10-hydroxy-2-decenoic acid prevents osteoarthritis by targeting aspartyl β hydroxylase and inhibiting chondrocyte senescence in male mice preclinically

Nana Geng; Mengtian Fan; Biao Kuang; Fengmei Zhang; Menglin Xian; Lin Deng; Cheng Chen; Yiming Pan; Jianqiang Chen; Naibo Feng; Li Liang; Yuanlan Ye; Kaiwen Liu; Xiaoli Li; Yu Du; Fengjin Guo

doi:10.1038/s41467-024-51746-3

. 2024 Sep 4;15:7712. doi: 10.1038/s41467-024-51746-3

10-hydroxy-2-decenoic acid prevents osteoarthritis by targeting aspartyl β hydroxylase and inhibiting chondrocyte senescence in male mice preclinically

Nana Geng ^1,^#, Mengtian Fan ^1,^#, Biao Kuang ², Fengmei Zhang ¹, Menglin Xian ¹, Lin Deng ¹, Cheng Chen ³, Yiming Pan ¹, Jianqiang Chen ¹, Naibo Feng ¹, Li Liang ¹, Yuanlan Ye ¹, Kaiwen Liu ¹, Xiaoli Li ¹, Yu Du ², Fengjin Guo ^1,^✉

PMCID: PMC11375154 PMID: 39231947

Abstract

Osteoarthritis is a degenerative joint disease with joint pain as the main symptom, caused by fibrosis and loss of articular cartilage. Due to the complexity and heterogeneity of osteoarthritis, there is a lack of effective individualized disease-modifying osteoarthritis drugs in clinical practice. Chondrocyte senescence is reported to participate in occurrence and progression of osteoarthritis. Here we show that small molecule 10-hydroxy-2-decenoic acid suppresses cartilage degeneration and relieves pain in the chondrocytes, cartilage explants from osteoarthritis patients, surgery-induced medial meniscus destabilization or naturally aged male mice. We further confirm that 10-hydroxy-2-decenoic acid exerts a protective effect by targeting the glycosylation site in the Asp_Arg_Hydrox domain of aspartyl β-hydroxylase. Mechanistically, 10-hydroxy-2-decenoic acid alleviate cellular senescence through the ERK/p53/p21 and GSK3β/p16 pathways in the chondrocytes. Our study uncovers that 10-hydroxy-2-decenoic acid modulate cartilage metabolism by targeting aspartyl β-hydroxylase to inhibit chondrocyte senescence in osteoarthritis. 10-hydroxy-2-decenoic acid may be a promising therapeutic drug against osteoarthritis.

Subject terms: Molecular medicine, Osteoarthritis

Chondrocyte senescence is known to play a role in the occurrence and progression of osteoarthritis. Here, the authors demonstrate that 10-hydroxy-2-decenoic acid (10-HDA), a small molecule derived from the food nutrient royal jelly, can prevent osteoarthritis by delaying chondrocyte senescence through targeting aspartyl β-hydroxylase.

Introduction

Osteoarthritis (OA) is one of the most common forms of joint disease¹. The main pathological changes in OA involve cartilage degeneration, decreased anabolism, subchondral bone remodeling, synovium inflammation, and osteophyte formation, resulting in limited joint movement and pain^2,3. With the growing aging population, the incidence of OA is increasing annually. Currently, pharmacological treatments mostly aim to relieve the OA symptoms associated with inflammation and pain⁴. To date, effective disease-modifying osteoarthritis drugs are still lacking^5–7. Artificial joint replacement is an effective strategy for end-stage OA, while there is a lack of effective prevention or treatment for early-stage OA⁸.

Cellular senescence, a stress response causing a permanent arrest of the cell cycle, is thought to be one of the important factors affecting joint tissue homeostasis and aggravating OA progression^9–12. Chondrocytes are the only cells in the mature cartilage that contributed to the maintenance of the integrity and homeostasis of the joint articular tissue¹³. However, the proliferation rate of chondrocytes is very low, thus leading to the gradual decline in cartilage anabolism with age, increasing the risk of OA. The gradual accumulation of senescent chondrocytes leads to alterations in cell metabolism and function, ultimately resulting in an imbalance in chondrocyte homeostasis^14,15.

Senescent chondrocytes can also secrete senescence-associated secretory phenotype (SASP) factors in an autocrine manner, leading to increased levels of catabolic cytokines, such as interleukin-1β (IL-1β), interleukin 6 (IL6), and tumor necrosis factor-α (TNF-α), which further induce the secretion of metal matrix proteins (MMPs), among which matrix metallopeptidase 13 (MMP13) is considered to be the core of factor for the irreversible degradation of cartilage type II collagen (COL2) lattice in OA^14,16,17. In addition, the SASP factors can also contribute to the communication between senescent chondrocytes and neighboring healthy cells, thereby leading to the senescence of the latter¹⁸. Undoubtedly, targeting the cellular senescence of chondrocytes is considered a promising approach for the treatment of OA. To date, the underlying mechanisms of chondrocyte senescence in OA have not been fully elucidated.

