A wogonin-rich-fraction of Scutellaria baicalensis root extract exerts chondroprotective effects by suppressing IL-1β-induced activation of AP-1 in human OA chondrocytes

Abstract

Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13 and ADAMTS-4 in IL-1β-treated OA chondrocytes. OA chondrocytes treated with F4 in the presence of IL-1β showed significantly enhanced expression of anabolic genes ACAN and COL2A1. In an in vitro model of cartilage degradation treatment with F4 inhibited s-GAG release from IL-1β-treated human cartilage explants. The inhibitory effect of F4 was not mediated through the inhibition of MAPKs and NF-κB activation but was mediated through the suppression of c-Fos/AP-1 activity at transcriptional and post transcriptional levels in OA chondrocytes. Purified Wogonin mimicked the effects of F4 in IL-1β-stimulated OA chondrocytes. Our data demonstrates that a Wogonin-rich fraction of SBE exert chondroprotective effects through the suppression of c-Fos/AP-1 expression and activity in OA chondrocytes under pathological conditions.

Figure 1. SBE Fraction 4 (F4) showed potent chondroprotective effects in human OA chondrocytes.

(A) HPLC chromatogram of ethanolic extract of S. baicalensis. Fractions were collected based on indicated retention time. (B) SBE fraction (s) was not toxic to human OA chondrocytes. Human OA chondrocytes were treated with indicated concentration (1–50 μg/ml) of SBE fraction (F1 or F2 or F3 or F4) for 24 h and cells viability were measured by MTT assays. Chondrocytes treated with 0.1% DMSO served as control. Viability was expressed relative to control cells. Data points represent mean ± SD from three subjects. (C) SBE fraction 4 (F4) inhibited IL-1β induced ROS production in OA chondrocytes. Human OA chondrocytes were treated with SBE fraction (F1 or F2 or F3 or F4) (50 μg/ml) for 2 h, then stained with DHR-123 (5 μM) for 0.5 h at 37 °C, and stimulated with IL-1β (1 ng/ml) for 5 minutes at 37 °C. Fluorescence emission was measured at 535 nm. Bar graph shows relative fluorescence units indicating ROS levels. (D–E) SBE fraction 4 (F4) inhibited IL-1β induced mRNA expression of IL-6 and MMP-13 in OA chondrocytes. Primary human OA chondrocytes were pre-treated with SBE fraction (s) for 2 h followed by treatment with IL-1β (1 ng/ml) for 16 h. At the end of treatment chondrocytes were harvested, total RNA were isolated, reverse transcribed to cDNA and mRNA expression of IL-6 (D) and MMP-13 (E) were measured by quantitative PCR using the TaqMan assay system. Expression of β-actin was used as endogenous expression control. Bar graph represent mean ± SD from three subjects **p ≤ 0.01, as compared to control, # ≤ 0.01, as compared to IL-1β.

Figure 2. F4 contains Wogonin as a major component.

(A) HPLC identification of Wogonin in F4. Fraction 4 (1 mg/ml) was subjected to HPLC on reversed-phase C 18 column as described in methods section. The major peak in HPLC chromatogram corresponds to Wogonin. (B) LC-MS/MS identification of Wogonin in F4. Fraction 4 (1 mg/ml) were directed to a mass spectrometer set up to run in Q1 scan using negative mode. Major peak at m/z 283.2 corresponds to Wogonin. (C) Quantification of Wogonin content in F4. F4 were directed to a mass spectrometer set up to run in negative multiple reaction (MRM-) mode detecting the transition pairs m/z 283.00 (precursor ion)/162.9 (product ion) for Wogonin. Quantification of Wogonin in F4 was calculated by four point calibration curves of Wogonin using MRM analysis. (D) Cellular uptake of F4 in OA chondrocytes. OA chondrocytes were treated with F4 (50, 100 μg/ml) for 4 or 24 h. Cell lysate were prepared and subjected to LC-MS/MS analysis as described in method section.

Figure 3. F4 inhibited the IL-1 β induced oxidative and nitrosative stress in OA chondrocytes.

