Continuous mild stimulation with advanced glycation end products reduce aggrecan and type II collagen production via the RAGE without inducing cell death in human OUMS-27 chondrosarcoma cells

Continuous mild stimulation with advanced glycation end products reduce aggrecan and type II collagen production via the RAGE without inducing cell death in human OUMS-27 chondrosarcoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Continuous mild stimulation with advanced glycation end products reduce aggrecan and type II collagen production via the RAGE without inducing cell death in human OUMS-27 chondrosarcoma cells Omer Faruk Hatipoglu, Takashi Nishinaka, Kursat Oguz Yaykasli, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4173286/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chondrocytes are responsible for the production of extracellular matrix (ECM) components of cartilage, such as collagen type II alpha-1 (COL2A1) and aggrecan, which are loosely distributed in articular cartilage. Chondrocyte dysfunction has been implicated in the pathogenesis of rheumatic diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA). Advanced glycation end products (AGEs) accumulate in all tissues and body fluids, including cartilage and synovial fluid, with aging. Their accumulation in vivo is one of the major factors that cause and accelerate pathological changes in some chronic diseases, such as OA. Glycolaldehyde-derived AGEs (AGE3), known as toxic AGEs, have the strongest effect on cartilage compared to other AGEs. Studies conducted to date to demonstrate the effects of AGEs on chondrocytes have used very high doses (100 µg/mL) and collagen and aggrecan were reduced in the short term (24 h) due to decreased chondrocyte cell viability. However, it is assumed that AGEs stimulate cells for a longer period of time in vivo without causing cell death. Therefore, we stimulated a human chondrosarcoma cell line (OUMS-27) with 10 µg/mL AGE3 for four weeks. As a result, the expression of COL2A1 and aggrecan was significantly downregulated in OUMS-27 cells without inducing cell death, but the expression of proteases that play an important role in cartilage destruction was not affected. In addition, the receptor for advanced glycation end products (RAGE) inhibitors suppressed the AGE3-induced reduction in cartilage component production, suggesting the involvement of RAGE in the action of AGE3. advanced glycation end products aging cartilage collagen aggrecan Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Rheumatic diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA), are degenerative joint conditions that affect millions of people worldwide(Goldring and Marcu 2009 ; Eakin et al. 2017 ). They lead to progressive degeneration of articular cartilage, resulting in joint pain, stiffness, and reduced range of motion(Van de Stadt et al. 2023 ). Chondrocytes, the predominant cell type in cartilage tissue, are silent and rarely divide under physiological conditions(Charlier et al. 2016 ). In rheumatic diseases, chondrocytes act as target cells for inflammatory mediators, such as cytokines leading to chondrocyte dysfunction(Tseng et al. 2020 ; Zheng et al. 2021 ). Chondrocytes are responsible for synthesizing components of the extracellular matrix (ECM), including collagens and aggrecan, which provide mechanical support to cartilage during joint motion(Knudson and Knudson 2001 ). An imbalance between the anabolic and catabolic processes of the ECM plays a substantial role in cartilage degradation. Altered synthesis and turnover of ECM components, such as collagen type II alpha-1 (COL2A1) and aggrecan, contribute to the loss of cartilage integrity(Mobasheri et al. 2017 ; Fujii et al. 2022 ). The degradation of cartilage matrix in OA is mediated by proteases. COL2A1 is primarily degraded by MMP1, -3, and − 13, whereas aggrecan is primarily degraded by ADAMTS4, -5, and − 9 in OA(Hatipoglu et al. 2015 ; Yaykasli et al. 2015 ). Advanced glycation end products (AGEs) are formed by the non-enzymatic reaction of reducing sugars with proteins, lipids, or nucleic acids(Prasad et al. 2019 ) and the receptor for AGEs (RAGE) is a major receptor for AGE signaling(Ramasamy et al. 2011 ). Among the AGEs, glycolaldehyde-derived AGE (AGE3), known as toxic AGE, plays a role in the pathogenesis of diabetic complications, and toxic AGEs exhibit stronger activity on cartilage compared with that of others, such as glucose-derived AGE (AGE1)(Aleksandra Kuzan; Takeuchi and Yamagishi 2004 ; Kitaura et al. 2021 ). In addition, in OA there is a strong relationship between AGE3 and RAGE in an increasing pattern with the degree of OA, and that AGE3 is the most important AGE for cartilage degeneration through the RAGE pathway(Hirose et al. 2011 ). Moreover type 2 diabetes has been reported to contribute to the progression and pathogenesis of OA, including a mechanism involving AGEs, in dysregulation of articular cartilage metabolism(Chowdhury et al. 2022 ). Accumulation of AGEs is an inevitable part of the aging process in the human body, and recent studies have linked AGEs to complications of age-related diseases, such as cardiovascular and Alzheimer's disease(Luevano-Contreras and Chapman-Novakofski 2010 ). In addition, limiting dietary AGEs provides a protective effect in conditions, such as wound healing and insulin resistance. Some studies have reported an increase in lifespan in animal models following restricted AGE intake(Zgutka et al. 2023 ). It has also been suggested that AGEs accumulate in articular cartilage with aging and affect cartilage function(Gkogkolou and Böhm 2012 ; Gouldin et al. 2023 ). However, the influence of prolonged direct exposure to AGEs on articular cartilage remains unclear. In the present study, we investigated the potential effects of four weeks of exposure to 10 µg/mL AGE3, on COL2A1 and aggrecan expression in human chondrocytes and the role of RAGE in mediating these effects. 2. Methods 2.1. Reagents and Antibodies The anti-COL2A1 and anti-aggrecan antibodies from Santa Cruz Biotechnology (sc-52658, sc-166951, Santa Cruz, CA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Millipore (MAB374, Bedford, MA, USA), and horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG from Sigma-Aldrich from SeraCare Life Sciences, Inc. (5220 − 0341, Milford, MA, USA) were used in the study. AGE3 was prepared as described in the previously described(Yamazaki et al. 2021 ; Nishinaka et al. 2022 ), in short BSA (FUJIFILM Wako, Osaka, Japan) was incubated with glycolaldehyde dimer (Sigma-Aldrich) in 0.2 M phosphate buffer (pH 7.4) at 37°C for 7 d under sterile conditions. Then, AGEs were dialyzed for 2 d against phosphate-buffered saline (PBS) at 4°C. RAGE antagonist, FPS-ZM1 was purchased from Millipore. 