摘要
目的基于网络药理学方法探究桑寄生治疗牙周炎的作用机制并通过分子对接和体内实验验证,探究桑寄生乙醇提取物对牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)生长作用的影响以及对实验性大鼠牙周炎的治疗效果。方法在中药系统药理学数据库与分析平台(Traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP)中检索桑寄生有效作用成分,通过Swiss Target Prediction数据库预测作用靶点,经GeneCards、DisGeNET和OMIM数据库检索得到牙周炎相关靶点,通过Venny分析获得共同靶点。采用STRING数据库构建PPI网络并筛选关键基因,经DAVID数据库进行GO和KEGG富集分析,采用Cytoscape 3.10.0软件构建桑寄生“药物-成分-靶点-通路”网络,经分子对接验证药物成分与靶点的结合情况。采用倍比稀释法测定桑寄生乙醇提取物对牙龈卟啉单胞菌的最低抑菌浓度(Minimum inhibitory concentration,MIC)和最低杀菌浓度(Minimum bactericidal concentration,MBC)。将42只雄性SD大鼠随机分为空白组、溶剂组、模型组、盐酸米诺环素组、桑寄生高、中、低剂量组。丝线结扎左上第一磨牙并接种P.g构建实验性大鼠牙周炎模型,每天对各组大鼠进行相应药物干预并记录体重、探诊深度(Probing depth,PD)、龈沟出血指数(Sulcus bleeding index,SBI)。14 d后拍摄X线片确定建模成功,处死各组大鼠获取血清、颌骨及牙龈组织标本。RT-PCR检测白细胞介素6(Interleukin 6,IL-6)、肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)、基质金属蛋白酶9(Matrix metalloproteinase 9,MMP-9)、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨钙素(Osteocalcin,OCN)、骨保护素(Osteoprotegerin,OPG)、核因子κB受体激活配体(Receptor activator of nuclear factor-κB ligand,RANKL)mRNA表达水平。ELISA检测血清中IL-6、TNF-α、MMP-9表达水平。HE染色观察炎症浸润情况。免疫组化染色观察RUNX2、OCN蛋白表达情况。结果筛选得到4个桑寄生有效成分,545个药物靶点,4151个牙周炎治疗靶点,Venny分析得到186个桑寄生治疗牙周炎的潜在靶点。PPI网络分析得到IL-6、TNF-α、MMP9等10个核心靶点。富集分析得到的主要通路为PI3K-Akt信号通路等。分子对接结果表明桑寄生有效成分与关键靶点具有较好的结合能力。桑寄生乙醇提取物对P.g的MIC为0.5 g/L,MBC为2 g/L。桑寄生乙醇提取物对实验性大鼠牙周炎的PD、SBI有良好的改善作用,可降低血清中IL-6、TNF-α、MMP-9表达,缓解牙周组织炎症情况。可促进牙周组织中RUNX2、OCN、OPG的表达,抑制RANKL的表达,调节骨稳态,缓解牙周骨组织流失。结论桑寄生乙醇提取物对P.g具有抑制和杀灭作用,可减轻炎症反应、调节骨稳态,通过多组分、多靶点、多通路的协同作用治疗牙周炎。
Objective Based on network pharmacology methods to explore the mechanism of action of Taxilli Herba in the treatment of periodontitis,and to verify through molecular docking and in vivo experiments,to investigate the effect of Taxilli Herba extract on the growth of P.g and its therapeutic effect on experimental periodontitis in rats.Methods The effective components of Taxilli Herba were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)database and the targets were predicted from Swiss Target Prediction database.GeneCards,DisGeNET and OMIM databases were used to retrieve periodontitis-related targets.The common targets were obtained by Venny analysis.The PPI network was constructed by STRING database.and key genes were screened.The GO function and KEGG signal pathway enrichment analysis was performed by DAVID database.The"drug-component-target-pathway"network of Taxilli Herba was constructed by Cytoscape 3.10.0 software.Molecular docking was used to verify the binding of drug components and targets.The minimum inhibitory concentration(MIC)and Minimum bactericidal concentration(MBC)of ethanol extract of Taxilli Herbaon P.g were determined by the ratio dilution method.42 male SD rats were randomly divided into 7 groups:blank group,solvent group,model group,minocycline hydrochloride group,Taxilli Herba high-dose group,Taxilli Herba medium-dose group and Taxilli Herba low-dose group.The experimental rat periodontitis model was constructed by ligating the left upper first molar with silk thread and inoculating P.g.The rats in each group were given corresponding drug intervention every day,and their weight,probing depth(PD)andsulcus bleeding index(SBI)were recorded.After 14 days,X-ray film was taken to confirm the success of modeling,and the rats in each group were sacrificed to obtain serum,jawbone and gingival tissue specimens.The mRNA expression levels of Interleukin 6(IL-6),tumor necrosis factorα(TNFα),tumor necrosis factorα(MMP-9),runt-related transcription factor 2(RUNX2),osteocalcin(OCN),osteoprotegerin(OPG),and receptor activator of nuclear factor-κB ligand(RANKL)were detected by RT-PCR.The expression levels of IL-6,TNF-α,and MMP-9 in serum were detected by ELISA.The inflammatory infiltration was observed by HE staining.The expression of RUNX2 and OCN protein was observed by immunohistochemical staining.Results 4 effective components of Taxilli Herba,545 drug targets and 4151 periodontitis treatmenttargets were obtained by screening,and 186 potential targets in treating periodontitis were obtained by Venny analysis.PPI network analysis obtained 10 core targets such as IL-6,TNF-αand MMP9.The main pathways obtained by enrichment analysis were PI3K-Akt signal pathway and so on.The results of molecular docking showed that the effective components of Taxilli Herba had good binding ability with the screened key targets.The MIC of the ethanol extract of Taxilli Herba to P.g was 0.5 g/L,and the MBC was 2 g/L.The ethanol extract of Taxilli Herba had a good effect on improving PD and SBI of experimental periodontitis in rats,and it can reduce the expression of IL-6,TNF-αand MMP-9 in serum,thus alleviating the inflammation of periodontal tissues.At the same time,it can promote the expression of RUNX2,OCN and OPGin periodontal tissue,and inhibit the expression of RANKL to regulate bone homeostasis,thus alleviating periodontal bone tissue loss.Conclusion The ethanol extract of Taxilli Herba can inhibit and kill P.g,reduce inflammatory reaction,regulate bone homeostasis,and treat periodontitis through multi-component,multi-target and multi-pathway.
作者
冯凯
张文杰
吴泽钰
姬晓炜
赵今
FENG Kai;ZHANG WenJie;WU Zeyu;JI Xiaowei;ZHAO Jin(Department of Cariology and Endodontics,the First Affiliated Hospital of Xinjiang Medical University/Affiliated Stomatology Hospital;Department of Prosthodontics and Dental Implantology,the First Affiliated Hospital of Xinjiang Medical University/Affiliated Stomatology Hospital;Institute of Stomatology of Xinjiang Uyghur Autonomous Region,Urumqi 830054,China)
出处
《新疆医科大学学报》
CAS
2024年第4期461-470,共10页
Journal of Xinjiang Medical University
基金
新疆维吾尔自治区自然科学基金重点项目(2022D01D57)
新疆维吾尔自治区“天山英才”科技领军创新人才项目(2023TSYCLJ0032)。
关键词
桑寄生
牙周炎
网络药理学
分子对接
牙龈卟啉单胞菌
动物模型
作用机制
Taxilli Herba
periodontitis
network pharmacology
molecular docking
Porphyromonas gingivalis
animal models
mechanism
