Mechanism and in Vitro Experiment of Wogonin in Treatment of Hepatocellular Carcinoma Based on Network Pharmacology

doi:10.12114/j.issn.1007-9572.2023.0238

Abstract

Abstract:

Background

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths. The current prevention and treatment situation remains critical. It is of scientific significance to explore new therapeutic agents for HCC.

Objective

To analyze the mechanism of wogonin on HCC by network pharmacology and to verify it in vitro.

Methods

The drug targets of wogonin were searched in TCMSP database, and the disease targets of HCC were collected from TTD, GenCard, OMIM, DisGent databases. The collected drug targets and disease targets were intersected as potential targets for drug intervention in diseases. R software was used for enrichment analysis of intersection targets, STRING database and Cytoscape software were used to construct protein interaction network and screen core targets. The core targets were further analyzed in GIEPA database. Finally, the preliminary analysis results were verified by in vitro experiments, including cell activity determination using CCK-8 kit, cell proliferation determination using plate clone formation experiment, cell migration determination using scoring test, protein expression level determination using Western-blotting (WB) assay.

Results

The AMDE characteristics of wogonin were found to be in accordance with the rules for small molecule drug formation and the toxicity analysis showed no toxicity. A total of 135 wogonin targets and 8 238 HCC targets were collected, and 113 targets were intersected. Through the analysis of the core genes of TOP10 screened by the constructed protein interaction network, it was found that the mRNA levels of CDK1 and SRC in liver cancer tissues were higher than those in normal liver tissues (P<0.05), and the high expression levels in liver cancer patients were related to poor prognosis (P<0.05). KEGG enrichment analysis showed that the intersection genes were enriched in the PI3K/AKT signaling pathway, and the molecular docking results showed that wogonin had strong binding configuration activity with CDK1 and SRC. The results of CCK-8 kit showed that the activity of HepG2 cells in the 75.0, 150.0, and 300.0 μmol/L wogonin groups was lower than that in the control group (P<0.05). The results of plate clone formation experiment showed that the number of colony formation of HepG2 cells in the 37.5, 75.0, 150.0 μmol/L wogonin groups was lower than that in the control group (P<0.05). The results of scoring test showed that the migration rate of HepG2 cells in the 37.5, 75.0 and 150.0 μmol/L wogonin groups was lower than that in the control group (P<0.05). The results of the WB assay showed that the expression levels of PI3K, P-AKT/AKT, CDK1 and SRC proteins in the 75.0 and 150.0 μmol/L wogonin groups were lower than those in the control group (P<0.05) .

Conclusion

Wogonin inhibits the proliferation and migration of HCC cells and induces apoptosis by down-regulating the expression of CDK1 and SRC proteins and attenuating the PI3K/AKT pathway signaling, to achieve the purpose of interfering with the occurrence and progression of HCC.

Key words: Carcinoma, hepatocellular, Wogonin, Network pharmacology, In vitro experiments

摘要：

背景

肝细胞癌是癌症相关死亡的主要原因，目前的防治形势依然严峻，对新的肝细胞癌治疗药物进行探索研究具有科学意义。

目的

通过网络药理学方法分析汉黄芩素干预肝细胞癌的作用机制，并进行体外实验验证。

方法

在TCMSP数据库中检索汉黄芩素的药物靶点，从TTD、GenCard、OMIM、DisGent数据库中收集肝细胞癌的疾病靶点。将收集的药物靶点和疾病靶点取交集，作为药物干预疾病的潜在靶点。对交集靶点运用R软件进行富集分析，使用STRING数据库和Cytoscape软件对交集靶点构建蛋白互作网络和筛选核心靶点。在GIEPA数据库对核心靶点行进一步分析。最后通过体外实验对前期分析结果进行验证：采用CCK-8试剂盒测定细胞活性；采用平板克隆形成实验测定细胞增殖；采用划痕实验测定细胞迁移；采用Western-blotting（WB）实验测定蛋白质表达水平。

结果

分析结果发现汉黄芩素的吸收、分布、代谢、排泄特性符合小分子药物成药规则并且毒性分析结果表明无毒性。收集到汉黄芩素靶点135个，肝细胞癌靶点8 238个，两者交集靶点113个。通过对构建的蛋白互作网络筛选出的前10位的核心基因进行分析，发现细胞周期蛋白依赖性激酶1（CDK1）、原癌基因酪氨酸蛋白激酶Src（SRC）在肝细胞癌组织中mRNA水平较正常肝组织上调（P<0.05），并且在肝细胞癌患者中高表达与不良预后相关（P<0.05）。KEGG富集分析发现交集基因富集在PI3K/AKT信号通路上最多，分子对接结果显示汉黄芩素与CDK1、SRC结合构型活力较强。CCK-8试剂盒检测结果显示，加入汉黄芩素75.0、150.0、300.0 μmol/L组HepG2细胞活性均低于对照组（P<0.05）；平板克隆形成实验结果显示，加入汉黄芩素37.5、75.0、150.0 μmol/L组HepG2细胞克隆形成数均低于对照组（P<0.05）；划痕实验结果表明，加入汉黄芩素37.5、75.0、150.0 μmol/L组HepG2细胞迁移率均低于对照组（P<0.05）；WB实验结果表明，加入汉黄芩素75.0、150.0 μmol/L组PI3K、P-AKT/AKT、CDK1、SRC蛋白表达水平均低于对照组（P<0.05）。