2.2 Cell Culture and Treatment We used a OUMS-27 cells in our study because chondrocytes become unstable and lose properties in vitro(Lafont 2010 ), and many previous studies have used OUMS-27 instead of primary chondrocytes(Yaykasli et al. 2009 ; Gun Bilgic et al. 2020 ). OUMS-27 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), and maintained at 37°C in a humidified chamber with 5% CO 2 and 20% O 2 . OUMS-27 cells (2.0 x 10 5 ) were seeded per well in six-well tissue culture plates, followed by culture in the presence or absence of AGE3 for 7 d. 2.3 Cell Viability Assay OUMS-27 cell viability was assessed using the CCK-8 kit (Dojindo, Tokyo, Japan). Briefly, OUMS-27 cells were seeded in 96-well plates (5×10 4 cells) and exposed to AGE3. The cells were then incubated for 1–8 d at 37°C under 5% CO 2 and the optical density was measured at 450 nm using a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA). Cell viability was determined as a percentage of proliferation compared with that in control cells. 2.4 RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qPCR) TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract and quantify total RNA from cell samples according to the manufacturer's guidelines. The RNA concentration was measured at 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The extracted total RNA was then subjected to reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan). The reaction was carried out at 25°C for 10 min, 42°C for 60 min, and 97°C for 5 min with random primers (Toyobo). qPCR was performed on a StepOnePlus system (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems). The PCR reaction mixture consisted of 5 µL of 2X SYBR Green mix, 0.5 µL (10 uM) of each target gene primer, and GAPDH as an endogenous internal control gene. The reaction was performed under the following conditions 10 min at 95°C, followed by 40 cycles of one-step thermal cycling, consisting of 3 s at 95°C and 30 s at 60°C, in a 96-well reaction plate. The mRNA expression levels of the target genes were determined by the comparative Ct (ΔΔCT) method and normalized to GAPDH. 2.5 Western Blot Analysis 2.5 Western Blot Analysis OUMS-27 cells were cultured at a density of 1×10 6 cells/well in six-well plates using DMEM supplemented with 10% FBS at 37°C and 5% CO 2 . Following AGE3 stimulation, the cells were washed with cold PBS and lysed in radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors and protein extracted as described previously(Hatipoglu et al. 2023 ). The inhibitors used were antipain, leupeptin, and aprotinin, with a concentration of 2.0 µg/mL each, from the Peptide Institute Inc. (Osaka, Japan) and Nacalai Tesque (Kyoto, Japan). After centrifugation, the total protein concentration was determined using a Bradford protein assay kit from Bio-Rad Laboratories. The cell lysates were then mixed with 6X reducing sample buffer, denatured at 95°C for 5 min and separated on 8% SDS-PAGE gels. The separated proteins were then transferred to polyvinylidene difluoride membranes (Merck Millipore Ltd). The primary antibodies used in the experiment were anti-COL2A1, anti-aggrecan, and anti-β-actin, with dilutions of 1:1,000 and 1:10,000. Secondary antibody, anti-mouse IgG, was used at dilutions of 1:2,000 and 1:10,000. To obtain accurate results, the membranes were blocked with 5% skim milk in TBS-0.05% Tween 20 (TBS-T) for 1 h at room temperature. The membranes were then incubated with primary antibodies overnight at 4°C. After three washes with 1X TBS-T buffer, the membranes were incubated with secondary antibody for 1 h at room temperature. Signals were visualized by chemiluminescence using Amersham™ ECL™ Prime (Cytiva, WA, USA), then imaged using an Amersham Imager 600 and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA). 2.6 Statistical Analysis Data are shown as the mean ± standard deviation of the mean. Differences between groups were analyzed by unpaired Student's t-test or by ANOVA followed by Dunnett's post hoc comparison. All experiments were repeated at least three times. In all analyses, p < 0.05 was considered statistically significant. 3. Results 3.1 Evaluation of the time- and dose-dependent cytotoxicity of AGE3 on OUMS cells using the CCK-8 assay To investigate the effect of AGE3 on the viability of OUMS cells, we incubated the cells with different concentrations of AGE3 (10, 20, 50, 100, and 200 µg/mL) for different time intervals (1, 2, 4 and 8 d) and evaluated cell viability using the CCK-8 assay. AGE3 concentrations below 50 µg/mL have no significant effect on OUMS cell proliferation as shown in Fig. 1 . This observation suggests that OUMS cells are able to maintain resistance to AGE-induced toxicity by maintaining viability in the presence of the AGE3 levels used in this study. 3.2 COL2A1 and aggrecan expression reduced by AGE3 stimulation in OUMS-27 for four weeks We investigated the four-week effects of AGE3 on chondrocytes by isolating mRNA from OUMS cells by treating them with different concentrations of AGE3 (5, 10, 20, and 50 µg/mL) for different durations (1, 3, 6, and 24 h, and one, two, four, and eight weeks), and using RT-PCR to assess the expression levels of COL2A1 and aggrecan. For a clearer understanding of our experimental design, please refer to Online Resource 1. Our results showed that the expression levels of COL2A1 and aggrecan were significantly decreased after four weeks of AGE3 stimulation compared with that in the control group (Fig. 2 a, b). However, we found that incubation for 1, 3, 6, and 24 h with AGE3 (10 µg/mL) did not show any effect on the expression level of COL2A1 and aggrecan in our experiments using the OUMS-27 human cell line (Fig. 2 c). We then performed western blotting to measure the protein expression levels of COL2A1 and aggrecan in chondrocytes treated with different concentrations of AGE3 (0, 5, 10, 20, and 50 µg/mL for four weeks). AGE3 exposure significantly decreased the protein expression levels of COL2A1 and aggrecan from 10 µg/mL compared with that in the control group (Fig. 2 d–f). 3.3 The RAGE-specific inhibitor FPS-ZM1 attenuated AGE-induced degradation of COL2A1 and aggrecan AGEs and their receptors are considered important components of AGE-related complications. We verified that RAGE, TLR4, FEEL1 and LOX1, known as AGE-related receptors(Ott et al. 2014 ), are expressed in chondrocytes (data not shown) and then investigated whether this receptor is affected by AGE stimulation. The results of quantitative real-time PCR analysis showed that 10 µg/mL of AGE3 stimulation (one, two, four, and eight weeks) significantly upregulated the expression of RAGE, while the expression of TLR4, FEEL1, and LOX1 remained unaffected (Fig. 3 a). Based on these findings, we conducted a study to investigate the possibility of reversing the AGE3-induced decrease in COL2A1 and aggrecan in OUMS by using a small molecule inhibitor of RAGE, FPS-ZM1. Notably, our results show that pretreatment with 1 µM FPS-ZM1 significantly attenuated all AGE3-mediated effects on COL2A1 and aggrecan expression (Fig. 3 b–d). These results indicate the involvement of RAGE in AGE3 action. 