基于网络药理学研究青蒿鳖甲汤合调营达卫布津方对衰老角质形成细胞的调控作用

To study the regulatory effect of Qinghao Biejia Decoction combined with Tiaoying Dawei Bujin Decoction on senescent keratinocytes based on network pharmacology

关键词:  青蒿鳖甲汤  调营达卫布津方  皮肤衰老  机制  体外实验

Key words:  Qinghao Biejia Decoction  Tiaoying Dawei Bujin Decoction  skin aging  mechanism  vitro experiment

摘要:

[目的] 基于网络药理学研究青蒿鳖甲汤合调营达卫布津方对衰老角质形成细胞的调控作用。[方法] 通过中药系统药理学数据库与分析平台（TCMSP）数据库获得青蒿鳖甲汤合调营达卫布津方的主要活性成分及其对应靶点信息；通过TTD、OMIM、Disgenet、GeneCards、Drugbank数据库获得皮肤衰老的相关靶点；在Cytoscape 3.7.2软件中绘制交集基因蛋白互作（PPI）网络图；利用Metascape数据库对核心靶点进行基因本体（GO）功能富集和京都基因与基因组百科全书（KEGG）通路富集分析；通过体外实验检测磷酸化蛋白激酶B（p-AKT）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、磷酸化细胞外信号调节激酶（p-ERK）、过氧化物酶体增殖物激活受体γ（PPAR-γ）、聚丝蛋白（FLG）、β-葡萄糖脑苷脂酶（GCASE）表达，验证其改善衰老角质形成细胞（HaCat）的作用机制。[结果] 通过网络构建获得青蒿鳖甲汤合调营达卫布津方的主要活性成分包括槲皮素、山柰酚、芍药苷、哈巴俄苷、牡丹皮苷H；经PPI网络分析获得5个关键靶点，包括肿瘤坏死因子（TNF）、蛋白激酶B1（AKT1）、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）和血管内皮生长因子A（VEGFA）；GO功能和KEGG通路富集显示青蒿鳖甲汤合调营达卫布津方改善皮肤衰老所作用的信号通路包括缺氧诱导因子-1（HIF-1）、丝裂原活化蛋白激酶（MAPK）、磷酸肌醇3-激酶/蛋白激酶B（PI3K/AKT）信号通路。体外实验证实与模型组比较，40 μg/mL的青蒿鳖甲汤合调营达卫布津方可以抑制p-AKT、p-mTOR（P＜0.05）、p-ERK（P＜0.01）蛋白质的磷酸化，促进PPAR-γ（P＜0.05）、FLG、GCASE蛋白表达。[结论] 研究初步揭示了青蒿鳖甲汤合调营达卫布津方从“多成分-多靶点-多途径”改善皮肤衰老的作用机制，其路径可能是基于中西医皮肤组织生物同一性与功能趋同性，以补肾益阴及调营达卫布津法改善皮毛疏泄与润泽的功能及状态，影响调控了PI3K/AKT和细胞外信号调节激酶（ERK）信号通路及其下游与皮肤屏障功能相关的蛋白，达到延缓皮肤衰老的作用。

Abstract:

[Objective] To study the regulatory effect of Qinghao Biejia Decoction combined with Tiaoying Dawei Bujin（QHBJ） Decoction on senescent keratinocytes based on network pharmacology. [Methods] The main active ingredients and their corresponding target information were obtained through Traditional Chinese Medicine Systematic Pharmacology（TCMSP） database. The targets related to skin aging were obtained through TTD，OMIM，Disgenet，GeneCards，and Drugbank databases. Network diagram of protein interaction（PPI） of intersection genes were drawed in Cytoscape3.7.2 software. Gene ontology（GO） functional enrichment and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis of core targets were performed using Metascape database. By detecting the protein expression of Phosphorylated protein kinase B（p-AKT），phosphorylated mammalian target of rapamycin（p-mTOR），phosphorylated extracellular signal-regulated kinase（p-ERK），peroxisome proliferator-activated receptor-γ（PPAR-γ），filaggrin（FLG），β-glucosidase（GCASE） to verify the mechanism of QHBJ Decoction to improve the aging HaCat cells. [Results] The main active ingredients of QHBJ Decoction include quercetin，kaempferol，paeoniflorin，paeonidanin；mudanpioside-h. Five key targets were obtained by PPI network analysis including tumor necrosis factor（TNF），protein kinase B1（AKT1），interleukin 6（IL-6），interleukin 1β（IL-1β），and vascular endothelial growth factor A（VEGFA）. GO function and KEGG pathway enrichment show that QHBJ Decoction act on hypoxia-inducible factor-1（HIF-1），phosphatidylinositol 3-kinase/protein kinase B（PI3K/AKT），mitogen-activated protein kinase（MAPK） and other signaling pathways，and then play a role in improving skin aging. Compared with the model group，40 μg/mL QHBJ Decoction can inhibit the phosphorylation of p-AKT，p-mTOR（P<0.05） and p-ERK（P<0.01） proteins，and promote the expression of PPAR-γ（P<0.05），FLG and GCASE proteins. [Conclusion] This study preliminarily revealed the mechanism of QHBJ Decoction improving skin aging from “multi-component-multi-target-multi-pathway”. The path may be based on the biological identity and functional convergence of skin tissues in traditional Chinese and Western medicine. To improve the function and state of “dispersion” and “moistening” by “reinforce in the kidney and nourishing yin” and “adjusting ying and wei and circulating body fluid and its metabolism”. The mechanism of action may be to delay the effect of skin aging through the regulation of PI3K/AKT and ERK signaling pathway and its downstream proteins related to the skin barrier function.