凤, 刘 晓菊, 曾 晓丽. NLRP3炎症小体与肺部疾病. 国际呼吸杂志. 2014;34(2):115–8. doi: 10.3760/cma.j.issn.1673-436X.2014.02.008. [王金凤, 刘晓菊, 曾晓丽. NLRP3炎症小体与肺部疾病[J].国际呼吸杂志, 2014, 34(2): 115-8.] [DOI] [Google Scholar]
- 19.Chen H, Ding SF, Tan JC, et al. Characterization of the Japanese flounder NLRP3 inflammasome in restricting Edwardsiella piscicida colonization in vivo. Fish Shellfish Immunol. 2020;103:169–80. doi: 10.1016/j.fsi.2020.04.063. [Chen H, Ding SF, Tan JC, et al. Characterization of the Japanese flounder NLRP3 inflammasome in restricting Edwardsiella piscicida colonization in vivo[J]. Fish Shellfish Immunol, 2020, 103: 169-80.] [DOI] [PubMed] [Google Scholar]
- 20.Kang MJ, Jo SG, Kim DJ, et al. NLRP3 inflammasome mediates interleukin-1β production in immune cells in response to Acinetobacter baumanniiand contributes to pulmonary inflammation in mice. Immunology. 2017;150(4):495–505. doi: 10.1111/imm.12704. [Kang MJ, Jo SG, Kim DJ, et al. NLRP3 inflammasome mediates interleukin-1β production in immune cells in response to Acinetobacter baumanniiand contributes to pulmonary inflammation in mice[J]. Immunology, 2017, 150(4): 495-505.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.蒋 磊, 赵 鸣雁. NLRP3炎性体在肺损伤的作用进展. 中华急诊医学杂志. 2017;26(7):829–33. doi: 10.3760/cma.j.issn.1671-0282.2017.07.024. [蒋磊, 赵鸣雁. NLRP3炎性体在肺损伤的作用进展[J].中华急诊医学杂志, 2017, 26(7): 829-33.] [DOI] [Google Scholar]
- 22.Li XY, Gong YL, Li D, et al. Low-dose radiation therapy promotes radiation pneumonitis by activating NLRP3 inflammasome. Int J Radiat Oncol. 2020;107(4):804–14. doi: 10.1016/j.ijrobp.2020.02.643. [Li XY, Gong YL, Li D, et al. Low-dose radiation therapy promotes radiation pneumonitis by activating NLRP3 inflammasome[J]. Int J Radiat Oncol, 2020, 107(4): 804-14.] [DOI] [PubMed] [Google Scholar]
- 23.Tang FY, Fan KF, Wang KL, et al. Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways. J Pharmacol Sci. 2018;136(4):203–11. doi: 10.1016/j.jphs.2017.11.010. [Tang FY, Fan KF, Wang KL, et al. Atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome and TLR4 pathways[J]. J Pharmacol Sci, 2018, 136 (4): 203-11.] [DOI] [PubMed] [Google Scholar]
- 24.Yang G, Lee HE, Moon SJ, et al. Direct binding to NLRP3 pyrin domain as a novel strategy to prevent NLRP3-driven inflammation and gouty arthritis. Arthritis Rheumatol. 2020;72(7):1192–202. doi: 10.1002/art.41245. [Yang G, Lee HE, Moon SJ, et al. Direct binding to NLRP3 pyrin domain as a novel strategy to prevent NLRP3-driven inflammation and gouty arthritis[J]. Arthritis Rheumatol, 2020, 72(7): 1192-202.] [DOI] [PubMed] [Google Scholar]
- 25.Wang D, Duncan B, Li XZ, et al. The role of NLRP3 inflammasome in infection-related, immune-mediated and autoimmune skin diseases. J Dermatol Sci. 2020;98(3):146–51. doi: 10.1016/j.jdermsci.2020.03.001. [Wang D, Duncan B, Li XZ, et al. The role of NLRP3 inflammasome in infection-related, immune-mediated and autoimmune skin diseases[J]. J Dermatol Sci, 2020, 98(3): 146-51.] [DOI] [PubMed] [Google Scholar]
- 26.Hu BL, Zhang YN, Jia L, et al. Binding of the pathogen receptor HSP90AA1 to Avibirnavirus VP2 induces autophagy by inactivating theAKT-MTOR pathway. Autophagy. 2015;11(3):503–15. doi: 10.1080/15548627.2015.1017184. [Hu BL, Zhang YN, Jia L, et al. Binding of the pathogen receptor HSP90AA1 to Avibirnavirus VP2 induces autophagy by inactivating theAKT-MTOR pathway[J]. Autophagy, 2015, 11(3): 503-15.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Wei W, Ren J, Yin WW, et al. Inhibition of Ctsk modulates periodontitis with arthritis via downregulation of TLR9 and autophagy. Cell Prolif. 2020;53(1):1–14. doi: 10.1111/cpr.12722. [Wei W, Ren J, Yin WW, et al. Inhibition of Ctsk modulates periodontitis with arthritis via downregulation of TLR9 and autophagy[J]. Cell Prolif, 2020, 53(1): 1-14.