3.4 Protease expression was not affected by AGE3 induction in OUMS-27 Proteases are involved in the degradation of cartilage matrix in OA. It has been reported that the cartilage matrix degradation is due to the activity of catabolic enzymes and inflammatory mediators, including MMPs and COX2(Fei et al. 2019 ; Mehana et al. 2019 ). Therefore, we investigated the mRNA expression levels of MMP1, MMP2, MMP3, MMP9, MMP13, ADAMTS4, ADAMTS9, and COX2 in response to AGE3 (10 µg/mL) treatment for different durations (1, 3, 6, 24 h, and one, two, four, and eight weeks) to evaluate the effect of AGE3 on the expression of these enzyme genes. Our results show that 10 µg/mL AGE3 had no effect on the expression of these catabolic enzymes at 1, 3, 6, and 24 h (Fig. 4 a) or at one, two, four, and eight weeks (Fig. 4 b). 4. Discussion AGEs accumulate in articular cartilage over time, impairing cartilage function and leading to arthritis, and this accumulation of AGEs causes cartilage browning in fluorescence(DeGroot et al. 2001 ). The action mechanism of AGEs involves cross-linking with joint proteins, including collagen and aggrecan, and RAGE binding, which activates a number of intracellular signaling pathways(DeGroot et al. 2004 ). Collagen and aggrecan, key components of the cartilage matrix, are essential for maintaining normal cartilage cells and decline with age; their dysfunction accelerates the progression of rheumatic diseases, such as OA and RA(Goldring and Marcu 2009 ; Rapp and Zaucke 2023 ). Therefore, inhibiting the loss of collagen and aggrecan in rheumatic disease may have the potential to prevent cartilage degeneration. Previous in vitro studies have shown that treatment with 100 µg/mL of AGEs for 24 h reduces the expression of COL2A1 and aggrecan in human SW1353 chondrocytes(Li et al. 2020 ). However, Zhou et al. showed that 100 µg/mL of AGEs after 24 h reduced the viability in human SW1353 chondrocytes(Zhou et al. 2022 ). In addition, 100 µg/mL AGE3 reduced the viability of human OUMS27 chondrocytes after 48 h in our study (Fig. 1 ). Furthermore, even in patients with advanced diabetes, serum AGE levels were 82.8 ± 9.4 µg/mL(Makita et al. 1991 ) and it can be speculated that intra-articular levels would be significantly lower. In the present study, we showed for the first time that four weeks of stimulation with 10 µg/mL AGE3 in human chondrocytes led to a decrease in the expression of COL2A1 and aggrecan via RAGE without affecting chondrocyte viability, whereas the 1, 3, 6, and 24 h of 10 µg/mL AGE3 had no effect on the expression level of COL2A1 and aggrecan in chondrocytes in our experiments using the human OUMS-27 cell line (Fig. 2 ). These findings are critical in demonstrating the dramatic catabolic effect of low doses of AGEs, closer to the biological environment, on aggrecan and COL2A1 after prolonged stimulation. RAGE is well known and is the most studied receptor in AGE-related studies(Yue et al. 2022 ). Additionally, RAGE knockout mice are considerably protected against the development of OA(Larkin et al. 2013 ). TLR4, FEEL1, and LOX1 receptors have also been identified to be AGE-binding receptors(Yonei et al. 2010 ). In this study, we found that the expression level of RAGE was upregulated after two weeks of 10 µg/mL AGE3 stimulation, while other receptors, including TLR4, FEEL1, and LOX1, remained unaffected (Fig. 3 A). To further substantiate our findings, we used FPS-ZM1, a high-affinity antagonist of RAGE. Pretreatment with FPS-ZM1 almost completely abolished the degradation of COL2A1 and aggrecan by AGE3 (Fig. 3 b–d). Similarly, other RAGE inhibitors, such as Salicin(Gao and Zhang 2019 ) and Saxagliptin(Hu et al. 2019 ), have been successfully used to prevent cartilage degradation in the mouse model of OA and in patients with OA. Additionally, it was concluded that AGEs in the ECM promote inflammation and cartilage degradation via RAGE. Evidence from in vivo and in vitro studies indicated that AGEs are one of the major players responsible for disrupting cartilage hemostasis by inducing inflammatory mediators, including proteases(Goldring and Otero 2011 ; Sanchez-Lopez et al. 2022 ). It is reported that 24 or 48 h of 100 µg/mL AGE3 stimulation increases the expression levels of MMP1, MMP3, MMP13, ADAMTS4, and ADAMTS5 up to four-fold(Gao and Zhang 2019 ; Zhang et al. 2020 ). In our experimental setup, 10 µg/mL AGE3 did not alter protease expression for 1–24 h or for one to eight weeks (Fig. 4 a, b). Although there was no change even at 100 µg/mL AGE3, AGE3 stimulation above 500 µg/mL resulted in an increase in MMP changes (data not shown). Further studies should be conducted on the effect of AGEs on MMP expression. Our study shows that four weeks of 10 µg/mL AGE3 stimulation has deleterious effects on cartilage, highlighting the importance of evaluating the duration and amount of AGE exposure, while providing valuable insights into the underlying mechanisms of rheumatic diseases, such as OA and RA pathogenesis. Declarations Contributions OFH, TN, HW, and HT designed and performed the experiments, analyzed the data, and wrote the manuscript. MN and SM analyzed the data and edited the manuscript. KOY analyzed the data and wrote the manuscript. TT and MW adjusted the analyzed data and revised the figures. All the authors have read and approved the final version of the manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Data Availability All data included in this study are available upon request from the corresponding author. Acknowledgements This work was supported in part by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (nos. 21K06588 to O.F.H., 21K15354 to T.N., 20K07290 to H.W., and 23K10465 to H.T.) and funded by Kindai University Research Enhancement Grant 2021 (KD2102 to HW) and Kindai University Research Enhancement Grants 2023 (IPO11 to TN and IPO12 to OFH). References Aleksandra Kuzan Toxicity of advanced glycation end products (Review). https://www.spandidos-publications.com/10.3892/br.2021.1422. Accessed 21 Dec 2023 Charlier E, Relic B, Deroyer C, et al (2016) Insights on Molecular Mechanisms of Chondrocytes Death in Osteoarthritis. Int J Mol Sci 17:. https://doi.org/10.3390/IJMS17122146 Chowdhury T, Bellamkonda A, Gousy N, Roy PD (2022) The Association Between Diabetes Mellitus and Osteoarthritis: Does Diabetes Mellitus Play a Role in the Severity of Pain in Osteoarthritis? 