基于网络药理学和实验验证探究糖肾宝复方治疗糖尿病肾病的作用机制

doi:10.19586/j.2095-2341.2023.0142

生物技术进展 ›› 2024, Vol. 14 ›› Issue (1): 160-171.DOI: 10.19586/j.2095-2341.2023.0142

• 研究论文 • 上一篇

基于网络药理学和实验验证探究糖肾宝复方治疗糖尿病肾病的作用机制

苏芳林¹(), 罗鑫¹, 唐赛男¹, 黄乐韵¹, 胡维²^,³, 陆世龙²^,³, 冉龙娇²^,³, 向少伟²^,³()

^1.广西中医药大学研究生院，南宁 530200
^2.广西中医药大学附属瑞康医院肾内科，南宁 530011
^3.广西中西医结合肾脏疾病临床医学研究中心，南宁 530011

收稿日期:2023-11-01 接受日期:2023-12-12 出版日期:2024-01-25 发布日期:2024-02-05
通讯作者: 向少伟
作者简介:苏芳林 E-mail：849875812@qq.com；
基金资助:
广西自然科学基金面上项目(2020JJA140235);广西科技基地和人才专项(桂科AD22035122)

The Mechanism Research of Tangshenbao Compound in Treating Diabetic Nephropathy Based on Network Pharmacology and Experimental Verification

Fanglin SU¹(), Xin LUO¹, Sainan TANG¹, Leyun HUANG¹, Wei HU²^,³, Shilong LU²^,³, Longjiao RAN²^,³, Shaowei XIANG²^,³()

^1.Graduate School of Guangxi University of Traditional Chinese Medicine，Nanning 530200，China
^2.Department of Nephrology，Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine，Nanning 530011，China
^3.Guangxi Clinical Research Center of Kidney Diseases of Integrated Traditional Chinese and Western Medicine，Nanning 530011，China

Received:2023-11-01 Accepted:2023-12-12 Online:2024-01-25 Published:2024-02-05
Contact: Shaowei XIANG

摘要/Abstract

摘要：

网络药理学方法结合分子生物学实验探究糖肾宝复方（Tangshenbao compound，TSB）治疗糖尿病肾病（diabetic nephropathy, DN）的作用机制。使用TCMSP、Pubchem、Swiss Target Prediction数据库，结合相关文献，得到TSB相关活性成分与靶点；通过GeneCards、DisGeNET、OMIM数据库，获得TSB治疗DN候选靶点;利用在线作图软件绘制“TSB-DN”候选靶点Venn图，并结合 Cytoscape 3.7.2 软件进行GO和KEGG 富集分析；然后使用PyMOL、AutoDock Tools和AutoDock Vina软件完成活性成分配体与靶蛋白晶体结构对接；再通过体外培养小鼠肾小球系膜细胞Mcs进行细胞实验验证，实时荧光定量PCR检测各组细胞中STAT3、PIK3CD、PIK3R1、MAPK8及关联性靶点Ets-1 mRNA表达水平，CCK-8法检测24 h后的细胞活性。结果发现，Swiss Target Prediction数据库合并处理后共获得TSB作用靶点835个，联合GeneCards、DisGeNET、OMIM等疾病数据库后共获得443个“TSB-DN ”交集靶点，而PPI网络分析得出Degree前10靶点分别为SRC、PIK3R1、STAT3、PIK3CA等；GO、KEGG富集分析结果显示TSB治疗DN可能与蛋白磷酸化、细胞信号转导、基因表达正调控等功能相关；最后体外细胞实验表明，TSB复方能有效降低SV40-MES-13细胞中PIK3CD、PIK3R1、ETS-1的mRNA表达量，药物作用于细胞后能起到“抑制、镇静”的作用。结果表明，TSB复方可通过抑制PIK3R1、PIK3CD和MAPK8、STAT3、ETS-1等靶基因的mRNA表达，实现对PI3K-Akt、AGE-RAGE等信号通路的调控并发挥对DN的治疗作用。

关键词: 网络药理, 糖尿病肾病, 糖肾宝复方, 肾小球系膜细胞

Abstract:

The research investigated the mechanism of action of Tangshenbao compound （TSB） in treating diabetic nephropathy （DN） by combining network pharmacology methods with molecular biology experiments. The active ingredients and targets related to TSB were obtained using TCMSP， Pubchem， and Swiss Target Prediction databases， along with relevant literature. Candidate targets for TSB in treating DN were obtained through GeneCards， DisGeNET， and OMIM databases. A Venn diagram of the "TSB-DN" candidate targets was drawn using online plotting software， followed by GO and KEGG enrichment analysis using Cytoscape 3.7.2 software. Subsequently， molecular docking of active ingredient ligands with target protein crystal structures was completed using PyMOL， AutoDock Tools， and AutoDock Vina software. Cell experiments were then conducted using Mcs mesangial cells cultured in vitro to validate the findings. Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of STAT3， PIK3CD， PIK3R1， MAPK8， and their associated targets in the cells of each group， while cell viability after 24 hours was assessed using the CCK-8 method. Results showed that a total of 835 TSB target genes were obtained after processing the Swiss Target Prediction database. When combined with GeneCards， DisGeNET， OMIM， and other disease databases， there were 443 intersecting target genes for “TSB-DN”. Protein-protein interaction （PPI） network analysis identified the top 10 degree targets as SRC， PIK3R1， STAT3 and PIK3CA. GO and KEGG enrichment analysis results indicated that TSB treatment for DN may be related to functions such as protein phosphorylation， cell signal transduction， and positive regulation of gene expression. Finally， cell experiments in vitro demonstrated that TSB could effectively reduce the mRNA expression levels of PIK3CD， PIK3R1， and ETS-1 in SV40-MES-13 cells， resulting in an “inhibitory， sedative” effect following drug action on the cells. The results indicated that the TSB compound can inhibit the mRNA expression of target genes such as PIK3R1， PIK3CD， MAPK8， STAT3， and ETS-1， thereby regulating the PI3K-Akt and AGE-RAGE signaling pathways and exerting a therapeutic effect on DN.

Key words: network pharmacology, diabetic nephropathy, glycokidney treasure compound, renal bulb mesangial cells

中图分类号:

Q291

苏芳林, 罗鑫, 唐赛男, 黄乐韵, 胡维, 陆世龙, 冉龙娇, 向少伟. 基于网络药理学和实验验证探究糖肾宝复方治疗糖尿病肾病的作用机制[J]. 生物技术进展, 2024, 14(1): 160-171.

Fanglin SU, Xin LUO, Sainan TANG, Leyun HUANG, Wei HU, Shilong LU, Longjiao RAN, Shaowei XIANG. The Mechanism Research of Tangshenbao Compound in Treating Diabetic Nephropathy Based on Network Pharmacology and Experimental Verification[J]. Current Biotechnology, 2024, 14(1): 160-171.

图/表 12

表1 引物序列

Table 1 Primers sequence

引物名称	引物序列（5’→3’）	扩增产物长度/bp
PIK3CD	5’- GATCCGGGGAACTGTACGAA-3’	20
PIK3CD	5’-CACAGCAAATAGAGCATCTGGG-3’	22
PIK3R1	5’- CCAATACTTGATGTGGCTGACG-3’	22
PIK3R1	5’- TTCCCTCGCAATAGGTTCTCC-3’	21
STAT3	5’- TGGCTGACTGGAAGAGGCGG-3’	20
STAT3	5’-TTGGCGGGTCTGAAGTTGAG-3’	20
MAPK8	5’-TCTCTATCAAATGCTGTGTGGAATC-3’	25
MAPK8	5’-CATAAGGCGTCATCATAAAACTCG-3’	24
Ets-1	5’-TTGATGCCCTTTCTCCCTA-3’	19
Ets-1	5’-ATAAACCACCCGCCACTC-3’	18
GADPH	5’-GGTTGTCTCCTGCGACTTCA-3’	20
GADPH	5’-TGGTCCAGGGTTTCTTACTCC-3’	21