10-hydroxy-2-decenoic acid (10-HDA) is a natural drug and food homologous compound originating from royal jelly (RJ)¹⁹. As a unique medium-chain fatty acid, 10-HDA comprises approximately 40% of the total fatty acids in royal jelly (RJ)²⁰. Interestingly, 10-HDA has been reported to prevent and treat skin photo-aging through inhibiting UVA-induced cellular senescence and the generation of reactive oxygen species (ROS)²¹. The pharmacological and biological activities of 10-HDA have also been reported against other diseases, such as rheumatoid arthritis (RA)²², colon cancer^23,24, colorectal carcinoma²⁵, neuro-inflammation²⁶, skeletal muscle atrophy²⁷, bacterial infections²⁸, colitis^29,30, various solid tumors³¹, and osteoporosis³².

In the current study, we aimed to explore whether and how 10-HDA administration can alleviate chondrocyte senescence and prevent articular cartilage degradation. This study broadens the therapeutic prospect of natural medicines derived from food in OA.

Results

10-HDA boosted proliferation and anabolism, whereas inhibited apoptosis and catabolism in chondrocytes

To study the effect of 10-HDA (Fig. 1a) on cell proliferation and metabolism in cartilage, we used primary chondrocytes extracted from the cartilage of wild-type C57BL/6 J mice, as well as human C28/I2 chondrocytes. First, we found that compared with the phosphate-buffered saline (PBS) group, 10-HDA alone promoted cell viability (Supplementary Fig. 1a, b) and the expression of proliferation genes such as cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), cyclinA1, cyclinB1, cyclinD1, cyclinE1, and proliferating cell nuclear antigen (PCNA) (Supplementary Figs. 2a, 3a), and that of cartilage anabolism genes including SRY-box transcription factor 9 (SOX9), COL2, and aggrecan (ACAN) (Supplementary Figs. 2b, 3b) in the mouse primary chondrocytes or C28/I2 cells in a dose-dependent manner in the absence of IL-1β. In addition, when 10-HDA reached a certain treatment time exceeds 4 days, chondrocyte proliferation will enter a plateau phase without further expansion (Supplementary Fig. 4a). Hyaline cartilage is composed of collagen fibers (mainly COL2) and glycosaminoglycans (GAGs). However, fibrocartilage in the joint plays an important role in the development of osteoarthritis and cartilage injuries³³. Previous research reported that cartilage fibrosis marker genes collagen type I alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), collagen type III alpha 1 chain (COL3A1), α-smooth muscle actin (α-SMA), fibromodulin (FMOD), fibronectin type III domain containing 1 (FNDC1) were up-regulated in the injured cartilage³⁴. Notably, we found treatment with 10-HDA increased synthesis of hyaline cartilage anabolism genes SOX9, COL2, ACAN (Supplementary Fig. 4b), while inhibited cartilage fibrosis-related genes COL1A1, COL1A2, COL3A1, α-SMA, FMOD, FNDC1 (Supplementary Fig. 4c) mRNA levels in a time-sensitive and did not exhibit features of chondrocyte dedifferentiation due to early promotion of chondrocyte proliferation, while still preserving the differentiation characteristics of chondrocytes.

Pro-inflammatory cytokines have been reported to decrease cartilage anabolism and induce the catabolism of chondrocytes, thus playing a role in OA pathogenesis^6,35. Next, we evaluated the ability of 10-HDA to promote cell proliferation and anabolism, whereas inhibit apoptosis and catabolism in the presence of IL-1β (10 ng/mL). We found that 10-HDA (2, 5, and 10 nM) in the presence of IL-1β increased cell viability (Fig. 1b), the expression of cell proliferation genes CDK1, CDK2, CDK4, CDK6, cyclinA1, cyclinB1, cyclinD1, cyclinE1, and PCNA (Fig. 1c, Supplementary Data 1; Supplementary Fig. 5a), proliferation rate (Fig. 1e), and that of anabolism markers SOX9, COL2 and ACAN (Fig. 1d and Supplementary Fig. 5b), whereas inhibited apoptosis rate (Fig. 1f), the expression of the catabolism gene MMP13 and IL6 (Fig. 1d and Supplementary Fig. 5b), compared with those in the IL-1β-treated control group in the mouse primary chondrocytes or C28/I2 cells.