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Ageing Res Rev 66:101249. https://doi.org/10.1016/J.ARR.2020.101249 Zhou Y, Li J, Wang C, Pan Z (2022) Fumitremorgin C alleviates advanced glycation end products (AGE)-induced chondrocyte inflammation and collagen II and aggrecan degradation through sirtuin-1 (SIRT1)/nuclear factor (NF)-κB/ mitogen-activated protein kinase (MAPK). Bioengineered 13:3867–3876. https://doi.org/10.1080/21655979.2021.2024387 Additional Declarations No competing interests reported. Supplementary Files LongtermAGE3FINALOnlineResource1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4173286","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284513111,"identity":"275bf2ce-7e57-4a43-959e-98a4330bf5da","order_by":0,"name":"Omer Faruk Hatipoglu","email":"","orcid":"","institution":"Kindai University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Omer","middleName":"Faruk","lastName":"Hatipoglu","suffix":""},{"id":284513112,"identity":"fa28ba31-7dfa-46c2-98ea-924b88b0d3f8","order_by":1,"name":"Takashi Nishinaka","email":"","orcid":"","institution":"Kindai University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Nishinaka","suffix":""},{"id":284513113,"identity":"6226ff6d-f09f-4c06-ac96-ae90e0d4772e","order_by":2,"name":"Kursat Oguz Yaykasli","email":"","orcid":"","institution":"Friedrich-Alexander-University Erlangen- Nürnberg (FAU) and Universitätsklinikum Erlangen","correspondingAuthor":false,"prefix":"","firstName":"Kursat","middleName":"Oguz","lastName":"Yaykasli","suffix":""},{"id":284513114,"identity":"7252719f-52ff-4ea9-a1e1-6a1ef995b6fe","order_by":3,"name":"Shuji Mori","email":"","orcid":"","institution":"Shujitsu University School of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Shuji","middleName":"","lastName":"Mori","suffix":""},{"id":284513115,"identity":"b70586bf-b67b-4759-aed6-e27f67281ad2","order_by":4,"name":"Masahiro Watanabe","email":"","orcid":"","institution":"Shujitsu University School of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Watanabe","suffix":""},{"id":284513116,"identity":"23ac1dd8-77f2-43c0-a2b3-88ea57093b28","order_by":5,"name":"Takao Toyomura","email":"","orcid":"","institution":"Shujitsu University School of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Takao","middleName":"","lastName":"Toyomura","suffix":""},{"id":284513118,"identity":"d2155ae3-6c2f-45ff-b544-19c8ea1dc324","order_by":6,"name":"Masahiro Nishibori","email":"","orcid":"","institution":"Okayama University Graduate School of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Nishibori","suffix":""},{"id":284513120,"identity":"29b6c610-3000-47d5-bfca-85478e743997","order_by":7,"name":"Satoshi Hirohata","email":"","orcid":"","institution":"Okayama University Graduate School of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Hirohata","suffix":""},{"id":284513123,"identity":"995e8afc-0b88-4384-a922-925e2168d323","order_by":8,"name":"Hideo Takahashi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACAx4GxgcMDMwMEswMDBIMBmDBBAJamJkNSNbCJgHWwsAAxoSBOc/5Y1U3KqwZJNt5D974UcAgz9/A8OwBPi2Wvc1st3POpDNIM/MlW/YYMBjOOMCQboDXYeeZ2W7nth1mkGPmMZPgMWBg3MDAkIbXhSAtxbn/IFok/xgw2BPWcraZjTm34TDQYTxm0kBbEglrOXPYWDrnWDqPZDOPsbWMgUTyjMOE/HIm8eHnnBprOYnzZwxvvvljY9vf3pP2AJ8WGOCB0qA44kkjRgcKYD9GspZRMApGwSgY1gAAF4U8M/0CkBoAAAAASUVORK5CYII=","orcid":"","institution":"Kindai University Faculty of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Hideo","middleName":"","lastName":"Takahashi","suffix":""},{"id":284513124,"identity":"e515383a-509d-402a-adc2-de83a17e3279","order_by":9,"name":"Hidenori Wake","email":"","orcid":"","institution":"Kindai University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hidenori","middleName":"","lastName":"Wake","suffix":""}],"badges":[],"createdAt":"2024-03-27 03:59:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4173286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4173286/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53853664,"identity":"25b8120a-5aaf-4a5a-9097-d7d01c0d4f18","added_by":"auto","created_at":"2024-04-01 10:39:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":235361,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the cytotoxic effects of different AGE3 doses on OUMS-27 cells.\u003cstrong\u003e \u003c/strong\u003eIncubation of different concentrations of AGE3 (10, 20, 50, 100, and 200 μg/mL) for different time intervals (1, 2, 4 and 8 d), low concentrations of AGE3 (≤50 μg/mL) had no significant effect on OUMS cell proliferation. Each bar represents the mean ± SD of the three independent experiments. Differences were determined using two-way ANOVA followed by Dunnett post-hoc test. * P\u0026lt;0.05 vs. control\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4173286/v1/0ba52f345e8aa27ef8a8155a.png"},{"id":53853665,"identity":"bcb95374-73f7-4b7b-a54a-7eabe9b2e42a","added_by":"auto","created_at":"2024-04-01 10:39:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":207288,"visible":true,"origin":"","legend":"\u003cp\u003eCOL2A1 and aggrecan expression in OUMS-27 long-term stimulated AGE3 assessed by quantitative real-time PCR. Continuous stimulation of AGE3 in OUMS-27 decreased COL2A1 and aggrecan expression from week 4. (a) COL2A1 (b) aggrecan expression. (c) Short-term incubation (1, 3, 6, and 24 h) of 10 μg/mL AGE3 had no effect on COL2A1 and aggrecan expression. (d) Western blot analysis revealed a significant downregulation of COL2A1 and aggrecan expression induced by AGE3 for four weeks in OUMS-27 cells compared with that in controls. GAPDH was used as an internal control. (e) Relative COL2A1 and (f) aggrecan protein expression was measured by ImageJ. Each bar represents the mean ± SD of the three independent experiments. Data were analyzed using a one-way ANOVA followed by Dunnett’s post hoc test. * P\u0026lt;0.05, ** P\u0026lt;0.01 vs. control\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4173286/v1/66580260b8ea1d7d0941ba3c.png"},{"id":53853666,"identity":"b2fe1c72-f1e2-463e-8e97-d52213f3feda","added_by":"auto","created_at":"2024-04-01 10:39:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85796,"visible":true,"origin":"","legend":"\u003cp\u003eAGE3 stimulation significantly upregulated the expression of RAGE and RAGE-specific inhibitor FPS-ZM1 attenuated AGEs-induced decrease of COL2A1 and aggrecan in OUMS-27 cells (a) Stimulation with 10 μg/mL of AGE3 from two weeks significantly increased the expression of increased the expression of RAGE while not affecting the expression of TLR4, FEEL1 and LOX1. (b) Western blot analysis revealed that pretreatment with 1 µM FPS-ZM1 significantly reduced all four-week AGE3-mediated effects on COL2A1 and aggrecan expression in OUMS-27 cells compared with that in controls. GAPDH was used as an internal control. (c) Relative COL2A1 and (d) aggrecan protein expression was measured using ImageJ. Each bar represents the mean ± SD of the three independent experiments. Statistical data analyses were performed using one-way ANOVA followed by Dunnett's or Tukey’s post hoc test. ** P\u0026lt;0.01. vs. control\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4173286/v1/defa671454285eda2d1d9070.png"},{"id":53853668,"identity":"4e0037f9-00c9-4323-975f-91563e307b22","added_by":"auto","created_at":"2024-04-01 10:39:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":471450,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of