Globular Adiponectin Attenuated H2O2-Induced Apoptosis in Rat Chondrocytes by Inducing Autophagy Through the AMPK/ mTOR Pathway
Cellular Physiology and Biochemistry
Background/Aims: Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Global adiponectin (gAPN), secreted from adipose tissue, possesses potent anti-inflammatory and antiapoptotic properties in various cell types. This study aimed to investigate the role of autophagy induced by gAPN in the suppression of H 2 O 2induced apoptosis and the potential mechanism of gAPN-induced autophagy in chondrocytes. Methods: H 2 O 2 was used to induce apoptotic injury in
... apoptotic injury in rat chondrocytes. CCK-8 assay was performed to determine the viability of cells treated with different concentrations of gAPN with or without H 2 O 2 . Cell apoptosis was detected by flow cytometry and TUNEL staining. Mitochondrial membrane potential was examined using JC-1 fluorescence staining assay. The autophagy inhibitors 3-MA and Bafilomycin A1 were used to treat cells and then evaluate the effect of gAPN-induced autophagy. To determine the downstream pathway, chondrocytes were preincubated with the AMPK inhibitor Compound C. Beclin-1, LC3B, P62 and apoptosisrelated proteins were identified by Western blot analysis. Results: H 2 O 2 (400 μM)-induced chondrocytes apoptosis and caspase-3 activation were attenuated by gAPN (0.5 μg/mL). gAPN increased Bcl-2 expression and decreased Bax expression. The loss of mitochondrial membrane potential induced by H 2 O 2 was also abolished by gAPN. Furthermore, the antiapoptotic effect of gAPN was related to gAPN-induced autophagy by increased formation of Beclin-1 and LC3B and P62 degradation. In particular, the inhibition of gAPN-induced autophagy by 3-MA prevented the protective effect of gAPN on apoptosis induced by H 2 O 2 . Moreover, gAPN increased p-AMPK expression and decreased p-mTOR expression. Compound C partly suppressed the expression of autophagy-related proteins and restored the expression of p-mTOR suppressed by gAPN. Thus, the AMPK/mTOR pathway played an important role Hu et al.: gAPN Attenuated Apoptosis via Autophagy in the induction of autophagy and protection of H 2 O 2 -induced chondrocytes apoptosis by gAPN. Conclusions: gAPN protected chondrocytes from H 2 O 2 -induced apoptosis by inducing autophagy possibly associated with AMPK/mTOR signal-pathway activation. Introduction Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive loss of articular cartilage, destruction of cartilage matrix, sclerosis of the subchondral bone, and formation of osteophyte  . OA occurs as the result of a variety of factors, including ageing, overload stress, and oxidative stress, and alters the physiological and biomechanical environment of joints [2, 3] . Reported studies have well demonstrated that cartilage matrix degradation and chondrocyte apoptosis induced by certain proteases and apoptotic factors are the two crucial pathogenic events occurring in OA    . Ageing-related oxidative stress has been considered to be a major causative factor for OA development [5, 6] . Hydrogen peroxide (H 2 O 2 ) formed from a superoxide anion through superoxide dismutase, which produces reactive oxygen species (ROS), is closely associated with the induction of chondrocyte apoptosis in vivo  and in vitro  . H 2 O 2 alters mitochondrial membrane permeability that allows for the release of cytochome c into the cytoplasm  and is involved in the activation of caspase-3, one of the key mediators in apoptotic signaling pathways, thus ultimately results in cell apoptosis. Autophagy, another type of self-destructive process from apoptosis, is a major cellular pathway essential for cell survival  . This process is crucial in the development and differentiation of cells and the maintenance of cytoplasmic organelle turnover  . Emerging evidence has shown that autophagy provides an efficient cellular defense mechanism and plays a cytoprotective role in various stressful conditions, including starvation, mitochondrial injury, and pathogen infection    . Growing evidence also highlights that the cytoprotective function of autophagy is mediated at least in part by the negative modulation of apoptosis    . For example, autophagy activation leads to the negative modulation of Bax [14, 15] . Beclin-1 mediated autophagy is also known to inhibit caspase-8 activity, thereby preventing apoptosis [16, 17] . Although autophagy is recognized as a critical role in cell death and survival, little is known about the