Pharmacological Mechanisms of Kirenol Against Ovarian Carcinoma: A Network Pharmacology and Experimental Validation Study in vitro

Materials and methodsNetwork pharmacologyPrediction and screening of kirenol targetsThe potential target of kirenol was forecasted using the SwissTarget Prediction database (http://www.swisstargetprediction.ch/). This online platform is renowned for its capability in predicting small molecule drug targets. The screening criteria employed were based on the following parameters: "high" gastrointestinal absorption (GI absorption) and positive results in at least 2 out of the following criteria: Lipinski, Ghose, Veber, Egan, and Muegge rules. These stringent criteria ensure a robust and reliable prediction of kirenol's target, enhancing our understanding of its pharmacological mechanism of action.Prediction of OC targetsThe search terms "Ovarian Cancer," "Ovarian Neoplasms," "Ovarian Carcinoma," and "Ovarian Tumor" were input into several renowned databases, including Genecards (https://www.genecards.org/), Drugbank (https://go.drugbank.com/), PharmGKB (https://www.pharmgkb.org/), TTD (http://db.idrblab.net/ttd/), and OMIM (https://omim.org/), with the purpose of identifying crucial targets for the treatment of ovarian cancer. Subsequently, the retrieved targets from these databases were amalgamated, and duplicate entries were meticulously eliminated to ensure a comprehensive and non-redundant collection of core targets for further analysis in the context of ovarian cancer therapy.Protein–protein interaction networkThe intersection of drug targets and disease targets presents a valuable approach to identify potential targets for kirenol in the context of ovarian cancer. To construct the PPI network, the predicted targets were submitted to the String database (https://string-db.org/), specifying "Homo sapiens" as the species, and applying the following options: "high confidence > 0.9" and "hide disconnected nodes in the network." Default settings were maintained for other parameters during network construction. Subsequently, topological parameters of the PPI network, including betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), network centrality (NC), and local edge connectivity (LAC), were analyzed using the CytoNCA plug-in in Cytoscape 3.9.0 software.The topological parameters of the PPI network were further assessed based on the screening criterion, whereby values exceeding the median value of all nodes were considered. Targets meeting this criterion were subjected to two consecutive screenings, resulting in the identification of core targets for kirenol's action in ovarian cancer. These core targets hold potential significance for further exploration and evaluation as therapeutic candidates in the treatment of ovarian cancer.Molecular dockingThe 2D structures of the core compounds were obtained from the PubChem database. The 3D structures of the core targets, namely MTOR (PDB ID: 1aue), EGFR (PDB ID: 1ivo), IL6 (PDB ID: 1alu), JAK2 (PDB ID: 2b7a), MAP2K1 (PDB ID: 1s9j), MMP9 (PDB ID: 1gkc), CDK4 (PDB ID: 2w96), and AR (PDB ID: 1e3g), were sourced from the Protein Data Bank (www.rcsb.org/) as predictive structures.The protein structures were prepared for molecular docking using PyMol 2.3.4 (https://pymol.org/). The preparation involved the removal of water molecules, addition of nonpolar hydrogen atoms to the structures, and saving the processed files in PDBQT format. The binding energy, serving as a quantitative measure of the molecular compound's affinity to its target, was employed to evaluate the strength of the binding interactions.Molecular docking was carried out utilizing Autodock Vina 1.1.2 (https://vina.scripps.edu/), a computational method allowing the docking of ligands to their respective target molecules. This approach offered significant insights into the potential binding interactions, shedding light on the possible molecular mechanisms underlying the observed effects of the core compounds on the selected targets.GO enrichment analysis and KEGG pathway enrichment analysisThe predicted targets were subjected to comprehensive analysis using the biological information annotation database DAVID (http://david.nifcrf.gov/). This analysis involved conducting enrichment studies to elucidate the Gene Ontology (GO) function and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway associated with the identified targets. Through this approach, we aimed to gain insights into the biological processes and signaling pathways in which the predicted targets are implicated, shedding light on the potential mechanisms of action of kirenol in the context of ovarian cancer treatment.Cell experimentsCell cultureHuman ovarian cancer cell lines