Herb couplet medicines (Erxian) protect osteoblasts from high glucose-induced damage by reducing cell apoptosis in diabetic osteoporosis: A network pharmacology and experimental verification-based study

Traditional Chinese medicine (TCM) has unique advantages in treating diabetic osteoporosis (DOP). This study aimed to explore the mechanisms underlying the protective effects of herb couplet medicines (Erxian) on DOP using network pharmacology and verify these mechanisms using cell experiments. We used chromatography to analyze the bioactive components of serum containing Erxian couplet medicines (ECMs) and searched for possible targets of the active ingredients in the Traditional Chinese Medicine Systems Pharmacology database. The target proteins of DOP were retrieved from the GeneCards and Online Mendelian Inheritance in Man databases. A component-target network diagram was then constructed using the screened drug components and target information for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In vitro, cell morphological changes were observed using alkaline phosphatase staining, and the effect of the ECMs on osteoblast viability was assessed using a cell counting kit-8 assay. Finally, the pathway information predicted by network pharmacology was validated using flow cytometry, real-time polymerase chain reaction, and western blot experiments. Twelve types of active ingredients in serum containing ECMs were obtained via chromatography, and 96 potential targets were identified using network pharmacology analysis. Furthermore, GO and KEGG pathway enrichment analyses revealed that the ECMs might participate in the apoptosis signaling pathway in the treatment of DOP. In vitro tests showed that treatment with ECM serum increased osteoblasts' survival rate and improved the HG group's cell morphology (P < 0.05). BCL2-associated X protein (BAX) expression increased in the HG group but decreased after ECM serum addition (P < 0.05). The expression of B-cell lymphoma-2 (BCL-2) was found to be reduced in the HG group but increased after ECM serum addition (P < 0.05). ECMs can treat DOP via multiple targets and multiple signaling pathways. Moreover, the mechanism underlying the effects of ECMs on osteoblasts was verified by inhibiting apoptotic. The complete signaling pathway requires future study.