Osteoarthritis (OA) as a chronic joint disease can affect multiple joints and cause severe joint pain due to the gradual destruction of articular cartilage (AC) in synovial joints (Loeser et al., 2012). The etiology of OA is multifactorial, including aging, obesity, traumatic joint injury, and genetic predisposition, and involves an imbalance in homeostasis via increased catabolism and decreased anabolism in the synovial joint (He et al., 2020). Although the etiology of OA remains largely unknown, it has recently been categorized as a chronic age-related disease accompanied with low-grade chronic systemic inflammation (Loeser, 2011).

Cholesterol as an essential structural molecule maintains animal cell membrane stability by modulating membrane fluidity and acts as a precursor of various physiological molecules, including steroid hormones, vitamin D, and bile acid (Centonze et al., 2022). It maintains physiological homeostasis, including sterol metabolism and cellular signaling associated with cell growth, proliferation, and migration (Mok and Lee, 2020). In a recent study, the amount of cholesterol and oxysterols in the synovial fluid of patients with arthritis was higher than that in synovial fluid from healthy people (Farnaghi et al., 2017). The cholesterol metabolic pathway composed of cholesterol-25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily B member 1 (CYP7B1) regulates OA pathogenesis (Choi et al., 2019). Some oxysterols can induce oxiapoptophagy that is mediated by a complex action of oxidative stress, apoptosis, and autophagy (Nury et al., 2021). Although recent studies have reported that 25-hydroxycholesterol (25-HC) derived from cholesterol by CH25H and reactive oxygen species (ROS) induces apoptosis in various cell types, including chondrocytes (Olivier et al., 2017; Seo et al., 2020), but the pathophysiological mechanism of 7α,25-dihydroxycholesterol (7α,25-DHC) formed from 25-HC by CYP7B1 and ROS in OA pathogenesis and progression remains unclear.

In our previous study, mRNAs of ch25h and cyp7b1 were upregulated in chondrocytes under inflammatory conditions (Seo et al., 2020). Hence, we explored the pathophysiological mechanism of 7α,25-DHC as a metabolic OA risk factor, to provide an additional biological link between cholesterol metabolism and OA pathogenesis in primary chondrocytes, ex vivo organ-cultured AC explants, and an OA animal model in the present study.

Isolation and maintenance of chondrocytes

This study was performed under protocol (CIACUC2017- A0055) approved by the Institutional Animal Care and Use Committee (IACUC) of Chosun University (Gwangju, Korea). Chondrocytes (1 × 10₆ cells/ml) isolated from the AC of the knee joints dissected from 5-day-old Sprague-Dawley (SD) rats were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12) culture medium (Welgene, Korea) mixed with 10% fetal bovine serum (FBS; Welgene), 1% penicillin-streptomycin (Welgene), and 1% ascorbic acid (Sigma-Aldrich, USA).

RNA analysis

The concentration of total RNA isolated from chondrocytes using TRIzol reagent (Invitrogen, USA) was determined by a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA was synthesized from quantified total RNA using ThermoScript_TM RT-PCR system (Invitrogen) in TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio, Japan). For conventional PCR, PCR products amplified from cDNA using 2× TOP simple DyeMIX-nTaq (Enzynomics, Korea) and specific primers of target gene (summarized in Table 1) in TaKaRa PCR Thermal Cycler Dice were loaded on an agarose gel to elucidate the alteration of target gene. For quantitative real-time PCR (qRT-PCR), cDNA was amplified by qPCRBIO SyGreen Blue Mix (PCR Biosystems, UK) and specific primers of target gene (summarized in Table 1) in Eco Real-Time PCR system (Illumina, USA). Relative ratio of target gene induction was measured by the ^ΔΔCT method (Illumina).