10-HDA enhanced anabolism and repressed catabolism of chondrocytes in human cartilage explants

To further explore the effect of 10-HDA on OA, we treated the knee cartilage tissues from OA patients with 10-HDA under the stimulation of IL-1β (10 ng/mL) in vitro. First, we detected that the mRNA level of all cell proliferation marker genes CDK1, CDK2, CDK4, CDK6, cyclinA1, cyclinB1, cyclinD1, cyclinE1, and PCNA was down-regulated in the IL-1β-treated control group. Conversely, we observed an upregulation in the expression of the above proliferation markers after treatment with 10-HDA (Fig. 2a, Supplementary Data 2). Then we explored the effect of 10-HDA on anabolism and catabolism in the human cartilage explants. It was found that IL-1β significantly reduced the mRNA levels of SOX9, COL2, and ACAN, increased MMP13 expression, while the cartilage explants treated with 5 nM and 10 nM 10-HDA exhibited an increase in the transcriptional levels of SOX9, COL2, and ACAN, whereas a decrease in the expression of MMP13, compared to those in the IL-1β-treated control group (Fig. 2b). In addition, western blot and immunofluorescence (IF) analysis further validated the upregulation in the protein levels of COL2, ACAN, and down-regulation in those of MMP13 in the cartilage explants treated with 10-HDA, compared with the IL-1β control group, revealing that 10-HDA promoted anabolism and suppressed catabolism in OA (Fig. 2c, d, and Supplementary Fig. 6). The pathology of OA is also characterized by the degradation of the extracellular matrix of cartilage. We hence examined the loss of cartilage matrix in the human cartilage explants treated as described above. Staining with toluidine blue and safranin O/fast green demonstrated that IL-1β caused cartilage degradation, whereas treatment with 10-HDA (5 nM and 10 nM) partially rescued this phenotype (Fig. 2d).

Fig. 2 — a *Cyclin-dependent kinase 1*(*CDK1*)*, cyclin-dependent kinase 2* (*CDK2*)*, cyclin-dependent kinase 4* (*CDK4*)*, cyclin-dependent kinase 6* (*CDK6*)*, cyclinA1, cyclinB1, cyclinD1, cyclinE1, proliferating cell nuclear antigen* (*PCNA*) mRNA levels in human cartilage explants treated with 10 ng/ml interleukin-1β (IL-1β) in the absence or presence of 0, 5, 10 nM 10-HDA for 7 d. n = 3 donors (3 female). Significant differences and p values were in Supplementary Data 2. b SRY-box transcription factor 9 (*SOX9*), type II collagen (*COL2*), aggrecan (*ACAN*), matrix metallopeptidase 13 (*MMP13*) mRNA levels in human cartilage explants treated with 10 ng/ml IL-1β in the absence or presence of 0, 5, 10 nM 10-HDA for 7 d. n = 3 donors (3 female). c COL2, ACAN, MMP13 protein levels in human cartilage explants treated with 10 ng/ml IL-1β in the absence or presence of 0, 5, 10 nM 10-HDA for 7 d. n = 3 donors (3 female). Quantification was in Supplementary Fig. 6. d Representative images of hematoxylin-eosin (HE), toluidine blue, safranin O staining, COL2, and MMP13 protein levels in the human cartilage explants treated with 10 ng/ml IL-1β in the absence or presence of 0, 5, 10 nM 10-HDA for 7 d. Superficial area (S), deep area (D). COL2, MMP13 were stained red, DAPI (4,6-diamino-2-phenyl indole) were blue, and merged images were displayed. Experiments in (d) were repeated independently three times with similar results. Scale bar = 200 μm. Data were shown as means ± SD. P values in (a, b, c) were determined using one-way ANOVA followed by Tukey’s HSD multiple comparison test. Source data and uncropped blots are provided as a Source Data file.

Moreover, we found 10-HDA (5 and 10 nM) can also increase the expression of COL2, ACAN proteins and partially relieve the injury of knee cartilage tissues from OA patient without the stimulation of IL-1β in vitro, compared with the 0 nM 10-HDA group, according to the results of toluidine blue and safranin O/fast green staining (Supplementary Fig. 7).

Intra-articular injection of 10-HDA relieved OA and pain in a surgically induced DMM model in vivo

To advance our understanding of the effect of 10-HDA on OA in vivo, we used an experimental medial meniscal destabilization (DMM) mouse model, which has similar pathological characteristics to those of OA patients and is widely used in examining the therapeutic efficacy of various compounds against OA^36,37. We collected the knee joints of mice 8 weeks after DMM surgery (Fig. 3a). General observation of knee joints and cartilage tissues following DMM surgery, revealed the presence of joint swelling (Fig. 3b) and cartilage imperfection (Fig. 3c). Whereas, we found that intra-articular (IA) injection of 10-HDA effectively prevented the above joint damage (Fig. 3b, c).

