基于血清药物化学、网络药理学的正骨丸治疗骨不连作用机制研究OA

Objective:To identify the chemical components and blood-absorbed components of Zhenggu Pills by ultra-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS),predict the potential treatment mechanism of Zhenggu Pills for bone nonunion by network pharmacology,and verify the efficacy of Zhenggu Pills by a mouse model of fracture.Methods:The water extract of Zhenggu Pills,rat blank plasma,and drug-containing plasma were detected by UPLC-Q-TOF-MS/MS to identify the blood-absorbed components.SwissTargetPrediction,SEA,and HERB databases were used to screen potential targets of the blood-absorbed components of Zhenggu Pills in the treatment of bone nonunion.A protein-protein interaction(PPI)network was constructed via the STRING database,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses of the core targets.A mouse model of femoral fracture was established.Micro-CT scanning and hematoxylin-eosin(HE)staining were performed on days 7,14,and 21 after administration to evaluate the fracture healing.Results:A total of 63 chemical components were identified from Zhenggu Pills,among which 30 were identified as blood-absorbed components.Through network pharmacology screening,a total of 33 common targets were obtained,along with 13 core components(e.g.,ferulic acid,caffeic acid,and chrysoeriol)and 14 core targets[e.g.,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),β1-catenin(CTNNB1),and hypoxia-inducible factor 1 alpha(HIF1A)].These were found to be mainly involved in the hypoxia-inducible factor 1(HIF-1),tumor necrosis factor(TNF),mitogen-activated protein kinase(MAPK),and nuclear factor-kappa B(NF-κB)signaling pathways.Micro-CT results showed that compared with the model group,the Zhenggu Pill group exhibited more pronounced callus formation and bone remodeling on days 7,14,and 21.On day 14,the bone volume fraction in the Zhenggu Pill group was higher than that in the model group(P<0.05).On day 21,although the bone volume fraction in the Zhenggu Pill group was lower than that in the model group(P<0.05),the fracture line disappeared,accompanied by the restored continuity of the cortical bone and markedly improved bone mineral density and trabecular bone structure.HE staining results indicated that in the Zhenggu Pill group,cartilage tissue formed in the fracture area on day 7,which transitioned into woven bone by day 14 and then gradually transformed into mature lamellar bone on day 21.Conclusion:This study identified 30 blood-absorbed components of Zhenggu Pills and revealed that components such as ferulic acid and paeoniflorin acted on core targets including GAPDH and CTNNB1 to promote fracture healing by regulating the HIF-1,TNF,MAPK and NF-κB signaling pathways.The therapeutic efficacy of Zhenggu Pills in promoting callus formation and ameliorating bone nonunion was further confirmed by animal experiments.