骨肉瘤（OS）通常被认为是一种起源于成骨间充质干细胞的恶性肿瘤，约占肉瘤的20%。黄芩苷是从黄芩中分离出来的一种生物活性黄酮苷，已被证明具有有效的抗炎和神经保护特性。 探讨黄芩苷在体外发挥抗骨肉瘤作用和促进成骨作用的潜在机制。细胞计数试剂盒（CCK） -8)、划痕实验和Transwell实验分别评估不同浓度(20、40和80μM)黄芩苷对U2OS细胞增殖、侵袭和迁移的影响。通过检测骨钙素 (OCN)、骨桥蛋白 (OPN) 和 runt 相关转录因子 2 (RUNX2) 等成骨细胞标志物，进行蛋白质印迹和 qRT-PCR 分析，以评估黄芩苷对 OS 细胞成骨潜力的影响，如以及核因子 kappa B 配体 (RANKL) 的破骨细胞标记受体激活剂。此外，黄芩苷对上皮间质转化 (EMT) 标记物（N-钙粘蛋白、E-钙粘蛋白、波形蛋白）和核因子 κB (NF-κB) 信号通路相关蛋白（p-p65、p-IκBα、通过蛋白质印迹分析评估 OS 细胞中的 p65、IκBα）。通过ALP染色和茜素红S（ARS）染色检查黄芩苷处理细胞中碱性磷酸酶（ALP）的活性和矿化能力。黄芩苷显着抑制OS细胞U2OS侵袭（p < 0.01）、迁移（p < 0.01） ）和不同浓度下的增殖（p < 0.05）。此外，黄芩苷治疗显着增加了E-钙粘蛋白的水平，同时降低了波形蛋白和N-钙粘蛋白的表达水平（p < 0.01），从而促进了EMT。黄芩苷处理后，RUNX2、OPN和OCN的蛋白和mRNA表达水平显着升高，而RANKL蛋白的表达水平降低（p < 0.05），表明成骨分化增强。用黄芩苷处理的组表现出更高的 ALP 活性和矿化能力 (p < 0.01)。此外，黄芩苷处理显着降低了p-IκBα和p-p65蛋白的表达水平，以及p-IκBα/IκBα和p-p65/p65的比率(p < 0.01)。黄芩苷的这些作用具有浓度依赖性，浓度越高，作用越强。在体外，黄芩苷具有抗 OS 作用，并可能通过抑制 NF-κB 通路活性来促进成骨分化。

Osteosarcoma (OS) is commonly recognized as a malignant cancer originating from bone-forming mesenchymal stem cells, comprising approximately 20% of sarcomas. Baicalin, a bioactive flavonoid glycoside isolated from Scutellaria baicalensis, has been demonstrated to possess potent anti-inflammatory and neuroprotective properties.To explore the potential mechanisms through which baicalin exerts anti-osteosarcoma effects and facilitates osteogenesis in vitro.Cell Counting Kit-8 (CCK-8), scratch assay, and transwell assay were employed to assess the effects of baicalin at varying concentrations (20, 40, and 80 μM) on U2OS cell proliferation, invasion, and migration, respectively. Western blot and qRT-PCR analyses were conducted to evaluate the influence of baicalin on the osteogenic potential of OS cells by examining osteoblast markers such as osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor 2 (RUNX2), as well as the osteoclast marker-receptor activator of nuclear factor kappa B ligand (RANKL). Additionally, the impact of baicalin on epithelial-mesenchymal transition (EMT) markers (N-cadherin, E-cadherin, Vimentin) and proteins related to the Nuclear factor κB (NF-κB) signaling pathway (p-p65, p-IκBα, p65, IκBα) in OS cells was evaluated via western blot analysis. The activity and mineralization capacity of Alkaline Phosphatase (ALP) in baicalin-treated cells were examined through ALP staining and Alizarin Red S (ARS) staining.Baicalin exhibited significant suppression of OS cell U2OS invasion (p < 0.01), migration (p < 0.01), and proliferation (p < 0.05) at various concentrations. Additionally, baicalin treatment notably increased the E-cadherin protein level, while decreasing the expression levels of Vimentin and N-cadherin proteins (p < 0.01), thus promoting EMT. Following baicalin treatment, there was a marked elevation in the protein and mRNA expression levels of RUNX2, OPN, and OCN, while the expression level of RANKL protein was reduced (p < 0.05), indicating enhanced osteogenic differentiation. The groups treated with baicalin exhibited higher ALP activity and mineralization ability (p < 0.01). Moreover, baicalin treatment significantly reduced the expression levels of p-IκBα and p-p65 proteins, as well as the ratios of p-IκBα/IκBα and p-p65/p65 (p < 0.01). These effects of baicalin were concentration-dependent, with higher concentrations yielding stronger effects.In vitro, baicalin demonstrates anti-OS effects and facilitates osteogenic differentiation, potentially by inhibiting NF-κB pathway activity.