Fig. 3 — a Experimental flow chart. 8-week-old male C57BL/6 J mice were performed destabilization of the medial meniscus (DMM) surgery (male, n = 18) or sham operation (male, n = 9), then administered vehicle (DMSO, DMM, male, n = 9; sham, male, n = 9) or 10-HDA (10 mg/kg, DMM, male, n = 9) twice a week for 6 weeks through intra-articular injection 1 week and sacrificed 8 weeks after surgery. The joints were collected 8 weeks after surgery. b Representative knee joint images at 8 weeks after surgery. c Representative knee joint cartilage images at 8 weeks after surgery. d Representative knee joint sections stained by hematoxylin-eosin (HE) and safranin O/fast green, OA severity was analyzed by chondrocyte numbers, cartilage thickness, and Osteoarthritis Research Society International score system (OARSI). Scale bar = 200 μm. n = 6 mice. e Representative immunofluorescence images of type II collagen (Col2), aggrecan (Acan), matrix metallopeptidase 13 (Mmp13), and quantification of positive cells in the knee joint. Col2, Acan, Mmp13 were stained green (middle boxes), DAPI (4,6-diamino-2-phenyl indole) were blue (upper boxes) and merged images were shown in the lower boxes. Scale bar = 50 μm. n = 6 mice. f *Col2, Acan, Mmp13* mRNA levels in knee joint cartilage. n = 3 mice. g Mechanical sensitivity was measured using von Frey filaments once a week after surgery. n = 6 mice. h Representative images and quantification of gait analysis parameters 8 weeks after surgery. Values are presented as the ratio of the right hind (RH, DMM applied knee)/the left hind (LH, contralateral knee). LF left front, FF right front. n = 6 mice. Data were shown as means ± SD. P values for chondrocyte numbers and cartilage thickness (d), positive cells (e), mRNA expression (f) were determined with one-way ANOVA followed by Tukey’s HSD multiple comparison test. P values in (h) were determined with unpaired two-tailed Student’s t-test. P values for OARSI score (d), paw withdrawal thresholds (g) were determined with the two-sided Kruskal–Wallis test followed by the Mann–Whitney U test. Source data are provided as a Source Data file.

Hematoxylin and eosin (HE) staining of knee joint sections revealed a reduction in the number of chondrocytes in the DMM surgery group, compared to the sham group; however, 10-HDA treatment elevated cell density (Fig. 3d, top and right). Cartilage erosion and loss of the cartilage superficial zone in the knee joints are prominent pathological features of OA¹³. Staining of knee joint sections with safranin O, which is a marker of mucopolysaccharides, indicated that treatment with 10-HDA largely ameliorated these pathological changes. Analysis using the Osteoarthritis Research Society International (OARSI) scores semiquantitative system³⁸ revealed reduced scores in the 10-HDA-treated group, compared to those from the vehicle group (Fig. 3d, middle, bottom, and right). Furthermore, we observed that administration of 10-HDA also promoted cartilage thickness, revealing its role in suppressing cartilage degradation in DMM-operated mice (Fig. 3d, bottom and right). Immunofluorescence staining further indicated a decrease in the protein levels of anabolism markers Col2 and Acan in the cartilage tissues after DMM surgery; however, this effect was reversed after administration of 10-HDA (Fig. 3e). Furthermore, treatment with 10-HDA reduced the protein level of catabolism marker Mmp13, compared to the vehicle group. (Fig. 3e). Consistent with the histopathological results, administration of 10-HDA increased the mRNA levels of Col2 and Acan, whereas decreased the transcriptional level of Mmp13 in the whole joint after 8 weeks (Fig. 3f).

Joint pain is also one of the main clinical symptoms in OA. The von Frey test is broadly used to test pain sensitivity in OA models³⁹. As expected, paw withdrawal thresholds were reduced after DMM surgery. Interestingly, we found that administration of 10-HDA increased paw withdrawal thresholds, indicating reduced OA-associated pain (Fig. 3g). Additionally, we also examined the pain-related behaviors of mice in response to DMM surgery using gait analysis, an effective assay for evaluating behavioral changes in pre-clinical arthritis models⁴⁰. We observed that after injection of 10-HDA, both the print area and the max contact area were increased, compared to those in the vehicle group, suggesting that 10-HDA alleviated the pain-related behavior induced by DMM surgery (Fig. 3h, Supplementary Fig. 8a). Taken together, these results demonstrated that IA injection of 10-HDA promoted cartilage anabolism, inhibited cartilage degradation, and relieved knee joint pain in OA in vivo.