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Alegre NS, Garcia CC, Billordo LA, et al. Limited expression of TLR9 on T cells and its functional consequences in patients with nonalcoholic fatty liver disease. Clin Mol Hepatol. 2020;26(2):216–26. doi: 10.3350/cmh.2019.0074. [Alegre NS, Garcia CC, Billordo LA, et al. Limited expression of TLR9 on T cells and its functional consequences in patients with nonalcoholic fatty liver disease[J]. Clin Mol Hepatol, 2020, 26(2): 216-26.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Ashley SN, Somanathan S, Giles AR, et al. TLR9 signaling mediates adaptive immunity following systemic AAV gene therapy. Cell Immunol. 2019;346:103997. doi: 10.1016/j.cellimm.2019.103997. [Ashley SN, Somanathan S, Giles AR, et al. TLR9 signaling mediates adaptive immunity following systemic AAV gene therapy[J]. Cell Immunol, 2019, 346: 103997.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Lima BHF, Marques PE, Gomides LF, et al. Converging TLR9 and PI3Kgamma signaling induces sterile inflammation and organ damage. Sci Rep. 2019;9:19085. doi: 10.1038/s41598-019-55504-0. [Lima BHF, Marques PE, Gomides LF, et al. Converging TLR9 and PI3Kgamma signaling induces sterile inflammation and organ damage[J]. Sci Rep, 2019, 9: 19085.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Stanbery AG, Newman ZR, Barton GM. Dysregulation of TLR9 in neonates leads to fatal inflammatory disease driven by IFN-Γ. PNAS. 2020;117(6):3074–82. doi: 10.1073/pnas.1911579117. [Stanbery AG, Newman ZR, Barton GM. Dysregulation of TLR9 in neonates leads to fatal inflammatory disease driven by IFN-Γ[J]. PNAS, 2020, 117(6): 3074-82.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Mallavia B, Liu F, Lefrançais E, et al. Mitochondrial DNA stimulates TLR9-dependent neutrophil extracellular trap formation in primary graft dysfunction. Am J Respir Cell Mol Biol. 2020;62(3):364–72. doi: 10.1165/rcmb.2019-0140OC. [Mallavia B, Liu F, Lefrançais E, et al. Mitochondrial DNA stimulates TLR9-dependent neutrophil extracellular trap formation in primary graft dysfunction[J]. Am J Respir Cell Mol Biol, 2020, 62(3): 364-72.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Balasuriya N, Davey NE, Johnson JL, et al. Phosphorylationdependent substrate selectivity of protein kinase B (AKT1) J Biol Chem. 2020;295(24):8120–34. doi: 10.1074/jbc.RA119.012425. [Balasuriya N, Davey NE, Johnson JL, et al. Phosphorylationdependent substrate selectivity of protein kinase B (AKT1)[J]. J Biol Chem, 2020, 295(24): 8120-34.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Nie YJ, Hu YD, Yu KK, et al. Akt1 regulates pulmonary fibrosis via modulating IL-13 expression in macrophages. Innate Immun. 2019;25(7):451–61. doi: 10.1177/1753425919861774. [Nie YJ, Hu YD, Yu KK, et al. Akt1 regulates pulmonary fibrosis via modulating IL-13 expression in macrophages[J]. Innate Immun, 2019, 25(7): 451-61.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Marina Casalino-Matsuda S, Chen F, Gonzalez-Gonzalez FJ, et al. Hypercapnia suppresses macrophage antiviral activity and increases mortality of influenza A infection via Akt1. bioRxiv. 2020 doi: 10.1101/2020.02.13.946400. [Marina Casalino-Matsuda S, Chen F, Gonzalez-Gonzalez FJ, et al. Hypercapnia suppresses macrophage antiviral activity and increases mortality of influenza A infection via Akt1[J]. bioRxiv, 2020, DOI: 10.1101/2020.02.13.946400.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.李 红蓉, 常 丽萍, 魏 聪, et al. 连花清瘟治疗新型冠状病毒肺炎的理论研究基础和临床疗效. 世界中医药. 2020;15(3):332–6. doi: 10.3969/j.issn.1673-7202.2020.03.006. [李红蓉, 常丽萍, 魏聪, 等.连花清瘟治疗新型冠状病毒肺炎的理论研究基础和临床疗效[J].世界中医药, 2020, 15(3): 332-6.] [DOI] [Google Scholar]