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

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,MMP13)mRNA和蛋白的表达。结果PharmMapper与HERB数据库检索得到AIF化合物靶点300个,GeneCards、OMIM、DisGeNET和PharmGKB数据库检索得到TMJOA疾病靶点378个,将化合物与疾病靶点交集得33个潜在靶点,将其上传至STRING数据库得31个关键靶点,主要与细胞凋亡和细胞外基质降解相关。这一过程可能涉及丝裂原活化蛋白激酶(mitogen⁃activated protein kinase,MAPK)、雌激素及肿瘤坏死因子(tumor necrosis factor,TNF)等信号通路。分子对接结果表明AIF与MAPK和雌激素信号通路中的关键靶点细胞外信号调节激酶1/2(extracellular sig⁃nal⁃regulated kinase 1/2,ERK1/2)和雌激素受体基因1/2(estrogen receptor gene 1/2,ESR1/2)具有较好的结合活性。CCK8结果表明AIF对髁突软骨细胞无明显细胞毒性。与IL⁃1β组相比,IL⁃1β+10μmol/L AIF组中AIF可抑制髁突软骨细胞凋亡;与IL⁃1β组相比,IL⁃1β+10μmol/L AIF组和IL⁃1β+30μmol/L AIF组中AIF可上调Bcl2和下调Caspase⁃3 mRNA和蛋白表达,抑制ADAMTS4、MMP3和MMP13 mRNA和蛋白表达。结论AIF可通过上调Bcl2与下调Caspase⁃3 mRNA和蛋白表达抑制TMJOA细胞模型中髁突软骨细胞凋亡,同时抑制由IL⁃1β诱导的细胞外基质降解,延缓TMJOA进展。

Objective 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 the same manner as before.The mRNA and protein expression of apoptosisassociated Bcell leukemia/lymphoma2(Bcl2),cysteinyl aspartate specific protease 3(caspase3),matrix degradationassociated a disintegrin and metalloproteinase with thrombospondin motifs 4(ADAMTS4),matrix metalloproteinase 3(MMP3),and matrix metalloproteinase 13(MMP13)were detected by qPCR and western blot,by AIF in the TMJOA cell model.Results The PharmMapper and HERB database search yielded 300 AIF compound targets.The GeneCards,OMIM,DisGeNET,and PharmGKB databases yielded 378 TMJOA disease targets.Thirtythree potential common targets were obtained by intersecting compounds with disease targets.The common targets were uploaded into the STRING database to obtain 31 key targets that were mainly associated with apoptosis and extracellular matrix degradation.This process may be associated with the MAPK,estrogen,and TNF signaling pathways.The molecular docking results showed that AIF has good binding activity with extracellular signalregulated kinase 1/2(ERK1/2)and estrogen receptor gene 1/2(ESR1/2),which are key targets in the MAPK and estrogen signaling pathways.The CCK8 assay showed that AIF had no obvious cytotoxicity to condylar chondrocytes.The cell experiments showed that AIF inhibited apoptosis in the IL1β+10μmol/L AIF group compared to the IL1βgroup.Compared to the IL1βgroup in the IL1β+10μmol/L AIF group and the IL1β+30μmol/L AIF group,AIF upregulated Bcl2 and downregulated caspase3 mRNA and protein expression and inhibited ADAMTS4,MMP3,and MMP13 mRNA and protein expression.Conclusion AIF inhibited apoptosis in the TMJOA cell model by upregulating Bcl2 and downregulating caspase3 mRNA and protein expression,and inhibited extracellular matrix degradation induced by IL1β,thereby delaying TMJOA progression.