中医正骨2026，Vol.38Issue(5)：9-23,15.
基于网络药理学分析和动物实验探讨益气活血通络方治疗膝骨关节炎的作用机制
Investigation of the mechanism of Yiqi Huoxue Tongluo Fang(益气活血通络方)in the treatment of knee osteo-arthritis based on the network pharmacology analysis and animal experiments
摘要
Abstract
Objective:To investigate the mechanism of Yiqi Huoxue Tongluo Fang(益气活血通络方,YHTF)in the treatment of knee oste-oarthritis(KOA).Methods:①Network pharmacology analysis.The chemical ingredients of YHTF were identified using ultra-high perfor-mance liquid chromatography-Orbitrap mass spectrometry(UHPLC-Orbitrap-MS).Furthermore,the network pharmacology approach was em-ployed to screen the core ingredients and key targets of YHTF against KOA.GO function and KEGG pathway enrichment analyses were then performed on the identified key target genes,and the molecular docking validation was conducted between key targets and core ingredients.②Animal experiment.Forty-eight SPF-grade female SD rats were randomized into blank group,model group,celecoxib group,low-dose YHTF(L-YHTF)group,medium-dose YHTF(M-YHTF)group,high-dose YHTF(H-YHTF)group,with 8 ones in each group.All rats but the ones in the blank group were subjected to anterior cruciate ligament transection(ACLT)on the left hind limbs to build KOA model.At 4 weeks post-modeling procedure,the rats in L-YHTF group,M-YHTF group,and H-YHTF group were intervened by intragastric administra-tion with YHTF solution(formula granules dissolved in double-distilled water)at concentrations of 0.65,1.30,and 2.60 g/mL,respective-ly,the ones in celecoxib group with celecoxib solution(celecoxib dissolved in double-distilled water)at a concentration of 2.065 mg/mL,while the ones in the blank group and model group with an equal volume of double-distilled water.All rats in the 6 groups were intervened once a day,2 mL at a time for consecutive 4 weeks.Following the end of intervention,the left hind knee joints were harvested from the rats in each group for Micro-CT examination and histopathological observation(HE staining,safranin O-fast green(SO-FG)staining,and tolui-dine blues taining).Additionally,the protein expression levels of sex determining region Y-box transcription factor 9(SOX9)and tumor nec-rosis factor-α(TNF-α)were detected using immunofluorescence and Western Blot.Results:①Findings from network pharmacology analy-sis.The core ingredients of YHTF against KOA were identified as α-linolenic acid,naringenin,liquiritigenin,apigenin,and luteolin.The key targets included SRC,signal transducer and activator of transcription 3(STAT3),Akt kinase(Akt)1,phosphatidylinositol-3-kinase regulato-ry subunit 1(PI3KR1),and phosphatidylinositol-3-kinase catalytic subunit alpha(PI3KCA).GO function enrichment analysis revealed that the screened key targets were mainly involved in biological processes such as signal transduction,inflammatory response,and positive regu-lation of cell proliferation.KEGG signaling pathway enrichment analysis revealed that YHTF may exert its anti-KOA effects through regu-lating hypoxia-inducible factor 1(HIF-1),phosphatidylinositol-3-kinase(PI3K)-Akt,TNF-α,and mitogen-activated protein kinase(MAPK)signaling pathways.Molecular docking results showed that the apigenin,liquiritigenin,and naringenin among the core ingredients exhibited good binding affinity with the screened key targets.②Findings from animal experiment.Micro-CT revealed significant subchondral bone loss in the model group(decreased trabecular bone volume/tissue volume(Tb.BV/TV),trabecular number(Tb.N),and trabecular thickness(Tb.Th),and increased trabecular separation(Tb.Sp)).The aforementioned bone microstructural indices were significantly improved in the H-YHTF group,with efficacy superior to the celecoxib group.The improvements in the L-,M-and H-YHTF groups showed a dose-dependent trend.Histopathological examination showed severe cartilage surface erosion,fissure formation,marked proteoglycan loss,and re-duced matrix metachromasia in the model group.The aforementioned pathological damages were improved in all YHTF groups,showing a dose-dependent trend,with the H-YHTF group achieving the best effect,which was comparable to that of the celecoxib group.Immunofluo-rescence and Western blot results showed that the protein expression level of SOX9 was downregulated,while that of TNF-α was upregulated in the model group.whereas,compared with the model group,the protein expression level of SOX9 was upregulated,while that of TNF-α was downregulated in H-YHTF group,with effects comparable to those of the celecoxib group.Conclusion:The core active ingredients of YHTF against KOA are apigenin,liquiritigenin,and naringenin,acting on the targets of SRC,STAT3,Akt1,PI3KR1,and PI3KCA.It may exert the effects by regulating HIF-1,PI3K-Akt,TNF-α,and MAPK signaling pathways,thereby inhibiting inflammatory responses,promoting cartilage repair,and improving subchondral bone microstructure.关键词
骨关节炎,膝/益气活血通络方/网络药理学/分子对接模拟/液相色谱-质谱法/动物实验Key words
osteoarthritis,knee/Yiqi Huoxue Tongluo Fang/network pharmacology/molecular docking simulation/liquid chromatography-mass spectrometry/animal experimentation引用本文复制引用
韩明丽,何敏聪,杨帆,吴淮,魏秋实,何宪顺,韩龙飞,罗贵锋,田龙,林锟,陆舜,侯文渊,李美霞..基于网络药理学分析和动物实验探讨益气活血通络方治疗膝骨关节炎的作用机制[J].中医正骨,2026,38(5):9-23,15.基金项目
2025年度深圳市基础研究专项自然科学基金计划项目(JCY120250604190412017) （JCY120250604190412017）