SKOV3 and A2780, as well as the human normal ovarian epithelial cell line IOSE-80, were procured from iCell Bioscience Inc (Shanghai, China) for experimental use. A2780 and IOSE-80 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (BI) (Solarbio Science Technology Co., Ltd., Beijing, China) and 1% penicillin/streptomycin (P/S) at 37°C in a humidified atmosphere with 5% CO2. SKOV3 cells were maintained in McCOY's 5A medium (Boster Biological Technology Co., Ltd., Wuhan, China), while the other culture conditions remained consistent with the aforementioned cells.Kirenol (purity ≥99.6%) and LY294002 (purity ≥99.76%) were obtained from Tsbiochem (#T4S1943; #T2008). These compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 mmol/L. The cells were treated with various doses of Kirenol in fresh medium, with DMSO (at the same volume as the high dose group) serving as the solvent control group for comparison.Cell viability assaySKOV3, A2780 and IOSE-80 cells were separately seeded at a density of 2000,1000 and 1000 cells per well, respectively, in 96-well plates. After 6 hours of incubation, various concentrations of Kirenol-containing medium (0, 100, 200, 300, 400, 500, 600, 700 μmol/L) were added to the cells, and they were further cultured for 72 hours. To assess cell viability, the CCK-8 assay kit (Abbkine Scientific Co., Ltd., Wuhan, China) was utilized, with a mixture of complete medium and CCK-8 at a ratio of 9:1. The cells were then incubated at 37°C for 1 hour. Subsequently, the optical density (OD) was measured at 450 nm using a microplate reader to determine the IC50 value. Additionally, the cells were seeded onto a 96-well plate and allowed to adhere for 6 hours. The original medium was then replaced with Kirenol-containing medium at various concentrations (0, 100, 200, 300, 400, 500, 600, 700 μmol/L). At 24, 48, and 72 hours, the original medium was replaced with a CCK-8 mixed solution. The 96-well plate was further incubated at 37°C for 1 hour, after which the optical density was measured at 450 nm.These experiments were conducted to evaluate the cytotoxic effect of Kirenol on SKOV3, A2780 and IOSE-80 cells and to determine the half-maximal inhibitory concentration (IC50) of the compound in these cell lines. The CCK-8 assay allowed for the assessment of cell viability at different time points and Kirenol concentrations, providing valuable insights into its potential anti-cancer activity.Colony formation experimentPrior to the clonogenic assay, SKOV3 and A2780 cells were subjected to a 72-hour pretreatment with varying concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells. Following this pretreatment, the cells were plated at a density of 500 cells per Petri dish and allowed to grow for a period of 10–14 days.After the incubation period, the cells were fixed using 100% methanol for 30 minutes and then stained with 0.2% crystal violet solution for 1 hour. Subsequently, the cells were thoroughly washed and left to dry.The clonogenic assay was performed to assess the long-term proliferative potential and survival of SKOV3 and A2780 cells after treatment with different concentrations of Kirenol. This experimental approach allows for the observation of colony formation and provides valuable information about the compound's ability to inhibit cell growth and colony formation, thereby contributing to the evaluation of its potential as an anti-cancer agent.Wound-healing assaySKOV3 and A2780 cells were cultured in 6-well plates, forming a monolayer. Subsequently, the cell monolayers were intentionally scratched using a 200 μl pipette tip to create wounds. Afterward, the cells were carefully washed with PBS.Next, SKOV3 and A2780 cells were treated with different concentrations of Kirenol, namely 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells. The wound healing process was closely monitored and recorded at 0 hours (immediately after scratching) and at 48 hours using microscopy, and photographs were captured.The wound healing assay was performed to assess the migratory capability of SKOV3 and A2780 cells in response to Kirenol treatment. By observing and documenting the closure of the scratched wounds over time, this assay provides valuable insights into the compound's potential to impede cell migration and motility, which are important aspects of cancer cell metastasis and invasion.Cell cycle by flow cytometrySKOV3 and A2780 cells were subjected to a 72-hour pretreatment with varying concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells. After the pretreatment period, the cells were detached, centrifuged, and resuspended in pre-chilled PBS. Subsequently, they were fixed with 70% ethanol at -4°C overnight. For cell cycle analysis, the fixed cells