Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair

Abstract

Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes including Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 was downregulated following Icariin treatment for 14 days. In a differentiation assay using bone marrow mesenchymal stem cells (MSCs) carrying HIF-1α floxed allele, the promotive effect of Icariin on chondrogenic differentiation is largely decreased following Cre recombinase-mediated deletion of HIF-1α in MSCs as indicated by Alcian blue staining for proteoglycan synthesis. In an alginate hydrogel 3D culture system, Icariin increases Safranin O positive (SO+) cartilage area. This phenotype is accompanied by upregulation of HIF-1α, increased proliferating cell nuclear antigen positive (PCNA+) cell numbers, SOX9+ chondrogenic cell numbers, and Col2 expression in the newly formed cartilage. Coincide with the micromass culture, Icariin treatment upregulates mRNA levels of Sox9, Col2α1, aggrecan and Col10α1 in the 3D cultures. We then generated alginate hydrogel 3D complexes incorporated with Icariin. The 3D complexes were transplanted in a mouse osteochondral defect model. ICRS II histological scoring at 6 and 12 weeks post-transplantation shows that 3D complexes incorporated with Icariin significantly enhance articular cartilage repair with higher scores particularly in selected parameters including SO+ cartilage area, subchondral bone and overall assessment than that of the controls. The results suggest that Icariin may inhibit PHD activity likely through competition for cellular iron ions and therefore it may serve as an HIF-1α activator to promote articular cartilage repair through regulating chondrocyte proliferation, differentiation and integration with subchondral bone formation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Icariin upregulates HIF-1α expression in chondrocytes by inhibiting PHDs activity through competition for iron ions.

(A) The chemical formula of Icariin. (B) Hypoxia response element luciferase reporter assay in C2C12 cells treated with Icariin at indicated concentrations. (C) Western blot analysis for HIF-1α protein expression in primary culture-derived chondrocytes under normoxia or hypoxia or treated with or without Icariin (10−6 M) for 8 h. β-actin used as the loading control. (D) Detection of HIF-1α nuclear localization in Icariin (10−6 M)-treated chondrocytes by immunofluorescence staining under confocal microscope. (E, F) Chondrocytes were cultured and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10−6 M) for 7 or 14 days. HIF-1α mRNA levels were detected by real-time PCR in Icariin-treated chondrocytes compared with that of the control cells. *P < 0.05, **P < 0.01, n = 3. (G) UV-Vis spectra of the Icariin, FeSO4 and their mixture (nIcariin: nFeSO4 = 3: 1, CIcariin = 0.5mM) in aqueous solution after incubation at 37°C for 12 h; CIcariin = 0.5mM; CFeSO4 = 1mM; The inset shows the visual appearance of each species. (H) Western blot analysis for HIF-1α protein expression in chondrocytes treated with or without Icariin (10−6 M) and FeSO4 (100 μM) for 12 h. (I) Western blot analysis for PHDs and HIF-1α protein expression in chondrocytes treated with or without Icariin (10−6 M) for 12 h. In all Figs, ICA, Icariin.

Fig 2. Icariin increases chondrocytes proliferation.

(A) MTT assay for cell viability of chondrocytes treated with or without Icariin (0 M, 10−7 M, 10−6 M, 10−5 M) for 3 days. Treated groups compared with control group, *P < 0.05, **P < 0.01, n = 3. (B) BrdU incorporation assay for chondrocytes treated with or without Icariin (0 M, 10−7 M, 10−6 M, 10−5 M) for 1 day or 3 days. Treated groups compared with control group, *P < 0.05; ***P < 0.001, n = 3. (C) Colony formation assay for chondroprogenitor cells treated with Icariin (10−7 M, 10−6 M, 10−5 M) for 24 h followed by 14 days sub-culture. (D) Quantitation of the colony numbers from (C), *P < 0.05, n = 3.

Fig 3. Icariin enhances chondrogenic marker expression and cartilage matrix synthesis while the effect is limited by knockdown of HIF-1α.

(A) Chondrocytes were processed for micromass culture and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10−7 M, 10−6 M, 10−5 M). The cell masses were stained with Alcian blue after 7 or 14 days culture, respectively. Note that Icariin (10−6 M) increased proteoglycan synthesis. (B) Quantitation of the value of integral optical density (IOD) from (A). Treated groups compared with control group, *P < 0.05; **P < 0.01; ***P < 0.01, n = 3. (C, D) ELISA assays for production of aggrecan (C) and hydroxypoline (D) in chondrocytes. Icariin treated groups versus control groups, *P < 0.05; **P < 0.01; ***P < 0.001, n = 3. (E, F) Chondrocytes were cultured and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10−6 M) for 7 (E) or 14 (F) days. Sox9, Col2α1 and Aggrecan mRNA expression was detected by real-time PCR in Icariin treated chondrocytes and compared with that in the control cells. *P < 0.05, **P < 0.01; ***P < 0.001, n = 3.

Fig 4. Icariin inhibits catabolic marker genes expression in chondrocytes.

Chondrocytes were cultured and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10−6 M) for 7 (A) or 14 (B) days. Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 mRNA expression was detected by real-time PCR in Icariin treated chondrocytes compared with that of the control cells. *P < 0.05; **P < 0.01; n = 3.

Fig 5. Deletion of HIF-1α eliminates the positive effects of Icariin on chondrocytes.

