Number of DEPs in each group.

AimsThe aim of this study was to investigate the mechanism of action of SB on TRP-induced abnormalities of bone metabolism in LNCaP cells.MethodsThe effects of different concentrations of SB and TRP alone and in combination on the proliferation of LNCaP cells were examined by CCK-8 assay, and the half maximal inhibitory concentration were screened for the subsequent experiments. Transwell migration and invasion assays were used to further investigate the effects of SB and TRP alone and in combination on the migration and invasion ability of LNCaP cells. Based on Tandem Mass Tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, the differentially expressed proteins (DEPs) of LNCaP cells in the control group, SB group, TRP group and combination group were quantified. Bioinformatics technology was used to analyze the DEPs of LNCaP cells in each group. At last, the achieved key targets were verified by western blot.ResultsThe inhibitory effects of different concentrations of SB and TRP alone and in combination on the proliferation, migration and invasion of LNCaP cells showed both time-dependent and concentration-dependent effects, and the inhibitory effects of the combination of drugs on LNCaP cells were more significant. The proteomics results showed that a total of 153 DEPs were identified in the SB group and the control group, 100 DEPs were identified in the TRP group and the control group, and 524 DEPs were identified in the combination group and the control group.Bioinformatics analysis showed that a higher number of DEPs were enriched in the IL-17 signaling pathway in the SB and combined treatment groups compared to the control group. These findings suggest a potential role of SB and the combined treatment in modulating the IL-17 signaling pathway. In contrast, the same DEPs were found to be enriched in both the Circadian entrainment and Apelin signaling pathways in the TRP versus control and combined treatment versus control comparisons.ConclusionsSB may regulate TRP-induced bone metabolism abnormalities in LNCaP cells through the IL-17 signaling pathway as well as five DEPs: p-ERK2, RELA, HSP90B1, GNAI1and GNAI3.研究目的：本研究旨在探究SB对TRP诱导的LNCaP细胞骨代谢异常的作用机制。研究方法：采用CCK-8法检测不同浓度SB、TRP单独及联合使用对LNCaP细胞增殖的影响，并筛选出半数抑制浓度用于后续实验。采用Transwell迁移与侵袭实验进一步探究SB、TRP单独及联合使用对LNCaP细胞迁移与侵袭能力的作用。基于串联质量标签（TMT）标记结合液相色谱-串联质谱（LC-MS/MS）技术，对对照组、SB组、TRP组及联合给药组LNCaP细胞的差异表达蛋白（DEPs）进行定量分析。采用生物信息学技术对各组LNCaP细胞的差异表达蛋白进行分析。最后通过蛋白质印迹（western blot）验证筛选得到的关键靶点。研究结果：不同浓度的SB、TRP单独及联合使用对LNCaP细胞增殖、迁移与侵袭的抑制作用均呈时间依赖性与浓度依赖性，且联合给药组的抑制效果更为显著。蛋白质组学结果显示，SB组与对照组相比共鉴定出153个差异表达蛋白，TRP组与对照组相比共鉴定出100个差异表达蛋白，联合给药组与对照组相比共鉴定出524个差异表达蛋白。生物信息学分析显示，相较于对照组，SB组与联合给药组中更多的差异表达蛋白富集于白介素-17（IL-17）信号通路，提示SB及联合给药可能通过调控该通路发挥作用。与之相反，在TRP组与对照组、联合给药组与对照组的比较中，相同的差异表达蛋白均富集于节律同步（Circadian entrainment）与Apelin信号通路。研究结论：SB可能通过白介素-17（IL-17）信号通路以及p-ERK2、RELA、HSP90B1、GNAI1、GNAI3这5个差异表达蛋白，调控TRP诱导的LNCaP细胞骨代谢异常。