The FASEB Journal article fj.201700265R. Published online October 25, 2017. THE JOURNAL • RESEARCH • www.fasebj.org Ghrelin protects against osteoarthritis through interplay with Akt and NF-kB signaling pathways Ruize Qu,*,†,1 Xiaomin Chen,*,†,1 Wenhan Wang,†,‡,1 Cheng Qiu,†,1 Miaomiao Ban,† Linlin Guo,† Krasimir Vasilev,§ Jianying Chen,{ Weiwei Li,*,2 and Yunpeng Zhao‡,3 *Department of Pathology and ‡Department of Orthopedics, Qilu Hospital, and †Medical School of Shandong University, Shandong University, Jinan, China; §School of Engineering, University of South Australia, Mawson Lakes, South Australia, Australia; and {Institute of Biopharmaceuticals of Shandong Province, Jinan, China ABSTRACT: Osteoarthritis (OA) is a common chronic degenerative disease characterized by degeneration in the joints and subsequent destruction of cartilage and bone, yet much remains to be elucidated regarding its molecular mechanism. Ghrelin is a recently discovered neuropeptide with anti-inflammatory actions, but it is unknown whether ghrelin is involved in OA. Human primary chondrocyte and cartilage samples were collected from patients with OA, and the expression pattern of ghrelin was assessed. Human chondrocyte and cartilage samples were stimulated with IL-1b and TNF-a, and exogenous ghrelin-alleviated disorganization of catabolism and anabolism were mediated by IL-1b and TNF-a. Destabilization of the medial meniscus and anterior cruciate ligament transection models were established in wild-type mice that were administered ghrelin or PBS. Severity of inflammation and degeneration in the joints were determined by measuring the levels of various inflammatory cytokines and degeneration-associated molecules. Ghrelin down-regulated the production of various inflammatory cytokines, inhibited apoptosis of chondrocytes, decreased the levels of metalloproteinases (including matrix metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin motif-5), and maintained the expression of critical matrix components, such as aggrecan and collagen 2. Moreover, suppression of the Akt signaling pathway and activation of NF-kB signaling in chondrocytes during OA development was antagonized by ghrelin administration. This supports the assessment of ghrelin as a potential therapeutic approach to treat degenerative cartilage diseases, including OA.—Qu, R., Chen, X., Wang, W., Qiu, C., Ban, M., Guo, L., Vasilev, K., Chen, J., Li, W., Zhao, Y. Ghrelin protects against osteoarthritis through interplay with Akt and NF-kB signaling pathways. FASEB J. 32, 000–000 (2018). www.fasebj.org KEY WORD: inflammatory cytokines • IL-1b • TNF-a • Osteoarthritis (OA), characterized by destruction of cartilage, an inflammatory reaction of synovium, and abnormal turnover of subchondral bone, is a prevalent type of arthritis and has become a main cause of disability in aging people. OA affects millions of patients all over the world ABBREVIATIONS: ACLT, anterior cruciate ligament transection; ADAMTS, ADAM metallopeptidase with thrombospondin type 1 motif; Col, collagen; DMM, destabilization of medial meniscus; GAG, glycosaminoglycan; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; micro-CT, microcomputed tomography; MMP, matrix metallopeptidase; OA, osteoarthritis; OARSI, Osteoarthritis Research Society International; WT, wild type 1 These authors contributed equally to this work. Correspondence: Department of Pathology, Qilu Hospital, Shandong University, 107 Wenhuaxi Rd., Jinan 250012, China. E-mail: [email protected] 163.com 3 Correspondence: Department of Orthopaedic Surgery, Qilu Hospital, Shandong University, 107 Wenhuaxi Rd., Jinan 250012, China. E-mail: [email protected] 2 doi: 10.1096/fj.201700265R This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information. 0892-6638/18/0032-0001 © FASEB cartilage • chondrocyte (1–3). However, much is left to be elucidated regarding the inductive factors and underlying mechanisms, and there is no efficient cure for OA in clinic (4, 5). The drugs used in clinic mainly attenuate symptoms, and arthroplasty surgery is almost inevitable for many patients (3). Extensive studies have reported that maintenance of homeostasis in chondrocytes might provide a potential target for treatment of OA (6, 7). Recent data have reported that various degenerative and inflammatory cytokines are involved in OA (8–10). Among them, IL-1b and TNF-a are critical proinflammatory cytokines that can induce other inflammation-associated molecules (11–13). IL-1b and TNF-a are known to induce critical catabolic biomarkers in cartilage, such as matrix metallopeptidase (MMP)-13 and ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-5 (8, 14). Moreover, these cytokines mediate activation of the NF-kB signaling pathway, which has been widely reported to disturb chondrocyte homeostasis (15, 16). Furthermore, IL-1b and TNF-a impair chondrocyte anabolism, which Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 1 leads to diminished production of extracellular matrix in cartilage (17, 18). Impaired chondrocyte anabolism in OA has been reported, which is achieved partially through antagonization of the Akt signaling pathway (19). IL-1b and TNF-a have become key molecular targets for OA investigation (20, 21). Ghrelin is a recently discovered neuropeptide that plays a potential role in various conditions, including neuronal activity and cell proliferation (22, 23). Ghrelin is expressed widely in humans, and previous data have implied that ghrelin might be a regulatory factor in several systems (24). Ghrelin is expressed in chondrocytes and plays an important role in chondrocyte differentiation (25). Previous studies have indicated that ghrelin is closely associated with various rheumatic diseases (26, 27). Furthermore, ghrelin is reported to antagonize the expression and function of IL-1b and TNF-a in several diseases (28–30). Additionally, ghrelin is reported to suppress activation of the NF-kB signaling pathway mediated by IL-1b and TNF-a (30, 31). However, whether ghrelin is involved in OA is unknown. The objectives of this research are to demonstrate the expression pattern and potential therapeutic effect of ghrelin in OA development. In our study, we found that treatment with ghrelin suppresses OA development. The therapeutic effect of ghrelin was exerted at multiple levels and was associated with the alleviation of inflammatory and degenerative response in OA. MATERIALS AND METHODS ligament and the anterior cruciate ligament of the right knee joint were transected. PBS or ghrelin (1 mg/g body weight, i.p.) was administered every day as previously reported with minor modification (33, 34). Sham treatment was performed in the left knee without transection of any ligament. Seven mice were used at each time point in every group. The mice were euthanized 4 or 8 wk