Curcumin Inhibits Apoptosis of Chondrocytes through Activation ERK1/2 Signaling Pathways Induced Autophagy

Abstract

Osteoarthritis (OA) is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective in treating pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe, and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Curcumin, the principal curcuminoid and the most active component in turmeric, is a biologically active phytochemical. Evidence from several recent in vitro studies suggests that curcumin may exert a chondroprotective effect through actions such as anti-inflammatory, anti-oxidative stress, and anti-catabolic activity that are critical for mitigating OA disease pathogenesis and symptoms. In the present study, we investigated the protective mechanisms of curcumin on interleukin 1β (IL-1β)-stimulated primary chondrocytes in vitro. The treatment of interleukin (IL)-1β significantly reduces the cell viability of chondrocytes in dose and time dependent manners. Co-treatment of curcumin with IL-1β significantly decreased the growth inhibition. We observed that curcumin inhibited IL-1β-induced apoptosis and caspase-3 activation in chondrocytes. Curcumin can increase the expression of phosphorylated extracellular signal-regulated kinases 1/2 (ERK1/2), autophagy marker light chain 3 (LC3)-II, and Beclin-1 in chondrocytes. The expression of autophagy markers could be decreased when the chondrocytes were incubated with ERK1/2 inhibitor U0126. Our results suggest that curcumin suppresses apoptosis and inflammatory signaling through its actions on the ERK1/2-induced autophagy in chondrocytes. We propose that curcumin should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

Keywords: ERK; apoptosis; autophagy; curcumin; osteoarthritis.

Figure 1

Chemical structures of curcumin. Curcumin is derived from the rhizomes of turmeric (Curcuma longa).

Figure 2

Effects of curcumin and interleukin 1β (IL-1β) on the viability and proliferation of primary chondrocytes in vitro. To evaluate the effect of curcumin or IL-1β-induced cytotoxicity, primary chondrocytes were treated with 10 ng/mL, 15 ng/mL, 20 ng/mL, and 25 ng/mL IL-1β or 5 μM/L, 10 μM/L, 15 μM/L, and 20 μM/L curcumin for the following times: 6 h (A), 12 h (B), 24 h (C), and 48 h (D). IL-1β has a significant cytotoxic effect on chondrocytes. Results are expressed as means ± SD for experiments performed in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with controls. (E) Immunofluorescence identification of collagen II (red) in primary chondrocytes.

Figure 3

Effects of curcumin on IL-1β-induced apoptosis in primary chondrocytes. (A) Apoptotic cells were visualized using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) fluorescence immunocytochemistry (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue); (C) Flow cytometric detection of apoptosis in the chondrocytes. Apoptotic cells labeled with Annexin V and propidium iodide (PI) fluorescence were estimated by flow cytometry. The percentages of cells in each quadrant is indicative of: upper left, necrotic cells; lower left, live cells; lower right, early apoptotic cells; and upper right, late apoptotic cells; (D) Accumulation of cleaved caspase 3 and bcl-2 was visualized by western blot. Results shown in (B,E,F) are expressed as means ± SD for experiments performed in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001.Con, control; Rap, rapamycin; Cur, curcumin; 3-MA, 3-Methyladenine.

Figure 4

Curcumin activated autophagy within chondrocytes. The curcumin induced autophagy was detected by monodansylcadaverine (MDC) staining ((A), quantified in (B)) and transmission electron microscope (TEM) (C) at 24 h. Accumulation of autophagosomes was observed in chondrocytes that were pretreated with curcumin (C). Accumulation of beclin1 and light chain 3 (LC3)-II upon autophagy activation was visualized by western blot ((D), quantified in (E) and (F)). Results in (B,E,F) are expressed as means ± SD for experiments performed in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001. Cur, curcumin; Rap, rapamycin; 3-MA, 3-Methyladenine.

Figure 5

Curcumin reverses U0126-induced inhibition of extracellular signal-regulated kinases 1/2(ERK1/2) in primary chondrocytes. (A) p-ERK was visualized by western blot in U0126-treated chondrocytes. Serum-starved chondrocytes were preincubated with 10 μm curcumin for indicated time points and co-treated with 10 μm U0126 for 10 min, 20 min, 30 min, and 60 min. Curcumin pretreatment reversed U0126-induced ERK1/2 inhibition in a time-dependent manner. The pan ERK1/2 was not affected (and quantified in (B)). (C) Effects of curcumin on IL-1β-induced inhibition of mitogen-activated protein kinase (MAPK)/ERK1/2 pathway in primary chondrocytes in vitro. Serum-starved chondrocytes were pre-stimulated with 10 μm curcumin alone for 4 h and then co-treated with IL-1β (10 ng/mL) and/or 10 μm U0126 for 24 h. Some cultures were left untreated and evaluated after 24 h. Results in (B,D) are expressed as means ± SD for experiments performed in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001. Cur, curcumin.

Figure 6

Curcumin could activate autophagy for U0126-induced inhibition of ERK1/2 in primary chondrocytes. Accumulation of beclin-1 and LC3-II upon autophagy activation was visualized by western blot ((A), quantified in (B) and (C)). Results in (B,C) are expressed as means ± SD for experiments performed in triplicate.* p < 0.05, ** p < 0.01, and *** p < 0.001. Rap, rapamycin; 3-MA, 3-Methyladenine.

Figure 7

Curcumin induced autophagy via the ERK1/2 signal pathway to protect chondrocytes from apoptosis. IL-1β stimulates the IL-1β receptor, initiating an intracellular signal transduction cascade, which inhibits the cytoplasmic MAPK/ERK1/2 signaling pathway, then activates proinflammatory and pro-apoptotic gene production. ERK1/ERK2, a downstream kinase of the MAPK pathway, regulates the expression and activity of various transcription factors. Specific inhibition of ERK1/ERK2 by U0126 or IL-1β results in cleavage of caspase-3 in primary chondrocytes in vitro. Since activation of caspase-3 and DNA fragmentation are common features of apoptosis, the specific inhibition of the Ras-mitogen-activated kinase leads to chondrocyte apoptosis. Curcumin blocks the inhibition effect of U0126 or IL-1β on the MAPK pathway and activates autophagy in chondrocytes. Beclin-1 promotes LC3 activation. Recruitment and integration of LC3B-II into the growing phagophore, eventually results in the formation of autophagosomes.