Western blotting

Total protein extracted from chondrocytes using PRO-PREP (iNtRON Biotechnology, Korea) was quantified by a BCA protein assay (Thermo Fisher Scientific). Equal amounts of each protein sample loaded on a 12% sodium dodecyl sulfate polyacrylamide gel were transferred to a polyvinylidene fluoride membrane (Millipore, USA) for western blot using primary antibodies diluted 1:1,000 at 4°C for 12 h. Following antibodies purchased from Santa Cruz Biotechnology (USA), Cell Signaling Technology (USA), Abcam (UK), or Invitrogen were used in the present study: matrix metalloproteinase (MMP)-3 (sc-21732), MMP-13 (ab28691), Fas Ligand (FasL; sc-19681), cleaved-caspase-8 (#9496S), β-actin (sc-47778), BH3 interacting-domain death agonist (Bid; 44-433G), B-cell lymphoma-2 (Bcl-2; sc-7382), B-cell lymphoma-extra-large (Bcl-xL; sc-8392), Bcl-2-associated X protein (Bax; #2772), Bcl-2-associated agonist of cell death (Bad; #9239), caspase-9 (#9508S), cleaved-caspase-3 (#9664T), poly(ADP-ribose) polymerase (PARP; #9542S), inducible nitric oxide synthase (iNOS; #13120S), cyclooxygenase-2 (COX-2; #2282), beclin-1(sc-48341), microtubule-associated protein 1A/1B-light chain (LC3; #12741), p53 (sc-126), phospho-serine-threonine kinase (p-Akt; #4060), total Akt (#4685), phospho-mammalian target of rapamycin (p-mTOR, sc-293133), and total mTOR (sc-517464). Immunoreactive bands detected by ECL System (Amersham Biosciences, USA) were exposed under MicroChemi 4.2 (Dong-Il Shimadzu, Korea).

Ex vivo organ-culture of AC explants

AC explants dissected from the knee joints of 5-day-old SD rats were maintained with 0, 25, or 50 µg/ml of 7α,25-DHC in DMEM/F12 culture medium for 14 days. Thereafter, to perform histological analysis, AC explants were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 72 h.

Gelatin-zymography

Conditioned media loaded on a 10% polyacrylamide gel copolymerized with 0.2% gelatin from porcine skin (Sigma-Aldrich) was maintained in zymogram developing buffer to renature MMPs. Thereafter, gelatinolytic bands on the gel stained with 0.1% Coomassie Brilliant Blue R250 were imaged using a digital camera (D3200; Nikon, Japan).

Measurement of prostaglandin E₂ (PGE₂) and nitric oxide (NO)

Conditioned media were collected from chondrocytes treated with 0, 25, or 50 µg/ml 7α,25-DHC for 48 h. PGE₂ and NO were assessed by a Parameter^TM PGE₂ assay kit (R&D Systems, USA) and a NO assay kit (Thermo Fisher Scientific), respectively.

Cell viability assay

Cell viability assay using dimethyl thiazolyl diphenyl tetrazolium bromide (MTT; Sigma-Aldrich) was performed in chondrocytes treated with 1, 10, 25, or 50 µg/ml 7α,25-DHC for 48 h. Optical density was assessed at 570 nm using a spectrophotometer (BioTek, USA).

Cell survival assay

Cell survival assay using a LIVE/DEAD_TM Viability/Cytotoxicity kit (Invitrogen) was performed in chondrocytes cultured with 0, 25, or 50 µg/ml of 7α,25-DHC for 48 h. Images were acquired by a fluorescence microscope (Eclipse TE2000; Nikon Instruments, USA).

Nuclear staining

Chondrocytes cultured with 0, 25, or 50 µg/ml of 7α,25-DHC for 48 h were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) to observe condensed nucleus. Images were acquired by a fluorescence microscope.

Flow cytometry analysis

Chondrocytes cultured with 0, 25 or 50 µg/ml of 7α,25-DHC for 48 h were resuspended in binding buffer, followed by the addition of annexin V-fluorescein isothiocyanate and propidium iodide (PI; Cell Signaling Technology). Apoptotic proportions were assessed by BD Cell Quest version 3.3 (Becton Dickinson, USA).

Histological analysis

H&E staining was performed to evaluate the morphological alteration of chondrocytes cultured with 0, 25, or 50 µg/ml of 7α,25-DHC for 48 h. Safranin-O & fast green (S&F) staining was performed to evaluate the proteoglycan loss in both ex vivo organ-cultured AC explants and AC of knee joint dissected from OA animals. AC explants prepared as 8-µm-thickness and chondrocyte monolayers were stained using the Vectastain_® ABC Kit (Vector Laboratories, USA) and antibodies, such as CYP7B1 (bs-5052R; Bioss Antibodies, USA), caspase-3, and beclin-1. Images were acquired by a DM750 microscope (Leica Microsystems, Germany).

