消癥抑癌方对卵巢癌SKOV3细胞增殖、迁移的影响

Effects of Xiaozheng Yiai decoction on the proliferation and migration of ovarian cancer SKOV3 cells

目的 探讨消癥抑癌方对人卵巢癌细胞SKOV3增殖和迁移的影响及潜在作用机制。方法 利用网络药理学分析消癥抑癌方抗卵巢癌的关键活性成分和核心靶点,并通过基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析筛选其相关生物学过程和通路。制备消癥抑癌方含药血清,用含药血清处理卵巢癌SKOV3细胞。采用CCK-8法和EdU染色法检测消癥抑癌方含药血清对SKOV3细胞增殖的影响;采用划痕实验和Transwell迁移实验检测消癥抑癌方含药血清对SKOV3细胞迁移的影响;采用Western blotting法检测消癥抑癌方含药血清对SKOV3细胞自噬蛋白(P62)、自噬相关蛋白3(ATG3)、微管相关蛋白1轻链3 B(LC3B)及腺苷酸活化蛋白激酶(AMPK)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白的影响。结果 网络药理学研究显示,消癥抑癌方抗卵巢癌的关键活性成分是槲皮素、山柰酚、木犀草素、汉黄芩素、柚皮素等,核心靶点是MAPK3、AKT1、SRC、MAPK1、TP53等。KEGG富集分析共得到171条通路(P<0.01),其中Degree值排名较靠前且与卵巢癌相关的通路主要涉及癌症通路、PI3K-AKT通路、MAPK通路、AMPK通路、自噬、mTOR通路等。体外实验结果表明,与对照组相比,10%和20%含药血清组SKOV3细胞存活率和EdU阳性细胞率均降低(P<0.001),细胞迁移率下降(P<0.001),迁移细胞数也减少(P<0.01或P<0.001)。Western blotting结果表明,10%和20%含药血清干预后,P62、ATG3、LC3B-Ⅱ蛋白表达水平均上调(P<0.05或P<0.01或P<0.001),p-AMPK/AMPK的表达水平上调(P<0.001),p-AKT/AKT和p-mTOR/mTOR的表达水平降低(P<0.05或P<0.01)。结论 消癥抑癌方抑制卵巢癌SKOV3细胞的增殖和迁移,其作用机制可能与调控AMPK/AKT/mTOR通路,进而阻断自噬流有关。

Objective To investigate the effects of Xiaozheng Yiai decoction on the proliferation and migration of ovar-ian cancer SKOV3 cells and to explore the underlying mechanism.Methods The key active components and core targets of the decoction against ovarian cancer were analyzed with network pharmacology,and the related biological processes and pathways were screened with gene ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)enrichment analysis.Serum containing Xiaozheng Yiai decoction was prepared and SKOV3 cells were treated with it.Its effects on the proliferation of SKOV3 cells was detected with CCK-8 and EdU staining;its effects on the mi-gration of SKOV3 cells was determined with wound healing assay and Transwell migration assay;its effects on the auto-phagy protein(P62),autophagy-related protein 3(ATG3),microtubule-associated protein 1 light chain 3 Beta(LC3B)and adenosine 5'-monophosphate-activated protein kinase(AMPK)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway related proteins in SKOV3 cells were assessed with Western blotting.Results Network pharmacology studies showed that the key active components of Xiaozheng Yiai decoction against ovarian canc-er were quercetin,kaempferol,luteolin,wogonin,naringenin,and the core targets were MAPK3,AKT1,SRC,MAPK1,TP53,and so on.KEGG enrichment analysis identified 171 pathways(P<0.01).The pathways with the high-est Degree ranking and associated with ovarian cancer mainly involved the cancer pathway,PI3K-AKT pathway,MAPK pathway,AMPK pathway,autophagy,and mTOR pathway.The results of in vitro experiments showed that compared with the control group,the 10%and 20%drug-containing serum groups had decreased cell viability,EdU positive cell rate,cell migration,and number of migrating cells(all P<0.001).The results of Western blotting showed that the protein expression levels of P62,ATG3 and LC3B-II were up-regulated after treatment with 10%and 20%drug-containing serum(all P<0.05),the p-AMPK/AMPK ratio was up-regulated(P<0.001),and the expression levels of p-AKT/AKT and p-mTOR/mTOR decreased(P<0.05 or P<0.01).Conclusion Xiaozheng Yiai decoction can inhibit the proliferation and migration of ovarian cancer SKOV3 cells,possibly via the regulation of AMPK/AKT/mTOR path-way and the blocking of autophagic flux.