were stained with PI/RNase staining buffer (Keygentec, JiangSu, China). Each flow cytometer tube was loaded with 500 μL of cell suspensions and allowed to stain for 30 minutes in the dark. Flow cytometry, using BD Biosciences equipment, was employed to determine the cell cycle distribution. Through flow cytometry, the distribution of cells in different phases of the cell cycle (G0/G1, S, and G2/M) was assessed. This analysis provides valuable information on how Kirenol influences the cell cycle progression and proliferation of SKOV3 and A2780 cells, offering insights into its potential as a regulator of cell growth and a candidate for ovarian cancer treatment.Apoptosis by flow cytometryThe Annexin V-FITC/PI Apoptosis Detection Kit (Keygentec, JiangSu, China) was employed for flow cytometry analysis to assess apoptosis. SKOV3 and A2780 cells were subjected to a 72-hour pretreatment with different concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells. Following the pretreatment period, the cells were detached using EDTA-free trypsin, centrifuged at 800 rpm for 5 minutes, and subsequently re-suspended in pre-cooled PBS.Next, the supernatant was removed, and the cells were suspended in 300 μL of binding buffer, along with 3 μL of Annexin V-FITC and 3 μL of Propidium Iodide reagent. The cell suspension was then incubated in the dark for 15 minutes. Flow cytometry, utilizing BD Biosciences equipment, was employed to measure and analyze apoptosis. This experimental setup enabled the detection and quantification of apoptotic events in SKOV3 and A2780 cells upon exposure to different concentrations of Kirenol. By utilizing Annexin V-FITC and Propidium Iodide, apoptotic cells can be differentiated from live and dead cells, providing valuable insights into the compound's pro-apoptotic effects and potential as a therapeutic agent for ovarian cancer.Western blotSKOV3 and A2780 cells were subjected to a 72-hour pretreatment with different concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells. In the subsequent response experiment, the cells were treated with LY294002 (10 μmol/L) alone or in combination with Kirenol (100 μmol/L) for an additional 48 hours.Cell lysates were prepared using RIPA lysis buffer (Beyotime Biotech, Jiangsu, China) supplemented with phenylmethylsulphonyl fluoride (PMSF; Solarbio, Beijing, China) in a ratio of RIPA to PMSF as 100:1. After cell lysis, the protein concentration was determined using a BCA Protein assay Kit (Beyotime Biotechnology Co., LTD., Shanghai, China). Protein samples were then denatured by heating at 99°C for 15 minutes. Subsequently, polyacrylamide gel electrophoresis (PAGE) rapid preparation kit (Servicebio Technology Co., Ltd., Wuhan, China) was utilized to separate and resolve the protein samples, followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, California, United States). The PVDF membranes were blocked with 5% skimmed milk for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies, and subsequently incubated with secondary antibodies for 2 hours at room temperature. Finally, the protein bands were visualized using a Tanon 5200 Chemiluminescence Imaging System, and ECL western blotting substrate (Tianneng Life Science Co., LTD., Shanghai, China) was employed for signal detection. ACTIN served as the internal control for protein normalization. The main antibodies included ACTIN (#YM3028;Immunoway, China), Matrix metalloproteinase (MMP)-2 (#ab92536; Abcam, Cambridge, United States), Bcl-2 (#T40056; Abmart, China), Bax (#T40051; Abmart, China), PI3K(#T40064; Abmart), Phospho-PI3K (Tyr467/199) (#T40065; Abmart), AKT(#T55561; Abmart), and Phospho- AKT (Ser473) (#T40067; Abmart), CCND1(#YM0176; Immunoway), CDK4(#ab108357; Abcam), and Phospho- RB (Ser 807) (#ab184796); Abcam). Image J (version, 1.51J8) was used to determine blot’s intensity.This series of experiments facilitated the investigation of protein expression and alterations in response to Kirenol treatment, alone or in combination with LY294002, to elucidate potential molecular mechanisms underlying Kirenol's effects on SKOV3 and A2780 cells. Western blotting analysis allowed for the characterization of protein changes and signaling pathways involved in the response to treatment.Statistical analysisStatistical analyses were carried out using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, United States). The data are presented as the mean ± standard deviation (SD). For comparisons between two groups, t-test was employed, while one-way ANOVA was used for comparisons among multiple groups. P-values less than 0.05 were considered statistically significant. All experiments were performed in triplicate to ensure robustness and reproducibility of the findings.

