背景及目的：激素性股骨头坏死（Glucocorticoid-induced Osteonecrosis of the femoral head, GIONFH）是非创伤性股骨头坏死中最常见的类型，其确切发病机制仍不明确，骨微血管内皮细胞（Bone microvascular endothelial cells, BMECs）损伤引起的股骨头微循环障碍在GIONFH发病中起重要作用。细胞程序性死亡是一种由特定基因调控的自主、有序的细胞死亡方式，包括凋亡、坏死性凋亡和铁死亡等类型，可以维持细胞内环境的稳定。在骨代谢调节中，细胞程序性死亡通过影响骨细胞的活性发挥着重要作用。中药骨碎补是中医“肾主骨”理论指导下补肾法的代表性药物，具有促进成骨、抑制骨吸收、促进血管形成、调节免疫及抗炎作用，已被广泛应用于治疗骨质疏松等骨科疾病。骨碎补有效成分为山奈酚、木犀草素和儿茶素等，其在激素诱导BMECs损伤及GIONFH中的作用仍有待探索。本文拟探究骨碎补对地塞米松诱导BMECs损伤的影响和对GIONFH的保护作用，以及程序性细胞死亡在其中的作用。

方法：研究采用转录组高通量测序、网络药理学、分子对接和体外实验验证相结合的方法。转录组高通量测序用于识别GIONFH差异表达基因。网络药理学用于预测骨碎补及其有效活性成分治疗GIONFH的潜在靶点及信号通路。分子对接预测有效活性成分与其靶点的结合模式及结合力。CCK-8实验筛选地塞米松抑制BMECs活性的最佳浓度和改善地塞米松抑制作用的最佳药物浓度，然后分组进行后续实验。EdU实验检测BMECs增殖，TUNEL实验和Annexin-FITC/PI实验检测BMECs凋亡，划痕实验及Transwell实验检测BMECs迁移，血管生成实验用于检测BMECs成血管能力，Western Blotting检测蛋白表达。

结果：1、网络药理学分析表明，山奈酚可调控PI3K/AKT/FoxO1通路，对GIONFH起保护作用；木犀草素可通过RIPK1/RIPK3/MLKL通路调控坏死性凋亡干预GIONFH。分子对接证实山奈酚和木犀草素与靶基因结合良好。2、体外实验表明，地塞米松干预后，BMECs增殖率、划痕愈合率、细胞迁移数、成管长度和分支点数目显著降低，TUNEL阳性细胞率和细胞凋亡率显著升高；山奈酚和木犀草素预处理后，BMECs增殖率、划痕愈合率、细胞迁移数、成管长度和分支点数目显著升高，TUNEL阳性细胞率和细胞凋亡率显著降低，PI3K抑制剂显著逆转山奈酚的上述作用。3、地塞米松可以显著促进Cleaved caspase-3和Bax表达，抑制MMP-2、Cyclin D1、Cyclin E1、VEGFA和Bcl2表达，山奈酚可以逆转地塞米松引起的蛋白表达改变。此外，地塞米松可以显著抑制PI3K、P-AKT、P-FoxO1、Bcl2表达，促进FoxO1、Bim、Bax表达，山奈酚可以减弱地塞米松引起的蛋白表达改变，而PI3K抑制剂可以逆转山奈酚的上述作用。4、地塞米松可以显著降低P-RIPK1/RIPK1、P-RIPK3/RIPK3和P-MLKL/MLKL比值，木犀草素可以逆转地塞米松的上述作用。5、转录组测序及生信分析结果表明，GIONFH的发病可能与细胞周期阻滞密切相关，CCNB1和CDK1是关键靶点。6、网络药理学分析表明，儿茶素是骨碎补调节GIONFH中铁死亡的重要成分，VEGFA是关键靶点，Hippo通路是最重要的信号通路。7、儿茶素可显著增强BMECs细胞活性，逆转地塞米松对细胞活性的抑制作用。地塞米松干预后，VEGFA和CCNB1（Cyclin B1）的表达显著降低，儿茶素可以逆转地塞米松引起的蛋白表达改变。

结论：1、山奈酚和木犀草素可以提高激素诱导的BMECs细胞活力、增殖、迁移、成血管和抗凋亡能力。2、山奈酚通过PI3K/AKT/FoxO1信号通路保护GIONFH中的BMECs，特别是抑制地塞米松引起的凋亡。3、地塞米松会激活RIPK1/RIPK3/MLKL信号通路，引起坏死性凋亡，但木犀草素可以通过抑制这一过程改善GIONFH中的BMECs损伤。4、GIONFH的发病可能与细胞周期阻滞密切相关，CCNB1和CDK1是潜在的药物治疗靶点。5、儿茶素可以显著增强BMECs活性，逆转激素对BMECs活性的抑制作用。儿茶素可能通过调控细胞周期对GIONFH起到保护作用，同时可能通过靶向VEGFA调控GIONFH中的铁死亡，具体机制仍需深入探究。

