Background and Objectives: Hesperetin (HSE) is a natural flavonoid derived from the hydrolysis of Hesperidin, which is mainly found in traditional natural Chinese herbs, such as Chenpi and Hovenia caryophyllus. HSE displays anti-inflammatory and antioxidant activities. However, its potential mechanism of action on bladder cancer (BLCA) remains unknown. The aim of this study was to investigate the potential mechanism of action of HSE on BLCA cells. background: Hesperetin (HSE) is a natural flavonoid derived from the hydrolysis of Hesperidin, which is mainly found in traditional natural Chinese herbs such as Chenpi and Hovenia caryophyllus. It has been shown that HSE displays anti-inflammatory, antioxidant activity. However, the potential mechanism of action on bladder cancer (BLCA) remains unknown. Methods: Network pharmacology analysis was used to construct a composite target network, combined with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify HSE-induced cell death patterns and signaling pathway alterations. Cytotoxicity evaluation was determined by CCK-8 assay. A clone formation assay was performed to assess cell proliferative capacity. Scratch and Transwell assays were performed to evaluate cell migration and invasion ability. Hoechst 33342 staining was visualized to observe morphological features of apoptosis. Apoptosis, cycle distribution, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) changes were examined by flow cytometry. Western blot analysis was performed to analyze the expression of key proteins associated with cell proliferation, apoptosis, cycle block, PI3K/AKT/FoxO3a and endoplasmic reticulum (ER) stress-mitochondrial pathways. objective: The aim of this study was to investigate the potential mechanism of action of HSE on BLCA cells. Results: Network pharmacology analysis was performed to identify 155 potential candidate targets of HSE-BLCA, and further topological analysis was performed to obtain 34 hub-gene. Enrichment analysis yielded patterns of death and key pathways, revealing that the anti-BLCA effect of HSE may be related to the positive regulation of PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. in vitro results showed that HSE blocked cell proliferation, migration, and invasion in a concentration-dependent manner and triggered apoptosis, G0/G1 phase blockade, ROS production, and MMP depolarization. In addition, Western blot results showed that HSE downregulated phosphorylated (p)-3-phosphoinositide-dependent kinase-1 (PI3K), phosphorylated (p)-AKT serine/threonine kinase 1 (AKT), phosphorylated (p)-Forkhead box O 3a (FoxO3a), anti-apoptotic proteins, proliferation-associated proteins, and cell cycle promoters, whereas the levels of proteins related to the expression of cell cycle regulators, pro-apoptotic proteins, and ER stress-mitochondrial pathway were up-regulated in BLCA cells by the intervention of HSE. PI3K agonist (YS-49) and ER stress inhibitor (4-PBA) partially or completely reversed HSE-mediated proliferation, apoptosis, and cycle blockade in BLCA cells. method: Network pharmacology analysis was used to construct a composite target network, combined with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify HSE-induced cell death patterns and signaling pathway alterations. Cytotoxicity evaluation was determined by CCK-8 assay. Clone formation assay to assess cell proliferative capacity. Scratch and Transwell assays were performed to evaluate cell migration and invasion ability. Hoechst 33342 staining was visualized to observe morphological features of apoptosis. Apoptosis, cycle distribution, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) changes were examined by flow cytometry. Western blot analysis was performed to analyze the expression of key proteins associated with cell proliferation, apoptosis, cycle block, PI3K/AKT/FoxO3a and endoplasmic reticulum (ER) stress-mitochondrial pathways. Conclusion: The anticancer effects of HSE in BLCA may be attributed to its coordination of actions, inhibiting cell proliferation, migration, and invasion, inducing apoptosis, G0/G1 phase arrest, generating reactive oxygen species, causing MMP loss, and engaging the caspase protein family. These actions are likely mediated through the PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. Thus, our findings suggest that HSE is a promising novel therapeutic candidate for the prevention and treatment of BLCA.

