Mechanism of Fangji Huangqi Tang against Nephrotic Syndrome Based on Network Pharmacology and Transcriptomics Methods
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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。 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. -
Key words:
- Fangji Huangqi Tang /
- nephrotic syndrome /
- transcriptomics /
- network pharmacology /
- mechanism
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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
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/bpAKT F: GCTTCTACGGTGCGGAGATTGTGT
R: GTCCGTTATCTTGATGTGCCCGTC125 AMPK F: GACCTCGGTCAAGTGTCGATTC
R: AACGGGCTAAAGCAGTGATAAGA168 CPT1B F: ATGTAAGTGACTGGTGGGAAGA
R: TGGGATGCGTGTAGTGTTGAA260 NF-κB1 F: AGATGTGGTGGAGGACTTGCT
R: CCGTTGGGGTGGTTGATAA162 P53 F: AAGGAAATCCGTATGCTGAGTAT
R: GCACAAACACGAACCTCAAAG235 TGF-β1 F: CATTTGGAGCCTGGACACACA
R: GCTTGCGACCCACGTAGTAGAC136 TLR4 F: AAGACTATCATCAGTGTATCGGTGG
R: CGTTTCTCACCCAGTCCTCATT181 GAPDH F: CGTATCGGACGCCTGGTT
R: AGGTCAATGAAGGGGTCGTT83 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 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 -
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