结论

汉黄芩素通过下调CDK1、SRC蛋白表达，减弱PI3K/AKT通路信号，抑制肝癌细胞增殖和迁移，诱导细胞凋亡，从而达到干预肝细胞癌发生和进展的目的。

关键词: 癌，肝细胞, 汉黄芩素, 网络药理学, 体外实验

CLC Number:

R 730.261

YANG Anyin,LIU Hongli,CHEN Miaoyang, et al. Mechanism and in Vitro Experiment of Wogonin in Treatment of Hepatocellular Carcinoma Based on Network Pharmacology[J]. Chinese General Practice, 2024, 27(32): 4040-4049. DOI: 10.12114/j.issn.1007-9572.2023.0238.
杨安银,刘红丽,陈妙洋等. 基于网络药理学方法探析汉黄芩素治疗肝细胞癌作用机制和体外实验研究[J]. 中国全科医学, 2024, 27(32): 4040-4049. DOI: 10.12114/j.issn.1007-9572.2023.0238.

Figures/Tables 16

References 34

[1]	YANG J D，HAINAUT P，GORES G J，et al. A global view of hepatocellular carcinoma：trends，risk，prevention and management[J]. Nat Rev Gastroenterol Hepatol，2019，16（10）：589-604. DOI：10.1038/s41575-019-0186-y.
[2]	TORIMURA T，IWAMOTO H. Treatment and the prognosis of hepatocellular carcinoma in Asia[J]. Liver Int，2022，42（9）：2042-2054. DOI：10.1111/liv.15130.
[3]	WEN N Y，CAI Y L，LI F Y，et al. The clinical management of hepatocellular carcinoma worldwide：a concise review and comparison of current guidelines：2022 update[J]. Biosci Trends，2022，16（1）：20-30. DOI：10.5582/bst.2022.01061.
[4]	SULLIVAN L B，GUI D Y，VANDER HEIDEN M G. Altered metabolite levels in cancer：implications for tumour biology and cancer therapy[J]. Nat Rev Cancer，2016，16（11）：680-693. DOI：10.1038/nrc.2016.85.
[5]	VALKENBURG K C，DE GROOT A E，PIENTA K J. Targeting the tumour stroma to improve cancer therapy[J]. Nat Rev Clin Oncol，2018，15（6）：366-381. DOI：10.1038/s41571-018-0007-1.
[6]	ROSENBLUM D，JOSHI N，TAO W，et al. Progress and challenges towards targeted delivery of cancer therapeutics[J]. Nat Commun，2018，9（1）：1410. DOI：10.1038/s41467-018-03705-y.
[7]	WAITKUS M S，DIPLAS B H，YAN H. Biological role and therapeutic potential of IDH mutations in cancer[J]. Cancer Cell，2018，34（2）：186-195. DOI：10.1016/j.ccell.2018.04.011.
[8]	BUYEL J F. Plants as sources of natural and recombinant anti-cancer agents[J]. Biotechnol Adv，2018，36（2）：506-520. DOI：10.1016/j.biotechadv.2018.02.002.
[9]	CHENG F X，LIANG H，BUTTE A J，et al. Personal mutanomes meet modern oncology drug discovery and precision health[J]. Pharmacol Rev，2019，71（1）：1-19. DOI：10.1124/pr.118.016253.
[10]	SLIWOSKI G，KOTHIWALE S，MEILER J，et al. Computational methods in drug discovery[J]. Pharmacol Rev，2013，66（1）：334-395. DOI：10.1124/pr.112.007336.
[11]	NOOR F，TAHIR UL QAMAR M，ASHFAQ U A，et al. Network pharmacology approach for medicinal plants：review and assessment[J]. Pharmaceuticals，2022，15（5）：572. DOI：10.3390/ph15050572.
[12]	BANIK K，KHATOON E，HARSHA C，et al. Wogonin and its analogs for the prevention and treatment of cancer：a systematic review[J]. Phytother Res，2022，36（5）：1854-1883. DOI：10.1002/ptr.7386.
[13]	GU Y Q，ZHENG Q，FAN G F，et al. Advances in anti-cancer activities of flavonoids in Scutellariae radix：perspectives on mechanism[J]. Int J Mol Sci，2022，23（19）：11042. DOI：10.3390/ijms231911042.
[14]	LIPINSKI C A，LOMBARDO F，DOMINY B W，et al. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings[J]. Adv Drug Deliv Rev，2001，46（1/2/3）：3-26. DOI：10.1016/s0169-409x(00)00129-0.
[15]	XU Z，XU M，LIU P，et al. The mTORC2-Akt1 cascade is crucial for c-Myc to promote hepatocarcinogenesis in mice and humans[J]. Hepatology，2019，70（5）：1600-1613. DOI：10.1002/hep.30697.
[16]	ZHANG H Y. CCND1 silencing suppresses liver cancer stem cell differentiation through inhibiting autophagy[J]. Hum Cell，2020，33（1）：140-147. DOI：10.1007/s13577-019-00295-9.
[17]	DING H，WANG Y J，ZHANG H Y. CCND1 silencing suppresses liver cancer stem cell differentiation and overcomes 5-Fluorouracil resistance in hepatocellular carcinoma[J]. J Pharmacol Sci，2020，143（3）：219-225. DOI：10.1016/j.jphs.2020.04.006.