mechanism of autophagy in OA. Paloma et al. found that the activation of autophagy significantly protected human chondrocytes against mitochondrial dysfunction. Caramés et al.  demonstrated that aged and OA articular cartilage were associated with the reduced expression of ULK1, Beclin-1, and LC3-II, suggesting that autophagy may play a protective role against chondrocyte death. Adiponectin (APN) predominantly secreted from adipose tissue shows strong abilities in the regulation of various biological responses, including lipid and glucose metabolism  and also possesses potent anti-inflammatory, antiatherogenic, and antiapoptotic properties    . Apart from its well-characterized role in fat tissue metabolism and insulin resistance , APN played an important role in the survival and proliferation of the several types of cells. Much of the beneficial effect was attributed to the antiapoptotic actions of APN    . Meanwhile, a growing number of evidence suggests that autophagy negatively regulates the apoptotic process in various cellular conditions  , adiponectin possesses potent antiapoptotic properties, and the effect of adiponectin on chondrocytes autophagy and its role in the suppression of H 2 O 2 -induced chondrocytes apoptosis is not explored yet. AMP-activated protein kinase (AMPK), a conserved energy sensor in eukaryotic cells, is well recognized as a key mediator of various biological responses induced by adiponectin  . In addition to the established role of AMPK in metabolism, the AMPK signaling pathway has been intensively studied in recent years because it coordinates metabolism with both apoptosis and autophagy  . AMPK controls autophagy through the mammalian target of rapamycin (mTOR) and Unc-51-like kinase 1 (ULK1) signaling  . mTOR, a conserved Ser/ Thr protein kinase, is a potent inhibitor of autophagy. In addition, chondrocyte autophagy is known to be a constitutive homeostatic mechanism in articular cartilages  , thus can be Hu et al.: gAPN Attenuated Apoptosis via Autophagy promoted by AMPK signaling [31, 32] through mTOR suppression and the cartilage-specific genetic deletion of mTOR  . Therefore, we assumed that the underlying autophagic activation effect of APN on rat chondrocytes against chondrocyte apoptosis induced by H 2 O 2 may be related to AMPK/mTOR signaling pathways. Thus, we investigated the role of autophagy induced by globular adiponectin (gAPN) to better understand the mechanisms underlying the suppression of apoptosis by adiponectin in H 2 O 2 -treated chondrocytes. In the present study, we demonstrated that gAPN activates the autophagy process in rat chondrocytes that is implicated in the suppression of H 2 O 2 -induced apoptosis. Furthermore, we identified that AMPK/mTOR signaling pathway plays a critical role in the gAPN-induced expression of proteins related to autophagy. Materials and Methods Collection, isolation, and culture of rat articular chondrocytes All experiments were approved by the Ethical Committee of Nanjing Medical University. The cartilage of 1-week-old Sprague-Dawley rats was harvested and minced before being digested with 0.25% trypsin (Gibco, USA). The trypsin was then removed, and the cartilage was washed with phosphate-buffered saline (PBS) (Gibco, USA) three times. Subsequently, 0.2% collagenase II (Gibco, USA) was added for digestion at 37 °C for 4-5 h. A 200 μm mesh strainer was used to filter the above solution, and the cells were collected by centrifugation. The cells were then cultured in Dulbecco's modified Eagle's medium (DMEM)-F12 medium (Gibco,USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1%-2% penicillin/streptomycin (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., China) and incubated with 5% CO 2 at 37 °C. The F1 generation of chondrocytes was used for the experiments in this report. Determination of experimental concentrations of H 2 O 2 and gAPN by CCK-8 assay 1) The rat chondrocytes of the F1 generation were prepared into 1×10 5 /mL single-cell suspension that was seeded into 96-well plate, 10 4 cells in each well. After the cells adhered to the wall, the cells were starved for 24 h by adding 100 µL serum-free culture medium. Five wells were randomly selected, and the culture media containing different concentrations of H 2 O 2 (0, 200 µM, 400 µM, 600 µM, 800 µM) (Sea Derun Pharmaceutical, Beijing China) were added to treat the cells for 6 h. Subsequently, the cells were incubated at 37 °C for 30 min by the addition of 10 µL CCK-8 solution. Absorbance was measured at 450 nm (BioTek, Vermont, USA). 