网络药理学结合转录组学方法探讨防己黄芪汤治疗肾病综合征作用机制

万浩婷; 刘晴; 刘路瑶; 谢林彤; 朱慧; 刘晓

doi:10.14148/j.issn.1672-0482.2023.0346

网络药理学结合转录组学方法探讨防己黄芪汤治疗肾病综合征作用机制

doi: 10.14148/j.issn.1672-0482.2023.0346

万浩婷^{1, 2, 3,},
刘晴^{1, 2, 3},
刘路瑶^{1, 2, 3},
谢林彤^{1, 2, 3},
朱慧^{1, 2, 3},
刘晓^{1, 2, 3, ,}

1.
南京中医药大学药学院, 江苏南京 210023
2.
江苏省中药炮制重点实验室, 江苏南京 210023
3.
南京中医药大学国家教育部中药炮制规范化及标准化工程研究中心, 江苏南京 210023

基金项目:

江苏省高等学校基础科学(自然科学)重大项目 21KJA360008

详细信息

作者简介:
万浩婷, 女, 硕士研究生, E-mail: 15962763705@163.com

通讯作者:
刘晓, 女, 副教授, 主要从事中药炮制机理与饮片质量控制研究, E-mail: 300999@njucm.edu.cn

中图分类号: R285.5
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- HTML全文浏览量: 59
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- 被引次数: 0
出版历程
- 收稿日期: 2022-11-01
- 网络出版日期: 2023-04-17
- 发布日期: 2023-04-10

Mechanism of Fangji Huangqi Tang against Nephrotic Syndrome Based on Network Pharmacology and Transcriptomics Methods

WAN Hao-ting^{1, 2, 3
,},
LIU Qing^{1, 2, 3},
LIU Lu-yao^{1, 2, 3},
XIE Lin-tong^{1, 2, 3},
ZHU Hui^{1, 2, 3},
LIU Xiao^{1, 2, 3
, ,}

1.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Jiangsu Key Laboratory of Chinese Medicine Processing, Nanjing 210023, China
3.
Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的基于转录组学与网络药理学方法探讨防己黄芪汤治疗肾病综合征的作用机制。方法收集网络药理学和转录组学的共同靶点, 进行GO/KEGG功能富集分析。构建蛋白质-蛋白质相互作用(PPI)并进行网络拓扑分析, 确定潜在核心靶点。构建通路-靶点网络图, 确定发挥关键作用的信号通路。采用qPCR、Western blot法和免疫荧光染色验证大鼠肾脏组织中核心靶点的mRNA和蛋白表达水平。结果 PPI分析得到7个潜在核心靶点AKT1、AMPK、CPT1B、NF-κB1、P53、TGF-β1和TLR4, 主要信号通路为AMPK信号通路、PI3K-Akt信号通路、PPAR信号通路、NF-κB信号通路、TGF-β信号通路、P53信号通路、MAPK信号通路、JAK-STAT信号通路和FoxO信号通路。经动物体内实验验证, 防己黄芪汤显著下调AKT1、CPT1B、NF-κB1、P53、TGF-β1、TLR4的mRNA和蛋白表达水平(P < 0.05), 上调AMPK的mRNA和蛋白表达水平(P < 0.05)。结论从网络药理学和转录组学角度初步阐明了防己黄芪汤治疗肾病综合征多成分、多靶点、多途径的整体调节特点, 可为后续的药理学研究和临床应用提供依据与参考。
- 防己黄芪汤 /
- 肾病综合征 /
- 转录组学 /
- 网络药理学 /
- 作用机制
Abstract: OBJECTIVE To explore the mechanism of Fangji Huangqi Tang (FHT) in the treatment of nephrotic syndrome (NS) based on transcriptomics and network pharmacology. METHODS Intersection target genes of network pharmacology and transcriptomics were collected for GO/KEGG functional enrichment analysis. Protein-protein interactions (PPI) were constructed and network topology analysis was performed to identify potential core targets. The pathway-target network map was constructed to identify the key signaling pathways. Finally, real-time quantitative PCR (qPCR), western blot and immunofluorescence staining were used to verify the mRNA and protein expression levels of core targets in kidney tissues of rats. RESULTS Seven potential core targets AKT1, AMPK, CPT1B, NF-κB1, P53, TGF-β1 and TLR4 were identified by PPI analysis. The main signaling pathways were AMPK, PI3K-Akt, PPAR, NF-κB, TGF-β, P53, MAPK, JAK-STAT and FoxO. In animal experiments, FHT significantly down-regulated the mRNA and protein expression levels of AKT1, CPT1B, NF-κB1, P53, TGF-β1, TLR4 (P < 0.05), and up-regulated the mRNA and protein expression levels of AMPK (P < 0.05). CONCLUSION From the perspective of network pharmacology and transcriptomics, the overall regulation features of multi-component, multi-target and multi-pathway of FHT against NS were preliminaries elaborated, which could provide the basis and reference for subsequent pharmacological research and clinical application of FHT.
- Fangji Huangqi Tang /
- nephrotic syndrome /
- transcriptomics /
- network pharmacology /
- mechanism