MMP1, MMP2, MMP3, MMP9, MMP13, ADAMTS4, ADAMTS9 and COX2 expression in OUMS-27-stimulated AGE3 (a) Stimulation with 10 μg/mL AGE3 for up to 24 h does not affect the expression of target genes. (b) Stimulation with 10 μg/mL AGE3 for up to eight weeks does not affect the expression of target genes. Data were analyzed using a one-way ANOVA followed by Dunnett’s post hoc test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4173286/v1/0d35eeda1d275b982dcb9018.png"},{"id":54395374,"identity":"0a74ae7a-1270-491d-b884-42847bae93a8","added_by":"auto","created_at":"2024-04-09 21:29:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1538729,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4173286/v1/37824222-07c6-42c4-ba81-4f46afb6480a.pdf"},{"id":53853667,"identity":"9899e71f-92a2-4629-8896-86577a2b80e5","added_by":"auto","created_at":"2024-04-01 10:39:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":215784,"visible":true,"origin":"","legend":"","description":"","filename":"LongtermAGE3FINALOnlineResource1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4173286/v1/4236f8456cb8d9ee2895cf4b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Continuous mild stimulation with advanced glycation end products reduce aggrecan and type II collagen production via the RAGE without inducing cell death in human OUMS-27 chondrosarcoma cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRheumatic diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA), are degenerative joint conditions that affect millions of people worldwide(Goldring and Marcu \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Eakin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). They lead to progressive degeneration of articular cartilage, resulting in joint pain, stiffness, and reduced range of motion(Van de Stadt et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChondrocytes, the predominant cell type in cartilage tissue, are silent and rarely divide under physiological conditions(Charlier et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In rheumatic diseases, chondrocytes act as target cells for inflammatory mediators, such as cytokines leading to chondrocyte dysfunction(Tseng et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Chondrocytes are responsible for synthesizing components of the extracellular matrix (ECM), including collagens and aggrecan, which provide mechanical support to cartilage during joint motion(Knudson and Knudson \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). An imbalance between the anabolic and catabolic processes of the ECM plays a substantial role in cartilage degradation. Altered synthesis and turnover of ECM components, such as collagen type II alpha-1 (COL2A1) and aggrecan, contribute to the loss of cartilage integrity(Mobasheri et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Fujii et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The degradation of cartilage matrix in OA is mediated by proteases. COL2A1 is primarily degraded by MMP1, -3, and \u0026minus;\u0026thinsp;13, whereas aggrecan is primarily degraded by ADAMTS4, -5, and \u0026minus;\u0026thinsp;9 in OA(Hatipoglu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yaykasli et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdvanced glycation end products (AGEs) are formed by the non-enzymatic reaction of reducing sugars with proteins, lipids, or nucleic acids(Prasad et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and the receptor for AGEs (RAGE) is a major receptor for AGE signaling(Ramasamy et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Among the AGEs, glycolaldehyde-derived AGE (AGE3), known as toxic AGE, plays a role in the pathogenesis of diabetic complications, and toxic AGEs exhibit stronger activity on cartilage compared with that of others, such as glucose-derived AGE (AGE1)(Aleksandra Kuzan; Takeuchi and Yamagishi \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kitaura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, in OA there is a strong relationship between AGE3 and RAGE in an increasing pattern with the degree of OA, and that AGE3 is the most important AGE for cartilage degeneration through the RAGE pathway(Hirose et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover type 2 diabetes has been reported to contribute to the progression and pathogenesis of OA, including a mechanism involving AGEs, in dysregulation of articular cartilage metabolism(Chowdhury et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccumulation of AGEs is an inevitable part of the aging process in the human body, and recent studies have linked AGEs to complications of age-related diseases, such as cardiovascular and Alzheimer's disease(Luevano-Contreras and Chapman-Novakofski \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, limiting dietary AGEs provides a protective effect in conditions, such as wound healing and insulin resistance. Some studies have reported an increase in lifespan in animal models following restricted AGE intake(Zgutka et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It has also been suggested that AGEs accumulate in articular cartilage with aging and affect cartilage function(Gkogkolou and B\u0026ouml;hm \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gouldin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the influence of prolonged direct exposure to AGEs on articular cartilage remains unclear.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the potential effects of four weeks of exposure to 10 \u0026micro;g/mL AGE3, on COL2A1 and aggrecan expression in human chondrocytes and the role of RAGE in mediating these effects.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents and Antibodies\u003c/h2\u003e \u003cp\u003eThe anti-COL2A1 and anti-aggrecan antibodies from Santa Cruz Biotechnology (sc-52658, sc-166951, Santa Cruz, CA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Millipore (MAB374, Bedford, MA, USA), and horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG from Sigma-Aldrich from SeraCare Life Sciences, Inc. (5220\u0026thinsp;\u0026minus;\u0026thinsp;0341, Milford, MA, USA) were used in the study. AGE3 was prepared as described in the previously described(Yamazaki et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nishinaka et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in short BSA (FUJIFILM Wako, Osaka, Japan) was incubated with glycolaldehyde dimer (Sigma-Aldrich) in 0.2 M phosphate buffer (pH 7.4) at 37\u0026deg;C for 7 d under sterile conditions. Then, AGEs were dialyzed for 2 d against phosphate-buffered saline (PBS) at 4\u0026deg;C. RAGE antagonist, FPS-ZM1 was purchased from Millipore.