2) The cells were starved for 24 h using the above-mentioned method. Then five wells were randomly selected. The gAPN (Biovision, USA) of different concentrations (0, 0.1, 0.5, 1, and 2 μg/mL) was added into each well to treat the cells for 24 h, followed by 400 µM H 2 O 2 treatment for 6 h. One group was randomly selected as the control group in which no treatment was performed. Absorbance was measured by CCK-8 assay at 450 nm. Cell apoptosis detection by TUNEL /DAPI staining The chondrocytes of the F1 generation were seeded into 24-well plate. The cells in sub-aggregation state were starved for 24 h by using serum-free culture medium. After the cells in each treatment were dried, they were fixed in 4% paraformaldehyde (GuGe Biotechnology, Wuhan, China) for 1 h. Then, the cells were sealed for 10 min using the confining liquid (3% H 2 O 2 , dissolved in methanol). After transparentization for 2 min using 0.1% Triton X-100 (Biosharp, Hefei, China), the cells were sealed for 1 h in TUNEL reaction mixture (Roche, Switzerland) at 37 °C in dark box. The cells were incubated with DAPI (Beyotime Biotechnology, China) for 5 min and were observed under an inverted fluorescence microscope (Olympus, Tokyo, Japan) at 2009 magnification. Randomly selected fields were photographed, and representative pictures were chosen to count apoptotic cells and total cells. The rate of TUNEL-positive cells in each field was calculated. Detection of cell apoptosis rate by flow cytometry: Annexin V/PI double staining The rat chondrocytes of the F1 generation were inoculated to six-well plate. After the cells adhered to the wall, they were divided into different treatment groups. When the treatment was over, the cells were collected and centrifuged for 5 min at 1500 r/min. The supernatant was discarded and then washed twice Hu et al.: gAPN Attenuated Apoptosis via Autophagy 6, gAPN significantly increased the expression of the phosphorylation of AMPK (p-AMPK), accompanying with the increased expression of autophagy-related proteins LC3-II and Beclin-1 and P62 degradation in H 2 O 2 -treated cells, as shown in Fig. 7A . However, this effect was significantly inhibited by pretreatment with 10 µM Compound C (a potent, reversible, and selective AMPK inhibitor). To address how p-AMPK regulate autophagy in H 2 O 2 -treated chondrocytes, we examined the phosphorylation of the downstream regulator of AMPK. We found that gAPN inhibited the phosphorylation of mTOR (p-mTOR), whereas the use of Compound C restored the expression of p-mTOR (Fig. 6) . The results of immunostaining was consistent with that of the Western blot shown in Fig. 7B . The inhibition of AMPK significantly decreased bright LC3-II puncta compared with uninhibited chondrocytes. These data suggested gAPN that mediated the induction of autophagy was associated with the AMPK/mTOR signaling pathway in H 2 O 2 -treated chondrocytes. Discussion We demonstrated for the first time that gAPN protects chondrocytes from H 2 O 2 -induced apoptosis through the activation of autophagy. Moreover, we have also presented that the AMPK/mTOR signaling pathway plays an important role in the gAPN-induced expression of proteins related to autophagy in chondrocytes and the prevention of H 2 O 2 -induced apoptosis by gAPN (Fig. 8) . OA is the most common chronic arthritis in the elderly, characterized by the gradual degradation of articular cartilage, synovial inflammation and pain, resulting in significant disability  . Reported studies have well demonstrated that cartilage matrix degradation and chondrocyte apoptosis induced by certain proteases and apoptotic factors are the two primary pathogenic events occurring in OA [3, 4] . Among all the reasons, the ageing-related Fig. 6 . Involvement of AMPK/mTOR signaling pathway in gAPN-induced autophagy in chondrocytes. Chondrocytes were treated with 0.5 µg/mL gAPN or pretreated with Compound C (10 µM) for 20 min before gAPN treatment prior to H 2 O 2 . The expression of phosphorylated and total AMPK and its downstream transcriptional mediator mTOR were assessed by Western blot. GAPDH was used as a loading control. Results are presented as the ratio of p-AMPK to AMPK and p-mTOR to mTOR. Data are represented as the mean ± SEM of three independent experiments. # P<0.05 versus the H 2 O 2 group and *P<0.05 versus the gAPN+H 2 O 2 group. H 2 O 2 , hydrogen peroxide; gAPN, globular adiponectin; Compound C, an inhibitor of AMPK; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.