Involvement of ubiquitination-dependent process in EGb-mediated c-Jun degradation.

<p>CHON-002 cells were transfected by lentiviruses carrying Flag-c-Jun or HA-ubiquitin gene. Six days after transfection, the cells were treated with or without EGb for various time points and the total cell lysates were immunoprecipitated with antibodies against FLAG and then western blotted with antibodies against HA (A). Meanwhile, the total lysates were immunoblotted with antibodies against c-Jun or β-actin (A). The fold inductions of levels of c-Jun-ubiquitin conjugates at various time points in which the conjugate in un-treated cell lysates was taken as one fold are shown (B). Similar to (B), the levels of total c-Jun in different conditions are shown (C). The representative results from at least 3 independent experiments are shown.</p

Suppression of IL-1-induced AP-1, but not NF-κB, DNA-binding activity by EGb.

<p>Chondrocytes, 1–2×10⁶ for each condition, were pretreated with or without various doses of EGb for 72 h and then stimulated with IL-1 for another 24 h. The cells were collected and the nuclear extracts were prepared for EMSA analysis. For competition studies, the competitors with unradiolabeled wild-type (Wt.) or mutant (Mt.) AP-1 oligonucleotides were added 30 min before addition of the radiolabeled AP-1 probe. The final reaction mixture was analyzed on a 6% native polyacrylamide gel (A). In (B), the IL-1-stimulated cellular nuclear extracts were incubated with or without 200 µg/ml EGb for 30 min before EMSA analysis. In (C), the experiments were performed similarly with (A), except that monoclonal antibodies against c-Jun, c-Fos, p65 or p50 were used instead of oligonucleotide competitors. EMSA analysis was also conducted to determine the NF-κB DNA-binding activity (D). NS, non-specific; SS, supershifts.</p

EGb treatment inhibited IL-1-induced chemokine production and cell migration.

<p>Chondrocytes, 3×10⁵ for each condition, were treated with various concentrations of EGb for 72 h and then stimulated with IL-1 (5 ng/ml) for another 24 h. The supernatants were collected and the concentrations of RANTES, MCP-2, MIP-1α and MIP-1β were measured by ELISA (A). Equal numbers of THP-1 cells were loaded in the upper chambers of the transwell cassette and the lower chambers were loaded with IL-1-treated cells that had been pretreated with or without EGb. Migration was carried out at 37°C in 5% CO₂ for 4 h. Then cells migrating from the upper chambers to the lower chambers were counted by flow cytometry (B). <b>*</b>Denotes statistical significance (P<0.05) vs stimulated control cells. V: vehicle (DMSO in this study).</p

EGb caused c-Jun degradation via the proteosome pathway.

<p>CHON-002 cells were pretreated with 200 µg/ml EGb in the presence or absence of various doses of MG-132 for 72 h and then stimulated with or without IL-1 (5 ng/ml) for another 15 min. The total cell lysates were prepared and the expressions of phosphorylated c-Jun, total c-Jun and β-actin were determined by western blot (A). Similarly, the effects of the proteosome inhibitor MG-132 on EGb-induced degradation of c-Jun were examined in primary human chondrocytes (B). The representative results from 3 independent experiments are shown.</p

Inhibition of IL-1-induced NO production and iNOS expression by EGb.

<p>Chondrocytes, 1-2×10⁶ for each condition, were pretreated with various concentrations of EGb for 72 h and then stimulated with IL-1 for another 24 h. The culture supernatants were collected for NO measurement by Griess assays (A). The cells were collected for the determination of mRNA expression by RT-PCR (B) or protein expression by western blot (C). The representative results of at least three independent experiments using different donor cells are shown. V: vehicle.</p

EGb independently and individually blocked IL-1-mediated activation of JNK and induced degradation of c-Jun in chondrocytes.

<p>Binding of IL-1 to its receptor causes activation of MAPK upstream kinases (MKKs) and subsequently MAPKs, including JNK, p38 and ERK. Activated MAPKs then stimulate AP-1. EGb through inhibiting JNK activity and inducing ubiquitination-dependent degradation of c-Jun suppressed IL-1-mediated effects in chondrocytes.</p

EGb induced c-Jun degradation.

<p>Chondrocytes were pretreated with or without various concentrations of EGb for 72-1 for another 15 min. The total cell lysates were prepared and the levels of c-Jun, phosphorylated c-Jun, c-Fos and β-actin were determined by western blot (A). In (B), the fold inductions of relative densitometric intensity of c-Jun, phosphorylated c-Jun and c-Fos, adjusted by β-actin intensity, from more than 3 independent experiments using different donor cells are shown. In (C), total RNA was prepared from the collected cells and RT-PCR was performed to measure c-Jun and GAPDH mRNA expression. The pooled results from 4 independent experiments examining different donor cells are shown.</p

Effects of HS-Ck on the expression levels of MMP-13 in TNF-α-induced porcine chondrocytes.

<p>(A) Porcine chondrocytes were pretreated with various doses of HS-Ck or the DMSO solvent as vehicle (V) control for 2 h and then stimulated with TNF-α (5 ng/mL) for 24 h. The activities of pro-MMP-13 released into the culture supernatants were determined by using Western Blotting assay. (B) The expression levels of pro-MMP-13 activities were expressed as the relative band intensity. The representative data out of at least three independent experiments are shown. *<i>P</i> < 0.05 compared to the TNF-α-stimulated in the absence of HS-Ck treatment.</p

Effects of HS-Ck on TNF-α-induced NO production and cell viability in porcine chondrocytes.

<p>(A) Porcine chondrocytes were pretreated with various doses of HS-Ck or the DMSO solvent as vehicle (V) control for 2 h and then stimulated with TNF-α for 24 h. The production of NO was determined by using the Griess reagent. (B) To determine potential cytotoxic effects of HS-Ck, porcine chondrocytes were treated with various concentrations of HS-Ck for 24 h. The cells and culture supernatants were collected and determined by using MTT assay. The representative data out of at least three independent experiments are shown. *<i>P</i> < 0.05 compared to the TNF-α-stimulated in the absence of HS-Ck treatment.</p

Effects of HS-Ck on TNF-α-stimulated activation of NF-κB and STAT-3 transcriptional factors in porcine chondrocytes.

<p>For determining the effects of HS-Ck on TNF-α-induced DNA-binding activity of NF-κB (A) and STAT-3 (B), various doses of HS-Ck were pretreated with nuclear extracts for 30 min before the addition of radiolabeled oligonucleotides. The effects of HS-Ck on TNF-α-induced DNA-binding activity of NF-κB and STAT-3 were determined by using EMSA. The expression levels of NF-κB (A) and STAT-3 (B) were expressed as the relative band intensity. DMSO solvent was showed as vehicle (V) control. The representative data out of at least three independent experiments are shown. **<i>P</i> < 0.01 compared to the TNF-α-stimulated in the absence of HS-Ck treatment.</p