later. Knee joint tissues were isolated and processed for further evaluation. Histology and immunohistochemistry Human cartilage samples and knee joints of mice in each experimental group were collected and fixed with 4% paraformaldehyde for 72 h, decalcified in 10% w/v EDTA for 14 d, dehydrated, and embedded with paraffin. Sections (5 mm thick) were cut, and serial sections of each sample were stained with Safranin-O/fast green/iron hematoxylin. For immunohistochemistry of the indicated biomarkers, sections were pretreated with 0.1% trypsin for 30 min at 37°C. The other matrix proteins in the cartilage sections were pretreated with chondroitinase ABC (0.25 U/ml) (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37°C and with hyaluronidase (1 U/ml) (Sigma-Aldrich) for 60 min at 37°C. To reduce nonspecific staining, 10% normal goat serum was used for protein blocking at room temperature for 30 min. Thereafter, slices were incubated with MMP-13 antibody (1:200 dilution) (ab3208; Abcam, Cambridge, United Kingdom), ADAMTS-5 pAb (1:100 dilution) (ab41037; Abcam), anti-ghrelin antibody (1:200 dilution) (ab129383; Abcam), collagen (Col) 10 pAb (diluted 1:100) (Santa Cruz Biotechnology), and anti–p-IkBa antibody (diluted 1:100) (Santa Cruz Biotechnology) overnight at 4°C. Detection was performed using the Vectastain Elite ABC kit (Vector, Burlingame, CA, USA), and 0.5 mg/ml 3,3-diaminobenzidine in 50 mM Tris-Cl substrate (SigmaAldrich) was used for visualization. Sections were counterstained with 1% hematoxylin. Mice All of the animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee of Shandong University. C57BL/6 background age-matched male wild-type (WT) mice were purchased from the Laboratory Animal Centre of Shandong University for in vivo experiments of the current study. Destabilization of medial meniscus–induced OA model In the surgically induced destabilization of medial meniscus (DMM) model, surgery was performed in 10-wk-old WT mice. Animals were anesthetized with isoflurane. The right knee joint was destabilized by transection of the medial meniscotibial ligament to generate destabilization of the medial meniscus, as previously reported (32). Thereafter, intra-articular injection of PBS or 2 mg ghrelin (sc-364689; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was performed every 3 d as previously reported with minor modification (8). The left knee joint was sham treated, in which the joint was prepared using the same approach as that for the right knee joint but without ligament transection. Nine mice were used per time point in each group. Mice were killed at 4 or 8 wk after surgery. Knee joint tissues were processed for histologic evaluation or other indicated experiments. Histopathological and quantificational evaluation of OA To grade the proteoglycan content of the articular cartilage on Safranin-O–stained sections, the Osteoarthritis Research Society International (OARSI) histology scoring system was performed for medial tibia plateau as previously reported (8, 35, 36). To further determine whether the OA changes in mice of each group were associated with a loss of chondrocytes in cartilage, articular chondrocytes were counted per unit area, and the articular cartilage thickness was analyzed through Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA). Proteoglycan loss of articular cartilage in surgically induced OA models was scored using a scoring system as previously reported (37). Briefly, proteoglycan loss was graded as follows: 0, normal; 1, minimal loss; 2, moderate loss; 3, marked loss; 4, severe, diffuse loss. Five random regions of interest were chosen from each sample, and the thickness of cartilage within each region of interest was determined. All the mentioned parameters were determined and averaged in all sections from each mouse, and 4 mice were analyzed from each group. For each mentioned parameter of the present study, at least 5 sections of each sample underwent double-blind examinations by 2 authors independently (R.Q. and X.C.). ELISA assays for circulating TNF-a and IL-6 Anterior cruciate ligament transection OA model To demonstrate the protective effect of ghrelin in OA, 10-wk-old WT mice were generally anesthetized, and the medial collateral 2 Vol. 32 February 2018 In the current study, serum was collected from each group of mice in the OA models, and circulating levels of TNF-a and IL-6 were measured through ELISA as previously reported (38). The FASEB Journal x www.fasebj.org Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 QU ET AL. Briefly, TNF-a and IL-6 were assessed using a commercial kit (eBioscience, San Diego CA, USA) according to the manufacturer’s instructions. All the samples were assayed in triplicate and repeated at least 3 times. Primary cultures of human chondrocytes Human cartilage samples were harvested from patients undergoing total knee joint replacement surgery for OA at Qilu Hospital, Shandong University. Informed consent was collected from the patients in this study before surgery. The process was approved by the Institutional Review Board of Shandong University, and all experiments were performed in accordance with the relevant guidelines and regulations. Primary human articular chondrocytes were isolated from the articular cartilage by enzymatic digestion as previously reported (39). Briefly, cartilage slices were minced finely and washed several times with 1% PBS. They were then digested in a mixture of 0.25% collagenase II in DMEM without FBS and Penn Strep for 3–5 h in a spinner flask in an incubator at 37°C and in an atmosphere of 5% CO2. The cell suspension was used to establish cultures in the T175 dishes. Within 2–3 d of harvesting, primary chondrocytes were replated at 80% confluence in 6-well plates before being used in the experiments. and 59-TTGTCTCCAAGGGACCAGG-39 (iNOS); 59-CAGTGAGAAGGGCCGAAAGAC-39 and 59-CAGGGGCAGGGAGAAGGAG-39 for NF-kB2; 59-AGAAGGCTGGGGCTCATTTG-39 and 59-AGGGGCCATCCACAGTCTTC-39 (GAPDH). For mice: 59TGAGCCCTGAACACCAGAGAG-39 and 59-AAAGCCAGATGAGCGCTTCTA-39 (ghrelin); 5-AATGCTGGTACTCCAAACCC-3 and 5-CTGGATCGTTATCCAGCAAACAGC-3 (aggrecan); 59-ACTAGTCATCCAGCAAACAGCCAGG-39 and 59-TTGGCTTTGGGAAGAGAC-39 (Col II); 59-AATCTCACAGCAGCACATCA-39 and 59-AAGGTGCTCATGTCCTCATC-39 (IL-1b); 59CCTTCCTACCCCAATTTCCAAT-39 and 59-GCCACTCCTTCTGTGACTCCAG-39 for IL-6; 59-ACAGGAGGGGTTAAAGCTGC-39 and 59-TTGTCTCCAAGGGACCAGG-39 (iNOS); 59GCATTGACGCATCCAAACCC-39 and 59-CGTGGTAGGTCCAGCAAACAGTTAC-39 (ADAMTS-5); 59-ACTTTGTTGCCAATTCCAGG-39 and 59-TTTGAGAACACGGGGAAGAC-39 (MMP-13); 59-AATGCTGACTATGGCTACAAAA-39 and 59AAAACTGATGCGTGAAGTGCTG-39 (COX-2); 59-CAGTGAGAAGGGCCGAAAGAC-39 and 59-CAGGGGCAGGGAGAAGGAG-39 (NF-kB2); 59-AGAACATCATCCCTGCATCC-39 and 59-AGTTGCTGTTGAAGTCGC-39 (GAPDH). The production of a single specific PCR product was measured through melting curve analysis. The