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

Abstract

Objective: To explore whether the modified Qing' e Pills (MQEP) exerts anti-osteoporotic effects and prevents bone loss by enhancing angiogenesis.

Methods: Network pharmacology was used to assess whether MQEP has a pro-angiogenic capacity and to predict its potential targets. Human umbilical vein endothelial cells were treated with glucocorticoids and MQEP to assess cell viability. The expression of angiotensin II type 1 receptor, angiotensin II type 2 receptor, and angiotensin converting enzyme, which are associated with the activation of the renin-angiotensin-aldosterone system, and the expression of vascular endothelial growth factor and hypoxia-inducible factor 1 alpha, which are associated with the formation of type H blood vessels, were examined by western blot and RT-qPCR. Thereafter, the glucocorticoid-induced osteoporosis model was established and intervened with MQEP. Femur scanning was performed with micro-computed tomography; trabecular spacing, trabecular thickness, and trabecular number were observed and calculated; the expression of nuclear factor-kappa B ligand and osteoprotegerin was detected by ELISA, and the ratio was calculated to evaluate the degree of bone resorption. Finally, type H blood vessels that were highly coupled to osteogenic cells were identified by immunohistochemistry staining and flow cytometry.

Results: This is the first study to reveal and confirm that MQEP could prevent bone loss in glucocorticoid-induced osteoporosis by promoting the expression of hypoxia-inducible factor 1 alpha and vascular endothelial growth factor, which are highly associated with type H blood vessel formation. In vitro experiments confirmed that MQEP could effectively promote the proliferation of vascular endothelial cells and alleviate glucocorticoids-induced activation of the renin-angiotensin-aldosterone system, thereby reducing vascular injury.

Conclusion: MQEP exerts anti-osteoporosis effects and prevents bone loss by alleviating vascular injury caused by renin-angiotensin-aldosterone system activation and promoting type H blood vessel formation.

Keywords: alternative medicine; angiotensin; bone loss; osteoporosis; type H blood vessels; vessel formation.

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.