基于网络药理学和分子对接研究高山金莲花素对颞下颌关节骨关节炎细胞模型的作用机制  

Network pharmacology and molecular docking to study the mechanism of action of alpinumisoflavone in a temporomandibular joint osteoarthritis cell model

作　　者：王泽杰 吴高义 WANG Zejie;WU Gaoyi(School of Stomatology,Jiamusi University,Heilongjiang Key Lab of Oral Biomedicine Materials and Clinical Application,Jiamusi 154000,China)

机构地区：[1]佳木斯大学口腔医学院,黑龙江省口腔生物医学材料及临床应用重点实验室,黑龙江佳木斯154000

出　　处：《口腔疾病防治》2024年第8期578-588,共11页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金(61871393)。

摘　　要：目的运用网络药理学与分子对接方法探讨高山金莲花素(alpinumisoflavone,AIF)对颞下颌关节骨关节炎(temporomandibular joint osteoarthritis,TMJOA)细胞模型的作用机制,为AIF治疗TMJOA提供研究基础。方法运用GeneCards、OMIM、DisGeNET和PharmGKB数据库获取TMJOA疾病靶点,PharmMapper和HERB获取AIF作用靶点,取化合物与疾病交集靶点上传至STRING数据库得到关键靶点后做GO和KEGG富集分析,分子对接评估相关信号通路中关键靶点。获得医院伦理委员会的审批,提取3周龄SD大鼠髁突软骨细胞。CCK8检测AIF对髁突软骨细胞的毒性。用10 ng/mL白细胞介素⁃1β(interleukin 1β,IL⁃1β)诱导髁突软骨细胞24 h构建TMJOA细胞模型。实验分为3组,其中,对照组:DMEM培养基培养髁突软骨细胞48 h;IL⁃1β组(TMJOA细胞模型):预使用DMEM培养基培养髁突软骨细胞24 h后,保留原培养基的情况下加入IL⁃1β使终浓度达10 ng/mL继续培养24 h;IL⁃1β+10μmol/L AIF组:预使用含10μmol/L AIF的DMEM培养基培养髁突软骨细胞24 h,保留原培养基情况下加入IL⁃1β使终浓度达10 ng/mL继续培养24 h,流式细胞术检测AIF对TM⁃JOA细胞模型中的髁突软骨细胞凋亡的影响。进一步,实验分为对照组、IL⁃1β组、IL⁃1β+10μmol/L AIF组和IL⁃1β+30μmol/L AIF组共4组,其中,IL⁃1β+30μmol/L AIF组:预使用含30μmol/L AIF的DMEM培养基培养24 h,保留原培养基情况下加入IL⁃1β使终浓度达10 ng/mL继续培养24 h;其余3组方法同前。以qPCR与Western blot分别检测AIF对TMJOA细胞模型中与细胞凋亡相关的B淋巴细胞瘤2(B⁃cell leukemia/lymphoma⁃2,Bcl2)、天冬氨酸特异的半胱氨酸蛋白酶3(cysteinyl aspartate specific protease 3,Caspase⁃3)及基质降解相关的解聚蛋白样金属蛋白酶4(a disintegrin and metalloproteinase with thrombospondin motifs 4,ADAMTS4)、基质金属蛋白酶3(matrix metalloproteinase 3,MMP3)和基质金属蛋白酶13(matrix metalloproteinase 13,MMP1Objective To explore the potential role of alpinumisoflavone(AIF)in the treatment of temporomandibular joint osteoarthritis(TMJOA)cell model through network pharmacology and molecular docking and to provide a research basis for AIF in the treatment of TMJOA.Methods GeneCards,OMIM,DisGeNET,and PharmGKB databases were used to screen TMJOA disease targets,and PharmMapper and HERB were used to retrieve AIFrelated targets.The intersection targets of the compounds and diseases were uploaded to the STRING database to obtain the key targets for GO and KEGG enrichment analysis,while the key targets in related signaling pathways were evaluated through molecular docking.Approval was obtained from the Ethics Committee to extract condylar chondrocytes from 3weekold SD rats.The CCK8 assay was used to detect AIF cytotoxicity on condylar chondrocytes.Condylar chondrocytes were induced with 10 ng/mL interleukin 1β(IL1β)for 24 h to construct a TMJOA cell model.The experiment was divided into three groups:control group,comprising condylar chondrocytes cultured in DMEM for 48 h;IL1βgroup,comprising condylar chondrocytes precultured in DMEM for 24 h,after which IL1βwas added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h;and the IL1β+10μmol/L AIF group,comprising condylar chondrocytes precultured in DMEM medium containing 10μmol/L AIF for 24 h,after which IL1βwas added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h.The effect of AIF on condylar chondrocyte apoptosis in the TMJOA cell model was detected by flow cytometry.The experiment was divided into four groups:control group,IL1βgroup,IL1β+10μmol/L AIF group,and IL1β+30μmol/L AIF group.The IL1β+30μmol/L AIF group was precultured in DMEM containing 30μmol/L AIF for 24 h,after which IL1βwas added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h.The remaining three groups were cultured in t

关 键 词：颞下颌关节 骨关节炎 颞下颌关节骨关节炎细胞模型 高山金莲花素 网络药理学 分子对接 黄酮类化合物 下颌髁突软骨细胞 细胞凋亡 细胞外基质降解 白细胞介素⁃1β B淋巴细胞瘤2 天冬氨酸特异的半胱氨酸蛋白酶3 

分 类 号：R78[医药卫生—口腔医学]