Table 1.
Sequences of Primers Used for Quantitative Real-time PCR.
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.