Oral administration of 10-HDA protected against OA in a DMM model in vivo

We further verified the effect of oral administration of 10-HDA on OA mice. At the third day after DMM surgery, mice were fed with a diet supplemented with 100 mg/kg/d 10-HDA or a control diet containing sodium carboxymethyl cellulose (CMC-Na) three times a week for 7 weeks (Fig. 4a). HE staining of knee joint sections showed an increased number of chondrocytes after oral administration of 10-HDA in mice, compared to that in the vehicle group (Fig. 4b, top and Fig. 4c, top). Likewise, staining with safranin O/ fast green revealed evident scuff and surface or whole-thickness loss of cartilage in the vehicle group, thus leading to decreased cartilage thickness and increased OARSI score. However, we observed that oral administration of 10-HDA partially alleviated DMM-induced cartilage degeneration and improved the OARSI scores after 7 weeks of treatment (Fig. 4b, bottom; Fig. 4c, middle and bottom). In addition, IF staining revealed that compared with the vehicle group, 10-HDA increased the protein levels of Col2 and Acan, whereas decreased those of Mmp13 in the cartilage (Fig. 4d). The von Frey test showed that oral administration of 10-HDA increased the paw withdrawal thresholds and relieved the OA-associated pain (Fig. 4e). Furthermore, we found that oral administration of 10-HDA decreased stand and single stance at 8 weeks, compared to those in the vehicle group, indicating that 10-HDA alleviated the increased pain-related behaviors after DMM surgery (Fig. 4f, Supplementary Fig. 8b).

Fig. 4 — a Experimental flow chart. Destabilization of the medial meniscus (DMM) surgery (male, n = 12) or sham operation (male, n = 6) were performed in 8-week-old C57BL/6 J mice, then administered vehicle (CMC-Na, DMM, male, n = 6; sham, male, n = 6) or 10-HDA (100 mg/kg, DMM, male, n = 6) three times a week for 7 weeks through oral administration 4 days and sacrificed 8 weeks after surgery. The joints were collected 8 weeks after surgery. b, c Representative hematoxylin-eosin (HE) and safranin O/fast green staining images in the knee joint 8 weeks after the surgery, then OA severity was analyzed by chondrocyte numbers, cartilage thickness and Osteoarthritis Research Society International score system (OARSI). Scale bar = 200 μm. n = 6 mice. d Representative immunofluorescence images of type II collagen (Col2), aggrecan (Acan), matrix metallopeptidase 13 (Mmp13), and quantification of positive cells in knee joint 8 weeks after surgery. Col2, Acan, Mmp13 were stained green (middle boxes), DAPI (4,6-diamino-2-phenyl indole) were blue (upper boxes) and merged images were shown in the lower boxes. Scale bar = 50 μm. n = 6 mice. e Mechanical sensitivity was measured using von Frey filaments once a week after surgery. n = 6 mice. f Representative images and quantification of gait analysis parameters 8 weeks after surgery. Values are presented as the ratio of the right hind (RH, DMM applied knee)/the left hind (LH, contralateral knee). LF left front, FF right front. n = 6 mice. Data were shown as means ± SD. P values for chondrocyte numbers and cartilage thickness (c), positive cells (d) were determined using one-way ANOVA followed by Tukey’s HSD multiple comparison test. P values in (f) were determined with unpaired two-tailed Student’s t-test. P values for OARSI score (c), paw withdrawal thresholds (e) were determined with two-sided Kruskal–Wallis test followed by Mann–Whitney U test. Source data are provided as a Source Data file.

To evaluate the safety of oral administration of 10-HDA, we monitored the body weight every two days and performed internal organs coefficient assessment and pathological examination in mice. We found that the body weight of mice was increased over time in the 10-HDA oral administration group, compared with that in the sham group, however, this increase was not statistically significant (Supplementary Fig. 9a). Furthermore, there was no significant difference between the 10-HDA and sham groups in the internal organ coefficient of heart, liver, spleen, lung, kidney, and brain (Supplementary Fig. 9b). Additionally, histological analyses of internal organs also revealed that compared with the sham group, oral administration of 10-HDA did not cause significant pathological changes in mice (Supplementary Fig. 9c). These results indicated that oral administration of 10-HDA was not associated with any significant toxicity, suggesting it to be safe for the clinical treatment of OA in the future.

