目的 利用網(wǎng)絡(luò)藥理學(xué)、分子對(duì)接技術(shù)探究姜黃素在非小細(xì)胞肺癌治療中的潛在作用機(jī)制，并進(jìn)行體外實(shí)驗(yàn)驗(yàn)證。方法 利用PubChem和Swiss Target Prediction數(shù)據(jù)庫(kù)篩選姜黃素的藥物靶點(diǎn)；在GeneCards數(shù)據(jù)庫(kù)中獲得非小細(xì)胞肺癌的疾病靶點(diǎn)。利用韋恩在線分析軟件獲得藥物和疾病的交集基因，通過(guò)STRING網(wǎng)站和Cytoscape軟件將交集基因進(jìn)行蛋白質(zhì)-蛋白質(zhì)相互作用網(wǎng)絡(luò)（PPI）分析，根據(jù)節(jié)點(diǎn)的度值篩選核心靶點(diǎn)。運(yùn)用DAVID數(shù)據(jù)庫(kù)對(duì)潛在作用靶點(diǎn)進(jìn)行了基因本體（GO）和京都基因與基因組百科全書(shū)（KEGG）通路分析。通過(guò)AutoDock等軟件將姜黃素和核心靶點(diǎn)進(jìn)行分子對(duì)接，并通過(guò)Pymol軟件對(duì)結(jié)果進(jìn)行可視化分析。體外培養(yǎng)非小細(xì)胞肺癌A549細(xì)胞，將不同濃度（0、20、40 μmol·L⁻¹）的姜黃素作用于A549細(xì)胞，通過(guò)MTT實(shí)驗(yàn)、克隆形成實(shí)驗(yàn)、劃痕、Transwell實(shí)驗(yàn)和DAPI染色實(shí)驗(yàn)檢測(cè)姜黃素對(duì)非小細(xì)胞肺癌A549細(xì)胞增殖、遷移和凋亡的影響，通過(guò)Western blotting法檢測(cè)姜黃素對(duì)核心靶點(diǎn)絲氨酸和蘇氨酸激酶1（AKT1）、絲裂原活化蛋白激酶1（MAPK1）、熱休克蛋白（HSP90AA1）、非受體酪氨酸蛋白激酶（SRC）、信號(hào)傳導(dǎo)及轉(zhuǎn)錄激活蛋白3（STAT3）以及酪氨酸蛋白激酶/STAT3（JAK/STAT3）信號(hào)通路相關(guān)蛋白表達(dá)水平的影響。結(jié)果 基于網(wǎng)絡(luò)藥理學(xué)，篩選獲得姜黃素治療非小細(xì)胞肺癌的潛在作用靶點(diǎn)159個(gè)。按照節(jié)點(diǎn)的度值篩選出前5位的核心靶點(diǎn)為：AKT1、MAPK1、Hsp90AA1、SRC和STAT3。GO富集結(jié)果顯示，姜黃素治療非小細(xì)胞肺癌涉及多個(gè)生物過(guò)程，如信號(hào)轉(zhuǎn)導(dǎo)、啟動(dòng)子轉(zhuǎn)錄、蛋白質(zhì)磷酸化、JAK-STAT級(jí)聯(lián)反應(yīng)信號(hào)轉(zhuǎn)導(dǎo)途徑和細(xì)胞凋亡。并通過(guò)KEGG富集篩出多個(gè)信號(hào)通路，如癌癥信號(hào)通路、趨化因子信號(hào)通路、催乳素信號(hào)通路、AGE-RAGE信號(hào)通路和JAK-STAT信號(hào)通路等。分子對(duì)接顯示：姜黃素與核心靶點(diǎn)AKT1、Hsp90AA1、SRC和STAT3的結(jié)合能均小于−20.9 kJ·mol⁻¹，說(shuō)明姜黃素可能通過(guò)以上靶點(diǎn)治療非小細(xì)胞肺癌。細(xì)胞實(shí)驗(yàn)結(jié)果顯示，姜黃素抑制A549細(xì)胞的增殖能力、遷移能力并促進(jìn)細(xì)胞凋亡，同時(shí)發(fā)現(xiàn)姜黃素處理A549細(xì)胞后，細(xì)胞中AKT1、MAPK1、SRC、STAT3和JAK2蛋白的表達(dá)下調(diào)，但HSP90AA1蛋白的表達(dá)沒(méi)有變化。結(jié)論 姜黃素可能通過(guò)多靶點(diǎn)、多信號(hào)通路在治療非小細(xì)胞肺癌過(guò)程中發(fā)揮重要作用，姜黃素可能通過(guò)抑制JAK2/STAT3信號(hào)通路的表達(dá)影響非小細(xì)胞肺癌的增殖、遷移和凋亡。

Objective To explore the potential mechanism of curcumin in the treatment of non-small cell lung cancer (NSCLC) using network pharmacology and molecular docking techniques, and to validate in vitro experiments. Methods Curcumin drug targets were screened using PubChem and Swiss Target Prediction databases; And disease targets of NSCLC were obtained in GeneCards database. The intersection genes of drugs and diseases were obtained by using the Venny2.1.0. The intersection genes were analyzed by protein-protein interaction (PPI) network through the STRING database and the Cytoscape software, and the core targets were screened according to the degree value of the nodes. Then, GO function and KEGG pathway analysis were performed using the DAVID database. Curcumin and core targets were molecular docked by AutoDock software, and the results were visualized by Pymol software. NSCLC A549 cells were cultured and treated with curcumin at different concentrations (0, 20, 40 μmol·L⁻¹) in vitro. The effect of curcumin on cell proliferation, migration and apoptosis of NSCLC were determined by MTT assay, clones formation assay, nicking, Transwell assay and DAPI staining assay, The effect of curcumin on the expression levels of the core targets AKT1, MAPK1, Hsp90AA1, SCR, STAT3, and JAK/STAT3 signaling-related proteins were tested by Western blotting assay. Results Based on network pharmacology, 159 potential targets for the treatment of NSCLC. The top five core targets were selected according to the degree values of the nodes: AKT1, MAPK1, Hsp90AA1, SRC, and STAT3. GO enrichment results showed that curcumin treatment in NSCLC involved several biological processes, such as signal transduction, promoter transcription, protein phosphorylation, signal transduction pathway of JAK-STAT cascade, and apoptosis. Several signaling pathways, such as cancer signaling pathway, chemokine signaling pathway, prolactin signaling pathway, AGE-RAGE signaling pathway and JAK-STAT signaling pathway, were screened out by KEGG enrichment. Molecular docking showed that the binding energy of curcumin and the core targets AKT1, Hsp90AA1, SRC and STAT3 was less than −20.9 kJ·mol⁻¹, indicating that curcumin may be treating NSCLC. The results of cell experiments showed that curcumin inhibited the proliferation, migration and promoted apoptosis of A549 cells. Meanwhile, it was found that the expression of AKT1, MAPK1, SRC, STAT3 and JAK2 was downregulated in A549, but the expression of HSP90AA1 protein was unchanged. Conclusion Curcumin may play an important role in the treatment of NSCLC through a multi-targets, multiple pathway, and curcumin may affect the proliferation, migration and apoptosis of NSCLC by inhibiting the expression of JAK2/STAT3 signaling pathway.

R285.5

國(guó)家自然科學(xué)基金資助項(xiàng)目(82003216);山西省科技廳青年科技研究基金(20210302124580);呂梁市科技計(jì)劃項(xiàng)目（2023SHFZ50）;山西省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目（20231798）