Modified Qing’ e Pills exerts anti-osteoporosis effects and prevents bone loss by enhancing type H blood vessel formation

Figures

• 

  Figure 1. Predicting the targets MQEP by network pharmacology. (A) Targets for osteoporosis were retrieved from four disease databases. (B) An intersection of the MQEP and osteoporosis targets is the target on which MQEP exerts anti-osteoporosis and bone loss prevention effects. (C) The main components of MQEP were searched, and the top 10 compounds were analyzed for interactions with their target proteins of action using the TCMSP and ETCM databases. (D) The String database and Cytospace software were used to construct protein interaction networks. (E, F) The target on which MQEP exerts the anti-osteoporosis effect was obtained, and GO enrichment and KEGG enrichment were performed to obtain the main biological processes and signaling pathways. (G) Molecular docking was used to verify the target proteins with high affinity to Isopsoralen, the main component in MQEP.

• 

  Figure 2. The intervention of MQEP reversed GC-induced activation of the RAAS and the downregulation of HIF-1a and VEGF expression. (A) The morphology of HUVEC cells was observed under the microscope after GC treatment and MQEP intervention. (B) CCK-8 assay was performed, the absorbance was measured at 450 nm, and cell viability was calculated. (C, D) The expression of several key factors of the RAAS and type H blood vessels were detected using Western blotting after treatment of HUVEC cells with MQEP for 48h. Images were quantified and statistically analyzed. (E) The expression of several key factors of the RAAS and type H blood vessels were detected using RT-qPCR after treating HUVEC cells with MQEP for 48h. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Figure 3. MQEP exerts an ameliorative effect on bone turnover regulatory factors and bone structure. (A) Micro-CT was used to evaluate the femur microstructure. The bone structure was observed from three perspectives and reconstructed in three dimensions. (B) Micro-structure measurements were calibrated to the manufacturer’s calcium hydroxyapatite phantom and analyzed using the manufacturer’s evaluation software. (C) Collection of serum and detection of relevant indicators using Elisa. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Figure 4. The intervention of MQEP promotes the formation of type H blood vessels. (A) Tissue immunofluorescence was performed to observe type H blood vessel formation, and the images were quantified and statistically analyzed. (B) Flow cytometry was performed to detect the specificity indexes of type H blood vessel formation. *p < 0.05, **p < 0.01.

• 

  Figure 5. Schematic depiction of the mechanism by which the MQEP exerts its anti-osteoporosis and bone loss prevention effects by enhancing vessel formation.