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2 Cell Culture and Treatment\u003c/h3\u003e\n\u003cp\u003eWe used a OUMS-27 cells in our study because chondrocytes become unstable and lose properties in vitro(Lafont \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and many previous studies have used OUMS-27 instead of primary chondrocytes(Yaykasli et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gun Bilgic et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). OUMS-27 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), and maintained at 37\u0026deg;C in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e and 20% O\u003csub\u003e2\u003c/sub\u003e. OUMS-27 cells (2.0 x 10\u003csup\u003e5\u003c/sup\u003e) were seeded per well in six-well tissue culture plates, followed by culture in the presence or absence of AGE3 for 7 d.\u003c/p\u003e\n\u003ch3\u003e2.3 Cell Viability Assay\u003c/h3\u003e\n\u003cp\u003eOUMS-27 cell viability was assessed using the CCK-8 kit (Dojindo, Tokyo, Japan). Briefly, OUMS-27 cells were seeded in 96-well plates (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells) and exposed to AGE3. The cells were then incubated for 1\u0026ndash;8 d at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e and the optical density was measured at 450 nm using a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA). Cell viability was determined as a percentage of proliferation compared with that in control cells.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qPCR)\u003c/h2\u003e \u003cp\u003eTRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract and quantify total RNA from cell samples according to the manufacturer's guidelines. The RNA concentration was measured at 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The extracted total RNA was then subjected to reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan). The reaction was carried out at 25\u0026deg;C for 10 min, 42\u0026deg;C for 60 min, and 97\u0026deg;C for 5 min with random primers (Toyobo).\u003c/p\u003e \u003cp\u003eqPCR was performed on a StepOnePlus system (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems). The PCR reaction mixture consisted of 5 \u0026micro;L of 2X SYBR Green mix, 0.5 \u0026micro;L (10 uM) of each target gene primer, and GAPDH as an endogenous internal control gene. The reaction was performed under the following conditions 10 min at 95\u0026deg;C, followed by 40 cycles of one-step thermal cycling, consisting of 3 s at 95\u0026deg;C and 30 s at 60\u0026deg;C, in a 96-well reaction plate. The mRNA expression levels of the target genes were determined by the comparative Ct (ΔΔCT) method and normalized to GAPDH.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.5 Western Blot Analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e2.5 Western Blot Analysis\u003c/div\u003e \u003cp\u003eOUMS-27 cells were cultured at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well in six-well plates using DMEM supplemented with 10% FBS at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Following AGE3 stimulation, the cells were washed with cold PBS and lysed in radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors and protein extracted as described previously(Hatipoglu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The inhibitors used were antipain, leupeptin, and aprotinin, with a concentration of 2.0 \u0026micro;g/mL each, from the Peptide Institute Inc. (Osaka, Japan) and Nacalai Tesque (Kyoto, Japan). After centrifugation, the total protein concentration was determined using a Bradford protein assay kit from Bio-Rad Laboratories. The cell lysates were then mixed with 6X reducing sample buffer, denatured at 95\u0026deg;C for 5 min and separated on 8% SDS-PAGE gels. The separated proteins were then transferred to polyvinylidene difluoride membranes (Merck Millipore Ltd). The primary antibodies used in the experiment were anti-COL2A1, anti-aggrecan, and anti-β-actin, with dilutions of 1:1,000 and 1:10,000. Secondary antibody, anti-mouse IgG, was used at dilutions of 1:2,000 and 1:10,000. To obtain accurate results, the membranes were blocked with 5% skim milk in TBS-0.05% Tween 20 (TBS-T) for 1 h at room temperature. The membranes were then incubated with primary antibodies overnight at 4\u0026deg;C. After three washes with 1X TBS-T buffer, the membranes were incubated with secondary antibody for 1 h at room temperature. Signals were visualized by chemiluminescence using Amersham\u0026trade; ECL\u0026trade; Prime (Cytiva, WA, USA), then imaged using an Amersham Imager 600 and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).\u003c/p\u003e\n\u003ch3\u003e2.6 Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of the mean. Differences between groups were analyzed by unpaired Student's t-test or by ANOVA followed by Dunnett's post hoc comparison. All experiments were repeated at least three times. In all analyses, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1 Evaluation of the time- and dose-dependent cytotoxicity of AGE3 on OUMS cells using the CCK-8 assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effect of AGE3 on the viability of OUMS cells, we incubated the cells with different concentrations of AGE3 (10, 20, 50, 100, and 200 \u0026micro;g/mL) for different time intervals (1, 2, 4 and 8 d) and evaluated cell viability using the CCK-8 assay. AGE3 concentrations below 50 \u0026micro;g/mL have no significant effect on OUMS cell proliferation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This observation suggests that OUMS cells are able to maintain resistance to AGE-induced toxicity by maintaining viability in the presence of the AGE3 levels used in this study.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 COL2A1 and aggrecan expression reduced by AGE3 stimulation in OUMS-27 for four weeks\u003c/h2\u003e \u003cp\u003eWe investigated the four-week effects of AGE3 on chondrocytes by isolating mRNA from OUMS cells by treating them with different concentrations of AGE3 (5, 10, 20, and 50 \u0026micro;g/mL) for different durations (1, 3, 6, and 24 h, and one, two, four, and eight weeks), and using RT-PCR to assess the expression levels of COL2A1 and aggrecan. For a clearer understanding of our experimental design, please refer to Online Resource 1. Our results showed that the expression levels of COL2A1 and aggrecan were significantly decreased after four weeks of AGE3 stimulation compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). However, we found that incubation for 1, 3, 6, and 24 h with AGE3 (10 \u0026micro;g/mL) did not show any effect on the expression level of COL2A1 and aggrecan in our experiments using the OUMS-27 human cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eWe then performed western blotting to measure the protein expression levels of COL2A1 and aggrecan in chondrocytes treated with different concentrations of AGE3 (0, 5, 10, 20, and 50 \u0026micro;g/mL for four weeks). AGE3 exposure significantly decreased the protein expression levels of COL2A1 and aggrecan from 10 \u0026micro;g/mL compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u0026ndash;f).