Caspase-3 activity assay

Caspase-3 activity staining was performed by cell-permeable fluorogenic substrate PhiPhiLux-G1D2 (OncoImmunin, USA) in chondrocytes cultured with 0, 25, or 50 µg/ml of 7α,25-DHC for 48 h. Images were acquired by a fluorescence microscope.

ROS detection assay

ROS detection assays were performed using 2’,7’-dichlorofluorescein-diacetate (H2DCF-DA; Sigma-Aldrich) in chondrocytes cultured with 0, 25, or 50 µg/ml of 7α,25-DHC for 48 h. Fluorescence was measured using a fluorescent microplate reader (excitation 485 nm and emission 520 nm; Thermo Fisher Scientific). Images were acquired by a fluorescence microscope.

Autophagy assay

Autophagy assay using an autophagy detection kit (ab139484; Abcam) was performed in chondrocytes cultured with 0, 25, or 50 µg/ml of 7α,25-DHC for 48 h. Images were acquired by a confocal laser scanning microscope (LSM 800 with Airyscan; Carl Zeiss, Germany).

OA animal model

OA mice were generated by the surgical destabilization of the medial meniscus (DMM) at knee joint, following a protocol approved as CIACUC2017-A0055. Thereafter, knee joints were dissected to the intermediate and late OA stages at three and eight weeks, respectively, to perform histological analysis using S&F staining and immunohistochemistry using CYP7B1, caspase-3 and beclin-1 antibodies.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics (ver. 26.0; IBM, USA), unless otherwise specified. Data were expressed as the mean ± SD for continuous variables. For gene expression and inflammatory mediators, repeated-measures ANOVA was applied with different treatment concentrations as repeat factors. Statistical significance was set at P < 0.05.

7α,25-DHC induces progressive AC degeneration through accelerated proteoglycan loss and upregulated degenerative enzyme expression

S&F staining of AC explants ex vivo organ-cultured with 0, 25, or 50 µg/ml of 7α,25-DHC for 14 days revealed that proteoglycan loss was greater in 7α,25-DHC-treated AC explants than controls (Fig. 1A). Consistent with the histological results, type II collagen (Col II) and aggrecan mRNA levels decreased in chondrocytes cultured with 7α,25-DHC for 48 h (Fig. 1B). Aggrecan mRNA levels in the 0, 25, or 50 µg/ml 7α,25-DHC-treated chondrocytes were 100.4% ± 9.7%, 14.8% ± 3.6%, and 10.6% ± 1.3%, respectively, while Col II mRNA levels were 101.1% ± 15.3%, 32.0% ± 3.7%, and 25.2% ± 3.5%, respectively (Fig. 1C).

The mRNA of degenerative enzymes, including MMP-3 and -13, were upregulated by 7α,25-DHC in chondrocytes (Fig. 2A). MMP-3 mRNA levels increased by 49 ± 3.1-fold and 64.3 ± 7.3-fold in chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively (Fig. 2B). Similarly, MMP-13 mRNA levels also increased by 11.2 ± 4.2-fold and 22.9 ± 9.2-fold in chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively. Sequentially, expressions of MMP-3 and -13 were upregulated in chondrocytes cultured with 7α,25-DHC (Fig. 2C). Zymography showed that the activities of degenerative enzymes were enhanced by 7α,25-DHC in chondrocytes (Fig. 2D). These findings demonstrate 7α,25-DHC-induced progressive AC degeneration by promoting proteoglycan loss through the increased expression and activation of degenerative enzymes.

7α,25-DHC increases apoptotic chondrocyte death

The relative viabilities of 1, 10, 25, and 50 µg/ml 7α,25-DHC-treated chondrocytes were 97.6% ± 1.3%, 75.9% ± 1.0%, 75.2% ± 1.5%, and 67.9% ± 0.8%, respectively, compared with that of the control (100% ± 1.5%) (Fig. 3A). Furthermore, 7α,25-DHC decreased the total number of chondrocytes and increased the number of red fluorescent dead cells (Fig. 3B, upper panel). H&E staining revealed that the chondrocytes with altered morphologies (Fig. 3B, middle panel) and condensed chromatin (Fig. 3B, lower panel) were increased by 25 and 50 µg/ml 7α,25-DHC. The relative rate of cell death in 25 and 50 µg/ml 7α,25-DHC-treated chondrocytes was 40.87% and 82.61%, respectively (Fig. 3C). The proportion of early-stage apoptotic cells was assessed by 12.28% and 10.82% in the chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively. While that of late-stage apoptotic chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC was 27% and 68.47%, respectively. The proportion of necrotic cells in 25 and 50 µg/ml 7α,25-DHC-treated chondrocytes was 1.59% and 3.32%, respectively. These findings demonstrated that apoptotic chondrocyte death is induced by 7α,25-DHC.

