Epimedium flavonoids counteract the side effects of glucocorticoids on hypothalamic-pituitary-adrenal axis

Abstract

Our previous studies demonstrated that the epimedium herb, when simultaneously used with GCs, counteracted suppressive effects of GCs on the HPA axis without adverse influence on the therapeutic action of GCs. Here, total flavones were extracted from the epimedium flavonoids (EFs) and then used to investigate whether EFs provide protective effects on the HPA axis. We found that GCs induced a significant decrease in body weight gain, adrenal gland weight gain, and plasma adrenocorticotropin (ACTH) and corticosterone levels. After treatment with EFs, body weight gain, adrenal gland weight gain, and plasma corticosterone level were significantly restored, whilst plasma ACTH level was partially elevated. EFs were also shown to promote cell proliferation in the outer layer of adrenal cortex and to enhance the migration of newly divided cells toward the inner layer. To elucidate the underlying mechanisms, the mRNA expression of insulin-like growth factor II (IGF-II) was measured, and EFs significantly upregulated IGF-II expression. Our results indicated that EFs counteract the suppression of the HPA axis induced by GCs. This may involve both the ACTH and IGF-II pathways and thereby promote regeneration of the adrenal cortex suggesting a potential clinical application of EFs against the suppressive effects of GCs on the HPA axis.

Figure 1

Content of epimedium flavonoids (EFs) by high-performance liquid chromatography (HPLC). (a) HPLC profiles of EFs; the content of two components is more than 10%. (b) HPLC profiles of standard samples of icariin. The content of icariin is 43.7% of the preparation.

Figure 2

Effects of EFs on body weight gain of rats. (a) The body weight gain on 7th day for control, corticosterone only, and combined use of corticosterone and EFs was shown. (b) The body weight gain on 14th day for control, corticosterone alone, and combined use of corticosterone and EFs was shown. **P < 0.01 versus control group. ^# P < 0.05, ^## P < 0.01 versus corticosterone-only group.

Figure 3

Effects of EFs on plasma ACTH and corticosterone level of rats. (a) Plasma ACTH level was measured by ELISA in control, corticosterone, and combined use of corticosterone and EFs. (b) Plasma corticosterone level was measured by ELISA after treatment with saline (control), corticosterone, and combined use of corticosterone and EFs. *P < 0.05, **P < 0.01 versus control group. ^# P < 0.05 versus corticosterone-only group.

Figure 4

Localization and the number of BrdU-incorporated nuclei in the adrenal gland. ((a), (b), (c)) Sections of the adrenal gland from rats of the control group, corticosterone only-treated group, and combined corticosterone and EFs-treated group, respectively, were stained with anti-BrdU antibody and visualized with microscope (magnification ×400). (d) The percentage of BrdU-positive against total cells in zona glomerulosa was calculated. Cells with brown nuclei are considered as BrdU-positive cells. Cap, G, and F denote the capsule, zona glomerulosa, and zona fasciculata, respectively. All sections were counterstained with hematoxylin. **P < 0.01 versus control group. ^## P < 0.01 versus corticosterone-only group.

Figure 5

Migration of BrdU-positive cells toward the inner layer of adrenal gland. Rats were injected with BrdU; 14 days later, rats were killed and adrenal glands were excised, and the BrdU incorporation assay was carried out. ((a), (b), (c)) Sections of the adrenal gland from rats of the control group, corticosterone only-treated group, and combined corticosterone and EFs-treated group, respectively, were stained with anti-BrdU antibody and visualized with microscope (magnification ×400). (d) The percentage of BrdU-positive cells in zona glomerulosa or zona fasciculata against total BrdU-positive cells was calculated, respectively. Cells with brown nuclei are considered as BrdU-positive cells. Cap, G, and F denote the capsule, and zona glomerulosa, zona fasciculata, respectively. All sections were counterstained with hematoxylin.