(A) F4 inhibited the IL-1β induced ROS production in OA chondrocytes. Human OA chondrocytes were treated with F4 (50 μg/ml) for 2 h, then stained with H2DCF-DA (20 μM) for 0.5 h at 37 °C, and stimulated with IL-1β (1 ng/ml) for 5 minutes at 37 °C. Fluorescence emission was measured at 525 nm. Bar graph shows relative fluorescence units indicating ROS levels. Data points represent mean ± SD from four replicates. *p ≤ 0.01, as compared to control, # ≤ 0.01, as compared to IL-1β.(B) IL-1β induced production of H2O2 and O2- in OA chondrocytes. OA chondrocytes were treated with catalase (1000 unit/ml) or SOD F4 (200 unit/ml) for 1 h, stained with DHR-123 (5 μM) for 0.5 h at 37 °C and then stimulated with IL-1β (1 ng/ml) for 5 minutes at 37 °C. Fluorescence emission was measured at 535 nm. Bar graph shows relative fluorescence units indicating ROS levels. (C) F4 inhibited the IL-1β induced production of H2O2 in OA chondrocytes. Human OA chondrocytes were treated with F4 (50 μg/ml) for 2 h, and the stimulated with IL-1β (1 ng/ml) for 5 minutes at 37 °C. H2O2 generation was estimated by Amplex red assay as described in method section. (D–E) F4 inhibited the IL-1β induced production of NO and expression of iNOS in OA chondrocytes. Primary human OA chondrocytes were pre-treated with F4 (50 μg/ml) for 2 h followed by treatment with IL-1β (1 ng/ml) for 16 h. At the end of treatment culture supernatant were collected and chondrocytes were harvested for RNA isolation. (D) NO was estimated in supernatant using Griess reagent as described in methods. (E) Expression of iNOS was measured by quantitative PCR using the TaqMan assay system (Life Technologies). β-actin was used as endogenous expression control. Bar graph represents mean ± SD from three subjects. *p ≤ 0.01, as compared to control, # ≤ 0.01, as compared to IL-1β.

Figure 4. F4 inhibited IL-1 β the induced inflammatory mediators in OA chondrocytes.

Primary human OA chondrocytes were pre-treated with F4 (50 μg/ml) for 2 h followed by treatment with IL-1β (1 ng/ml) for 16 h. At the end of treatment culture supernatant were collected and chondrocytes were harvested. Cell lysate were prepared using RIPA buffer for immunoblot analysis or RNA were isolated for real time PCR analysis. (A) Protein expression was investigated by immunoblotting using antibodies against indicated protein. β-actin was used as a control for equal loading. Specific signal intensities were quantified by ImageJ software. (B) Expression of COX-2 was measured by quantitative PCR using the TaqMan assay system. β-actin was used as endogenous expression control. (C,D) Secreted levels of IL-6 (C) and PGE2 production (D) were measured in the culture supernatant by ELISA. Bar graph represents mean ± SD from two subjects. *p ≤ 0.01, as compared to control, # ≤ 0.01, as compared to IL-1β.

Figure 5. F4 inhibited the IL-1 β induced matrix degradation in OA chondrocytes.

Primary human OA chondrocytes were pre-treated with F4 (50 μg/ml) for 2 h followed by treatment with IL-1β (1 ng/ml) for 16 h. At the end of treatment culture supernatant were collected and chondrocytes were harvested. RNA was isolated for real time PCR analysis. (A) Expression of MMP-3, MMP-9, and ADAMTS4 was measured by quantitative PCR using the TaqMan assay system. β-actin was used as endogenous expression control. (B) Secreted levels of MMP-13 were measured in the culture supernatant by ELISA. (C) Expression of COL2A1, and ACAN was measured by quantitative PCR using the TaqMan assay system. β-actin was used as endogenous expression control. (D). F4 inhibited the IL-1 β induced release of s-GAG in OA explants. Human OA cartilage pieces were incubated with F4 (50 μg/ml) for 2 h followed by treatment with IL-1β (25 ng/ml) for 72 h. The s-GAG release from cartilage explants in culture supernatants was quantified by using DMMB colorometric assay as described in methods section. Bar graph represents mean ± SD from two subjects. *p ≤ 0.01, as compared to control, # ≤ 0.01, as compared to IL-1β.

