Wang Zaizhong, Cheng Lulu, Shang Zhigang, Li Zhihui, Zhao Yuping, Jin Wenwen, Li Yingyue, Su Fangchu, Mao Xiaobo, Chen Chuanliang, Zhang Jianhua
Medical Engineering Technology and Data Mining Institute, Zhengzhou University, Zhengzhou, China.
Department of Neurosurgery, Zhumadian Central Hospital, Zhumadian, China.
Front Pharmacol. 2021 Apr 7;12:646187. doi: 10.3389/fphar.2021.646187. eCollection 2021.
To analyze the key targets and potential mechanisms underlying the volatile components of Georgi acting on gliomas through network pharmacology combined with biological experiments. We have extracted the volatile components of by gas chromatography-mass spectrometry (GC-MS) and determined the active components related to the onset and development of gliomas by combining the results with the data from the Traditional Chinese Medicine Systems Pharmacology database. We screened the same targets for the extracted active components and gliomas through network pharmacology and then constructed a protein-protein interaction network. Using a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we analyzed the protein effects and regulatory pathways of the common targets. Lastly, we employed ELISA and Western blot in verifying the key targets in the regulatory pathway. We ultimately determined that the active component in Georgi related to the onset and development of gliomas was Wogonin. The results of the network pharmacology revealed 85 targets for glioma and Wogonin. We used gene ontology to analyze these target genes and found that they involved 30 functions, such as phosphatidylinositol phosphokinase activation, while the KEGG analysis showed that there were 10 regulatory pathways involved. Through the following analysis, we found that most of the key target genes are distributed in the PI3K-Akt and interleukin 17 signaling pathways. We then cultured U251 glioma cells for the experiments. Compared with the control group, no significant change was noted in the caspase-3 expression; however, cleaved caspase-3 expression increased significantly and was dose-dependent on Wogonin. The expression of Bad and Bcl-2 with 25 μM of Wogonin has remained unchanged, but when the Wogonin dose was increased to 100 μM, the expression of Bad and Bcl-2 was noted to change significantly (Bad was significantly upregulated, while Bcl-2 was significantly downregulated) and was dose-dependent on Wogonin. The ELISA results showed that, compared with the control group, the secretion of tumor necrosis factor alpha, IL-1β, and IL-6 decreased as the Wogonin concentration increased. Tumor necrosis factor alpha downregulation had no significant dose-dependent effect on Wogonin, the inhibitory effect of 25 μM of Wogonin on IL-6 was not significant, and IL-1β downregulation had a significant dose-dependent effect on Wogonin. Wogonin might promote the apoptosis of glioma cells by upregulating proapoptotic factors, downregulating antiapoptotic factors, and inhibiting the inflammatory response, thereby inhibiting glioma progression.
通过网络药理学结合生物学实验分析地锦草挥发成分作用于胶质瘤的关键靶点及潜在机制。我们采用气相色谱 - 质谱联用（GC - MS）法提取了地锦草的挥发成分，并将结果与中药系统药理学数据库的数据相结合，确定了与胶质瘤发生发展相关的活性成分。通过网络药理学筛选出提取的活性成分与胶质瘤的共同靶点，进而构建蛋白质 - 蛋白质相互作用网络。利用基因本体论（Gene Ontology）和京都基因与基因组百科全书（KEGG）分析，我们分析了共同靶点的蛋白质作用及调控途径。最后，我们采用酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法（Western blot）验证调控途径中的关键靶点。我们最终确定，地锦草中与胶质瘤发生发展相关的活性成分是汉黄芩素。网络药理学结果显示，胶质瘤和汉黄芩素共有85个靶点。我们利用基因本体论分析这些靶基因，发现它们涉及30种功能，如磷脂酰肌醇磷酸激酶激活，而KEGG分析表明有10条调控途径。通过后续分析，我们发现大多数关键靶基因分布在PI3K - Akt和白细胞介素17信号通路中。然后我们培养U251胶质瘤细胞进行实验。与对照组相比，caspase - 3表达无显著变化；然而，裂解的caspase - 3表达显著增加，且对汉黄芩素呈剂量依赖性。25 μM汉黄芩素处理时，Bad和Bcl - 2的表达未发生变化，但当汉黄芩素剂量增加到100 μM时，Bad和Bcl - 2的表达发生显著变化（Bad显著上调，而Bcl - 2显著下调），且对汉黄芩素呈剂量依赖性。ELISA结果显示，与对照组相比，随着汉黄芩素浓度增加，肿瘤坏死因子α、IL - 1β和IL - 6的分泌减少。肿瘤坏死因子α下调对汉黄芩素无显著剂量依赖性效应，25 μM汉黄芩素对IL - 6的抑制作用不显著，而IL - 1β下调对汉黄芩素具有显著剂量依赖性效应。汉黄芩素可能通过上调促凋亡因子、下调抗凋亡因子并抑制炎症反应来促进胶质瘤细胞凋亡，从而抑制胶质瘤进展。
