摘要
目的:探讨参威骨痹汤治疗骨质疏松症(osteoporosis,OP)的作用机制.方法:①网络药理学研究.采用超高效液相色谱-质谱法鉴定参威骨痹汤中的化合物,采用网络药理学方法初步筛选参威骨痹汤治疗OP的关键活性成分和靶点.②分子对接验证.采用分子对接技术验证网络药理学研究确定的参威骨痹汤治疗OP的关键活性成分与靶点的结合效果,从中筛选结合效果最好的组合.③动物实验验证.将60只雌性C57BL/6JNifdc小鼠随机等分为5组,模型组和参威骨痹汤低、中、高剂量组小鼠通过双侧卵巢切除手术进行OP造模,假手术组小鼠仅切取卵巢周围等体积的脂肪组织.参威骨痹汤低、中、高剂量组小鼠分别按照14.95 g·kg-1·d-1、29.9 g·kg-1·d-1、59.8 g·kg-1·d-1以参威骨痹汤药液灌胃,假手术组和模型组小鼠以等体积生理盐水灌胃.药物干预8周后,取股骨进行Micro-CT检查和组织病理学观察(HE染色和抗酒石酸酸性磷酸酶染色),采用实时定量PCR技术测定胫骨组织中根据网络药理学和分子对接技术确定的关键靶点基因的mRNA表达水平.结果:①网络药理学研究结果.网络药理学研究结果显示,SRC、促分裂原活化的蛋白激酶3(mitogen-activated protein kinase 3,MAPK3)、MAPK1、磷脂醜肌醇3-激酶调节亚基1(phosphatidylinositol-3-kinase regulatory subunit 1,PI3KR1)、信号转导及转录活化因子3为参威骨痹汤治疗OP的关键靶点,五味子丙素、10-姜酚、对羟基苯甲酸丁酯、倍半萜内酯和香豆雌酚为参威骨痹汤治疗OP的关键活性成分.②分子对接验证结果.分子对接结果显示,参威骨痹汤治疗OP关键活性成分与关键靶点的结合能均小于-5 kJ·mol-1,表明结合效果良好,其中靶点SRC、MAPK3、PI3KR1与活性成分五味子丙素、10-姜酚、对羟基苯甲酸丁酯的结合效果更好.③动物实验验证结果.Micro-CT检查及组织病理学观察结果显示,参威骨痹汤能改善OP模型小鼠的骨质疏松程度,其中中剂量参威骨痹汤效果更好.实时定量PCR测定结果显示,参威骨痹汤中剂量组胫骨组织中SRC mRNA和MAPK3 mRNA表达水平低于模型组,PI3KR1 mRNA表达水平与模型组比较差异无统计学意义.结论:参威骨痹汤治疗OP的机制可能是通过五味子丙素、10-姜酚、对羟基苯甲酸丁酯等成分调控SRC、PI3KR1、MAPK3的表达来调节骨代谢.
Abstract
Objective:To explore the mechanism of Shenwei Gubi Tang(参威痹汤,SWGBT)against osteoporosis(OP).Methods:①Network pharmacology research.The ingredients in SWGBT were identified by using ultra-high performance liquid chromatography-qua-drupole-orbitrap mass spectrometry(UHPLC-QE-MS),and the key active ingredients and action targets of SWGBT against OP were prelimi-narily screened using the network pharmacology approach.②Validation by molecular docking.The binding effects between the key active in-gredients and the targets of SWGBT against OP determined by network pharmacology were validated by molecular docking techniques,and the combination with the best binding effect was screened.③Verification by animal experimentation.Sixty female C57BL/6JNifdc mice were selected and randomized into model group,low-dose SWGBT(L-SWGBT)group,medium-dose SWGBT(M-SWGBT)group,high-dose SWG-BT(H-SWGBT)group and sham-operated group.The mice in model group,L-SWGBT group,M-SWGBT group and H-SWGBT group were subjected to bilateral ovariectomy for inducing OP;while the ones in sham-operated group were merely removed an equal volume of peri-ovarian adipose tissues.After successful modeling,the mice in L-SWGBT group,M-SWGBT group and H-SWGBT group were intervened by intragastric administration with 14.95,29.9,59.8 g/(kg·d)SWGBT,respectively;while the ones in sham-operated group and model group with the same dose of normal saline.After 8-week drug intervention,the rats were sacrificed and their femurs were harvested for Micro-CT examination and observation on histopathological changes through HE staining and tartrate-resistant acid phosphatase(TRAP)staining.Fur-thermore,the mRNA expression levels of key target genes(determined by network pharmacology approach and molecular docking tech-niques)in tibia tissues were detected by using real-time quantitative PCR(RT-qPCR).Results:①The results of network pharmacology re-search showed that SRC,mitogen-activated protein kinase 3(MAPK3),MAPK1,phosphatidylinositol-3-kinase regulatory subunit 1(PI3KR1),and signal transducer and activator of transcription 3(STAT3)were the key targets of SWGBT against OP,and the schisandrin C,10-gingerol,butyl p-hydroxybenzoate,sesquiterpene lactone,and coumestrol were the key active ingredients of SWGBT against OP.②The results of molecular docking showed that the key active ingredients of SWGBT against OP had good binding ability to the key targets with the binding energy less than-5.0 kJ/mol,and the targets including SRC,MAPK3,and PI3KR1 exhibited better binding effects with the active ingredients including schisandrin C,10-gingerol,and butyl p-hydroxybenzoate.③The results of Micro-CT examination and histopathological observation showed that the SWGBT could improve the degree of OP in OP model mice,with the medium-dose SWGBT displaying better effects.The results of RT-qPCR showed that the mRNA levels of SRC and MAPK3 were lowly expressed in the tibia tissues of OP model mice in M-SWGBT group compared to model group,while,the mRNA expression level of P13KR1 was not significantly different from that of model group.Conclusion:The SWGBT may regulate bone metabolism for treatment of OP through adjusting the expression of SRC,PI3KR1 and MAPK3 via the ingredients such as schisandrin C,10-gingerol and butyl p-hydroxybenzoate.
基金项目
云南省科学技术厅-中医联合专项面上项目(202101AG070053)
云南省科学技术厅-中医联合专项面上项目(202301AZ070001-091)
NETL
NSTL
万方数据