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The RAGE-specific inhibitor FPS-ZM1 attenuated AGE-induced degradation of COL2A1 and aggrecan\u003c/h2\u003e \u003cp\u003eAGEs and their receptors are considered important components of AGE-related complications. We verified that RAGE, TLR4, FEEL1 and LOX1, known as AGE-related receptors(Ott et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), are expressed in chondrocytes (data not shown) and then investigated whether this receptor is affected by AGE stimulation.\u003c/p\u003e \u003cp\u003eThe results of quantitative real-time PCR analysis showed that 10 \u0026micro;g/mL of AGE3 stimulation (one, two, four, and eight weeks) significantly upregulated the expression of RAGE, while the expression of TLR4, FEEL1, and LOX1 remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Based on these findings, we conducted a study to investigate the possibility of reversing the AGE3-induced decrease in COL2A1 and aggrecan in OUMS by using a small molecule inhibitor of RAGE, FPS-ZM1. Notably, our results show that pretreatment with 1 \u0026micro;M FPS-ZM1 significantly attenuated all AGE3-mediated effects on COL2A1 and aggrecan expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u0026ndash;d). These results indicate the involvement of RAGE in AGE3 action.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Protease expression was not affected by AGE3 induction in OUMS-27\u003c/h2\u003e \u003cp\u003eProteases are involved in the degradation of cartilage matrix in OA. It has been reported that the cartilage matrix degradation is due to the activity of catabolic enzymes and inflammatory mediators, including MMPs and COX2(Fei et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mehana et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, we investigated the mRNA expression levels of MMP1, MMP2, MMP3, MMP9, MMP13, ADAMTS4, ADAMTS9, and COX2 in response to AGE3 (10 \u0026micro;g/mL) treatment for different durations (1, 3, 6, 24 h, and one, two, four, and eight weeks) to evaluate the effect of AGE3 on the expression of these enzyme genes. Our results show that 10 \u0026micro;g/mL AGE3 had no effect on the expression of these catabolic enzymes at 1, 3, 6, and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) or at one, two, four, and eight weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e "},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003cp\u003eAGEs accumulate in articular cartilage over time, impairing cartilage function and leading to arthritis, and this accumulation of AGEs causes cartilage browning in fluorescence(DeGroot et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The action mechanism of AGEs involves cross-linking with joint proteins, including collagen and aggrecan, and RAGE binding, which activates a number of intracellular signaling pathways(DeGroot et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCollagen and aggrecan, key components of the cartilage matrix, are essential for maintaining normal cartilage cells and decline with age; their dysfunction accelerates the progression of rheumatic diseases, such as OA and RA(Goldring and Marcu \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rapp and Zaucke \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, inhibiting the loss of collagen and aggrecan in rheumatic disease may have the potential to prevent cartilage degeneration.\u003c/p\u003e \u003cp\u003ePrevious in vitro studies have shown that treatment with 100 \u0026micro;g/mL of AGEs for 24 h reduces the expression of COL2A1 and aggrecan in human SW1353 chondrocytes(Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, Zhou et al. showed that 100 \u0026micro;g/mL of AGEs after 24 h reduced the viability in human SW1353 chondrocytes(Zhou et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, 100 \u0026micro;g/mL AGE3 reduced the viability of human OUMS27 chondrocytes after 48 h in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, even in patients with advanced diabetes, serum AGE levels were 82.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 \u0026micro;g/mL(Makita et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) and it can be speculated that intra-articular levels would be significantly lower. In the present study, we showed for the first time that four weeks of stimulation with 10 \u0026micro;g/mL AGE3 in human chondrocytes led to a decrease in the expression of COL2A1 and aggrecan via RAGE without affecting chondrocyte viability, whereas the 1, 3, 6, and 24 h of 10 \u0026micro;g/mL AGE3 had no effect on the expression level of COL2A1 and aggrecan in chondrocytes in our experiments using the human OUMS-27 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings are critical in demonstrating the dramatic catabolic effect of low doses of AGEs, closer to the biological environment, on aggrecan and COL2A1 after prolonged stimulation.\u003c/p\u003e \u003cp\u003eRAGE is well known and is the most studied receptor in AGE-related studies(Yue et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, RAGE knockout mice are considerably protected against the development of OA(Larkin et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). TLR4, FEEL1, and LOX1 receptors have also been identified to be AGE-binding receptors(Yonei et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, we found that the expression level of RAGE was upregulated after two weeks of 10 \u0026micro;g/mL AGE3 stimulation, while other receptors, including TLR4, FEEL1, and LOX1, remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo further substantiate our findings, we used FPS-ZM1, a high-affinity antagonist of RAGE. Pretreatment with FPS-ZM1 almost completely abolished the degradation of COL2A1 and aggrecan by AGE3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u0026ndash;d). Similarly, other RAGE inhibitors, such as Salicin(Gao and Zhang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Saxagliptin(Hu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), have been successfully used to prevent cartilage degradation in the mouse model of OA and in patients with OA. Additionally, it was concluded that AGEs in the ECM promote inflammation and cartilage degradation via RAGE. Evidence from in vivo and in vitro studies indicated that AGEs are one of the major players responsible for disrupting cartilage hemostasis by inducing inflammatory mediators, including proteases(Goldring and Otero \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sanchez-Lopez et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is reported that 24 or 48 h of 100 \u0026micro;g/mL AGE3 stimulation increases the expression levels of MMP1, MMP3, MMP13, ADAMTS4, and ADAMTS5 up to four-fold(Gao and Zhang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our experimental setup, 10 \u0026micro;g/mL AGE3 did not alter protease expression for 1\u0026ndash;24 h or for one to eight weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Although there was no change even at 100 \u0026micro;g/mL AGE3, AGE3 stimulation above 500 \u0026micro;g/mL resulted in an increase in MMP changes (data not shown). Further studies should be conducted on the effect of AGEs on MMP expression.