材料与方法
网络药理学
奇壬醇靶点的预测与筛选
采用SwissTarget Prediction数据库（http://www.swisstargetprediction.ch/）预测奇壬醇（kirenol）的潜在靶点。该在线平台以预测小分子药物靶点的能力著称。筛选标准基于以下参数：“高”胃肠道吸收（gastrointestinal absorption，GI absorption），且在Lipinski、Ghose、Veber、Egan和Muegge规则中至少满足2项阳性结果。这些严格标准确保了奇壬醇靶点预测的稳健性与可靠性，有助于加深对其药理作用机制的理解。
卵巢癌靶点预测
将“Ovarian Cancer”“Ovarian Neoplasms”“Ovarian Carcinoma”“Ovarian Tumor”作为检索词，输入Genecards（https://www.genecards.org/）、Drugbank（https://go.drugbank.com/）、PharmGKB（https://www.pharmgkb.org/）、TTD（http://db.idrblab.net/ttd/）和OMIM（https://omim.org/）等知名数据库，以识别卵巢癌（Ovarian Cancer，简称OC）治疗的关键靶点。随后合并各数据库检索到的靶点，仔细去除重复条目，确保获得全面无冗余的核心靶点集合，用于后续卵巢癌治疗相关分析。
蛋白质-蛋白质相互作用网络
药物靶点与疾病靶点的交集是识别奇壬醇作用于卵巢癌潜在靶点的有效方法。为构建蛋白质-蛋白质相互作用（Protein–Protein Interaction，简称PPI）网络，将预测靶点提交至String数据库（https://string-db.org/），指定物种为智人（Homo sapiens），并应用“高置信度＞0.9”和“隐藏网络中不连通节点”的参数，其他参数保持默认设置。随后利用Cytoscape 3.9.0软件中的CytoNCA插件分析PPI网络的拓扑参数，包括介数中心性（betweenness centrality，BC）、 closeness中心性（closeness centrality，CC）、度中心性（degree centrality，DC）、特征向量中心性（eigenvector centrality，EC）、网络中心性（network centrality，NC）和局部边连通性（local edge connectivity，LAC）。
进一步基于筛选标准评估PPI网络拓扑参数：节点值超过所有节点中位数的靶点被纳入考虑。通过两次连续筛选，确定奇壬醇作用于卵巢癌的核心靶点。这些核心靶点作为卵巢癌治疗候选靶点具有潜在重要性，值得进一步探索与评估。
分子对接
从PubChem数据库获取核心化合物的二维结构。核心靶点（MTOR，PDB ID：1aue；EGFR，PDB ID：1ivo；IL6，PDB ID：1alu；JAK2，PDB ID：2b7a；MAP2K1，PDB ID：1s9j；MMP9，PDB ID：1gkc；CDK4，PDB ID：2w96；AR，PDB ID：1e3g）的三维结构来源于蛋白质数据库（Protein Data Bank，简称PDB，网址www.rcsb.org/）。
使用PyMol 2.3.4（https://pymol.org/）预处理蛋白质结构：去除水分子、添加非极性氢原子，并将处理后的文件保存为PDBQT格式。以结合能作为分子化合物与靶点亲和力的定量指标，评估结合相互作用强度。
采用Autodock Vina 1.1.2（https://vina.scripps.edu/）进行分子对接，该计算方法可实现配体与靶点分子的对接。此方法为潜在结合相互作用提供重要见解，有助于揭示核心化合物对所选靶点作用的潜在分子机制。
GO富集分析与KEGG通路富集分析
利用生物信息注释数据库DAVID（http://david.nifcrf.gov/）对预测靶点进行综合分析，包括基因本体（Gene Ontology，简称GO）功能和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，简称KEGG）通路的富集研究。通过该方法阐明预测靶点参与的生物学过程与信号通路，加深对奇壬醇治疗卵巢癌潜在作用机制的理解。