ASPH is a unique target of 10-HDA

To explore the possible binding target of 10-HDA in the mediation of chondrocyte homeostasis, drug affinity responsive target stability (DARTS) assay was performed⁴¹. Coomassie blue (Fig. 5a) and silver (Fig. 5b) staining revealed a band with a molecular weight of 50-75 kDa that was protected by 10-HDA. We excised this band from the gel and sent it for protein identification using mass spectrometry, which identified aspartyl β-hydroxylase (ASPH) as a potential candidate binding target of 10-HDA (Fig. 5c). To further confirm whether ASPH is a binding target of 10-HDA, we performed western blot using DARTS samples treated with a series of thermophilic-bacterial proteases with or without 10-HDA. We accordingly found that 10-HDA protected ASPH protein from protease digestion (Fig. 5d). Additionally, we conducted a cellular thermal shift assay (CETSA), which is based on the principle of measuring the protein melting curves, in which binding of the ligand with the protein raises thermostability, allowing the quantification of the change in thermal denaturation temperature of target protein under different drug doses, thereby enabling the identification of drug-target interactions^42–44. We observed that when the temperature ranged from 40 to 64 °C, we detected higher levels of ASPH protein in the 10-HDA-treated group, compared to those in the DMSO control group (Fig. 5e, top). We also detected a prominent change in the melting temperature (Tm) of the melt curve, which was 51.24 °C and 48.47 °C for the 10-HDA and DMSO groups, respectively (Fig. 5e, bottom), indicating that 10-HDA strengthened the thermal stability of ASPH protein at a series of temperatures, and confirming the specific binding between 10-HDA and ASPH.

Fig. 5 — a Coomassie blue staining of drug affinity responsive target stability (DARTS) assay in C28/I2 cells. The arrow indicates the protein band that was subjected to mass spectrometry. b Silver staining of DARTS assay in C28/I2 cells. c Aspartyl β-hydroxylase (ASPH) adapted image from mass spectrometry. d C28/I2 cells were digested with several dosages of protease with or without various concentrations of 10-HDA, then ASPH protein levels were assayed by western blot. n = 3 biologically independent replicates. e C28/I2 cell lysate was denatured under various temperatures and ASPH protein levels in DMSO control, 10-HDA-treated group were assayed by western blot and densitometry analysis curve. f Overview of binding of 10-HDA with ASPH and scheme of encoding serial myc-tagged ASPH full-length (FL) and domain deletion mutants (Δ1-Δ4). g DARTS for ASPH FL and Δ1-Δ4. HEK-293T cells were transfected with plasmid expressing myc-tagged ASPH FL and Δ1-Δ4. n = 3 biologically independent replicates. Quantification was in Supplementary Fig. 11. h DARTS assay for catalytic site deletion mutant of ASPH. HEK-293T cells were transfected with plasmid expressing myc-tagged ASPH catalytic site deletion mutant. n = 3 biologically independent replicates. Quantification was in Supplementary Fig. 12a. i DARTS for glycosylation site deletion mutant of ASPH. HEK-293T cells were transfected with plasmid expressing myc-tagged ASPH glycosylation site deletion mutant. n = 3 biologically independent replicates. Quantification was in Supplementary Fig. 12b. j DARTS assay for catalytic and glycosylation sites deletion mutant of ASPH. HEK-293T cells were transfected with plasmid expressing myc-tagged ASPH catalytic and glycosylation site deletion mutant. n = 3 biologically independent replicates. Quantification was in Supplementary Fig. 12c. ASPH protein levels of all mutants were detected by myc antibody. Data were shown as means ± SD. P values in (d) were performed by two-way ANOVA followed by Tukey’s HSD multiple comparison test. Experiments in (a, b, e) were repeated independently three times with similar results. Source data and uncropped blots are provided as a Source Data file.

To further unravel the binding site of 10-HDA to ASPH, we designed several ASPH domain deletion mutants. The ASPH protein contains three domains critical for its function, Asp-B-Hydro_N (43-108), tpr_16 (345-413), and Asp_Arg_Hydrox (591-745) (from http://pfam.xfam.org/). We thus constructed and verified four domain deletion mutants (Δ1, 2, 3, 4) by either deleting the N- or C-terminal (Fig. 5f and Supplementary Fig. 10a, b), and then detected the binding of 10-HDA to these mutants by DARTS and western blot assays. We found that 10-HDA protected domain deletion mutants Δ1 and Δ2, both containing the C-terminal Asp_Arg_Hydrox (591-745) domain; however, 10-HDA did not protect domain deletion mutants Δ3 and Δ4, lacking the Asp_Arg_Hydrox (591-745) domain, indicating that the C-terminal Asp_Arg_Hydrox (591-745) domain of ASPH is the binding region of 10-HDA (Fig. 5g, Supplementary Fig. 11a–e).