7α,25-DHC induces chondrocyte death occurs via extrinsic and intrinsic apoptosis pathways

Proapoptotic molecules related to extrinsic apoptosis pathway, including FasL, pro- and cleaved caspase-8, were increased by 7α,25-DHC in chondrocytes (Fig. 4A). Whereas, antiapoptotic molecules related to intrinsic apoptosis, including Bcl-2 and Bcl-xL, were decreased by 7α,25-DHC in chondrocytes. However, proapoptotic molecules related to intrinsic apoptosis pathway, including Bid, Bax, Bad, and cleaved caspase-9, were upregulated by 7α,25-DHC in chondrocytes (Fig. 4B). Expressions of cleaved caspase-3 and PARP were upregulated by cleaved caspase-8 and -9 in chondrocytes cultured with 7α,25-DHC (Fig. 4C). In addition, 7α,25-DHC increased caspase-3 activity in chondrocytes (Fig. 4D). Furthermore, the immunoreactivity of caspase-3 was enhanced by 7α,25-DHC in both chondrocytes and ex vivo organ-cultured AC explants (Fig. 4E). These findings demonstrate 7α,25-DHC-induced chondrocyte death via the extrinsic and intrinsic apoptosis pathways.

7α,25-DHC upregulates the ROS production and inflammatory response via the increase of oxidative stress in chondrocytes

The relative production of ROS was increased by 127.8% ± 3.1% and 226.3% ± 6.5% in chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively, compared to control (100% ± 3.1%) (Fig. 5A). Conventional PCR revealed that iNOS and COX-2 mRNA levels were upregulated by 7α,25-DHC in chondrocytes (Fig. 5B, upper panel). qRT-PCR showed that iNOS mRNA levels were 28.1 ± 4.4-fold and 48.9 ± 7.4-fold in chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively. The COX-2 mRNA level also increased by 8.7 ± 1.1-fold and 12.1 ± 1.7-fold in chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively (Fig. 5B, lower panel). Furthermore, the expression of iNOS and COX-2 were increased by 7α,25-DHC in chondrocytes (Fig. 5C). The relative NO production increased by 483.6% ± 12.4% and 592.7% ± 11.9% in chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively, compared to the control (100% ± 2.8%) (Fig. 5D). Similarly, PGE₂ production increased by 527.9 ± 14.5 and 439.9 ± 12.9 pg/ml in chondrocytes cultured with 25 and 50 µg/ml 7α,25-DHC, respectively, while remaining relatively low in control (152.2 ± 46.5 pg/ml) (Fig. 5E). Our findings suggest that 7α,25-DHC induces progressive AC degeneration via ROS production and inflammatory response that mediated by the increase of oxidative stress in chondrocytes.

Autophagy is involved with 7α,25-DHC-mediated chondrocyte apoptosis through modulation of p53-Akt-mTOR axis

Autophagy assay revealed that autophagosomes were significantly increased in chondrocytes cultured with 0, 25, and 50 µg/ml of 7α,25-DHC for 48 h (Fig. 6A). Sequentially, the expression of specific autophagy-associated molecules, including beclin-1 and LC3, upregulated by 7α,25-DHC in chondrocytes (Fig. 6B). Moreover, 7α,25-DHC upregulated the immunoreactivity of beclin-1 in both chondrocytes and ex vivo organ-cultured AC explants (Fig. 6C). As shown in Fig. 6D, the expression of p53 significantly increased, while phosphorylation of Akt and mTOR decreased in chondrocyte treated with 7α,25-DHC. Our results demonstrate that autophagy is involved in 7α,25-DHC-mediated chondrocyte apoptosis through modulation of the p53-Akt-mTOR cellular signaling pathway.

