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

<p>(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. *<i>P</i> < 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. *<i>P</i> < 0.05, n = 3.</p

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

<p>(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, **<i>P</i> < 0.01, n = 3. (E) Quantitation of the positive SO staining area. Icariin treated group compared with control group, **<i>P</i> < 0.01, n = 3. (F-I) The alginate-chondrocyte 3D cultures were treated with Icariin (10⁻⁶ M) for 21 days. <i>Sox 9</i>, <i>Aggrecan</i>, <i>Col2α1</i> and <i>Col10α1</i> mRNA expression was quantified by real-time PCR and compared with that of control group with no Icariin treatment. **<i>P</i> < 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). *<i>P</i> < 0.05, n = 3. (L) Densitometric analysis of Col2 immunostaining in (J) using GraphPad Prism 5 software. *<i>P</i> < 0.05, n = 3.</p

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

<p>(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, *<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.01, n = 3. (C, D) ELISA assays for production of aggrecan (C) and hydroxypoline (D) in chondrocytes. Icariin treated groups versus control groups, *<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 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. <i>Sox9</i>, <i>Col2α1</i> and <i>Aggrecan</i> mRNA expression was detected by real-time PCR in Icariin treated chondrocytes and compared with that in the control cells. *<i>P</i> < 0.05, **<i>P</i> < 0.01; ***<i>P</i> < 0.001, n = 3.</p

Icariin inhibits catabolic marker genes expression in chondrocytes.

<p>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. <i>Mmp2</i>, <i>Mmp9</i>, <i>Mmp13</i>, <i>Adamts4</i> and <i>Adamts5</i> mRNA expression was detected by real-time PCR in Icariin treated chondrocytes compared with that of the control cells. *<i>P</i> < 0.05; **<i>P</i> < 0.01; n = 3.</p

Icariin increases chondrocytes proliferation.

<p>(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, *<i>P</i> < 0.05, **<i>P</i> < 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, *<i>P</i> < 0.05; ***<i>P</i> < 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), *<i>P</i> < 0.05, n = 3.</p

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

<p>(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, *<i>P</i> < 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, *<i>P</i> < 0.05, **<i>P</i> < 0.01; n = 3. Compared with Ad-GFP-treated ICA control group, ^##<i>P</i> < 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) <i>Sox9</i>, <i>Aggrecan</i> and <i>Col2α1</i> mRNA expression in chondrocytes was detected by real-time PCR. (F) <i>Adamts4</i>, <i>Mmp2</i>, and <i>Mmp9</i> mRNA expression in chondrocytes was detected by real-time PCR. Compared with Ad-GFP-treated control group,*<i>P</i> < 0.05, **<i>P</i> < 0.01; n = 3. Compared with Ad-Cre-treated control group, ^#<i>P</i> < 0.05; ^##<i>P</i> < 0.01; n = 3.</p

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

<p>(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. *<i>P</i> < 0.05, **<i>P</i> < 0.01, n = 3. (G) UV-Vis spectra of the Icariin, FeSO₄ and their mixture (<i>n</i>_Icariin: <i>n</i>_FeSO4 = 3: 1, <i>C</i>_Icariin = 0.5mM) in aqueous solution after incubation at 37°C for 12 h; <i>C</i>_Icariin = 0.5mM; <i>C</i>_FeSO4 = 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.</p