目的 采用網(wǎng)絡(luò)藥理學(xué)和分子對(duì)接方法,探究鼓槌石斛素治療宮頸癌的作用機(jī)制并進(jìn)行體外實(shí)驗(yàn)驗(yàn)證。方法 通過(guò)中藥系統(tǒng)藥理學(xué)數(shù)據(jù)庫(kù)與分析平臺(tái)(TCMSP)、PharmMaper和SwissTarget Prediction平臺(tái)獲得鼓槌石斛素的預(yù)測(cè)靶點(diǎn);在GeneCards、TTD、OMIM、Disgenet、Drugbank疾病數(shù)據(jù)庫(kù)篩選宮頸癌的潛在靶點(diǎn);使用R軟件Venn包獲取鼓槌石斛素與宮頸癌的交集靶點(diǎn)基因,利用STRING網(wǎng)站與Cytoscape軟件獲取蛋白質(zhì)-蛋白質(zhì)相互作用(PPI)網(wǎng)絡(luò),篩得核心靶點(diǎn),進(jìn)行基因本體(GO)和京都基因與基因組百科全書(KEGG)富集分析;運(yùn)用Autodock Vina 1.1.2軟件和Pymol軟件進(jìn)行鼓槌石斛素和核心靶點(diǎn)之間的分子對(duì)接和可視化分析。體外培養(yǎng)宮頸癌SiHa細(xì)胞,用0、50、100 μmol·L^-1鼓槌石斛素處理細(xì)胞,采用MTT法、Transwell實(shí)驗(yàn)分別檢測(cè)鼓槌石斛素對(duì)細(xì)胞增殖、遷移和侵襲的影響,Western blotting檢測(cè)鼓槌石斛素對(duì)PI3K/Akt/mTOR信號(hào)通路中p-PI3K、PI3K、p-Akt、Akt、mTOR關(guān)鍵蛋白的調(diào)控作用。結(jié)果 共得到79個(gè)鼓槌石斛素調(diào)控宮頸癌的潛在交集靶點(diǎn),按照網(wǎng)絡(luò)拓?fù)浞治鲋械亩?degree)值篩選出HSP90AA1、ESR1、PIK3CA、mTOR、MAPK1、ABL1、PARP1等多個(gè)核心靶點(diǎn);KEGG富集篩選出PI3K/Akt、Focal adhesion、細(xì)胞衰老、催乳素等30條信號(hào)通路;分子對(duì)接結(jié)果顯示,其中靶蛋白HSP90AA1、ESR1、PIK3CA、mTOR、MAPK1與鼓槌石斛素結(jié)合能絕對(duì)值較高,說(shuō)明可能是鼓槌石斛素治療宮頸癌的作用位點(diǎn)。細(xì)胞實(shí)驗(yàn)驗(yàn)證表明,鼓槌石斛素處理SiHa細(xì)胞后,SiHa細(xì)胞增殖明顯減少(P<0.01),且與鼓槌石斛素的濃度呈現(xiàn)負(fù)相關(guān),同時(shí)SiHa細(xì)胞的遷移和侵襲能力下降。鼓槌石斛素處理SiHa細(xì)胞后,細(xì)胞中p-PI3K、Akt、p-Akt表達(dá)及p-PI3K/PI3K和p-Akt/Akt下調(diào)(P<0.05)。結(jié)論 鼓槌石斛素可通過(guò)多靶點(diǎn)、多通路發(fā)揮治療宮頸癌的作用,鼓槌石斛素可能通過(guò)抑制PI3K/Akt/mTOR信號(hào)通路的表達(dá)來(lái)影響宮頸癌細(xì)胞的增殖、遷移和侵襲。

Objective To investigate the mechanism of chrysotoxine in the treatment of cervical cancer using network pharmacology and molecular docking and validate the results through cell-based experiments. Methods Predicted targets of chrysotoxine were obtained by TCMSP, PharmMaper and SwissTarget Prediction platform. The potential targets of cervical cancer were screened in GeneCards, TTD, OMIM, Disgenet and Drugbank disease databases. R software Venn package was used to obtain common target genes of chrysotoxine and cervical cancer. STRING website and Cytoscape software were used to obtain the PPI network, screened the core targets, and analyzed them for GO and KEGG enrichment. Molecular docking and visualization between chrysotoxine and the core targets were conducted using Autodock Vina 1.1.2 and Pymol software. Cervical cancer cells SiHa were cultured and treated with 0, 50, 100 μmol·L^-1 chrysotoxine in vitro, and the effects of chrysotoxine on cell proliferation, migration and invasion were detected by MTT assay and Transwell assay, respectively. The molecular mechanism of chrysotoxine against cervical cancer was performed by Western blotting, focusing on the regulatory proteins of p-PI3K, PI3K, p-Akt, Akt, and mTOR in the PI3K/Akt/mTOR signaling pathway. Results A total of 79 potential common targets of chrysotoxine against cervical cancer were obtained, and then screened multiple core targets according to the degree value, such as:HSP90AA1, ESR1, PIK3CA, mTOR, MAPK1, ABL1, PARP1, et al. KEGG enrichment screened 30 pathways, including PI3K/Akt signaling pathway, Focal adhesion signaling pathway, cellular senescence signaling pathway, prolactin signaling pathway, et al. Molecular docking results showed that chrysotoxine had a higher affinity for target proteins, such as HSP90AA1, ESR1, PIK3CA, mTOR, and MAPK1, suggesting their potential role in chrysotoxine's anti-cervical cancer effects. Cellular experiments demonstrated that chrysotoxine significantly reduced the proliferation of SiHa cells (P < 0.01) and decreased their migration and invasion ability. Western blotting results further confirmed the down-regulation (P < 0.05) of p-PI3K, Akt, p-Akt, p-PI3K/PI3K, and p-Akt/Akt expression after treatment with chrysotoxine. Conclusion Chrysotoxine can exert therapeutic effects on cervical cancer through multi-targets and multi-pathways, and chrysotoxine may affect the proliferation and migration of cervical cancer cells by inhibiting the expression of PI3K/Akt/mTOR signaling pathway. These provide a reliable theoretical basis for the treatment of cervical cancer with chrysotoxine, and provide a new idea for the traditional Chinese medicine treatment of cervical cancer in the future.

R285.5

湖南省教育廳項(xiàng)目(18B538,22C0682);長(zhǎng)沙醫(yī)學(xué)院ESI學(xué)科專項(xiàng)(2022CYY033);湖南省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃(湘教通[2022]174號(hào)-一般項(xiàng)目-4587);湖南省衛(wèi)生和計(jì)劃生育委員會(huì)科研基金項(xiàng)目(C20180139)