HTML全文

图 1 中药活性成分-靶点网络

Figure 1. Active ingredient-target network of traditional Chinese medicine

下载: 全尺寸图片幻灯片

图 2 防己黄芪汤对各组小鼠肾病综合征相关指标的影响

注; 与正常组比较, ^{* * *}P < 0.001;与模型组比较, ^##P < 0.01, ^###P < 0.001。x±s, n=10。

Figure 2. Effects of Fangji Huangqi Tang on indexes related to nephrotic syndrome

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图 3 防己黄芪汤对各组大鼠肾脏组织的病理学影响(HE, ×200;Masson, ×200)

Figure 3. Effects of Fangji Huangqi Tang on histopathological morphology of kidneys in all groups (HE, ×200; Masson, ×200)

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图 4 正常组vs.模型组、防己黄芪汤组vs.模型组差异基因表达的火山图

Figure 4. Volcano diagram of differential expression gene of control group vs. model group and Fangji Huangqi Tang group vs. model group

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图 5 转录组学差异基因表达热图

Figure 5. Heat map of differential expression genes by transcriptomics

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图 6 网路药理学和转录组学交集靶点基因的韦恩图

Figure 6. Venn diagram of intersection target genes of network pharmacology and transcriptomics

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图 7 GO功能富集分析和KEGG通路富集分析

Figure 7. GO functional enrichment analysis and KEGG pathway enrichment analysis

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图 8 蛋白质互作网络图

Figure 8. Protein-protein interaction (PPI) network

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图 9 潜在核心靶点-通路网络图

Figure 9. Potential core target-pathway network

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图 10 qPCR检测肾组织中AKT1、AMPK、CPT1B、NF-κB1、P53、TGF-β1、TLR4的mRNA表达

注; 与正常组比较, ^{* * *}P < 0.001;与模型组比较, ^##P < 0.01, ^###P < 0.001。x±s, n=3。

Figure 10. Expressions of AKT1, AMPK, CPT1B, NF-κB1, P53, TGF-β1 and TLR4 mRNA in kidney tissues by qPCR

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图 11 Western blot检测肾组织中AKT1、p-AKT1、AMPK、p-AMPK、CPT1B、NF-κB1、P53、TGF-β1、TLR4的蛋白表达

注; 与正常组比较, ^*P < 0.05, ^{* *}P < 0.01, ^{* * *}P < 0.001;与模型组比较, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001。x±s, n=3。

Figure 11. Expressions of AKT1, p-AKT1, AMPK, p-AMPK, CPT1B, NF-κB1, P53, TGF-β1 and TLR4 protein in kidney tissues by Western blot

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图 12 免疫荧光染色法检测肾组织中AKT1、AMPK、CPT1B、NF-κB1、P53、TGF-β1、TLR4的蛋白表达(×200)

Figure 12. Expressions of AKT1, AMPK, CPT1B, NF-κB1, P53, TGF-β1 and TLR4 protein in kidney tissues by immunofluorescence (×200)