experiments were repeated 3 times for each indicated molecule. Western blot Cartilage explant cultures Cartilage explants from humans were isolated and cultured as previously reported (40). Briefly, cartilage samples were isolated from human tibia plateau cartilage during total knee arthroplasty, which would otherwise be discarded. The collected samples were then dissected into tiny pieces (diameter, 1 mm; thickness, 1–2 mm). Thereafter, the cartilage pieces were cultured in tissue-culture flasks with serum-free DMEM (containing 25 mM HEPES, 2 mM glutamine, 100 mg/ml streptomycin, 100 IU/ml penicillin, and 2.5 mg/ml gentamicin) supplemented with or without IL-1b (10 ng/ml) and ghrelin (50 ng/ml). After the indicated incubating time, the conditioned medium was collected for glycosaminoglycan (GAG) synthesis analysis and other indicated experiments. All protein extracts were collected from human articular cartilage or cultured primary chondrocytes of each treatment group, resolved on a 10% SDS polyacrylamide gel, and electroblotted onto a nitrocellulose membrane. After blocking in 5% nonfat dry milk in Tris-buffered saline–Tween 20 (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 0.5% Tween 20), blots were incubated with polyclonal ghrelin [diluted 1:1000 (1:200 dilution, ab129383; Abcam), iNOS (diluted 1:1000, ab3523; Abcam), p-Akt (diluted 1:1000, ab38449; Abcam), total Akt (diluted 1:1000, sc-8312; Santa Cruz Biotechnology), COX-2 (diluted 1:1000, Santa Cruz Biotechnology), p-IkBa (diluted 1:1000, Cell Signaling Technology), or Col 2 (diluted 1:1000, Santa Cruz Biotechnology)] antibody for 1 h. After washing, the horseradish peroxidase–conjugated secondary antibody (1:2000 dilution) was added, and bound antibody was detected through an enhanced chemilumescent system. Real-time PCR Total RNA was collected from the articular cartilage or cultured primary chondrocytes of each experimental group with the RNeasy kit (Qiagen, Germantown, MD, USA) as previously reported, and first-strand cDNA was established through the ImProm-II reverse transcription system (Promega, Madison, WI, USA). For mice experiments, several pieces of cartilage tissue from 3 mice of the same group were pooled to perform real-time PCR successfully. Real-time PCR was performed with SYBR Green I dye to monitor DNA synthesis. Data from each sample were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers used for real-time RT-PCR were designed to generate products between 100 and 200 bp in length. Oligonucleotides used as the specific primers to amplify genes are as follows. For humans: 59-TGAGCCCTGAACACCAGAGAG-39 and 59-AAAGCCAGATGAGCGCTTCTA-39 (ghrelin); 59-AATGCTGGTACTCCAAACCC-39 and 59-CTGGATCGTTATCCAGCAAACAGC-39 (aggrecan); 59-ACTAGTCATCCAGCAAACAGCCAGG-39 and 59-TTGGCTTTGGGAAGAGAC-39 (collagen 2; Col 2); 59-GCAGTATGACAAGTGCGGAGT-39 and 59-CAGGGCTAAATAGGCAGTGAA-39 (ADAMTS-5); 5-ACTTTGTTGCCAATTCCAGG-3 and 5-TTTGAGAACACGGGGAAGAC-3 (MMP-13); 59-ACAGGAGGGGTTAAAGCTGC-39 ROLE OF GHRELIN IN OSTEOARTHRITIS Immunofluorescence staining Primary human chondrocytes were cultured on coverslips, and immunofluorescence staining of ghrelin was then performed on these cells. Chondrocyte was stimulated with 10 ng/ml IL-1b in the presence or absence of ghrelin for 1 h. Immunofluorescence staining of NF-kB p65 was then performed on these cells, as previously described, and cells were examined using a confocal fluorescence microscope system (38). TUNEL staining TUNEL staining of knee joint articular cartilage from each treatment group of the DMM model was performed according to previous reports (8) by using the Promega DeadEnd Colorimetric TUNEL System (Promega) following the manufacturer’s protocol. GAG assay for cartilage explants Cultured human cartilage explants were cultured as indicated. Conditioned media were collected from the human Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 3 cartilage explant culture of each group, and the level of GAG released from explants was assessed using dye DMMB (Polysciences, Warrington, PA, USA). Culture medium was pretreated with 0.5 U/ml of hyaluronidase (Seikagaku, Tokyo, Japan) at 37°C for 3 h to remove exogenous hyaluronic acid, which interferes with the DMMB assay. Digests (in duplicate) were mixed with DMMB in 96-well plates and read at 520 nm using SpectraMax 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The amount of GAG in the conditioned medium was extrapolated using chondroitin-6-sulfate sodium salt from shark cartilage (Sigma-Aldrich) as a standard. The average values of the duplicates were normalized to the wet weights of cartilage explants. The data were shown as the mean of GAG released into the condition media from three explants (treated in separate wells). Statistical analysis For comparison of various treatment groups, unpaired MannWhitney t tests, paired Student t tests, and 1-way or 2-way ANOVA (where appropriate) were performed. For ANOVA, Bonferroni post hoc analysis was used to compare treatment groups. Each statistical analysis was performed using GraphPad Prism, v.4.01 (GraphPad Software, La Jolla, CA, USA). Statistical significance was achieved at P , 0.05. RESULTS Ghrelin is detected in cartilage and is downregulated in IL-1b–stimulated chondrocytes To date, it is unknown whether the ghrelin expression level is associated with the development of OA. To investigate the expression pattern of ghrelin, human cartilage tissue was collected from patients with OA undergoing arthroplasty. Articular cartilage from patients with trauma undergoing amputation was used as a control. Immunohistochemistry for ghrelin was performed in cartilage samples, and ghrelin was detected in cartilage tissue. Ghrelin was largely expressed within the cytoplasm of chondrocytes (Fig. 1A, insert). Cartilage tissue was collected from patients with OA and from the control group, and the ghrelin expression level was determined using Western blot. The expression level of ghrelin was diminished in OA cartilage (Fig. 1B, C). Moreover, real-time PCR performed in human cartilage showed that the mRNA level of ghrelin was remarkably decreased in degenerative cartilage when compared with the control group (Fig. 1D). To further study the expression pattern of ghrelin in chondrocytes, immunostaining was performed for ghrelin in human chondrocytes. Ghrelin was expressed mainly in the cytoplasm of chondrocyte (Fig. 1E). To determine whether inflammatory cytokine IL-1b is associated with the expression level of ghrelin, primary human chondrocytes were stimulated with 10 ng/ml IL-1b. Thereafter, mRNA and protein were collected from chondrocytes to test ghrelin levels. As a result, IL-1b significantly reduced protein (Fig. 1F, G) and mRNA levels (Fig. 1H) of ghrelin in chondrocytes. Moreover, an anterior cruciate ligament transection (ACLT) mouse model was established, and mRNA as well as protein levels of ghrelin were detected by real-time PCR (Fig. 1I), Western blot (Fig. 1J, K), and 4 Vol. 32 February 2018 immunohistochemistry (Fig. 1L). The expression level of ghrelin was decreased in cartilage of ACLT mouse model. Exogenous ghrelin protects against OA development in vivo To investigate whether ghrelin has a protective effect against OA, an ACLT posttraumatic OA model was established. Intraperitoneal injection of PBS or ghrelin (1 mg/g body weight) was performed once every day for 4 or 8 wk. At the 4-wk time point, the knee joint of each group was collected from the PBS and ghrelin groups, and immunohistochemistry and Western blot were performed to detect ghrelin expression. The ghrelin level was elevated in the ghrelin treatment group compared with the PBS group, which implied that systemic administration of ghrelin might reach the articular cartilage of the knee joint (Supplemental Fig. S1). To determine the protective function of ghrelin in OA, micro-CT was performed in each group at the 8-wk time point. Microcomputed-tomography (microCT) analysis showed disorganization of bone structure and formation of osteophytes after ACLT surgery, which was attenuated by additional administration of ghrelin (Fig. 2A). The ACLT model displayed destruction of articular cartilage in the PBS treatment group, and ghrelin administration protected the structure of articular cartilage, which was detected by Safranin-O staining (Fig. 2B). Moreover, ghrelin significantly improved the OARSI score, attenuated proteoglycan loss, and protected chondrocyte number and cartilage thickness in cartilage, which were assessed by histologic grading analysis (Fig. 2C–F). Furthermore, mRNA was isolated and collected from articular cartilage of the PBS and ghrelin treatment groups at the 8-wk time point, and levels of MMP-13, ADAMTS-5, TNF-a, IL-6, iNOS, and COX-2 were tested through real-time PCR. All the mentioned degeneration-associated molecules induced in the ACLT OA model were abolished by ghrelin (Fig. 3A–F). Cartilage samples were collected, and immunohistochemistry was performed for critical metalloproteinases, including MMP-13 and ADAMTS-5. Expression levels of both molecules were enhanced in cartilage of the ACLT model, whereas ghrelin diminished their expression levels (Fig. 3G). Serum was collected from each group, and circulating levels of IL-6 and TNF-a were assayed with ELISA. Treatment of ghrelin markedly decreased the levels of both molecules in the ACLT model (Fig. 3H). Additionally, total protein was isolated from articular cartilage of the ACLT model, and iNOS and COX-2 expression levels were detected through Western blot. Ghrelin greatly attenuated induction of iNOS and COX-2 in cartilage tissue of the ACLT model (Fig. 3I–K). To further investigate the role of exogenous ghrelin in OA, the DMM model was established in WT mice, and local delivery of 2 mg ghrelin or PBS was performed through intra-articular injection every 3 d. Knee joint samples were collected from the PBS and ghrelin treatment groups, and Safranin-O staining implied that ghrelin protected the cartilage structure in the DMM model (Fig. 4A). OARSI score, proteoglycan loss, and chondrocyte number in cartilage as well as cartilage thickness were The FASEB Journal x www.fasebj.org Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 QU ET AL. Figure 1. Detection and expression pattern of ghrelin chondrocyte and articular cartilage. A) Detection of ghrelin in cartilage tissue as assayed by immunohistochemistry (n = 4). Scale bar, 200 mm. B, C ) Detection of ghrelin in primary human cartilage as assayed by Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). **P , 0.01 vs. control group. D) Dampened expression of ghrelin in primary human cartilage measured by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). **P , 0.01 vs. control group. E ) Diminished level of ghrelin in chondrocyte after treatment of IL-1b as detected by immunostaining (n = 4/group). Scale bars, 50 mm. F, G) Detection of ghrelin in primary human chondrocyte stimulated by IL-1b as assayed by Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). *P , 0.05 vs. control group. H ) Reduced level of ghrelin in primary human chondrocytes stimulated by IL-1b as compared with the control group as measured by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). *P , 0.05 vs. control group. I ) Dampened level of ghrelin in articular cartilage of the ACLT model as compared with the sham control group measured by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). **P , 0.01 vs. control group. J, K ) Decreased expression of ghrelin in cartilage after ACLT operation as detected through Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). **P , 0.01 vs. control group. L) Reduced level of ghrelin in articular cartilage of the ACLT model as detected by immunohistochemistry (n = 4/group). Scale bars, 100 mm. assessed by histologic grading analysis (Fig. 4B–E), and ghrelin treatment improved all the mentioned parameters in the DMM mouse model. Moreover, cartilage samples were collected from the sham control group and from the DMM groups, and immunohistochemistry was performed for ADAMTS-5 and MMP-13. Both molecules were elevated in the DMM model, and ghrelin suppressed this alteration in cartilage tissue (Supplemental Fig. S2A). Furthermore, protein was collected from each group, and Western blot was performed for iNOS. An enhanced iNOS level in the DMM model was inhibited by local delivery of ghrelin (Supplemental Fig. S2B, C). It has been reported that ghrelin suppresses cell apoptosis (41). In this study, cell death of chondrocytes in the DMM model was detected through TUNEL staining, which showed that ghrelin antagonized apoptosis of ROLE OF GHRELIN IN OSTEOARTHRITIS chondrocytes in the DMM model (Supplemental Fig. S3A, upper panels). Hypertrophy of chondrocytes is a feature of cartilage degeneration in OA development. In this study, chondrocyte hypertrophy in the DMM model was assayed through immunohistochemistry for Col 10 and Western blot for Col 2. As a result, ghrelin treatment inhibited expression of Col 10 (Supplemental Fig. S3A, lower panels) and maintained the expression level of Col 2 (Supplemental Fig. S3B, C). Ghrelin down-regulates IL-1b–mediated catabolism in chondrocytes To determine the role of ghrelin in catabolism of degenerative chondrocytes, human articular chondrocytes were isolated and stimulated with IL-1b (10 ng/ml) for Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 5 Figure 2. Effect of ghrelin on cartilage degeneration in the ACLT model. A) Ghrelin protected against destruction of bone structure in the ACTL mouse model as detected by micro-CT (n = 4/group). B) Ghrelin protected the structure of articular cartilage as detected by Safranin-O staining (n = 4/ group). Scale bars, 100 mm. C) OARSI score of OA based on the result of Safranin-O staining. After ACLT operation, PBS or ghrelin was intraperitoneally injected into the mice, and the samples were collected 4 wk later. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). *P , 0.05 vs. control group. D–F ) Comparison of OA severity between the PBS and ghrelin treatment groups in the ACLT model as assessed by proteoglycan loss, chondrocyte number, and articular cartilage thickness in the tibia. Significant differences are indicated (2-tailed Student’s t test; n = 4/group; means 6 SEM). *P , 0.05 vs. control group. 12 h with or without simultaneous stimulation of ghrelin (50 ng/ml). Ghrelin remarkably suppressed IL-1b–induced increases of MMP-13, ADAMTS-5, IL-6, and TNF-a, as assayed by real-time PCR (Fig. 5A–D). Chondrocytes were stimulated by IL-1b for 72 h. Protein extracts were collected from chondrocytes of each group, and iNOS expression was tested using Western blot. iNOS was markedly induced by IL-1b, which was abolished by additional treatment of ghrelin (Fig. 5E, F). Conditioned medium of each group was collected, and ELISA was performed. The release of IL-6 and TNF-a in the culture medium was greatly reduced after treatment with ghrelin (Fig. 5G). To further study the role of ghrelin alone in chondrocyte catabolis, primary human chondrocytes were cultured in the presence or absence of 50 ng/ml ghrelin for 12 h, and real-time PCR was performed for degeneration-associated biomarkers, including IL-1b, ADAMTS-5, and MMP-13. Ghrelin alone slightly reduced the levels of the catabolic and degenerative biomarkers in chondrocytes, but no significant change was observed (Supplemental Fig. S4A–C). Ghrelin reverses IL-1b repression of chondrocyte anabolism To investigate the role of ghrelin in impaired anabolism in chondrocyte degeneration, human articular chondrocytes were isolated and stimulated with IL-1b (10 ng/ml) for 12 h with or without simultaneous stimulation of ghrelin (50 ng/ml). Ghrelin remarkably suppressed IL-1b– induced reduction in the mRNA levels of aggrecan, Col 2, and Sox-9, as assayed by real-time PCR (Fig. 6A–C). Chondrocyte was stimulated by IL-1b for 72 h. Conditioned medium was collected from each group, and 6 Vol. 32 February 2018 GAG synthesis was tested. GAG content was markedly diminished by IL-1b, which was attenuated by ghrelin (Fig. 6D). Cell extract of each group was collected, and Western blot was performed. Col 2 level was remarkably maintained after treatment with ghrelin (Fig. 6E, F). To further demonstrate the role of ghrelin alone in chondrocyte anabolism, primary human chondrocytes were cultured in the presence or absence of 50 ng/ml ghrelin for 12 h, and real-time PCR was performed for anabolic biomarkers, including Sox-9, aggrecan, and Col 2. Ghrelin alone significantly promoted anabolism in chondrocytes (Supplemental Fig. S4D–F). To investigate whether the anabolic effect of ghrelin is dependent on the Akt signaling pathway, primary chondrocytes were stimulated by IL-1b in the presence or absence of ghrelin. IL-1b diminished activity of the Akt signaling pathway, whereas ghrelin largely maintained Akt signaling in chondrocytes (Fig. 6G–I). Moreover, MK-2206, a typical Akt inhibitor, markedly suppressed the role of ghrelin in anabolism of IL-1b–stimulated chondrocytes. Real-time PCR showed that ghrelin induction of aggrecan, Col 2, and Sox-9 was remarkably repressed by MK-2206 (Fig. 6J). Total protein was collected, and Western blot for Col 2 was performed. Maintenance of Col 2 by ghrelin was antagonized by MK-2206 (Fig. 6K, L). Additionally, GAG synthesis assay (Fig. 6M) showed that ghrelin-mediated GAG content in conditioned medium was dramatically inhibited by MK-2206. To study whether activation of the Akt signaling pathway alone can play a role similar to that of ghrelin in the attenuation of impaired anabolism during degeneration, SC-79, a novel Akt activator (42), was used to treat the IL-1b–stimulated chondrocytes for 12 h, and real-time PCR for anabolic biomarkers, including aggrecan, Col 2, and Sox-9, was performed. Activation of the The FASEB Journal x www.fasebj.org Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 QU ET AL. Figure 3. Effect of ghrelin on catabolic and inflammatory markers in ACLT model. A–F ) Ghrelin antagonized expression of MMP-13, ADAMTS-5, TNF-a, IL-6, iNOS, and COX-2 in cartilage of the mouse model as assayed by real-time PCR. Total RNA was isolated, followed by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05; **P , 0.01; ***P , 0.005 vs. indicated control group. G) Ghrelin inhibited expression of MMP-13 and ADAMTS-5 as detected by immunohistochemistry (n = 4/group). Scale bar, 100 mm. H ) Administration of ghrelin attenuated circulating levels of IL-6 and TNF-a as measured by ELISA. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group. I–K ) Levels of iNOS and COX-2 in cartilage of the ACLT mouse model were suppressed by ghrelin. Total protein from each group was extracted, followed by Western blot (n = 3/group). *P , 0.05, **P , 0.01, ***P , 0.005 vs. indicated control group. Akt signaling pathway through SC-79 greatly abolished IL-1b mediation of impaired anabolism (Supplemental Fig. S5). Ghrelin represses degeneration of cartilage explants induced by IL-1b ex vivo It is well established that IL-1b mediates cartilage destruction in OA (43). To demonstrate the function of ghrelin in cartilage destruction, cartilage samples from patients with OA were isolated and cultured with 10 ng/ml IL-1b in the presence or absence of 50 ng/ml ghrelin for 48 h, followed by Safranin-O staining. IL-1b enhanced the loss of proteoglycan, whereas the additional treatment of ghrelin largely rescued this effect of IL-1b (Fig. 7A). Total mRNA was collected from all the groups, followed by real-time PCR. Production of Col2 and aggrecan in cartilage was markedly diminished by IL-1b, whereas this effect was antagonized by ghrelin (Fig. 7B, C). Levels of catabolism-associated molecules, including MMP-13 and ADAMTS-5, were significantly higher in the IL-1b–treated group, and additional ghrelin administration greatly reduced the levels ROLE OF GHRELIN IN OSTEOARTHRITIS of these molecules (Fig. 7D, E). Total protein was collected from all the groups, and Western blot was performed for iNOS. IL-1b induction of iNOS was dramatically inhibited by ghrelin (Fig. 7F, G). Culture medium was collected from each group for testing the TNF-a level. Ghrelin significantly reduced expression of TNF-a in the conditioned medium (Fig. 7H). Culture medium was also collected to analyze the release of GAG using DMMB assay. Ghrelin treatment markedly reduced the release of GAG into the culture medium from IL-1b–treated human cartilage samples (Fig. 7I). Ghrelin attenuates TNF-a–induced disorganization of metabolism in chondrocytes Because TNF-a plays a detrimental role in OA development and chondrocyte degeneration (44) and ghrelin antagonized TNF-a in several conditions (45), we determined the role of ghrelin in TNF-a–mediated chondrocyte catabolism. Human articular chondrocytes were isolated and stimulated with TNF-a (10 ng/ml) for 12 h with or without simultaneous administration of ghrelin (50 ng/ml). Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 7 Figure 4. Function of ghrelin on destruction of cartilage structure in the DMM model. A) Ghrelin protected the structure of articular cartilage in the DMM model as detected by Safranin-O staining (n = 4/group). Scale bars, 100 mm. B) OARSI score of OA based on the result of Safranin-O staining. After the DMM operation, PBS or ghrelin was intraperitoneally injected into the mice, and the samples were collected 4 wk later. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/ group; means 6 SEM). *P , 0.05 vs. control group. C–E ) Comparison of OA severity between PBS and ghrelin treatment groups in the DMM model as assessed by proteoglycan loss, chondrocyte number, and articular cartilage thickness in the tibia. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. control group. Ghrelin markedly suppressed the TNF-a–mediated increase in the mRNA levels of MMP-13, ADAMTS-5, and IL-1b, as assayed by real-time PCR (Fig. 8A). IL-6 has been reported to be closely associated with cartilage degeneration (46). In the present study, chondrocytes were stimulated by TNF-a for 72 h with PBS or ghrelin. Conditioned medium was collected from each group, and IL-6 expression was tested using ELISA. IL-6 was markedly induced by TNF-a in chondrocytes, which was rescued by additional treatment of ghrelin (Fig. 8B). Protein extracts were collected, and Western blot was performed for iNOS. iNOS was Figure 5. Effect of ghrelin on catabolic and inflammatory markers in IL-1b–treated human articular chondrocyte. A–D) Human articular chondrocyte was treated with ghrelin, followed by treatment with IL-1b. Total RNA was isolated, and levels of mRNA for MMP-13, ADAMTS-5, IL-6, and TNF-a were detected by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; means 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. control group. E, F ) Treatment of ghrelin attenuated IL-1b–mediated expression of iNOS as assayed by Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 3/group). *P , 0.05; **P , 0.01 vs. control group. G) Levels of IL-6 as well as TNF-a were suppressed by ghrelin. Conditioned medium from each group was collected, and ELISA was performed. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; means 6 SEM). *P , 0.05 vs. indicated control group. 8 Vol. 32 February 2018 The FASEB Journal x www.fasebj.org Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 QU ET AL. Figure 6. Ghrelin reverses IL-1b repression of anabolism in chondrocyte through activation of the Akt signaling pathway. A–C ) Ghrelin remarkably suppressed the IL-1b–induced reduction of aggrecan, Col 2, and Sox-9 as assayed by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; means 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. control group. D) GAG content was markedly diminished by IL-1b, which was attenuated by ghrelin. Chondrocyte was stimulated by IL-1b for 72 h. Conditioned medium was collected from chondrocyte of each group, and GAG synthesis was tested. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. control group. E, F ) Ghrelin remarkably maintained Col 2 level suppressed by IL-1b. Chondrocyte was stimulated by IL-1b for 72 h, and cell extract of each group was collected, followed by Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. control group. G–I ) IL-1b diminished activity of the Akt signaling pathway, whereas ghrelin largely maintained Akt signaling in chondrocyte as measured by Western blot. Primary chondrocyte was stimulated by IL-1b in the presence or absence of ghrelin. Significant differences are indicated (2-tailed Student’s t test; n = 3/ group; means 6 SEM). ***P , 0.005 vs. indicated control group. NS, no significant difference. J ) Ghrelin induction of aggrecan, Col 2, and Sox-9 was remarkably repressed by MK-2206 as indicated by real time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. indicated control group. K, L) Maintenance of Col 2 by ghrelin was antagonized by MK-2206. Total protein was collected, and Western blot for Col 2 was performed. *P , 0.05, **P , 0.01 vs. indicated control group. M ) Ghrelin-mediated GAG content in conditioned medium was dramatically inhibited by MK-2206 as detected by GAG synthesis assay. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group. greatly reduced after treatment with ghrelin in a dosedependent manner (Fig. 8C). To investigate the role of ghrelin in disorganized anabolism mediated by TNF-a, human articular chondrocytes were isolated and stimulated with TNF-a (10 ng/ml) for 12 h with or without simultaneous stimulation of ghrelin (50 ng/ml). Real-time PCR indicated that ghrelin inhibited impairment of anabolism biomarkers induced by TNF-a, including aggrecan, Sox9, and Col 2 (Fig. 8D). Chondrocytes were stimulated by TNF-a for 72 h with PBS or ghrelin, and protein extracts were collected from chondrocytes of each group. Ghrelin attenuated the reduction of Col 2 mediated by TNF-a (Fig. 8E). In addition, chondrocytes were stimulated by TNF-a for 72 h, conditioned ROLE OF GHRELIN IN OSTEOARTHRITIS medium was collected from chondrocytes of each group, and GAG synthesis was tested. GAG content was markedly diminished by TNF-a but was greatly improved by ghrelin (Fig. 8F). Ghrelin antagonizes exaggerated catabolism in degenerative chondrocytes by inhibiting the NF-kB signaling pathway Disorganization of metabolism in OA involves impaired anabolism and abnormally enhanced catabolism, and activation of NF-kB signaling is known to play a predominant role in cartilage degradation of OA (47). It is reported that NF-kB signaling represses anabolism of Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 9 Figure 7. Protective role of ghrelin in IL-1b–induced degeneration of cartilage explants. A) IL-1b–mediated loss of proteoglycan in cartilage explants was rescued by ghrelin as detected by Safranin-O staining (n = 4–6/group). Scale bar, 100 mm. B, C ) Production of Col 2 and aggrecan in cartilage were markedly diminished by IL-1b, but this effect was antagonized by ghrelin as measured by real-time PCR. Total mRNA was isolated from each group of cartilage as assessed by realtime PCR. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. indicated control group. D, E ) Levels of MMP-13 and ADAMTS-5 induced by IL1b. Ghrelin treatment abolished this effect as assayed by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. indicated control group. F, G) IL-1b induction of iNOS was dramatically inhibited by ghrelin as detected by Western blot. Total protein was extracted from cartilage of all groups, and Western blot was performed for iNOS. Culture medium was collected for testing TNF-a. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. indicated control group. H ) Ghrelin reduced expression of TNF-a in culture medium as measured by ELISA. Culture medium was collected, and ELISA was performed. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group. I ) Ghrelin treatment markedly decreased the release of GAG into the culture medium from IL-1b–treated human cartilage samples. Culture medium was collected to analyze the release of GAG using DMMB assay. Significant differences are indicated (2tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group, ***P , 0.005 vs. indicated control group. chondrocytes (48) and that the NF-kB signaling pathway triggers production of catabolic and inflammatory biomarkers in chondrocytes (49). In the current study, the results suggest that ghrelin activated Akt signaling and promoted anabolism of chondrocytes while suppressing the expression of catabolic and inflammatory biomarkers. Based on these data, we determined whether treatment of ghrelin affected the signaling activity of NF-kB in surgically induced OA models. Total protein extracts were collected from cartilage of an ACLT mouse model. Ghrelin treatment greatly impaired phosphorylation of IkBa in the ACLT OA model (Fig. 9A, B). Immunohistochemistry of p-IkBa was also performed for cartilage of the ACLT model, and ghrelin markedly suppressed p-IkBa expression (Fig. 9C). mRNA and protein were collected from cartilage samples of the DMM model. Real-time PCR for NF-kB2 (Supplemental Fig. S6A) and Western blot for p-IkBa (Supplemental Fig. S6B) indicated that ghrelin treatment suppressed activation of the NF-kB signaling pathway. Furthermore, immunohistochemistry of p-IkBa revealed that ghrelin inhibited phosphorylation of IkBa in the DMM model (Supplemental Fig. S6C). 10 Vol. 32 February 2018 To further investigate whether ghrelin represses enhanced activity of the NF-kB signaling pathway in primary chondrocytes, human chondrocytes were isolated and treated with IL-1b in the presence or absence of ghrelin, and the NF-kB signaling pathway was assayed. The NF-kB2 level was measured with real-time PCR, and IL-1b–mediated NF-kB2 was antagonized with ghrelin treatment (Fig. 9D). Total protein was collected from chondrocytes. Ghrelin attenuated p-IkBa expression as compared with the PBS group (Fig. 9E, F). Nuclear translocation of NF-kB p65 is a parameter for activity of NF-kB signaling. In this study, cell immunostaining was performed for chondrocytes of each treatment group, and ghrelin greatly antagonized nuclear translocation of NF-kB p65 (Fig. 9G). These findings reveal that ghrelin treatment reduces the degeneration-associated activity of the NF-kB signaling pathway in chondrocytes. DISCUSSION OA is a common degenerative disease worldwide and is mainly characterized by destruction of articular cartilage (50). Proinflammatory cytokines, including The FASEB Journal x www.fasebj.org Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 QU ET AL. Figure 8. Protective role of ghrelin on TNF-a–mediated disorganization of metabolism in human articular chondrocytes. A) Human articular chondrocytes were treated with ghrelin, followed by treatment with TNF-a. Total RNA was isolated, and levels of mRNA for MMP-13, ADAMTS-5, and IL-1b were detected by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; mean 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. indicated control group. B) The TNFa–mediated elevation of IL-6 level was suppressed by ghrelin. Conditioned medium from each group was collected, and ELISA for IL-6 was performed. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; mean 6 SEM). *P , 0.05, **P , 0.01 vs. indicated control group. C ) Treatment of ghrelin attenuated IL-1b–mediated expression of iNOS as assayed by Western blot. Each experiment was repeated 3 times. D) TNF-a–induced impairment of Aggrecan, Col 2, and Sox-9 expression was remarkably repressed by ghrelin as indicated by real-time PCR. Significant differences are indicated (2-tailed Student’s t test; n = 4–6/group; mean 6 SEM). *P , 0.05, **P , 0.01, ***P , 0.005 vs. indicated control group. E ) TNF-a–induced reduction of Col 2 level in chondrocyte was antagonized by ghrelin. Total protein was collected, and Western blot for Col 2 was performed. Each experiment was repeated 3 times. F ) TNF-a inhibited GAG content in conditioned medium, which was antagonized by additional ghrelin as detected by GAG synthesis assay. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group. TNF-a and IL-1b, are well known to induce OA, and previous data have shown the potential antagonization between these cytokines and ghrelin in several conditions (30, 51, 52). In the current study, we demonstrated that ghrelin was detected in human articular cartilage and that the ghrelin expression level was remarkably diminished in degenerative cartilage, inflammatory cytokine–stimulated chondrocytes, and cartilage in a surgically induced arthritis model, which implies the potential involvement of ghrelin in the development of OA. Previous studies have reported the potential role of ghrelin in patients who underwent hip joint replacement (53). However, the function of ghrelin in the development of OA is unknown. Mouse models of surgically induced OA play a key role in studies of OA (54, 55). Among them, the DMM model and the ACLT model are widely used for identification of OA-related molecules (55). In the current study, both models were established, and systemic as well as local delivery methods of ghrelin were used. The halflife of ghrelin is short, and repeated intraperitoneal injection of ghrelin was required to achieve a therapeutic result in several cases (56–58). In this study, we performed ROLE OF GHRELIN IN OSTEOARTHRITIS repeated intraperitoneal injection and tested the expression level of ghrelin. An elevated ghrelin level was observed in articular cartilage tissue of the ACLT model, suggesting that systemic ghrelin administration reached the cartilage tissue. Development of OA was assayed through histology as well as other experiments. As a result, systemic and local delivery of ghrelin markedly alleviated the severity of OA in both surgically induced OA models, which implied that ghrelin might play a protective role in degeneration of articular cartilage. Exaggerated catabolism and inflammatory reaction has been extensively studied and widely reported during development of OA (59). It is known that inflammatory cytokines TNF-a and IL-1b induce expression of molecules associated with OA in human chondrocyte, including ADAMTS-5 and MMP-13, which degrade the critical matrix components aggrecan and Col 2, respectively (60). Furthermore, oxidative stress reaction and inflammatory response are observed in cartilage degeneration, during which process COX-2, IL-6, iNOS, and TNF-a levels are usually enhanced (61). Herein, we assessed all the mentioned biomarkers in IL-1b as well as TNF-a–induced chondrocytes and found that the additional use of ghrelin Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 11 Figure 9. Ghrelin antagonizes IL-1b induction of catabolism by inhibiting the NF-kB signaling pathway. A, B) Ghrelin treatment impaired phosphorylation of IkBa in OA models as detected by Western blot. Protein was collected from cartilage of ACLT models in the PBS and ghrelin treatment groups, and Western blot was performed. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05 vs. indicated control group. C ) Ghrelin markedly suppressed p-IkBa expression in cartilage in the ACLT model as shown by immunohistochemistry of p-IkBa (n = 3/group). Scale bar, 100 mm. D) IL-1b–mediated NF-kB2 expression in primary chondrocyte was antagonized by ghrelin treatment. Human chondrocytes were isolated and treated with IL-1b for 12 h in the presence or absence of ghrelin. Significant differences are indicated (2-tailed Student’s t test; n = 3/group; means 6 SEM). *P , 0.05, **P , 0.01 vs. indicated control group. E, F ) Ghrelin attenuated pIkB-a expression as compared with the PBS group with stimulation of IL-1b. Human chondrocyte was treated with IL-1b for 12 h in the presence or absence of ghrelin, followed by Western blot. Significant differences are indicated (2-tailed Student’s t test; n = 3/ group; means 6 SEM). **P , 0.01 vs. indicated control group. G) Ghrelin suppressed IL-1b induction of NF-kB p65 nuclear translocation as detected by cell immunostaining. Human chondrocytes were treated with IL-1b for 12 h in the presence or absence of ghrelin, and cell immunostaining was performed (n = 3/group). greatly reduced the mentioned molecules associated with OA. Exaggerated apoptosis of chondrocyte is commonly observed with cartilage degeneration, and how to antagonize apoptosis has become a therapeutic target in OA (62). In this study, ghrelin treatment suppressed chondrocyte apoptosis in the DMM OA mouse model. Chondrocyte hypertrophy is another parameter to detect the development of OA (63), and in the current study, exogenous ghrelin inhibited chondrocyte hypertrophy in the DMM model, supporting the therapeutic potential of ghrelin in OA. Diminished production of extracellular matrix components, including Col 2 and aggrecan, is a feature in the aging process of cartilage (64). A critical aspect of the therapeutic strategy of OA is to attenuate the loss of Col 2 and aggrecan (65). It is known that IL-1b and TNF-a lead to reduced matrix production and elevated loss of these extracellular matrix components in cartilage (16, 66). In the current study, to imitate degeneration, primary human chondrocyte and cartilage explants were cultured and stimulated with IL-1b or TNF-a in the presence or absence of ghrelin. As a result, Col 2 and aggrecan expression levels were protected, which suggested the protective role of ghrelin in disorganized 12 Vol. 32 February 2018 anabolism of cartilage. Moreover, activation of the Akt signaling pathway is reported to promote chondrocyte anabolism, and impaired activity of Akt signaling in chondrocyte suppresses chondrocyte anabolism in OA development (67). Ghrelin is reported to activate Akt signaling and to promote the regeneration procedure in several conditions (42, 68, 69). In this study, ghrelin maintained phosphorylation of the Akt signaling pathway, and a specific Akt inhibitor, MK-2206, repressed the protective role of ghrelin. Moreover, it is known that activation of Akt signaling maintains the impaired anabolism of chondrocyte during degeneration (70). In this study, a commercial Akt activator, SC-79 (54), was used to antagonize the detrimental role of IL-1b in anabolism, which had similar effect compared with ghrelin. This finding further suggested that ghrelin might suppress the disorganized anabolism through Akt signaling. The NF-kB signaling pathway is a key mediator of age-dependent cartilage degeneration (71). Studies have shown that activation of NF-kB signaling accelerates— whereas suppression of this signaling attenuates— cartilage-degenerative diseases associated with aging (15, 72). Phosphorylation of IkBa is a commonly tested parameter for the activation of the NF-kB signaling The FASEB Journal x www.fasebj.org Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017 QU ET AL. pathway (21). In our research, we found that the level of p-IkBa was markedly elevated in inflammatory cytokine–treated chondrocytes and degenerative cartilage, although this was largely abolished by exogenous ghrelin. Moreover, nuclear transaction of p65 is a well-established feature for activation of the NF-kB signaling pathway (73, 74), which was inhibited by ghrelin in the current study. Taken together, these data suggest that excessive activation of the NF-kB signaling pathway in chondrocytes induced by inflammatory cytokines or OA was repressed by ghrelin, which may lead to the attenuation of cartilage degeneration. Our results show that ghrelin inhibits cartilage degeneration through maintaining chondrocyte homeostasis, which may have implications for the treatment of OA. ACKNOWLEDGMENTS This work was supported by Key Research and Development Projects of Shandong Province (Grant 2015GSF118115), the Natural Science Foundation of Shandong Province (Grants BS2014YY048 and BS2015SW028), and the National Natural Science Foundation of China (Grants 81501880 and 26010105131643). The authors declare no conflicts of interest. AUTHOR CONTRIBUTIONS K. Vasilev, W. Li, and Y. Zhao, conceived and designed the experiments; R. Qu, W. Wang, C. Qiu, and L. Guo performed the experiments; R. Qu, M. Ban, and J. Chen, analyzed the data; and W. Li and Y. 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FASEB J published online October 25, 2017 Access the most recent version at doi:10.1096/fj.201700265R Supplemental Material http://www.fasebj.org/content/suppl/2017/10/25/fj.201700265R.DC1 Subscriptions Information about subscribing to The FASEB Journal is online at http://www.faseb.org/The-FASEB-Journal/Librarian-s-Resources.aspx Permissions Submit copyright permission requests at: http://www.fasebj.org/site/misc/copyright.xhtml Email Alerts Receive free email alerts when new an article cites this article - sign up at http://www.fasebj.org/cgi/alerts © FASEB Downloaded from www.fasebj.org to IP 134.148.10.13. The FASEB Journal Vol., No. , pp:, October, 2017
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