[18]	SHI L，CAO J，LEI X，et al. Multi-omics data identified TP53 and LRP1B as key regulatory gene related to immune phenotypes via EPCAM in HCC[J]. Cancer Med，2022，11（10）：2145-2158. DOI：10.1002/cam4.4594.
[19]	LONG J Y，WANG A Q，BAI Y，et al. Development and validation of a TP53-associated immune prognostic model for hepatocellular carcinoma[J]. EBioMedicine，2019，42：363-374. DOI：10.1016/j.ebiom.2019.03.022.
[20]	YANG H Y，SUN L J，GUAN A，et al. Unique TP53 neoantigen and the immune microenvironment in long-term survivors of hepatocellular carcinoma[J]. Cancer Immunol Immunother，2021，70（3）：667-677. DOI：10.1007/s00262-020-02711-8.
[21]	LUO X E，ZHOU N，WANG L，et al. Long noncoding RNA GATA3-AS1 promotes cell proliferation and metastasis in hepatocellular carcinoma by suppression of PTEN，CDKN1A，and TP53[J]. Can J Gastroenterol Hepatol，2019，2019：1389653. DOI：10.1155/2019/1389653.
[22]	LI B，LI A，YOU Z，et al. Epigenetic silencing of CDKN1A and CDKN2B by SNHG1 promotes the cell cycle，migration and epithelial-mesenchymal transition progression of hepatocellular carcinoma[J]. Cell Death Dis，2020，11（10）：823. DOI：10.1038/s41419-020-03031-6.
[23]	ZHOU J，HAN S，QIAN W C，et al. Metformin induces miR-378 to downregulate the CDK1，leading to suppression of cell proliferation in hepatocellular carcinoma[J]. Onco Targets Ther，2018，11：4451-4459. DOI：10.2147/OTT.S167614.
[24]	LIU H M，TAN H Y，LIN Y，et al. microRNA-1271-5p inhibits cell proliferation and enhances radiosensitivity by targeting CDK1 in hepatocellular carcinoma[J]. J Biochem，2020，167（5）：513-524. DOI：10.1093/jb/mvz114.
[25]	YIN S S，YANG S S，LUO Y M，et al. Cyclin-dependent kinase 1 as a potential target for lycorine against hepatocellular carcinoma[J]. Biochem Pharmacol，2021，193：114806. DOI：10.1016/j.bcp.2021.114806.
[26]	LIU Z，WANG Y，YAO Y T，et al. Quantitative proteomic and phosphoproteomic studies reveal novel 5-fluorouracil resistant targets in hepatocellular carcinoma[J]. J Proteomics，2019，208：103501. DOI：10.1016/j.jprot.2019.103501.
[27]	JIN A L，ZHANG C Y，ZHENG W J，et al. CD155/SRC complex promotes hepatocellular carcinoma progression via inhibiting the p38 MAPK signalling pathway and correlates with poor prognosis[J]. Clin Transl Med，2022，12（4）：e794. DOI：10.1002/ctm2.794.
[28]	ZHANG X，XU H，BI X Y，et al. Src acts as the target of matrine to inhibit the proliferation of cancer cells by regulating phosphorylation signaling pathways[J]. Cell Death Dis，2021，12（10）：931. DOI：10.1038/s41419-021-04221-6.
[29]	MA H Y，YAMAMOTO G，XU J，et al. IL-17 signaling in steatotic hepatocytes and macrophages promotes hepatocellular carcinoma in alcohol-related liver disease[J]. J Hepatol，2020，72（5）：946-959. DOI：10.1016/j.jhep.2019.12.016.
[30]	TANG B F，ZHU J Y，LI J，et al. The ferroptosis and iron-metabolism signature robustly predicts clinical diagnosis，prognosis and immune microenvironment for hepatocellular carcinoma[J]. Cell Commun Signal，2020，18（1）：174. DOI：10.1186/s12964-020-00663-1.
[31]	WANG G G，LI J H，YAO Y，et al. Small nucleolar RNA 42 promotes the growth of hepatocellular carcinoma through the p53 signaling pathway[J]. Cell Death Discov，2021，7（1）：347. DOI：10.1038/s41420-021-00740-5.
[32]	XU B，WEI Y G，LIU F，et al. Long noncoding RNA CERS6-AS1 modulates glucose metabolism and tumor progression in hepatocellular carcinoma by promoting the MDM2/p53 signaling pathway[J]. Cell Death Discov，2022，8（1）：348. DOI：10.1038/s41420-022-01150-x.
[33]	TEWARI D，PATNI P，BISHAYEE A，et al. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer：a novel therapeutic strategy[J]. Semin Cancer Biol，2022，80：1-17. DOI：10.1016/j.semcancer.2019.12.008.
[34]	HE Y，SUN M M，ZHANG G G，et al. Targeting PI3K/Akt signal transduction for cancer therapy[J]. Signal Transduct Target Ther，2021，6（1）：425. DOI：10.1038/s41392-021-00828-5.