细胞实验
细胞培养
从上海iCell生物科技有限公司获取人卵巢癌细胞系SKOV3、A2780及人正常卵巢上皮细胞系IOSE-80。A2780和IOSE-80细胞培养于含10%胎牛血清（fetal bovine serum，FBS，BI品牌；北京Solarbio科技有限公司）和1%青霉素/链霉素（penicillin/streptomycin，简称P/S）的RPMI1640培养基中，置于37℃、5%CO₂的 humidified培养箱中。SKOV3细胞培养于McCOY's 5A培养基（武汉Boster生物科技有限公司），其他培养条件与上述细胞一致。
奇壬醇（纯度≥99.6%）和LY294002（纯度≥99.76%）购自Tsbiochem公司（货号T4S1943；T2008）。将化合物溶于二甲基亚砜（dimethyl sulfoxide，简称DMSO）中，浓度为500 mmol/L。用含不同剂量奇壬醇的新鲜培养基处理细胞，以等体积DMSO（与高剂量组相同）作为溶剂对照组。
细胞活力测定
将SKOV3、A2780和IOSE-80细胞分别以每孔2000、1000和1000个细胞的密度接种于96孔板。孵育6小时后，加入含不同浓度奇壬醇（0、100、200、300、400、500、600、700 μmol/L）的培养基，继续培养72小时。采用CCK-8检测试剂盒（武汉Abbkine科学有限公司）评估细胞活力：将完全培养基与CCK-8以9:1比例混合，37℃孵育1小时后，用酶标仪在450 nm波长下测定光密度（optical density，简称OD），计算半数抑制浓度（half-maximal inhibitory concentration，简称IC50）。此外，将细胞接种于96孔板并贴壁6小时，更换为含不同浓度奇壬醇（0、100、200、300、400、500、600、700 μmol/L）的培养基。在24、48和72小时时更换为CCK-8混合液，37℃孵育1小时后测定450 nm处OD值。
这些实验用于评估奇壬醇对SKOV3、A2780和IOSE-80细胞的细胞毒性作用，并确定其在这些细胞系中的IC50值。CCK-8实验可在不同时间点和奇壬醇浓度下评估细胞活力，为其潜在抗癌活性提供有价值的见解。
集落形成实验
克隆形成实验前，用不同浓度奇壬醇预处理SKOV3和A2780细胞72小时：SKOV3细胞为0、100、150、200 μmol/L；A2780细胞为0、100、200、300 μmol/L。预处理后，以每皿500个细胞的密度接种于培养皿，培养10-14天。
孵育后，用100%甲醇固定细胞30分钟，再用0.2%结晶紫溶液染色1小时，随后彻底洗涤并干燥。
克隆形成实验用于评估不同浓度奇壬醇处理后SKOV3和A2780细胞的长期增殖潜能与存活能力。该实验可观察集落形成情况，为化合物抑制细胞生长和集落形成的能力提供信息，有助于评估其作为抗癌剂的潜力。
划痕愈合实验
将SKOV3和A2780细胞培养于6孔板中形成单层。用200 μl移液器吸头在单层细胞上划痕制造伤口，随后用PBS仔细洗涤细胞。
用不同浓度奇壬醇处理SKOV3和A2780细胞：SKOV3细胞为0、100、150、200 μmol/L；A2780细胞为0、100、200、300 μmol/L。分别在0小时（划痕后立即）和48小时通过显微镜观察并记录伤口愈合过程，拍摄照片。
划痕愈合实验用于评估SKOV3和A2780细胞对奇壬醇处理的迁移能力。通过观察和记录划痕伤口随时间的闭合情况，该实验为化合物抑制细胞迁移和运动的潜力提供见解，这是癌细胞转移和侵袭的重要方面。
流式细胞术检测细胞周期
用不同浓度奇壬醇预处理SKOV3和A2780细胞72小时：SKOV3细胞为0、100、150、200 μmol/L；A2780细胞为0、100、200、300 μmol/L。预处理后，消化细胞、离心、重悬于预冷PBS中，随后用70%乙醇在-4℃固定过夜。细胞周期分析时，用PI/RNase染色缓冲液（江苏Keygentec公司）染色固定细胞：每管流式管加入500 μL细胞悬液，避光染色30分钟。采用BD Biosciences设备进行流式细胞术，测定细胞周期分布。通过流式细胞术评估细胞在G0/G1、S和G2/M期的分布，为奇壬醇如何影响SKOV3和A2780细胞的细胞周期进程和增殖提供见解，有助于评估其作为细胞生长调节剂和卵巢癌治疗候选药物的潜力。
