Positive Effect of Gushukang on Type-H Vessel and Bone Formation

Abstract

Gushukang (GSK) is a traditional herbal compound used in Chinese medicine for the treatment of osteoporosis. Numerous studies have been conducted to elucidate the effects of GSK, but the mechanisms underlying these effects remain unclear. In the present study, we cultured osteoblasts and osteoclasts with low and high doses of GSK, and also administered 3-month-old mice with 4 and 8 g/kg/day of GSK solution. Gushukang was found to promote osteoblast differentiation and inhibit osteoclast differentiation in vitro. In vivo, mice in the GSK treatment groups showed an increase in bone mass, as measured by micro-computed tomography (Micro-CT). Tartrate resistant acid phosphatase (TRAP) staining and osteocalcin (OCN) staining experiments revealed decreased bone resorption and increased bone formation in the GSK treatment groups. In addition, we found a novel effect of GSK-it could induce type-H vessel formation in mice. The underlying mechanisms of these actions were further explored at the molecular level to investigate whether these effects were due to an overexpression of the hypoxia inducible factor-1 (HIF-1α). Our findings indicate the utility of GSK as a therapeutic for the prevention of osteoporosis.

Keywords: Gushukang; HIF-1α; bone formation; bone mass; type-H vessel.

FIGURE 1

GSK prevented bone loss in mice. (A) Representative μCT images of femurs collected from mice treated with low-dose (4 g/kg/day) and high-dose (8 g/kg/day) GSK or control. (B) Quantitation of Tb.BV/TV (trabecular bone volume per tissue volume); Tb.N (trabecular number); Tb.Th (trabecular thickness); and Tb.Sp (trabecular separation). Values were expressed as mean ± SD.*P < 0.05; all the assays were repeated more than three times.

FIGURE 2

GSK increases osteogenic differentiation and decreases osteoclast differentiation in vitro. (A) BMSCs were gathered from C57BL/6 mice (4 weeks) and cultured with GSK serum. Alizarin red staining was carried out at day 14 to assess osteogenic differentiation. Scale bar = 100 μm. (B) The Alizarin red staining optical density was quantified by ImageJ. *P < 0.05; the groups of GSK versus control. (C) BMMs were obtained from 4 weeks C57BL/6 mice and treated with M-CSF (100 ng/ml) and RANKL (50 ng/ml) (control), M-CSF, and RANKL added GSK serum. Osteoclast differentiation was evaluated at day 8 by TRAP staining. Scale bar = 100 μm. (D) The number of osteoclasts was quantified. Values were expressed as mean ± SD. *P < 0.05; all the assays were repeated more than three times.

FIGURE 3

GSK promoted the expressions of OCN in mice. (A,B) The protein expression of osteocalcin (OCN) of femurs gathered from mice treated with low-dose and high-dose GSK or control (saline) was determined by immunohistological staining. Scale bar = 100 μm. Values were expressed as mean ± SD. *P < 0.05; all the assays were repeated more than three times.

FIGURE 4

GSK decreases the number of osteoclasts in mice. (A) TRAP staining was performed on femurs collected from the group of low-dose and high-dose GSK or control (saline). Scale bar = 100 μm. (B) Osteoclast-covered surface over bone surface (OCs/BS%) of each group was quantified. (C) Osteoclast number (OC.N). Values were expressed as mean ± standard deviation (SD). *P < 0.05; all the assays were repeated more than three times.

FIGURE 5

GSK accelerated CD31hiEmcnhi vessels formation in mice. (A) Representative images of CD31 (green), Emcn (red) of femurs from the group of low-dose and high-dose GSK or control (saline) by immunofluorescence staining. Scale bar = 100 μm. (B) Type-H vessel surface was quantified. Values were expressed as mean ± SD. *P < 0.05; all the assays were repeated more than three times.

FIGURE 6

GSK induced overexpression of HIF-1a. (A) Representative images of WB and quantification of the protein expression level of HIF-1a. (B) Quantification of the mRNA expression level of HIF-1a. Values were expressed as mean ± SD. *P < 0.05; all the assays were repeated more than three times.