中文翻译：

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橙皮素通过 PI3K/AKT/FoxO3a 和 ER 应激线粒体途径抑制膀胱癌细胞增殖并促进细胞凋亡和细胞周期停滞

背景与目的：橙皮素（HSE）是一种由橙皮苷水解得到的天然黄酮类化合物，主要存在于陈皮、枳椇等传统天然中药材中。HSE 具有抗炎和抗氧化活性。然而，其对膀胱癌（BLCA）的潜在作用机制仍不清楚。本研究的目的是探讨 HSE 对 BLCA 细胞的潜在作用机制。背景：橙皮素（HSE）是由橙皮甙水解而成的天然黄酮类化合物，主要存在于陈皮、枳椇等传统天然中药材中。研究表明，HSE 具有抗炎、抗氧化活性。然而，对膀胱癌（BLCA）的潜在作用机制仍不清楚。方法：采用网络药理学分析构建复合靶点网络，结合基因本体论（GO）和京都基因与基因组百科全书（KEGG）富集分析来识别HSE诱导的细胞死亡模式和信号通路改变。通过CCK-8测定法测定细胞毒性评价。进行克隆形成测定以评估细胞增殖能力。进行划痕和Transwell实验来评估细胞迁移和侵袭能力。Hoechst 33342染色可视化观察细胞凋亡的形态特征。通过流式细胞术检查细胞凋亡、循环分布、活性氧（ROS）产生和线粒体膜电位（MMP）变化。进行蛋白质印迹分析来分析与细胞增殖、凋亡、周期阻滞、PI3K/AKT/FoxO3a 和内质网 (ER) 应激线粒体途径相关的关键蛋白的表达。目的：本研究的目的是探讨HSE对BLCA细胞的潜在作用机制。结果：通过网络药理学分析确定了HSE-BLCA的155个潜在候选靶点，并进一步进行拓扑分析获得了34个hub-gene。富集分析得出了死亡模式和关键通路，揭示了 HSE 的抗 BLCA 作用可能与 PI3K/AKT/FoxO3a 和 ER 应激线粒体通路的正调节有关。体外结果显示，HSE 以浓度依赖性方式阻断细胞增殖、迁移和侵袭，并引发细胞凋亡、G0/G1 期阻断、ROS 产生和 MMP 去极化。此外，Western blot结果显示，HSE下调磷酸化(p)-3-磷酸肌醇依赖性激酶-1 (PI3K)、磷酸化(p)-AKT丝氨酸/苏氨酸激酶1 (AKT)、磷酸化(p)-Forkhead box O 3a (FoxO3a)、抗凋亡蛋白、增殖相关蛋白和细胞周期启动子，而与细胞周期调节因子、促凋亡蛋白和 ER 应激线粒体途径表达相关的蛋白水平在BLCA细胞受HSE的干预。PI3K 激动剂 (YS-49) 和 ER 应激抑制剂 (4-PBA) 部分或完全逆转 BLCA 细胞中 HSE 介导的增殖、凋亡和周期阻断。方法：采用网络药理学分析构建复合靶点网络，结合基因本体论（GO）和京都基因与基因组百科全书（KEGG）富集分析，识别HSE诱导的细胞死亡模式和信号通路改变。通过CCK-8测定法测定细胞毒性评价。克隆形成测定评估细胞增殖能力。进行划痕和Transwell实验来评估细胞迁移和侵袭能力。Hoechst 33342染色可视化观察细胞凋亡的形态特征。通过流式细胞术检查细胞凋亡、循环分布、活性氧（ROS）产生和线粒体膜电位（MMP）变化。进行蛋白质印迹分析来分析与细胞增殖、凋亡、周期阻滞、PI3K/AKT/FoxO3a 和内质网 (ER) 应激线粒体途径相关的关键蛋白的表达。结论: BLCA中HSE的抗癌作用可能与其协同作用、抑制细胞增殖、迁移和侵袭、诱导细胞凋亡、G0/G1期阻滞、产生活性氧、引起MMP丢失以及与caspase蛋白结合有关。家庭。这些作用可能是通过 PI3K/AKT/FoxO3a 和 ER 应激线粒体途径介导的。因此，我们的研究结果表明，HSE 是预防和治疗 BLCA 的一种有前途的新型治疗候选药物。这些作用可能是通过 PI3K/AKT/FoxO3a 和 ER 应激线粒体途径介导的。因此，我们的研究结果表明，HSE 是预防和治疗 BLCA 的一种有前途的新型治疗候选药物。这些作用可能是通过 PI3K/AKT/FoxO3a 和 ER 应激线粒体途径介导的。因此，我们的研究结果表明，HSE 是预防和治疗 BLCA 的一种有前途的新型治疗候选药物。