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基于网络药理学和动物实验研究灵猫方治疗慢性乙型肝炎的机制
DOI: 10.3969/j.issn.1001-5256.2022.07.010
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：马利节、朱晓骏负责课题设计，资料分析，撰写论文；林佳成、王芳参与收集数据，修改论文；孙学华、高月求负责拟定写作思路，指导撰写文章并最后定稿。
Mechanism of Lingmao Formula in treatment of chronic hepatitis B based on network pharmacology and animal experiment
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摘要:
目的 探讨灵猫方治疗慢性乙型肝炎与Ⅰ型IFN的相关性。 方法 TCMSP数据库筛选灵猫方主要活性成分；Genecards数据库获取灵猫方、慢性乙型肝炎作用靶点；Bioinformatics获取药物与疾病靶点交集、绘制韦恩图、并进行GO富集分析；DAVID 6.8进行KEGG富集分析；CytoScape 3.8.0进行免疫过程分析。30只SPF级雄性C57BL/6N小鼠，随机分为正常组、模型组、灵猫方组，每组10只，除正常组10只外，余20只采用水动力转染技术建立HBV体内复制模型，灵猫方灌胃3 d，2次/d，模型组、正常组同体积生理盐水灌胃。结束后收集血清样本，ELISA检测HBsAg、HBeAg、Ⅰ型IFN水平变化。计量资料两组间比较采用独立样本t检验，组间多重比较采用One-way ANOVA结合最小显著性差异法LSD进行多重比较分析。 结果 网络药理学获取灵猫方22个活性成分、6059个作用靶点，481个慢性乙型肝炎相关靶点，387个药物-疾病交集靶点，并提出灵猫方通过调控Ⅰ型IFN参与乙型肝炎相关免疫应答的假设。动物实验证实与模型组比较，灵猫方组HBsAg(t=2.227, P=0.043)、HBeAg(t=2.488, P=0.026)显著下降，且HBsAg(t=-4.603, P＜0.001)、HBeAg(t=-2.224, P=0.043)下降幅度亦具有统计学差异；与正常组相比，模型组IFNα、IFNβ水平无明显变化(P值均＞0.05)，灵猫方组IFNα、IFNβ水平显著上升(P值均＜0.001)；与模型组相比，灵猫方组IFNα、IFNβ水平亦显著上升(P值均＜0.001)。 结论 灵猫方通过调控Ⅰ型IFN的分泌，达到抗HBV的作用。 Abstract:Objective To investigate the association between Lingmao Formula and type Ⅰ interferon in the treatment of chronic hepatitis B. Methods TCMSP database was used to obtain the main active components of Lingmao Formula; Genecards database was used to obtain the targets of Lingmao Formula and chronic hepatitis B; Bioinformatics was used to obtain the intersection of drug targets and disease targets, plot Venn diagram, and perform GO enrichment analysis, and DAVID 6.8 was used for KEGG enrichment analysis; CytoScape 3.8.0 was used to obtain immune response-related pathways. A total of 30 specific pathogen-free male C57BL/6N mice were randomly divided into normal group, model group, and Lingmao Formula group, with 10 mice in each group. All mice except those in the normal group were used to establish a model of HBV replication in vivo by hydrodynamic transfection; the mice in Lingmao Formula group were given Lingmao Formula by gavage, twice a day for 3 days, and those in the model group and the normal group were given an equal volume of normal saline by gavage. Serum samples were collected after the experiment ended and ELISA was used to measure the changes in the levels of HBsAg, HBeAg, and type Ⅰ interferon. The independent samples t-test was used for comparison of continuous data between two groups, and one-way ANOVA combined with the least significant difference t-test was used for multiple comparisons between groups. Results Network pharmacology obtained 22 active components and 6059 action targets of Lingmao Formula, 481 targets associated with chronic hepatitis, and 387 drug-disease intersecting targets and proposed the hypothesis that Lingmao Formula participated in hepatitis B-related immune response by regulating type Ⅰ interferon. Animal experiments confirmed that compared with the model group, the Lingmao Formula group had significant reductions in HBsAg (t=2.227, P=0.043) and HBeAg (t=2.488, P=0.026), with significantly greater reductions in HBsAg (t=-4.603, P < 0.001) and HBeAg (t=-2.224, P=0.043); compared with the normal group, the model group had no significant changes in the levels of IFNα and IFNβ (P > 0.05), and the Lingmao Formula group had significant increases in the levels of IFNα and IFNβ (P < 0.001); compared with the model group, the Lingmao Formula group also had significant increases in the levels of IFNα and IFNβ (P < 0.001). Conclusion Lingmao Formula exerts an anti-HBV effect by regulating the secretion of type Ⅰ interferon. -
Key words:
- Hepatitis B, Chronic /
- Lingmao Formula /
- Network Pharmacology /
- Animal Experiment /
- Interferon Type Ⅰ
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图 1 基于GeneCards数据库预测的灵猫方-CHB靶点基因交集
Figure 1. Prediction of Lingmao Formula-CHB gene intersection from GeneCards database
注：a，灵猫方-CHB靶点基因GO分析图；b，灵猫方-CHB靶点基因KEGG分析图。
图 2 灵猫方-CHB靶点基因GO及KEGG分析
Figure 2. GO and KEGG analysis of Lingmao Formula-CHB target genes
注：a，灵猫方-CHB免疫过程图；b，灵猫方-乙型肝炎免疫过程图。
图 3 药物-疾病免疫过程网络
Figure 3. Lingmao Formula-CHB immune process network
表 1 灵猫方22种活性成分
Table 1. 22 active ingredients in Lingmao Formula
分子代码 活性成分 OB(%) DL MOL000006 木樨草素(luteolin) 36.16 0.25 MOL000098 槲皮素(quercetin) 46.43 0.28 MOL000211 丁子香萜(mairin) 55.38 0.78 MOL000354 异鼠李素(isorhamnetin) 49.60 0.31 MOL000358 β-谷甾醇(beta-sitosterol) 36.91 0.75 MOL000359 谷甾醇(sitosterol) 36.91 0.75 MOL000392 芒柄花黄素(formononetin) 69.67 0.21 MOL000398 异黄烷酮(isoflavanone) 109.99 0.30 MOL000417 毛蕊异黄酮(calycosin) 47.75 0.24 MOL000422 山柰酚(kaempferol) 41.88 0.24 MOL000433 FA 68.96 0.71 MOL000449 豆甾醇(Stigmasterol) 43.83 0.76 MOL000953 CLR 37.87 0.68 MOL001645 乙酸亚油醇酯(linoleyl acetate) 42.10 0.20 MOL001803 甜橙黄酮(sinensetin) 50.56 0.45 MOL004328 柚皮素(naringenin) 59.29 0.21 MOL004425 灵猫方苷(icariin) 41.58 0.61 MOL004427 灵猫方次甙A7(icariside A7) 31.91 0.86 MOL004576 花旗松素(taxifolin) 57.84 0.27 MOL005190 圣草酚(eriodictyol) 71.79 0.24 MOL005438 菜油甾醇(campesterol) 37.58 0.71 MOL005828 川陈皮素(nobiletin) 61.67 0.52 
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表 2 KEGG通路富集结果
Table 2. Enrichment results of KEGG pathway
ID 通路名称 基因数量 P值 基因 hsa05161 乙型肝炎 32 7.81×10-14 PCNA, CXCL8, IFNA2, PTEN, FASLG, TNF, RELA, IFIH1, CCND1, STAT4, MAPK1, JAK1, JUN, TGFB1, IFNB1, STAT1, STAT3, MMP9, TGFBR1, NFKB1, MAVS, IL6, IRF3, IRF7, BCL2, BAX, FAS, TP53, TLR4, TLR3, TLR2, IFNAR1
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表 3 灵猫方对HBV模型鼠HBsAg、HBeAg的影响
Table 3. Effect of Lingmao Formula on HBsAg and HBeAg in HBV model mouse
组别 动物数(只) 治疗前 治疗后 下降幅度 HBsAg(IU/mL) 模型组 10 1 813.88±590.89 1 395.43±566.21 418.45±184.63 灵猫方组 10 1 767.53±496.81 882.25±323.06 885.28±219.56 t值 0.170 2.227 -4.603 P值 0.868 0.043 ＜0.001 HBeAg(S/CO) 模型组 10 58.69±12.10 29.89±5.95 28.80±9.08 灵猫方组 10 65.34±12.89 23.29±4.57 42.05±14.21 t值 -1.064 2.488 -2.224 P值 0.305 0.026 0.043
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表 4 灵猫方对HBV模型鼠Ⅰ型IFN的影响
Table 4. Effect of Lingmao formula on typeⅠIFN in HBV model mouse
组别 动物数(只) IFNα(ng/L) IFNβ(ng/L) 正常组 10 49.44±13.231) 171.80±8.751) 模型组 10 54.59±10.471) 178.37±14.281) 灵猫方组 10 85.62±13.34 243.19±21.73 F值 24.838 62.052 P值 ＜0.001 ＜0.001 注：与灵猫方组比较，1) P＜0.001。
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