UNASSIGNED: Network pharmacology methods were used to explore the potential targets and molecular mechanisms. LPS was used to stimulate RWPE-1 cells to construct an in vitro model of CP. An experimental autoimmune prostatitis (EAP) mice model was built. CCK-8 assay, EdU assay, BrdU detection, and Tunel assay were performed to evaluate the proliferation and apoptosis process of cells or tissues, respectively. DCFH-DA and Fluo-4 fluorescence probes were used to detect intracellular ROS and calcium concentrations. Von Frey filaments and open-field tests were utilized to evaluate pain response and depressive-like behavior of mice. Histopathology was evaluated through hematoxylin and eosin staining. RT-qPCR, Western blot, immunofluorescence, and immunohistochemistry were utilized to evaluate the transcription, expression, and location of related proteins. Molecular dynamics (MD) simulation and surface plasmon resonance (SPR) assay were performed to measure the binding capacity of sertraline and related proteins.
UNASSIGNED: Through a network pharmacology analysis, 27 potential targets of sertraline for CP were obtained, and 5 key targets (CHRM1, ADRA1B, HTR2B, HTR2A, and TRPV1) were finally identified. Functional experiments suggested that TRPV1 was involved in the proliferation, apoptosis inhibition, and ROS production of LPS-induced RWPE-1 cells. In vitro experiments showed that sertraline significantly inhibited cell proliferation, ROS generation, and transcription of inflammation cytokines of LPS-induced RWPE-1 cells. Additionally, sertraline markedly promoted the apoptosis level of LPS-stimulated RWPE-1 cells and elevated the expression level of BAX while reducing the expression levels of Bcl2 and Caspase-3. MD simulation and SPR assay confirmed the direct binding of sertraline to TRPV1. Moreover, sertraline significantly down-regulated the expression level of TRPV1 and inhibited calcium influx of LPS-induced RWPE-1 cells. TRPV1 agonist (Capsaicin) significantly restored the effects on proliferation, apoptosis, ROS production, and calcium influx of sertraline on LPS-induced RWPE-1 cells. Mice experiments demonstrated that sertraline treatment could reduce pain response, improve depression-like symptoms, and relieve local prostate inflammation of EAP mice, as well as down-regulated the expression level of TRPV1, inhibit the proliferation, and promote apoptosis of prostate tissues in EAP mice.
UNASSIGNED: The results revealed the anti-inflammatory effect of sertraline for RWPE-1 cells and EAP mice, and the potential mechanism was regulating the TRPV1 channel. It indicated that sertraline might serve as a complementary anti-inflammatory agent for CP.
■网络药理学方法用于探索潜在的靶标和分子机制。用LPS刺激RWPE-1细胞构建CP的体外模型。建立实验性自身免疫性前列腺炎(EAP)小鼠模型。CCK-8测定,EdU分析,BrdU检测,和Tunel实验用于评估细胞或组织的增殖和凋亡过程,分别。DCFH-DA和Fluo-4荧光探针用于检测细胞内ROS和钙离子浓度。使用VonFrey丝和开放场测试来评估小鼠的疼痛反应和抑郁样行为。通过苏木精和曙红染色评价组织病理学。RT-qPCR,蛋白质印迹,免疫荧光,和免疫组织化学被用来评估转录,表达式,以及相关蛋白质的位置。进行分子动力学(MD)模拟和表面等离子体共振(SPR)测定以测量舍曲林和相关蛋白的结合能力。
■通过网络药理学分析,获得了舍曲林对CP的27个潜在靶标,和5个关键目标(CHRM1、ADRA1B、HTR2B,HTR2A,和TRPV1)最终确定。功能实验表明TRPV1参与了细胞增殖,凋亡抑制,和LPS诱导的RWPE-1细胞的ROS产生。体外实验表明舍曲林显著抑制细胞增殖,ROS生成,和LPS诱导的RWPE-1细胞炎症细胞因子的转录。此外,舍曲林显着促进LPS刺激的RWPE-1细胞的凋亡水平,提高BAX的表达水平,同时降低Bcl2和Caspase-3的表达水平。MD模拟和SPR测定证实舍曲林与TRPV1的直接结合。此外,舍曲林显着下调TRPV1的表达水平,并抑制LPS诱导的RWPE-1细胞的钙内流。TRPV1激动剂(辣椒素)显着恢复对增殖的影响,凋亡,ROS生产,舍曲林对LPS诱导的RWPE-1细胞的钙流入。小鼠实验表明,舍曲林治疗可以减轻疼痛反应,改善抑郁症状,缓解EAP小鼠的局部前列腺炎症,以及下调TRPV1的表达水平,抑制细胞增殖,促进EAP小鼠前列腺组织凋亡。
■结果显示舍曲林对RWPE-1细胞和EAP小鼠的抗炎作用,可能的机制是调节TRPV1通道。这表明舍曲林可能作为CP的补充抗炎药。