基于虚拟筛选-分子对接-活性评价技术探讨四逆汤抗抑郁的药效物质基础

Pharmacological Substance Basis of Sini Decoction for Antidepressant Based onVirtual Screening-Molecular Docking-Activity Evaluation Technology

目的:基于虚拟筛选-分子对接-活性评价技术探讨四逆汤治疗抑郁症的药效物质基础。方法:通过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)检索四逆汤(甘草、干姜、附子)的活性成分,并根据类药五原则及相关条件进行二次筛选。通过RCSB PDB数据库检索抑郁症的作用靶点。借助Discovery StudioTM2016软件对四逆汤活性成分与抑郁症靶点进行分子对接,探索两者的亲和力和结合方式,进而筛选关键活性成分。HT22细胞分为空白组、模型组、(R)-去甲乌头碱组、甘草素组及山柰酚组。除空白组外,其余各组细胞加入皮质酮(1 mol·L^(-1),200μL)培养24 h建立体外抑郁模型。然后,加入相应药物(1 mol·L^(-1),100μL)干预24 h,光镜下观察细胞形态,TUNEL染色观察细胞凋亡情况。C57BL/6J小鼠随机分为空白组、模型组、(R)-去甲乌头碱组(10 mg·kg^(-1))、甘草素组(15 mg·kg^(-1))、山柰酚组(20 mg·kg^(-1))。除空白组外,其余小鼠采用慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)结合孤养的方式建立抑郁模型。造模首日开始给药,每日1次,连续28 d。末次灌胃后,通过糖水偏爱实验(sucrose preference test,SPT)、强迫游泳实验(forced swimming test,FST)与悬尾实验(tail suspension test,TST)检测各组小鼠的抑郁情绪和绝望情绪。结果:四逆汤活性成分493个,二次筛选得到91个化合物。抑郁症作用靶点4个,即单胺氧化酶A(monoamine oxidase A,MAO-A)、多巴胺转运体(dopamine transporter,DAT)、5-羟色胺转运体(serotonin transporter,SERT)、组胺H1受体(histamine receptor H1,H1R)。通过分子对接得到15个四逆汤抗抑郁的关键活性成分,进一步筛选得到3个核心成分,即(R)-去甲乌头碱、甘草素、山柰酚。细胞实验显示,空白组细胞光泽度好,突起交错分布且相互连接;模型组细胞光泽度较差,突起连接部分断裂消失,可见细胞变圆皱缩;与模型组比较,(R)-去甲乌头碱组、甘草素组、山柰酚组细胞形态有所改善,突起连接更紧密。空白组基本无细胞凋亡;与空白组比较,模型组细胞凋亡增加;与模型组比较,(R)-去甲乌头碱组、甘草素组、山柰酚组细胞凋亡减少。动物实验显示,与空白组比较,模型组小鼠糖水偏爱度降低(P<0.05);与模型组比较,(R)-去甲乌头碱组小鼠糖水偏爱度升高(P<0.05)。与空白组比较,模型组小鼠不动时间(TST和FST)增加(P<0.05);与模型组比较,甘草素组小鼠TST不动时间减少(P<0.05),(R)-去甲乌头碱组、甘草素组小鼠FST不动时间减少(P<0.05)。结论:本研究采用中药新药发现与评价的新模式“虚拟筛选-分子对接-活性评价”,深入挖掘四逆汤抗抑郁的物质基础主要为(R)-去甲乌头碱、甘草素及山柰酚。

Objective:To explore the pharmacological substance basis of Sini Decoction in the treatment of depression based on virtual screening-molecular docking-activity evaluation technology.Methods:The active ingredients of Sini Decoction[Gancao(liquorice),Ganjiang(rhizoma zingiberis),Fuzi(monkshood)]were retrieved through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and the secondary screening was carried out based on the five principles of drug classification and related conditions.Retrieve the targets of depression through the RCSB PDB database.Molecular docking was conducted between active ingredients of Sini Decoction and depression target with Discovery Studio TM 2016 software to explore the affinity and binding mode of the two to screen for key active ingredients.HT22 cells were divided into blank group,model group and noraconitine group(100μmol·L^(-1)),glycyrrhizin group(200μmol·L^(-1))and kaempferol group(100μmol·L^(-1)).Except for the blank group,all of the other groups of cells were treated with corticosterone(200μmol·L^(-1))to establish an in vitro depression model.After 24 hours of cultivation,observe the cell morphology under a light microscope and observe cell apoptosis with TUNEL staining.C57BL/6J mice were randomly divided into normal group,model group,noraconitine(10 mg·kg^(-1)),glycyrrhizin group(15 mg·kg^(-1)),and kaempferol group(20 mg·kg^(-1)).Except for the normal group,other mice were subjected to chronic unpredictable mild stress(CUMS)combined with solitary care to establish a depression model.Starting from the first day of modeling,the medication is administered once a day for 28 consecutive days.After the last gavage,the symptoms of depression and despair in each group of mice were detected through sucrose preference test(SPT),forced swimming test(FST),and tail suspension test(TST).Results:There were 493 active ingredients in Sini Decoction,and 91 compounds were obtained through secondary screening.There are four targets of depression,namely monoamine oxidase A(MAO-A),dopamine transporter(DAT),serotonin transporter(SERT)and histamine H1 receptor(H1R).Through molecular docking,10 key antidepressant components of Sini Decoction were obtained,in which 3 core components were revealed in further screening,namely(R)-noraconitine,glycyrrhizic and kaempferol.It is suggested with cell experiments that the blank group had good cell glossiness,with staggered and interconnected protrusions.The glossiness of the model group cells was poor,and the protrusion connections were broken and disappeared,indicating that the cells became round and wrinkled.Compared with that of the model group,the(R)-noraconitine group,glycyrrhizin group and kaempferol group showed improved cell morphology and tighter protrusion connections.There is almost no cell apoptosis in the blank group;while there is an increase in cell apoptosis in the model group compared with that of the blank group.Compared with that of the model group,there is a decrease in cell apoptosis in the(R)-noraconitine group,glycyrrhizin group and kaempferol group.In animal experiments it is indicated that compared with the blank group,the preference for sugar water in the model group mice decreased significantly(P<0.05).Compared with that of the model group,there is a significantly increased preference for glucose and water in mice in the(R)-noraconitine group(P<0.05).Compared with that of the blank group,the immobility time(TST and FST)significantly increased in the model group mice(P<0.05).Compared with that of the model group,the TST immobility time of mice in the glycyrrhizin group decreases significantly(P<0.05),while the FST immobility time of mice in the(R)-noraconitine group and glycyrrhizin group significantly decreased(P<0.05).Conclusion:By adopting a new model of traditional Chinese medicine discovery and evaluation,which is"virtual screening-molecular docking-activity evaluation",this study deeply explore substance basis of Sini Decoction′s antidepressant effects,mainly including(R)-noraconitine,glycyrrhizic acid and kaempferol.