【摘要】 目的 探讨柚皮素（NGN）治疗糖皮质激素诱导的激素性股骨头坏死（SONFH）的机制。方法 使用甲泼尼龙（MPS）处理构建SONFH大鼠模型，并行NGN及锌原卟啉（ZnPP）干预。采用Micro-CT分析股骨头组织形态学变化，检测大鼠血清骨钙素（OCN）及股骨头组织中血红素加氧酶-1(HO-1)、缺氧诱导因子-α(HIF-1α)水平。采用地塞米松（DEX）处理MC3T3-E1细胞构建细胞模型，随后进行NGN干预。生物信息学分析结合分子对接技术推定NGN的作用靶点，并行Pull-down实验验证。通过细胞转染敲低HO-1的表达。分析MC3T3-E1细胞活力、增殖、凋亡、迁移能力。采用血管生成实验评估人脐静脉内皮细胞（HUVECs）的血管生成能力。结果 Micro-CT分析发现，较对照组，MPS组大鼠骨小梁厚度、骨小梁数量明显降低，而骨表面积/骨体积比和骨小梁分离度显著增加（P<0.001）。体外实验结果表明，DEX可抑制MC3T3-E1细胞增殖，促进细胞凋亡，并增加活性氧生成（P<0.01）;DEX抑制成骨分化相关指标矿化结节形成及成骨相关基因（Runt相关转录因子2、骨桥蛋白、骨钙素）的表达（P<0.01）；然而，NGN处理部分逆转了上述影响。DEX显著抑制HUVECs迁移、血管生成和血管生成相关标志物（血小板内皮细胞黏附因子-1、血管内皮生长因子和血管性血友病因子）的表达（P<0.01），然而，NGN处理对上述效应无显著影响，但NGN条件培养基[CM(NGN)]处理部分逆转了上述效应（P<0.01）。生物信息学分析结合Pull-down实验结果表明，HO-1是NGN的作用靶点。DEX处理显著下调HO-1的表达，而NGN干预部分抵消DEX诱导的抑制效应（P<0.01）。HO-1敲低抵消了NGN的治疗效果（P<0.01）。与MPS单独给药相比，NGN和MPS联合给药上调大鼠股骨头组织中HO-1和HIF-1α的表达，而HO-1抑制剂ZnPP进一步上调HO-1的表达，但下调HIF-1α蛋白水平（P<0.01）。结论 NGN通过激活HO-1的表达和活性，调节HIF-1α/VEGF通路促进成骨细胞分化、骨形成和血管生成，发挥对SONFH的治疗作用。更多还原

【Abstract】 Objective To investigate the mechanism of Naringenin(NGN) in the treatment of steroid(glucosteroid)-induced osteonecrosis of the femoral head(SONFH). Methods A SONFH rat model was established using Methylprednisolone(MPS) treatment, followed by intervention with NGN and zinc protoporphyrin(ZnPP). Micro-CT was used to analyze the morphological changes in femoral head tissues, and the levels of osteocalcin(OCN) in rat serum as well as heme oxygenase-1(HO-1) and hypoxia-inducible factor-1α(HIF-1α) in femoral bone tissue were measured. A cellular model was constructed by treating MC3T3-E1 cells with Dexamethasone(DEX), followed by NGN intervention. Bioinformatics analysis combined with molecular docking technology was used to predict the target of NGN, and the Pulldown experiment was performed for validation. The expression of HO-1 was knocked down through cell transfection, to analyze the viability, proliferation, apoptosis, and migration of MC3T3-E1 cells, and angiogenesis assays were conducted to evaluate the angiogenic potential of human umbilical vein endothelial cells(HUVECs). Results Micro-CT analysis revealed that, compared with the control group, the trabecular thickness and trabecular number were significantly reduced in the MPS group, while the bone surface area/bone volume ratio and trabecular separation were significantly increased(P<0.001). In vitro experimental results indicated that DEX inhibited the proliferation of MC3T3-E1 cells, promoted cell apoptosis, and increased reactive oxygen species generation(P<0.01), and that DEX suppressed the formation of mineralized nodules, a key indicator of osteogenic differentiation, and downregulated the expression of osteogenesis-related genes(Runt-related transcription factor 2, osteopontin, osteocalcin)(P<0.01). However, NGN treatment partially reversed these effects. DEX significantly inhibited the migration of HUVECs, angiogenesis, and the expression of angiogenesis-related markers(platelet endothelial cell adhesion molecule-1, vascular endothelial growth factor, and von Willebrand factor)(P<0.01). In contrast, NGN treatment did not significantly affect the aforementioned effects, but the treatment with NGN conditioned medium [CM(NGN)] partially reversed these effects(P<0.01). Bioinformatics analysis combined with Pulldown assay results indicated that HO-1 was the target of NGN. DEX treatment significantly downregulated the expression of HO-1, while NGN intervention partially counteracted the inhibitory effect induced by DEX(P<0.01); knockdown of HO-1 negated the therapeutic effects of NGN(P<0.01). Compared with MPS administration alone, the combined administration of NGN and MPS upregulated the expression of HO-1 and HIF-1α in rat femoral head tissues. However, the HO-1 inhibitor ZnPP further upregulated the expression of HO-1 but downregulated the protein level of hypoxia-inducible factor-α(HIF-1α)(P<0.01). Conclusion NGN exerts its therapeutic effects on SONFH by activating the expression and activity of HO-1, which regulates the HIF-1α/VEGF pathway to promote osteoblast differentiation, bone formation, and angiogenesis.更多还原