组别	24 h	48 h	72 h
对照组	1.608±0.092	1.473±0.086	1.642±0.087
DMSO溶媒组	1.562±0.071	1.459±0.106	1.641±0.089
18.5 μmol/L	1.344±0.112^a	1.459±0.117	1.655±0.101
37.5 μmol/L	0.922±0.108^a	1.322±0.081^a	1.630±0.161
75.0 μmol/L	0.818±0.022^a	0.976±0.081^a	1.317±0.042^a
150.0 μmol/L	0.737±0.017^a	0.389±0.015^a	0.261±0.043^a
300.0 μmol/L	0.637±0.018^a	0.361±0.029^a	0.223±0.036^a
F值	148.5	178.1	271.2
P值	<0.000 1	<0.000 1	<0.000 1

组别	24 h	48 h	72 h
对照组	1.608±0.092	1.473±0.086	1.642±0.087
DMSO溶媒组	1.562±0.071	1.459±0.106	1.641±0.089
18.5 μmol/L	1.344±0.112^a	1.459±0.117	1.655±0.101
37.5 μmol/L	0.922±0.108^a	1.322±0.081^a	1.630±0.161
75.0 μmol/L	0.818±0.022^a	0.976±0.081^a	1.317±0.042^a
150.0 μmol/L	0.737±0.017^a	0.389±0.015^a	0.261±0.043^a
300.0 μmol/L	0.637±0.018^a	0.361±0.029^a	0.223±0.036^a
F值	148.5	178.1	271.2
P值	<0.000 1	<0.000 1	<0.000 1

组别	18.5 μmol/L	37.5 μmol/L	75.0 μmol/L	150.0 μmol/L	300.0 μmol/L
HepG2细胞活性	83.45±2.96	57.67±9.28	51.18±4.56	45.97±3.35	39.71±2.90
LO₂细胞活性	113.41±5.68	108.77±4.54	106.06±4.54	82.75±3.92	61.92±3.33
t值	10.06	11.06	17.57	15.96	11.23
P值	<0.000 1	<0.000 1	<0.000 1	<0.000 1	<0.000 1

组别	18.5 μmol/L	37.5 μmol/L	75.0 μmol/L	150.0 μmol/L	300.0 μmol/L
HepG2细胞活性	83.45±2.96	57.67±9.28	51.18±4.56	45.97±3.35	39.71±2.90
LO₂细胞活性	113.41±5.68	108.77±4.54	106.06±4.54	82.75±3.92	61.92±3.33
t值	10.06	11.06	17.57	15.96	11.23
P值	<0.000 1	<0.000 1	<0.000 1	<0.000 1	<0.000 1

组别	18.5 μmol/L	37.5 μmol/L	75.0 μmol/L	150.0 μmol/L	300.0 μmol/L
HepG2细胞活性	95.23±4.91	91.97±2.40	51.05±2.83	24.11±1.30	13.90±0.80
LO₂细胞活性	99.26±9.30	90.04±8.39	66.62±9.30	26.48±1.75	24.62±2.47
t值	0.857	0.496	3.587	2.428	9.237
P值	0.416	0.633	0.007	0.041	<0.001