流式细胞术检测凋亡
采用Annexin V-FITC/PI凋亡检测试剂盒（江苏Keygentec公司）进行流式细胞术分析以评估凋亡。用不同浓度奇壬醇预处理SKOV3和A2780细胞72小时：SKOV3细胞为0、100、150、200 μmol/L；A2780细胞为0、100、200、300 μmol/L。预处理后，用无EDTA胰酶消化细胞，800 rpm离心5分钟，重悬于预冷PBS中。
去除上清液，将细胞重悬于300 μL结合缓冲液中，加入3 μL Annexin V-FITC和3 μL碘化丙啶（Propidium Iodide，简称PI）试剂，避光孵育15分钟。采用BD Biosciences设备进行流式细胞术，测量并分析凋亡情况。该实验设置可检测和定量SKOV3和A2780细胞在不同浓度奇壬醇作用下的凋亡事件。通过Annexin V-FITC和PI可区分凋亡细胞与活细胞和死细胞，为化合物的促凋亡作用及其作为卵巢癌治疗剂的潜力提供见解。
蛋白质印迹法
用不同浓度奇壬醇预处理SKOV3和A2780细胞72小时：SKOV3细胞为0、100、150、200 μmol/L；A2780细胞为0、100、200、300 μmol/L。在后续响应实验中，用LY294002（10 μmol/L）单独或与奇壬醇（100 μmol/L）联合处理细胞48小时。
用含苯甲基磺酰氟（phenylmethylsulphonyl fluoride，简称PMSF；北京Solarbio公司）的RIPA裂解缓冲液（江苏Beyotime生物科技有限公司）制备细胞裂解液，RIPA与PMSF比例为100:1。细胞裂解后，用BCA蛋白定量试剂盒（上海Beyotime生物科技有限公司）测定蛋白浓度。蛋白样品在99℃加热15分钟变性，随后用聚丙烯酰胺凝胶电泳（polyacrylamide gel electrophoresis，简称PAGE）快速制备试剂盒（武汉Servicebio科技有限公司）分离蛋白，转移至聚偏二氟乙烯膜（polyvinylidene difluoride，简称PVDF；Bio-Rad公司，美国Hercules）。PVDF膜用5%脱脂牛奶室温封闭2小时，随后4℃孵育一抗过夜，再室温孵育二抗2小时。最后用Tanon 5200化学发光成像系统可视化蛋白条带，用ECL蛋白印迹底物（上海Tianneng生命科学有限公司）检测信号。肌动蛋白（ACTIN）作为蛋白归一化的内参。主要抗体包括：肌动蛋白（ACTIN，货号YM3028；Immunoway公司，中国）、基质金属蛋白酶2（Matrix metalloproteinase 2，简称MMP-2，货号ab92536；Abcam公司，美国Cambridge）、Bcl-2（货号T40056；Abmart公司，中国）、Bax（货号T40051；Abmart公司，中国）、PI3K（货号T40064；Abmart公司）、磷酸化PI3K（Tyr467/199，货号T40065；Abmart公司）、AKT（货号T55561；Abmart公司）、磷酸化AKT（Ser473，货号T40067；Abmart公司）、CCND1（货号YM0176；Immunoway公司）、CDK4（货号ab108357；Abcam公司）、磷酸化RB（Ser 807，货号ab184796；Abcam公司）。用Image J软件（版本1.51J8）测定条带强度。
该系列实验有助于研究奇壬醇单独或与LY294002联合处理对SKOV3和A2780细胞蛋白表达的影响及变化，以阐明奇壬醇作用的潜在分子机制。蛋白质印迹分析可表征处理响应中的蛋白变化和信号通路。
统计分析
采用GraphPad Prism 8.0软件（GraphPad Software公司，美国La Jolla）进行统计分析。数据以均数±标准差（mean ± standard deviation，简称SD）表示。两组比较采用t检验，多组比较采用单因素方差分析（one-way ANOVA）。P＜0.05被认为具有统计学意义。所有实验均进行三次重复，以确保结果的稳健性和可重复性。