[Liuwei Dihuang Pills alleviates postmenopausal osteoporosis and fatigue in rats by inhibiting the epigenetic regulatory molecule BRD4 pathway]

Abstract

Objective: To explore the role of epigenetic signal molecule bromodomain protein 4 (BRD4) in mediating the therapeutic effect of Liuwei Dihuang (LWDH) Pills on postmenopausal osteoporosis (PMOP) and fatigue.

Methods: Thirty rat models of PMOP induced by bilateral ovariectomy were randomized equally into two groups for treatment with normal saline (model group) or LWDH Pills (385.7 mg/kg), with another 15 sham-operated rats as the sham operation group. After 12 weeks of treatment, femoral samples were taken to determine the bone density and BRD4 protein expression. The weight-bearing exhaustive swimming time of the rat models was recorded, and serum levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured using enzyme-linked immunosorbent assay. In cultured primary osteoblasts the changes in the expressions of BRD4, MAPK and NF-κB proteins were detected by immunofluorescence staining following treatment with LWDH Pills.

Results: The rat models of PMOP showed significantly up-regulated expression of BRD4 protein in the femoral tissue (P < 0.05), which was obviously lowered by treatment with LWDH Pills. The rats treated with LWDH Pills also showed significant improvement of fatigue. Immunofluorescent staining of the osteoblasts showed that treatment with LWDH Pills significantly decreased the protein expressions of BRD4, MAPK and NF-κB. Analysis of the GSE56116 dataset revealed that that patients with kidney-yin deficiency had significantly higher BRD4 expression than those in the kidney-yang-deficiency group and non-kidney-deficiency group (P < 0.05). The upregulation of BRD4 expression involved multiple signaling pathways including neural ligand receptor response, cytoskeleton rearrangement, cytokine interaction, and granulocyte colony-stimulating factor chemotaxis pathways.

Conclusion: LWDH can alleviate PMOP and fatigue by decreasing BRD4 signaling pathway, suggesting that potential of BRD4 as a promising therapeutic target for PMOP.

Keywords: Liuwei Dihuang Pills; bromodomain protein 4; fatigue; postmenopausal osteoporosis.

图 1

六味地黄丸改善PMOP骨组织病理 Liuwei Dihuang (LWDH) Pills improve bone histopathology in PMOP rats. A: Representative bone density scan images. B: Comparison of bone density scans among the 3 groups. C: Comparison of bone strength among the groups. D: HE staining of the bone tissue in each group. **P < 0.01, ^##P < 0.01.

图 2

六味地黄丸对抗PMOP鼠模型疲劳症状 Effects of LWDH Pills on fatigue symptoms in PMOP rats shown by comparison of weight-bearing swimming time among the groups. **P < 0.01, ^##P < 0.01.

图 3

六味地黄丸减轻鼠PMOP血清cAMP和cGMP浓度 Effect of LWDH Pills on serum cAMP and cGMP levels in in each group. A: Serum cAMP levels. B: Serum cGMP levels. C: Ratio of cAMP to cGMP. **P < 0.01, ***P < 0.001, ^##P < 0.01, ^###P < 0.001.

图 4

股骨成骨细胞原代培养 Primary culture osteoblasts from femur (Original magnification: ×200).

图 5

六味地黄丸促进成骨细胞钙结节形成 LWDH Pills promote the formation of calcium nodules in osteoblasts (Alizarin red staining, ×200). **P < 0.01, ***P < 0.001, ^##P < 0.01.

图 6

六味地黄丸抑制BRD4蛋白表达 LWDH Pills inhibit the expression of BRD4 protein in femoral bone tissues. **P < 0.01, ^##P < 0.01.

图 7

六味地黄丸抑制成骨细胞中BRD4、MAPK和NF-κB蛋白表达 Immunofluorescence assay showing the effects of LWDH Pills on subcellular distribution of BRD4 (A), MAPK (B) and NF-κB (C) in the osteoblasts (×200). **P < 0.01, ###P < 0.001, ^##P < 0.01, ^#P < 0.05.

图 8

PMOP肾虚分型与BRD4 mRNA的差异表达 Analysis of the differential BRD4 mRNA expressions in PMOP kidney deficiency subtypes. *P < 0.05.

图 9

肾阴虚型PMOP中BRD4表达的富集分析 Enrichment analysis of BRD4 expression in PMOP. A: Map of the altered KEGG pathways between high and low BRD4 expression groups. B: Altered GO enrichment plot between high and low BRD4 expression groups.