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图 13 防己黄芪汤治疗肾病综合征的靶点通路分析图

Figure 13. Target pathway analysis of Fangji Huangqi Tang against nephrotic syndrome

下载: 全尺寸图片幻灯片

表 1 目的基因的引物序列

Table 1. Primer sequence of target genes

基因	序列(5’-3’)	产物长度/bp
AKT	F: GCTTCTACGGTGCGGAGATTGTGT R: GTCCGTTATCTTGATGTGCCCGTC	125
AMPK	F: GACCTCGGTCAAGTGTCGATTC R: AACGGGCTAAAGCAGTGATAAGA	168
CPT1B	F: ATGTAAGTGACTGGTGGGAAGA R: TGGGATGCGTGTAGTGTTGAA	260
NF-κB1	F: AGATGTGGTGGAGGACTTGCT R: CCGTTGGGGTGGTTGATAA	162
P53	F: AAGGAAATCCGTATGCTGAGTAT R: GCACAAACACGAACCTCAAAG	235
TGF-β1	F: CATTTGGAGCCTGGACACACA R: GCTTGCGACCCACGTAGTAGAC	136
TLR4	F: AAGACTATCATCAGTGTATCGGTGG R: CGTTTCTCACCCAGTCCTCATT	181
GAPDH	F: CGTATCGGACGCCTGGTT R: AGGTCAATGAAGGGGTCGTT	83

下载: 导出CSV

表 2 防己黄芪汤活性成分基本信息

Table 2. Basic information of active ingredients of Fangji Huangqi Tang

序号	成分名称	口服生物利用度(OB)/%	类药性(DL)	归属药物
1	Calycosin-7-O-D-glucoside	36.52	0.23	防己
2	Corydine	37.16	0.55	防己
3	Isoliguiritigenin	33.64	0.26	防己
4	Fangchinoline	41.73	0.21	防己
5	(-)-Tetrahydropalmatine	73.94	0.64	防己
6	(+)-Tetrandrine	36.64	0.20	防己
7	Liquiritigenin	32.76	0.18	防己
8	Isoliquiritin	38.61	0.60	防己
9	Glycycoumarin	43.56	0.44	黄芪
10	Atractylenolide Ⅲ	35.95	0.21	黄芪
11	Atractylenolide Ⅰ	37.37	0.19	黄芪
12	Isolicoflavonol	45.17	0.42	黄芪
13	Licoricone	63.58	0.47	黄芪
14	Atractylenolide Ⅱ	52.36	0.19	黄芪
15	Licoricidin	30.99	0.62	黄芪
16	(+)-Cassythicine	32.64	0.20	白术
17	Nantenine	35.49	0.74	白术
18	Cyclanoline	42.64	0.57	白术
19	Liquiritin	39.20	0.52	甘草
20	Astragaloside Ⅳ	41.78	0.63	甘草
21	Licochalcone B	76.76	0.19	甘草
22	Methylnissolin 3-O-glucoside	36.22	0.18	甘草
23	Calycosin	45.75	0.24	甘草
24	Fenfangjine F	43.30	0.23	甘草
25	Astragaloside Ⅶ	30.94	0.45	甘草
26	Formononetin	69.67	0.22	甘草
27	Vestitol	74.66	0.24	甘草
28	Glycyrrhizin	39.62	0.21	甘草
29	Astragaloside Ⅱ	46.06	0.23	甘草

下载: 导出CSV

表 3 潜在核心靶点在蛋白互作网络中的参数

Table 3. Parameters of potential core targets in protein-protein interaction (PPI)

基因名称	度值	接近中心性	中介中心性	拓扑系数
AKT1	57	0.793 8	0.154 3	0.261 8
AMPK	56	0.763 2	0.132 5	0.300 5
P53	52	0.725 4	0.113 6	0.313 0
TLR4	41	0.623 8	0.096 5	0.380 4
CPT1B	32	0.526 9	0.075 3	0.443 3
NF-κB1	30	0.503 2	0.072 1	0.435 8
TGF-β1	26	0.472 5	0.063 8	0.506 7

下载: 导出CSV

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