Traditional Chinese medicine Youguiyin decoction ameliorate glucocorticoid-induced osteonecrosis in rat by modulating ROS/PHD2/HIF-1α oxidative stress signaling pathway in bone marrow mesenchymal stem cells

Abstract

Background: The incidence of osteonecrosis is increasing annually due to the widespread use of glucocorticoids. Recent evidence suggests a significant association between glucocorticoid-induced osteonecrosis and oxidative stress. Youguiyin (YGY) decoction, a classic formula of traditional Chinese medicine, has been widely used for the prevention of glucocorticoid-induced osteonecrosis. However, its underlying pharmacological mechanisms are still not fully understood.

Methods: UPLC-Q-TOF-MS and network pharmacology were used to elucidate the material basis of YGY decoction and its mechanism for the treatment of glucocorticoid-induced osteonecrosis. The anti-oxidative stress and bone-enhancing effects in vivo were detected by hematoxylin-eosin (HE) staining, serum metabolomics, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and Western Blot (WB). Rat bone marrow mesenchymal stem cells (BMSCs) were induced with dexamethasone (DXMS) for 24 h, followed by YGY medicated serum for 24 h. Significantly up- and down-regulated genes were detected by RNA sequencing. Oxidative stress levels were detected by ROS fluorescence. Alizarin red S staining was used to detect osteogenic effects. WB and ELISA were used to detect the expression of proteins related to the ROS/PHD2/HIF-1a pathway.

Results: The application of YGY decoction significantly promoted bone repair and antagonized excess reactive oxygen species (ROS) generation in glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH) rats. In addition, YGY medicated serum antagonized DXMS-induced ROS production and promoted osteogenic differentiation in BMSCs. We also found that YGY medicated serum attenuated excess ROS generation while PHD2 expression was significantly increased, HIF-1α expression was significantly decreased and RUNX2 expression was significantly increased.

Conclusion: These results provide compelling in vivo and in vitro evidence that YGY decoction may play a role in promoting glucocorticoid-induced osteonecrosis bone repair by targeting the mediation of the ROS/PHD2/HIF-1α oxidative stress signaling pathway, thus providing a new theoretical basis for the clinical application of YGY decoction to glucocorticoid-induced osteonecrosis.

Keywords: Glucocorticoid-induced osteonecrosis; Oxidative stress; ROS/PHD2/HIF-1α pathway; Youguiyin (YGY) decoction.

Fig. 1

Base peak chromatograms of YGY decoction obtained by UPLC-Q-TOF–MS in both positive (A) and negative (B) ion mode

Fig. 2

Network pharmacology analysis results of YGY decoction and GA-ONFH. A Venn diagram of target genes associated with abundant components of YGY decoction and GA-ONFH. B PPI network diagram of potential target proteins for the treatment of GA-ONFH with YGY decoction. C Hub proteins and degree values closely associated with YGY treatment of GA-ONFH. D The enrichment results of GO-BP for YGY intervention with GA-ONFH

Fig. 3

The reparative effect of YGY decoction on the femoral head of GA-ONFH rats. A Specific procedure of animal experiment. B Representative micrograph of HE and Masson staining of the femoral head. Scale bar: 200/50 μm. C Quantitative analysis of the results of HE staining and Masson staining (n = 9 technical replicates). Statistical analysis: Unpaired t-test. ^###P < 0.001 compared vs Con group. ^nsP > 0.05,*P < 0.05 and ***P < 0.001 vs Mod group

Fig. 4

The effects of YGY decoction on ROS, PHD2, HIF-1α, and osteogenic expression in GA-ONFH rats. A Oxidative stress levels in the femoral head of rats in all groups. B Oxidative stress levels in the serum of rats in all groups. C Immunohistochemical staining results of the femoral head in each group of rats. Scale bar: 200 µm. D Western blot results of the femoral head in each group of rats. E Quantitative analysis of immunohistochemical positive expression in each group of rats (n = 9 technical replicates, 3 fields/sample). F Quantitative analysis of expression of western blot in each group of rats (n = 3 independent experiments). Statistical analysis: Unpaired t-test. ^##P < 0.01 and ^###P < 0.001 vs Con group. ^nsP > 0.05,*P < 0.05, **P < 0.01 and ***P < 0.001 vs Mod group

Fig. 5

RNA sequencing and qPCR results. A The volcano plot of different expressed genes. B Hierarchical clustering analysis of different expressed genes. C The relative gene expression levels of GCLC, SOD2, PHD2, HIF-1α and RUNX2 (n = 6 independent experiments). Statistical analysis: Unpaired t-test. **P < 0.01 and ***P < 0.001 vs Control group

Fig. 6

Promoting effect of YGY medicated serum on osteogenic differentiation of dexamethasone-induced BMSCs. A Specific procedure and grouping of cell experiments. B Alizarin red staining results of each group of cells. Scale bar: 200 μm. C ROS fluorescence results for each group of cells. Scale bar: 100 μm. D Levels of oxidative stress for each group of cells (n = 6 technical replicates). E Number of calcium nodule deposits for each group of cells (n = 9 technical replicates). F Western blot results for each group of cells. G Quantitative analysis results of Western Blot (n = 3 independent experiments). Statistical analysis: Unpaired t-test. ^nsP > 0.05,*P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 7

PHD2 knockdown hindered the promotion of osteogenic differentiation of dexamethasone-induced BMSCs by YGY medicated serum. A Specific procedure and grouping of cell experiments. B Alizarin red staining results for each group of cells. Scale bar: 200 μm. C ROS fluorescence results for each group of cells. Scale bar: 100 μm. D Western blot analysis of PHD2 knockdown in BMSCs. E Quantitative analysis results of Western Blot of PHD2 knockdown in BMSCs (n = 3 independent experiments). F Number of calcium nodule deposits for each group of cells (n = 9 technical replicates). G Western blot results for each group of cells. H Quantitative analysis results of western blot (n = 3 independent experiments). Statistical analysis: Unpaired t-test. ^nsP > 0.05, **P < 0.01 and ***P < 0.001

Fig. 8

HIF-1α knockdown promoted the promotion of osteogenic differentiation of dexamethasone-induced BMSCs by YGY-medicated serum. A Specific procedure and grouping of cell experiments. B Alizarin red staining results for each group of cells. Scale bar: 200 μm. C ROS fluorescence results for each group of cells. Scale bar: 100 μm. D Western blot analysis of HIF-1α knockdown in BMSCs. E Quantitative analysis results of Western Blot of HIF-1α knockdown in BMSCs (n = 3 independent experiments). F Number of calcium nodule deposits for each group of cells (n = 9 technical replicates). G Western blot results for each group of cells. H Quantitative analysis results of western blot (n = 3 independent experiments). Statistical analysis: Unpaired t-test. ^nsP > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 9

Schematic diagram of the mechanisms by which YGY ameliorates GA-ONFH by regulating the ROS/PHD2/HIF-1α pathway