The immunoreactivity of CYP7B1, caspase-3, and beclin-1 is increased in the degenerative AC of knee joint with OA

We determined the pathophysiological link between 7α,25-DHC-induced oxiapoptophagy and OA. S&F staining showed the loss of proteoglycan in the knee AC of OA animals eight weeks after DMM surgery (Fig. 7). At 8 weeks after DMM surgery, the immunoreactivity of CYP7B1, caspase-3, and beclin-1 was increased in degenerative AC of knee joints with OA. These results indicate that progressive AC degeneration might be involved with 7α,25-DHC-induced oxiapoptophagic chondrocyte death in degenerative AC derived from OA knee joint.

Previously, we showed 25-HC-induced chondrocyte death (Seo et al., 2020). Moreover, some oxysterols are directly involved with the pathogenesis of inflammatory diseases, including OA, via modulation of the CH25H–CYP7B1–retinoic acid–related orphan receptor α axis (Choi et al., 2019). Progressive AC degeneration and eventual complete chondrocyte death are major clinical symptoms of OA (Mehana et al., 2019). Degenerative enzymes, including MMP-3 and -13, are responsible for progressive AC degeneration in OA pathogenesis (Mehana et al., 2019). In this study, 7α,25-DHC accelerated the loss of proteoglycan in AC and suppressed the mRNA expression of the major extracellular matrix (ECM) components aggrecan and Col II (Fig. 1). Furthermore, the expression and activity of degenerative enzymes, including MMP-3 and -13, were upregulated in conditioned media harvested from 7α,25-DHC-treated chondrocytes, indicating that 7α,25-DHC accelerates the digestion of the ECM through the upregulation of degenerative enzymes (Fig. 2), in a manner similar with that of 25-HC (Seo et al., 2020). These findings demonstrate that 7α,25-DHC induces progressive AC degeneration through accelerated proteoglycan loss, ECM component suppression, and the expression of degenerative enzymes.

The dysregulation of apoptosis is a pathophysiological risk factor for the induction of cancer, developmental abnormalities, and degenerative diseases, including OA (Hwang and Kim, 2015). Chondrocytes are associated with ECM maintenance. Therefore, chondrocyte apoptosis leads to progressive AC degeneration, due to an imbalance between anabolism and catabolism (Hwang and Kim, 2015). Chondrocyte apoptosis occurs in osteoarthritic cartilage. We reported recently that 25-HC induced chondrocyte death via extrinsic and intrinsic apoptosis (Seo et al., 2020). Similarly, in the present study, 7α,25-DHC increased caspase-dependent apoptotic cell death through extrinsic and intrinsic pathways of apoptosis (Figs. 3 and 4). Our findings indicate that 7α,25-DHC induces OA pathogenesis by increasing apoptotic chondrocyte death.

Oxidative stress is related to the upregulation of ROS, lipid peroxidation, and protein carbonylation (Nury et al., 2021). In particular, intrinsic apoptosis is involved in the loss of mitochondrial membrane potential (^ΔΨm), which is mediated by mitochondrial membrane permeabilization through ROS generation (Marchi et al., 2012). Inflammatory mediators, including iNOS, COX-2, NO, and PGE₂, are related with degenerative enzyme upregulation and proteoglycan loss, due to increased production of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), during OA progression (Wojdasiewicz et al., 2014). In addition, 25-HC levels are related to the inflammatory response via the increase of proinflammatory cytokines, including TNF-α and IL-6, by activating α5β1 and αvβ3 integrin in macrophages and epithelial cells (Pokharel et al., 2019). In the present study, the expression and production of ROS and inflammatory mediators were upregulated by 7α,25-DHC in chondrocytes (Fig. 5). Hence, our findings indicate that 7α,25-DHC upregulates ROS and inflammatory mediators associated with oxidative stress to induce proinflammatory cytokine production during OA progression.