(A) Western blot analysis for HIF-1α protein expression in MSCs (HIF-1α Floxed) following treatment with Ad-GFP or Ad-Cre and treated with or without Icariin (10−6 M) for 12 h. β-actin used as the loading control. (B) Alcian blue staining for preoteoglycan synthesis in MSCs (HIF-1α floxed) following treatment with Ad-GFP or Ad-Cre and treated with or without Icariin (10−6 M) for 14 days. (C) Quantitation of the value of integral optical density (IOD) from (B). Compared with Ad-GFP-treated control group, *P < 0.05; n = 3. (D) BrdU incorporation assay for chondrocytes following treatment with Ad-GFP or Ad-Cre and treated with or without Icariin (10−6 M) for 48 h. Compared with Ad-GFP-treated control group, *P < 0.05, **P < 0.01; n = 3. Compared with Ad-GFP-treated ICA control group, ##P < 0.01; n = 3. (E, F) Chondrocytes following treatment with Ad-GFP or Ad-Cre were cultured under normal medium in the presence or absence of Icariin (10−6 M). (E) Sox9, Aggrecan and Col2α1 mRNA expression in chondrocytes was detected by real-time PCR. (F) Adamts4, Mmp2, and Mmp9 mRNA expression in chondrocytes was detected by real-time PCR. Compared with Ad-GFP-treated control group,*P < 0.05, **P < 0.01; n = 3. Compared with Ad-Cre-treated control group, #P < 0.05; ##P < 0.01; n = 3.

Fig 6. Icariin promotes chondrogenesis in alginate-chondrocyte 3D culture system.

(A-C) Representative H&E, Alcian blue and SO histological images for sections from alginate-chondrocyte 3D culture system treated with Icariin (10−6 M) for 21 days, with none treatment as control. Scale bar = 50 μm. (D) Quantitation of IOD for Alcian blue staining. Icariin treated group compared with control group, **P < 0.01, n = 3. (E) Quantitation of the positive SO staining area. Icariin treated group compared with control group, **P < 0.01, n = 3. (F-I) The alginate-chondrocyte 3D cultures were treated with Icariin (10−6 M) for 21 days. Sox 9, Aggrecan, Col2α1 and Col10α1 mRNA expression was quantified by real-time PCR and compared with that of control group with no Icariin treatment. **P < 0.01, n = 3. (J) Representative images of the immunostaining for SOX9 and Col2 in the sections. IgG was used as negative control. Scale bar = 50 μm. (K) Quantitation of SOX9+ chondrocytes was presented as percentage of total chondrocytes in the SOX9 stained sections from (J). *P < 0.05, n = 3. (L) Densitometric analysis of Col2 immunostaining in (J) using GraphPad Prism 5 software. *P < 0.05, n = 3.

Fig 7. Icariin increases chondrocyte proliferation accompanied by upregulation of HIF-1α in alginate-chondrocyte 3D culture system.

(A) Representative images of immunostaining for PCNA in 3D cultured sections from Icariin treated group and control group. Arrows indicate PCNA+ chondrocytes. IgG was used as negative control. Scale bar = 50 μm. (B) Quantitation of the percentage of PCNA positive cells in the Icariin treated group and control group. *P < 0.05, n = 3. (C) Representative images of immunostaining for HIF-1α in 3D cultured sections from Icariin treated group and control group. Arrows indicate HIF-1α positive (HIF-1α+) chondrocytes. IgG was used as negative control. Scale bar = 50 μm. (D) Quantitation of the percentage of HIF-1α+ cells in the sections from Icariin treated groups and control groups. *P < 0.05, n = 3.

Fig 8. Icariin promotes articular cartilage repair in the mouse osteochondral defect model.

3D complexes incorporated with or without Icariin were transplanted in osteochondral defect regions of the distal femur of mice (detailed in Material and Methods). The dashed lines surround the newly formed tissue in the osteochondral defect region. (A) H&E staining showed the osteochondral defect regions in Icariin treated group and control group at 2 weeks post-transplantation. Scale bar = 200 μm. (B) Immunostaining for PCNA in the sections from icariin treated group and control group at 2 weeks post-transplantation. Upper panel, low magnification, Scale bar = 200 μm. Lower panel, high magnification, Scale bar = 50 μm. (C) Quantitation of the percentage of PCNA+ cells in the PCNA-stained sections represented in (B). *P < 0.05, n = 5. (D, F) H&E staining (upper panels) showed the osteochondral defect regions and SO staining (lower panels) indicated proteoglycan synthesis in the osteochondral defects in Icariin treated group and control group at 6 weeks (D) and 12 weeks (F) post-transplantation. Scale bar = 200 μm. (E, G) Quantitation of ICRS II cartilage repair score at 6 weeks (E) and 12 weeks (G) post-transplantation. The parameters shown include matrix staining (SO staining), subchondral bone and overall assessment. Icariin-treated group compared with control group, *P < 0.05, **P < 0.01, n = 5. (H) Immunostaining for Col2 in the newly formed cartilage tissue in Icariin treated group and control group at 12 weeks post-transplantation. IgG was used as negative control. Scale bar = 50 μm. (I) Densitometric analysis of Col2 immunostaining in (H) using GraphPad Prism 5 software. *P < 0.05, n = 5.