Finally, we explored the possible amino acid sites to which 10-HDA binds in the Asp_Arg_Hydrox (591-745) domain of ASPH. The Asp_Arg_Hydrox (591-745) domain of ASPH contains two active sites, a catalytic (670-675) and a glycosylation (706) site. Therefore, we generated three ASPH amino acid site deletion mutants with deleting the catalytic (670-675) site, glycosylation (706) site, or both sites and further tested the binding of 10-HDA to these mutants by DARTS and western blot assays. Interestingly, 10-HDA protected the catalytic site (670-675) deletion mutant from protease digestion (Fig. 5h and Supplementary Fig. 12a), whereas totally or largely lost its protective effect on the glycosylation site (706) deletion mutant (Fig. 5i and Supplementary Fig. 12b) and double sites deletion mutant (Fig. 5j and Supplementary Fig. 12c), respectively. These results revealed that the glycosylation site (706) is the critical amino acid site for the interaction between 10-HDA and ASPH. Collectively, these results indicated that ASPH is a novel binding target of 10-HDA.

10-HDA regulated chondrocyte metabolism dependent on the Asp-Arg-Hydrox domain of ASPH

To investigate the effect of ASPH on OA, we measured the expression of ASPH in the undamaged and damaged cartilage of OA patients, human cartilage explants, and mice after DMM surgery. We found that the levels of both ASPH mRNA and ASPH protein were down-regulated in the damaged groups, compared with those in the undamaged groups of human OA cartilage (Fig. 6a, b). Consistent with the above results, ASPH protein levels were reduced in IL-1β-treated human cartilage explants (Fig. 6c) and knee joints of DMM mice (Fig. 6d). Interestingly, 10-HDA prominently inhibited the OA-induced down-regulation of ASPH expression, compared to the vehicle group (Fig. 6c, d) Furthermore, we found that 10-HDA also increased the levels of ASPH protein containing the binding C-terminal Asp_Arg_Hydrox domain (Supplementary Fig. 13).

Fig. 6 — a, b *ASPH* mRNA (a) and ASPH protein (b) levels were measured in the undamaged and damaged area of OA patient cartilage. Undamaged, n = 6 donors (1 male, 5 female); damaged, n = 6 donors (1 male, 5 female) (a). Undamaged, n = 3 donors (1 male, 2 female); damaged, n = 9 donors (1 male, 8 female) (b). c ASPH protein levels in human cartilage explant treated with 10 ng/ml IL-1β in the absence or presence of 0, 5, 10 nM 10-HDA for 7 d. n = 3 donors (3 female). d Immunofluorescence of Asph and positive cells quantification in the knee joint of mice administered with vehicle (DMSO, 8-week-old male C57BL/6 J mice, DMM, n = 6; sham, n = 6) or 10-HDA (10 mg/kg, 8-week-old male C57BL/6 J mice, DMM, n = 6) through intra-articular injection 8 weeks after DMM surgery. Asph was stained green (middle boxes), DAPI was blue (upper boxes) and merged images were shown in the lower boxes. Scale bar = 50 μm. n = 6 mice. e HEK-293T cells were co-transfected with siASPH and COL2 or MMP13 luciferase reporter plasmid in the absence or presence of 10 nM 10-HDA. n = 3 biologically independent replicates. f ASPH knockout (KO) confirmation in C28/I2 cells. Experiments in (f) were repeated independently three times with similar results. g *COL2* and *MMP13* mRNA expression in control and *ASPH* knockout C28/I2 cells with or without re-expression of ASPH-Δ2 treated with 10 nM 10-HDA and 10 ng/ml IL-1β for 48 h. n = 3 biologically independent replicates. h COL2 and MMP13 protein levels in control and *ASPH* knockout C28/I2 cells with or without re-expression of ASPH-Δ2 treated with 10 nM 10-HDA and 10 ng/ml IL-1β for 48 h. n = 3 biologically independent replicates. Data were shown as means ± SD. P values in (a, b) were performed by unpaired two-tailed Student’s t-test, in (c, d, e) were one-way ANOVA followed by Tukey’s HSD multiple comparison test, and in (g, h) were two-way ANOVA followed by Tukey’s HSD multiple comparison test. Source data and uncropped blots are provided as a Source Data file.