Figure 6. F4 did not inhibit the IL-1 β induced activation of MAPKs and NF-κB in OA chondrocytes.

Primary human OA chondrocytes were pre-treated with F4 (50 μg/ml) for 2 h followed by treatment with IL-1β (10 ng/ml) for 15 or 30 minutes. Cell lysate were prepared from harvested chondrocytes using RIPA lysis buffer or nuclear extracts were prepared using nuclear extract kit (Active Motif). (A) Activation of MAPKs was investigated by immunoblotting using primary antibodies specific for phospho-ERK1/2, phospho-JNK, phospho-p38. Expression of total ERK1/2, total JNK, or total-p38 was used as control. Immunoblot results are representatives of two blots performed on samples obtained from two individuals. (B) Binding activity of NF-κB from nuclear extracts of the cells treated as above to its consensus sequence was quantitated by a specific ELISA assay (Active Motif). (C) Degradation of Iκβα was investigated by immunoblotting in cell lysate prepared as above. β-actin was used as a control for equal loading. Bar graph represents mean ± SD from two subjects. *p ≤ 0.01, as compared to control.

Figure 7. F4 inhibited the IL-1β induced activation of c-Fos/AP-1.

Primary human OA chondrocytes were pre-treated with F4 (50 μg/ml) for 2 h followed by treatment with IL-1β (10 ng/ml) for 30 minutes. Cell lysate were prepared from harvested chondrocytes using RIPA lysis buffer or nuclear extracts were prepared using nuclear extract kit (Active Motif). (A,B) Binding activity of c-Fos/AP-1 from nuclear extracts of the cells treated as above to its consensus sequence was quantitated by a specific ELISA assay (Active Motif). (C) F4 (50 μg/ml) was added to nuclear extracts of the IL-1β treated cells for 0.5 h and binding activity of c-Fos/AP-1 was quantitated by a specific ELISA assay (Active Motif). (D) Protein expression was investigated by immunoblotting using antibodies against indicated protein. β-actin was used as a control for equal loading. (E) Expression of c-Fos was measured by quantitative PCR using the TaqMan assay system. GAPDH was used as endogenous expression control. Immunoblot results are representatives of two blots performed on samples obtained from two individuals. Bar graph represents mean ± SD from two subjects. *p ≤ 0.05, as compared to control, # ≤ 0.05, as compared to IL-1β.

Figure 8. Purified Wogonin exerts chondroprotective effects in OA chondrocytes.

(A) Human OA chondrocytes were treated with purified Wogonin (20 μg/ml) for 2 h, then stained with H₂DCF-DA (20 μM) for 0.5 h at 37 °C and stimulated with IL-1β (1 ng/ml) for 5 minutes at 37 °C. Fluorescence emission was measured at 525 nm. Bar graph shows relative fluorescence units indicating ROS levels. Data points represent mean ± SD from four replicates. *p ≤ 0.01, as compared to control, # ≤ 0.01, as compared to IL-1β. (B) Primary human OA chondrocytes were pre-treated with purified Wogonin (20 μg/ml) for 2 h followed by treatment with IL-1β (1 ng/ml) for 16 h. At the end of treatment culture chondrocytes were harvested and RNA was isolated for real time PCR analysis. Expression of IL-6 and MMP-13 was measured by quantitative PCR using the TaqMan assay system. β-actin was used as endogenous expression control. (C) Primary human OA chondrocytes were pre-treated with purified Wogonin (20 μg/ml) for 2 h followed by treatment with IL-1β (10 ng/ml) for 30 minutes and nuclear extracts were prepared using nuclear extract kit (Active Motif). Binding activity of c-Fos/AP-1 to its consensus sequence was quantitated by a specific ELISA assay (Active Motif). Bar graph represents mean ± SD from two subjects. *p ≤ 0.01, as compared to control, # ≤ 0.01, as compared to IL-1β.