Upregulation of ROS induces apoptosis in various cell types and is related to autophagosome formation (Caillot et al., 2021; Marioli-Sapsakou and Kourti, 2021; Michallet et al., 2011). Autophagy is a fundamental physiological process in all living cells that breaks down intracellular components, such as unnecessary or damaged organelles and protein aggregates, and transports them to the lysosomes for recycling (Chun and Kim, 2018). Autophagy is mediated by autophagy biomarkers, such as LC3 and beclin-1, through modulation of the p53-Akt-mTOR cellular signaling pathway (Chun and Kim, 2018). Upregulation of p53 suppresses the phosphorylation of Akt through an increase in phosphatase and tensin homolog. Suppression of Akt phosphorylation decreases the phosphorylation of mTOR, which initiates the formation of autophagosomes through interactions with LC3 and beclin-1 (Bhutia et al., 2019; Menon and Dhamija, 2018). Oxysterols induce autophagy in various cell types, such as vascular smooth muscle cells and murine oligodendrocytes (Nury et al., 2014). In the present study, autophagosome formation is mediated by the upregulation of beclin-1 and LC3 through the regulation of the p53-Akt-mTOR axis in both chondrocytes and AC explants cultured with 7α,25-DHC (Fig. 6). Our findings indicate 7α,25-DHC-induced chondrocyte death involves apoptosis accompanied by autophagy.

Although several studies have estimated 7α,25-DHC levels in mouse liver and plasma to be 4.7 ng/g and 0.5 ng/ml, respectively, and 0.4 ng/ml in human plasma (Sun and Liu, 2015), the level of 7α,25-DHC during OA remains unknown. However, the concentration of 7α,25-DHC used in the present study was higher than that under normal physiological conditions. Therefore, to investigate 7α,25-DHC-induced oxiapoptophagy in osteoarthritic cartilage, OA mice were generated via surgical DMM at the knee joint. To evaluate the link between oxiapoptophagy and OA severity, OA animals were euthanized at three (middle stage of OA) and eight weeks (terminal stage of OA) after DMM surgery. Immunoreactivity of CYP7B1, caspase-3, and beclin-1 were increased in the AC, with severe proteoglycan loss due to DMM in the knee joint (Fig. 7). Choi et al. (2019) reported that the production of oxysterols including 25-HC and 7α,25-DHC was upregulated by the expression of CH25H and CYP7B1, respectively, in the chondrocytes treated with pro-inflammatory cytokines such as IL-1β and TNFα. Furthermore, they showed that the production of 25-HC and 7α,25-DHC was upregulated in the chondrocytes infected with adenovirus containing ch25h or cyp7b1 genes, respectively (Choi et al., 2019). Hence, these data demonstrated a chondrocyte oxiapoptophagy that was mediated by the expression of CYP7B1 to the synthesis of 7α,25-DHC, the expression of caspase-3 to chondrocyte apoptosis, and the expression of beclin-1 to autophagy in the osteoarthritic knee joint dissected from OA animals. Furthermore, as shown in Fig. 7, the immunoreactivity of caspase-3 related to apoptosis was increased in the AC dissected from knee joint after DMM surgery at 8 weeks compared with that of 3 weeks. Hence, these data suggest that 7α,25-DHC-induced oxiapoptophagy is involved with the apoptosis of chondrocytes in the late-stage of OA. In the future, we aim to determine the utility of OA or RA biomarkers to verify the levels of oxysterols, including 25-HC and 7α,25-DHC, in serum collected from a metabolic OA animal and a patient with metabolic diseases and OA.

Here, our study demonstrated 7α,25-DHC-induced OA pathogenesis via the increases the catabolic molecules, including degenerative enzyme and inflammatory mediator expression, and oxiapoptophagic chondrocyte death (Fig. 8). To fully understand the pathophysiological link between cholesterol metabolism and OA, our further study is warranted to elucidate the pathophysiological mechanism of 7α,25-DHC in animals with metabolic syndromes and OA. The present study provides insights into the pathophysiological link between metabolic syndromes and OA and has identified 7α,25-DHC as a possible diagnostic or prognostic biomarker or therapeutic target for OA.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2019R1F1A104537913).

J.Y.S., T.H.K., and J.S.K. conceived and designed the study. J.Y.S., T.H.K., K.R.K., H.L., M.C.C., and J.S.K. developed the methodology. J.Y.S., T.H.K., D.K.K., H.S.C., H.J.K., S.K.Y., and J.S.K. analyzed and interpreted the data. J.Y.S., T.H.K., and J.S.K. wrote and reviewed the manuscript. D.K.K., H.S.C., and J.S.K. supervised the study.

The authors have no potential conflicts of interest to disclose.

Primer sequences for conventional PCR and quantitative real-time PCR (qRT-PCR)