Icariin Reduces Cartilage Degeneration in a Mouse Model of Osteoarthritis and is Associated with the Changes in Expression of Indian Hedgehog and Parathyroid Hormone-Related Protein

Abstract

BACKGROUND The aim of this study was to determine the role of icariin, a Chinese traditional herbal medicine extracted from Epimedium, in osteoarthritis (OA), using the murine anterior cruciate ligament transection (ACLT)-induced model of OA and micromass culture of murine chondrocytes. MATERIAL AND METHODS Twenty-four three-month-old C57/6J mice were randomly divided into three groups: the sham group (no surgery and joint injection with normal saline) (N=8); the ACLT + ICA group (ACLT surgery and icariin treatment) (N=8); and the ACLT group (ACLT surgery and joint injection with normal saline) (N=8). At 12 weeks after ACLT surgery, murine articular cartilage was harvested from all mice for histological evaluation of any differences in cartilage degeneration. In vitro micromass culture of mouse chondrocytes was used to study the effects of icariin on chondrocyte differentiation and growth from the three mouse groups. RESULTS Icariin treatment (mice in the ACLT + ICA group) significantly reduced degeneration of cartilage in OA with increased cartilage thickness, associated with increased expression of collagen type II alpha 1 (COL2A1), decreased chondrocyte hypertrophy, and decreased expression of collagen type X (ColX) and matrix metalloproteinase 13 (MMP13). In vitro, icariin promoted chondrocyte differentiation by upregulating the expression of agrrecan, Sox9 and parathyroid hormone-related protein (PHrP) and down-regulation of Indian hedgehog (Ihh) and genes regulated by Ihh. CONCLUSIONS In a mouse model of OA icariin treatment reduced destruction of cartilage, promoted chondrocyte differentiation, upregulated expression of PHrP and down-regulated the expression of Ihh.

Figure 1

Icariin treatment reduced the degeneration of articular cartilage in the murine anterior cruciate ligament transection (ACLT)-induced model of osteoarthritis (OA). (A) Photomicrographs of the histology of the murine articular cartilage of the three mouse groups: the sham group (no surgery and joint injection with normal saline), the ACLT + ICA group (ACLT surgery and icariin treatment) and the ACLT group (ACLT surgery and joint injection with normal saline). The yellow squares are the higher magnification of the upper images, black dots imply the separation lines among hyaline cartilage (HC), calcified cartilage (CC), and subchondral bone (SB). Hematoxylin and eosin (H&E). (B) Photomicrographs of the histology of the Safranin O staining of murine cartilage in the sham, ACLT + ICA, and ACLT mouse groups. (C) The degeneration of cartilage was assessed by using Osteoarthritis Research Society International (OARSI) scoring system. (D) The thickness of cartilage in three groups of mice, 12 weeks after surgery: the sham group, where the incision was made and sutured immediately; the ACLT + ICA group of mice underwent ACLT surgery and icariin treatment; the ACLT group underwent ACLT surgery and joint injection with normal saline. Data are presented as the mean ±SD. N=5. NS – nonsignificant. * P<0.05; ** P<0.01.

Figure 2

Icariin treatment promoted the expression of chondrogenic marker genes and inhibited the expression of chondrocyte hypertrophic genes in vivo. (A) The expression of Sox9, collagen type II alpha 1 (COL2A1), collagen type X (ColX), and matrix metalloproteinase 13 (MMP13) in articular cartilage, 12 weeks after murine anterior cruciate ligament transection (ACLT), were detected by immunohistochemical (IHC) staining. (B–E) The quantification of IHC staining of Sox9, Col2a1, ColX, and MMP13 in (A). The three mouse groups include: the sham group (no surgery and joint injection with normal saline), the ACLT + ICA group (ACLT surgery and icariin treatment) and the ACLT group (ACLT surgery and joint injection with normal saline). Data are presented as the mean ±SD. N=5. NS – nonsignificant. * P<0.05; ** P<0.01.

Figure 3

Icariin treatment of chondrocytes increased proteoglycan synthesis during chondrogenic differentiation. (A) Micromass culture of chondrocytes in the chondrogenic medium without or with different concentration of icariin: 10⁻⁷ M, 10⁻⁶ M, and 10⁻⁵ M. Alcian blue staining was used to detect the proteoglycans synthesis in day 7 and day 14 cells. (B) Quantification of the Alcian blue staining in (A). (C) Enzyme-linked immunosorbent assay (ELISA) results of the expression of collagen type II alpha 1 (COL2A1) of cells in culture on Day 7 and Day 14 treated with or without icariin. Data are presented as the mean ±SD. N=4. NS – nonsignificant * P<0.05; ** P<0.01.

Figure 4

Icariin treatment increased the expression of chondrogenic-related genes and inhibited the expression of chondrogen hypertrophy-related genes in micromass culture of chondrocytes in vitro. Chondrocytes were cultured in chondrogenic medium in the absence or presence of icariin (10⁻⁶ M). Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis was used to detect the mRNA expression level of Sox9 (A), Agrrecan (B), runt-related transcription factor (RUNX2) (C), MMP13 (D), and ColX (E). Data are presented as the mean ±SD. N=4. NS, nonsignificant * P<0.05; ** P<0.01; *** P<0.005.

Figure 5

The effects of icariin treatment on Indian hedgehog (Ihh) and parathyroid hormone-related protein (PHrP) signaling in promoting chondrogenesis in vitro. Mouse chondrocytes were cultured and induced to differentiation in the chondrogenic medium in the absence or presence of icariin (10⁻⁶ M). Immunofluorescence (IF) staining was used to detect the protein expression level of Indian hedgehog (Ihh) (A), parathyroid hormone-related protein (PHrP) (B), and runt-related transcription factor (RUNX2) (C). (D–F) The quantification of IF staining in A–C. Data are presented as the mean ±SD. N=4. NS – nonsignificant. * P<0.05.

Figure 6

Icariin treatment and the effects on Indian hedgehog (Ihh) and parathyroid hormone-related protein (PHrP) signaling in promoting chondrogenesis in vitro and in vivo in the murine anterior cruciate ligament transection (ACLT)-induced model of osteoarthritis (OA). (A) Chondrocytes in culture show differentiation in the chondrogenic medium in the absence or presence of icariin (10⁻⁶ M). Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis detects the mRNA expression levels of genes regulated by Indian hedgehog (Ihh): Cyclin D1, Ptch1, and Gli1. Data are presented as the mean ±SD. N=4. (B, C) Immunofluorescence (IF) staining detects the protein expression level of parathyroid hormone-related protein (PHrP) (B) and Indian hedgehog (Ihh) (C) in murine articular cartilage of in the three mouse groups: the sham group (no surgery and joint injection with normal saline), the ACLT + ICA group (ACLT surgery and icariin treatment) and the ACLT group (ACLT surgery and joint injection with normal saline). (D, E) The quantification of IF staining in A, B. Data are presented as the mean ±SD. N=5. NS – nonsignificant. * P<0.05; ** P<0.01.