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目的 基于网络药理学、分子对接及体外实验探究抗菌肽LL-37联合唑来膦酸（ZA）对核因子κB受体活化因子配体（RANKL）与脂多糖（LPS）联合诱导的体外炎性破骨细胞分化与功能的抑制作用，并评估其对牙龈卟啉单胞菌（P.gingivalis）活性的影响。方法 通过TargetNet和SEA数据库获取LL-37与ZA的作用靶点，并从GeneCards及OMIM数据库筛选种植体周炎相关靶点；利用Venn在线工具获得“药物-疾病”交集靶点，构建蛋白互作网络，进行富集分析及分子对接。随后采用抗酒石酸酸性磷酸酶（TRAP）染色及RT-qPCR技术，比较RANKL与LPS单独及联合诱导RAW264.7细胞所构建的体外破骨模型中的细胞分化情况；最后通过CCK-8实验与微量肉汤稀释法确定LL-37和ZA的安全浓度范围，并通过圆盘扩散法、TRAP染色、RT-qPCR、ELISA及Westernblot技术，检测相较于单独应用，LL-37与唑ZA联合应用在抑制P.gingivalis、抑制破骨细胞活性及缓解炎症反应方面的影响。结果 筛选获得28个潜在交集靶点，其中半胱天冬酶-3（CASP3）、信号转导与转录激活因子-3（STAT3）、基质金属蛋白酶-3（MMP3）、沉默调节蛋白-1（SIRT1）、内皮型一氧化氮合酶-3（NOS3）等为关键靶点，富集分析结果表明，LL-37联合ZA可能通过多靶点、多通路协同参与种植体周炎的病理调控。TRAP染色与RT-qPCR结果显示，相较于单一诱导，RANKL联合LPS诱导RAW264.7细胞可构建更优的体外炎性破骨细胞模型。体外实验证实，与单药治疗相比，LL-37联合ZA更能显著抑制破骨细胞分化与形成，并有效下调炎症因子表达；圆盘扩散法显示，LL-37联合ZA有良好的抑菌活性。结论 在炎性环境下，LL-37与ZA联合应用可有效抑制细菌生长、破骨细胞活性及炎症反应。
Abstract：Objective To explore the inhibitory effects of LL-37 combined with zoledronic acid on the differentiation and function of inflammatory osteoclasts induced by receptor activator of nuclear factor-κB ligand (RANKL)and Lipopolysaccharide (LPS)， as well as its activity against Porphyromonas gingivalis(P.gingivalis)， via network pharmacology， molecular docking， and in vitro experiments. Methods The targets of LL-37 and zoledronic acid (ZA)were retrieved from TargetNet and Similarity Ensemble Approach(SEA)databases， and implant peri-implantitis-related targets were screened out via GeneCards and Online Mendelian Inheritance in Man(OMIM)databases. The Venn online tool was used to acquire drug-disease intersecting targets， followed by the construction of protein-protein interaction network as well as enrichment analysis and molecular docking. Afterwards， tartrate-resistant acid phosphatase (TRAP) staining and RT-qPCR were adopted to compare the osteoclast differentiation in RAW264.7 cell models induced by RANKL alone， LPS alone and their combination in vitro. Finally， CCK-8 assay and microbroth dilution method were used to confirm the safe concentration ranges of LL-37 and ZA. Disc diffusion method， TRAP staining， RT-qPCR， ELISA and Western blot were applied to explore the superior effects of combined administration of LL-37 and ZA over single drug use in inhibiting P.gingivalis， suppressing osteoclast activity and alleviating inflammatory responses. Results A total of 28 potential intersecting targets were screened， among which CASP3， STAT3， MMP3， SIRT1 and NOS3 were identified as core targets. Enrichment analyses revealed that LL-37 combined with ZA might regulate the pathological progression of peri-implantitis via multiple targets and signaling pathways. TRAP staining and RT-qPCR results demonstrated that RAW264.7 cells induced by RANKL combined with LPS established a better in vitro inflammatory osteoclast model compared with single induction mode. In vitro experiments verified that combined treatment with LL-37 and ZA exerted more prominent effects on inhibiting osteoclast differentiation and formation and downregulating the expression of inflammatory factors than monotherapy. Disc diffusion assay further confirmed that the combination possessed favorable antibacterial activity. Conclusion Under inflammatory conditions， combined administration of LL-37 and ZA can effectively inhibit bacterial growth， osteoclast activity and inflammatory responses.
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基本信息:
中图分类号:R783.6
引用信息:
[1]席歆,石超,安哲庆,等.抗菌肽LL-37联合唑来膦酸调节炎性状态下破骨细胞功能的体外研究[J].安徽医科大学学报().
基金信息:
国家自然科学基金项目(编号：82260198); 贵州省高层次创新型人才项目（编号：黔科合平台人才-GCC[2023]080）
2026-09-01
2026-09-01
2026-09-01