表2 TSB组方药物主要活性成分信息

Table 2 Information of main active ingredients of TSB prescription drugs

MOL ID	化合物名称	OB	DL	Cas号	归属
MOL000398	异黄烷酮（isoflavanone）	109.99	0.30	4 737-27-3	黄芪
MOL000378	华良姜素（7-O-methylisomucronulatol）	74.69	0.30	3 301-49-3	黄芪
MOL000392	刺芒柄花素（formononetin）	69.67	0.21	485-72-3	黄芪/葛根
MOL000433	叶酸（folic acid）	68.96	0.71	33 609-88-0 59-30-3	黄芪/川芎
MOL000438	异微凸剑叶莎醇（isomucronulatol）	67.67	0.26	64 474-51-7	黄芪
MOL002140	川芎哚（perlolyrine）	65.95	0.27	29 700-20-7	川芎
MOL000211	白桦脂酸（mairin）	55.38	0.78	472-15-1	黄芪
MOL000417	毛蕊异黄酮（calycosin）	47.75	0.24	20 575-57-9	黄芪
MOL000098	槲皮素（quercetin）	46.43	0.28	117-39-5	黄芪
MOL001494	亚油酸乙酯（mandenol）	42.00	0.19	544-35-4	川芎

图1 TSB组方与糖尿病肾病交集Venn图

Fig. 1 Venn diagram of intersection of TSB prescription and diabetic nephropathy

图2 “TSB-DN”交集靶点PPI网络图

Fig. 2 PPI network diagram of "TSB-DN" intersection target

图3 GO功能富集分析

Fig. 3 GO functional enrichment analysis

图4 KEGG通路富集分析结果

Fig. 4 KEGG pathway enrichment analysis results

图5 “中药-成分-靶点-通路”网络图注：红色/黄色三角形节点代表组方单味药，绿色六边形节点代表中药化合物，蓝色圆形节点代表各化合物节点对应靶点，淡蓝色正方形节点代表通路靶点，黄色倒置三角形节点代表通路。

Fig. 5 “Chinese medicine-ingredient-target-pathway” network diagram

表3 核心活性成分与核心靶点分子对接结果

Table 3 Results of docking between core active ingredients and core target molecules

靶点	结合能/（kcal·mol^-1）
靶点	异微凸剑叶莎醇	3,9-di-O-methylnissolin	20(S)-原人参三醇	香豆雌酚	甘草素	华良姜素
STAT3	-8.1	-7.9	-10.1	-8.9	-9.8	-8.7
PIK3CD	-9.1	-8.7	-11.1	-10.3	-11.2	-8.3
PIK3R1	-8.5	-8.6	-10.8	-9.6	-10.9	-8.6
PIK3CA	-8.7	-9.3	-11.5	-10.0	-11.6	-8.5
ETS-1	-7.9	-8.6	-10.0	-8.9	-10.5	-8.2

图6 分子对接可视化结果

Fig. 6 Molecular docking visualization results

图7 SV40-MES-13细胞含药血清干预前后状态

Fig. 7 State of SV40-MES-13 cells before and after treatment with drug-containing serum

图8 TSB对SV40-MES-13细胞活性影响注：**和***分别表示两组之间的差异在P<0.01和P<0.001水平上有统计学意义。

Fig. 8 Effect of TSB on SV40-MES-13 cell activity

图9 TSB治疗DN相关信号通路靶基因结果注：*，**，***分别表示与正常组比较在P<0.05，P<0.001，P<0.01水平差异具有统计学意义；#，##，###分别表示与高糖组比较在P<0.05，P<0.001，P<0.01水平差异具有统计学意义；▲，▲▲▲分别表示与低剂量组比较在P<0.05，P<0.001水平差异具有统计学意义。

Fig. 9 Target gene results of DN-related signaling pathway treated by TSB

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基于网络药理学和实验验证探究糖肾宝复方治疗糖尿病肾病的作用机制

The Mechanism Research of Tangshenbao Compound in Treating Diabetic Nephropathy Based on Network Pharmacology and Experimental Verification

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