Background and Purpose: Glucocorticoids-induced osteonecrosis of the femoral head (GIONFH) is the most common type of non-traumatic osteonecrosis of the femoral head. The exact pathogenesis of GIONFH is still unclear, but microcirculation disorders of the femoral head caused by bone microvascular endothelial cell (BMEC) damage play an important role. Programmed cell death is a type of autonomous and orderly cell death regulated by specific genes, including apoptosis, necroptosis, and ferroptosis, which can maintain the stability of the intracellular environment. In bone metabolism regulation, programmed cell death plays an important role by affecting the activity of bone cells. Rhizoma Drynariae, a traditional Chinese medicine, is a representative drug for tonifying the kidney based on the Chinese medicine theory of “the kidney governs the bone”. It has been widely used to treat osteoporosis and other orthopedic diseases due to its effects in promoting bone formation, inhibiting bone resorption, promoting angiogenesis, regulating immunity, and exerting anti-inflammatory effects. The effective components of Rhizoma Drynariae include Kaempferol, Luteolin, and (+)-Catechin, and their effects on glucocorticoid-induced BMECs damage and GIONFH are still to be explored. This article aims to investigate the effects of Rhizoma Drynariae on dexamethasone-induced BMECs damage and its protective effect on GIONFH, as well as the role of programmed cell death in these processes.

Methods: This study employed a combination of high-throughput transcriptome sequencing, network pharmacology, molecular docking, and in vitro experiments for validation. High-throughput transcriptome sequencing was used to identify differentially expressed genes in GIONFH, while network pharmacology was used to predict potential targets and signaling pathways for the treatment of GIONFH with Rhizoma Drynariae and its active ingredients. Molecular docking was used to predict the binding mode and strength between active ingredients and their targets. The CCK-8 assay was used to determine the optimal concentration of dexamethasone for inhibiting BMECs activity and the optimal drug concentration for mitigating dexamethasone’s inhibitory effects, followed by grouping for subsequent experiments. The EdU assay was used to assess BMECs proliferation, while the TUNEL assay and Annexin-FITC/PI assay were used to detect BMECs apoptosis. Wound healing experiment and Transwell assay were used to investigate BMECs migration, and a tube formation experiment was used to evaluate BMECs angiogenic potential. Finally, Western blotting was used to measure protein expression.

Results: 1. Network pharmacology analysis revealed that Kaempferol has a protective effect on GIONFH by regulating the PI3K/AKT/FoxO1 pathway, while Luteolin can intervene in GIONFH by regulating necroptosis through the RIPK1/RIPK3/MLKL pathway. Molecular docking confirmed that Kaempferol and Luteolin bind well with their target genes. 2. In vitro experiments showed that after dexamethasone intervention, the proliferation rate, wound healing rate, number of migrating cells, tube length, and number of branch points of BMECs significantly decreased, while the rate of TUNEL-positive cells and cell apoptosis increased significantly. After pretreatment with Kaempferol and Luteolin, the proliferation rate, wound healing rate, number of migrating cells, tube length, and number of branch points of BMECs significantly increased, while the rate of TUNEL-positive cells and cell apoptosis decreased significantly. PI3K inhibitor significantly reversed the effects of Kaempferol mentioned above. 3. Dexamethasone can significantly promote the expression of Cleaved caspase-3 and Bax, inhibit the expression of MMP-2, Cyclin D1, Cyclin E1, VEGFA, and Bcl2, while Kaempferol can reverse the protein expression changes caused by dexamethasone. In addition, dexamethasone can significantly inhibit the expression of PI3K, P-AKT, P-FoxO1, and Bcl2, while promoting the expression of FoxO1, Bim, and Bax. Kaempferol can weaken the protein expression changes caused by dexamethasone, while PI3K inhibitor can reverse the effects of Kaempferol mentioned above. 4. Dexamethasone can significantly decrease the ratio of P-RIPK1/RIPK1, P-RIPK3/RIPK3, and P-MLKL/MLKL, while Luteolin can reverse the effects of dexamethasone mentioned above. 5. Transcriptome sequencing and bioinformatics analysis indicate that the pathogenesis of GIONFH may be closely related to cell cycle arrest, with CCNB1 and CDK1 as key targets. 6. Network pharmacology analysis shows that (+)-Catechin is an important component in regulating ferroptosis in GIONFH, with VEGFA as a key target, and the Hippo pathway as the most important signaling pathway. 7. (+)-Catechin can significantly enhance the activity of BMECs, and reverse the inhibitory effect of dexamethasone on cell activity. After dexamethasone intervention, the expression of VEGFA and CCNB1 (Cyclin B1) was significantly decreased, and (+)-Catechin can reverse the protein expression changes caused by dexamethasone.

Conclusion: 1. Kaempferol and Luteolin can enhance dexamethasone-induced cell viability, proliferation, migration, angiogenesis, and anti-apoptotic capacity in BMECs. 2. Kaempferol partially protects BMECs in GIONFH, particularly against dexamethasone-induced apoptosis, through the PI3K/AKT/FoxO1 signaling pathway. 3. Dexamethasone activates the RIPK1/RIPK3/MLKL signaling pathway, leading to necroptosis, but luteolin can improve BMECs damage in GIONFH by inhibiting this process. 4. The onset of GIONFH may be closely related to cell cycle arrest, and CCNB1 and CDK1 are potential drug therapy targets. 5. (+)-Catechin can significantly enhance BMECs activity and reverse the inhibitory effect of glucocorticoids on BMECs activity. (+)-Catechin may protect against GIONFH by regulating the cell cycle, and may also regulate ferroptosis in GIONFH by targeting VEGFA. The specific mechanism still requires further exploration.