\u003c/p\u003e \u003cp\u003eOur study shows that four weeks of 10 \u0026micro;g/mL AGE3 stimulation has deleterious effects on cartilage, highlighting the importance of evaluating the duration and amount of AGE exposure, while providing valuable insights into the underlying mechanisms of rheumatic diseases, such as OA and RA pathogenesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOFH, TN, HW, and HT designed and performed the experiments, analyzed the data, and wrote the manuscript. MN and SM analyzed the data and edited the manuscript. KOY analyzed the data and wrote the manuscript. TT and MW adjusted the analyzed data and revised the figures. All the authors have read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (nos. 21K06588 to O.F.H., 21K15354 to T.N., 20K07290 to H.W., and 23K10465 to H.T.) and funded by Kindai University Research Enhancement Grant 2021 (KD2102 to HW) and Kindai University Research Enhancement Grants 2023 (IPO11 to TN and IPO12 to OFH).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAleksandra Kuzan Toxicity of advanced glycation end products (Review). https://www.spandidos-publications.com/10.3892/br.2021.1422. 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Ann N Y Acad Sci 1243:88. https://doi.org/10.1111/J.1749-6632.2011.06320.X\u003c/li\u003e\n\u003cli\u003eRapp AE, Zaucke F (2023) Cartilage extracellular matrix-derived matrikines in osteoarthritis. Am J Physiol Cell Physiol 324:C377\u0026ndash;C394. https://doi.org/10.1152/AJPCELL.00464.2022\u003c/li\u003e\n\u003cli\u003eSanchez-Lopez E, Coras R, Torres A, et al (2022) Synovial inflammation in osteoarthritis progression. Nat Rev Rheumatol 18:258\u0026ndash;275. https://doi.org/10.1038/S41584-022-00749-9\u003c/li\u003e\n\u003cli\u003eTakeuchi M, Yamagishi S (2004) TAGE (toxic AGEs) hypothesis in various chronic diseases. Med Hypotheses 63:449\u0026ndash;452. https://doi.org/10.1016/j.mehy.2004.02.042\u003c/li\u003e\n\u003cli\u003eTseng CC, Chen YJ, Chang WA, et al (2020) Dual Role of Chondrocytes in Rheumatoid Arthritis: The Chicken and the Egg. Int J Mol Sci 21:. https://doi.org/10.3390/IJMS21031071\u003c/li\u003e\n\u003cli\u003eVan de Stadt LA, Haugen IK, Felson D, Kloppenburg M (2023) Prolonged morning stiffness is common in hand OA and does not preclude a diagnosis of hand osteoarthritis. Osteoarthr Cartil 31:529\u0026ndash;533. https://doi.org/10.1016/j.joca.2022.10.022\u003c/li\u003e\n\u003cli\u003eYamazaki Y, Wake H, Nishinaka T, et al (2021) Involvement of multiple scavenger receptors in advanced glycation end product-induced vessel tube formation in endothelial cells. Exp Cell Res 408:. https://doi.org/10.1016/J.YEXCR.2021.112857\u003c/li\u003e\n\u003cli\u003eYaykasli KO, Hatipoglu OF, Yaykasli E, et al (2015) Leptin induces ADAMTS-4, ADAMTS-5, and ADAMTS-9 genes expression by mitogen-activated protein kinases and NF-kB signaling pathways in human chondrocytes. 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Int J Mol Sci 2023, Vol 24, Page 9881 24:9881. https://doi.org/10.3390/IJMS24129881\u003c/li\u003e\n\u003cli\u003eZhang Z, Zha Z, Zhao Z, et al (2020) Lentinan Inhibits AGE-Induced Inflammation and the Expression of Matrix-Degrading Enzymes in Human Chondrocytes. Drug Des Devel Ther 14:2819. https://doi.org/10.2147/DDDT.S243311\u003c/li\u003e\n\u003cli\u003eZheng L, Zhang Z, Sheng P, Mobasheri A (2021) The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis. Ageing Res Rev 66:101249. https://doi.org/10.1016/J.ARR.2020.101249\u003c/li\u003e\n\u003cli\u003eZhou Y, Li J, Wang C, Pan Z (2022) Fumitremorgin C alleviates advanced glycation end products (AGE)-induced chondrocyte inflammation and collagen II and aggrecan degradation through sirtuin-1 (SIRT1)/nuclear factor (NF)-\u0026kappa;B/ mitogen-activated protein kinase (MAPK). Bioengineered 13:3867\u0026ndash;3876. https://doi.org/10.1080/21655979.2021.2024387\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"advanced glycation end products, aging, cartilage, collagen, aggrecan","lastPublishedDoi":"10.21203/rs.3.rs-4173286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4173286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChondrocytes are responsible for the production of extracellular matrix (ECM) components of cartilage, such as collagen type II alpha-1 (COL2A1) and aggrecan, which are loosely distributed in articular cartilage. Chondrocyte dysfunction has been implicated in the pathogenesis of rheumatic diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA). Advanced glycation end products (AGEs) accumulate in all tissues and body fluids, including cartilage and synovial fluid, with aging. Their accumulation in vivo is one of the major factors that cause and accelerate pathological changes in some chronic diseases, such as OA. Glycolaldehyde-derived AGEs (AGE3), known as toxic AGEs, have the strongest effect on cartilage compared to other AGEs. Studies conducted to date to demonstrate the effects of AGEs on chondrocytes have used very high doses (100 \u0026micro;g/mL) and collagen and aggrecan were reduced in the short term (24 h) due to decreased chondrocyte cell viability. However, it is assumed that AGEs stimulate cells for a longer period of time in vivo without causing cell death. Therefore, we stimulated a human chondrosarcoma cell line (OUMS-27) with 10 \u0026micro;g/mL AGE3 for four weeks. As a result, the expression of COL2A1 and aggrecan was significantly downregulated in OUMS-27 cells without inducing cell death, but the expression of proteases that play an important role in cartilage destruction was not affected. In addition, the receptor for advanced glycation end products (RAGE) inhibitors suppressed the AGE3-induced reduction in cartilage component production, suggesting the involvement of RAGE in the action of AGE3.\u003c/p\u003e","manuscriptTitle":"Continuous mild stimulation with advanced glycation end products reduce aggrecan and type II collagen production via the RAGE without inducing cell death in human OUMS-27 chondrosarcoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 10:39:52","doi":"10.21203/rs.3.rs-4173286/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd029ce7-6cb7-46c7-a70b-28d55da11bc9","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-09T21:29:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-01 10:39:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4173286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4173286","identity":"rs-4173286","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}