To explore whether 10-HDA regulates chondrocyte metabolism through ASPH, we knocked down the ASPH expression by siRNA in HEK-293T cells to test the activity of COL2 or MMP13 in the absence or presence of 10-HDA using a luciferase reporter gene assay. We observed that knocking down ASPH weakened the activated COL2 luciferase reporter gene (Fig. 6e, left), whereas promoted the inhibition of the MMP13 reporter gene (Fig. 6e, right) induced by 10-HDA. To further verify the importance of ASPH in mediating the regulation of chondrocyte metabolism by 10-HDA, we knocked out the ASPH gene in C28/I2 cells using the CRISPR-Cas9 genome-editing method (Fig. 6f). Under IL-β stimulus, we found that in ASPH-knockout (KO) cells, the 10-HDA-induced activation of cartilage anabolism was inhibited; more specifically, the expression of anabolism marker gene COL2 remained unchanged after treatment with 10-HDA (Fig. 6g, left). In addition, we noticed that the 10-HDA-mediated inhibition of cartilage catabolism was almost entirely abolished in ASPH-KO cells, leading to the increased expression of catabolism marker gene MMP13 (Fig. 6g, right). Subsequently, we re-expressed the ASPH Δ2 mutant, which contains the 10-HDA binding domain, in the ASPH-KO C28/I2 cells. Under IL-β stimulus, transfection of these cells with a Δ2-overexpressing pcDNA3.1 plasmid largely rescued the activation of cartilage anabolism and inhibition of cartilage catabolism induced by 10-HDA (Fig. 6g). Western blot analysis of COL2 and MMP13 protein levels were consistent with the qPCR results (Fig. 6h, left and right). Accordingly, these findings suggested that 10-HDA regulated chondrocyte metabolism dependent on the Asp-Arg-Hydrox domain of ASPH.

To understand the downstream pathways and in-depth mechanisms of how 10-HDA targets ASPH-mediated regulation of chondrocyte function, we sent both ASPH-KO and normal control (NC) C28/I2 cells for whole transcriptome RNA sequencing. We determined that 10913 genes were co-expressed between the ASPH-KO and NC cells, as shown in the Venn diagram (Supplementary Fig. 14a). Volcano map displayed that compared with the control, there were 483 differentially expressed genes (DEGs) in the ASPH-KO cells, including 217 up-regulated and 266 down-regulated genes with a p value < 0.05 (Supplementary Fig. 14b). According to differential gene clustering analysis, part of the 483 DEGs, which were plotted in a heat map, might regulate the same signaling pathway (Supplementary Fig. 14c).

To further investigate the function of these DEGs, we performed Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis. GO is a comprehensive database describing gene function that is divided into biological process (BP), cellular component (CC), and molecular function (MF). Supplementary Fig. 14d revealed the enriched GO functions of DEGs. GO terms, such as extracellular matrix organization, collagen fibril organization, proteoglycan binding, collagen binding, glycosaminoglycan binding, extracellular matrix binding, extracellular matrix structural constituent, and extracellular matrix component were functionally enriched in the ASPH-KO cells. KEGG analysis revealed that the mitogen-activated protein kinase (MAPK) and p53 signaling pathways were mainly activated in ASPH-KO compared with the control (Supplementary Fig. 14e). Gene Set Enrichment Analysis (GSEA) also indicated that these DEGs were related with cellular senescence, as well as p53 and MAPK signaling pathways (Supplementary Fig. 14f).

10-HDA alleviates OA by inhibiting chondrocytes senescence in vitro and in vivo

Since GSEA showed that a portion of DEGs were enriched in cellular senescence, we then further verified the effect of 10-HDA on alleviating chondrocyte senescence in vitro and in vivo. We first observed a decrease in senescence-associated β-galactosidase (SA-β-gal) staining in C28/I2 cells treated with 10-HDA (Fig. 7a, b). Meanwhile, we detected that the mRNA levels of cellular senescence marker genes p16 and p21 were decreased in human cartilage explants, human C28/I2 chondrocytes and mouse primary chondrocytes treated with a series of 10-HDA concentrations in the presence of IL-1β, compared with those in the IL-1β control, further indicating the anti-senescence effect of 10-HDA (Fig. 7c; Supplementary Fig. 15a, b). Notably, immunofluorescence staining displayed that the p16 and p21 protein levels were decreased in the knee joints of DMM mice treated with 10-HDA through IA injection in vivo (Fig. 7d).

Fig. 7 — a Representative images of β-gal staining and quantification (b) of C28/I2 cells treated with 10 ng/ml IL-1β in the absence or presence of 0, 2, 5, 10 nM 10-HDA for 48 h. n = 3 biologically independent replicates. Scale bar = 100 μm. c *p21, p16* mRNA levels in human cartilage explants treated with 10 ng/ml IL-1β in the absence or presence of 0, 5, 10 nM 10-HDA for 7 d. n = 3 donors (3 female). d Representative images of immunofluorescence staining of p21 and p16 proteins and quantification of positive cells in knee joint of mice administered with vehicle (DMSO, 8-week-old male C57BL/6 J mice, DMM, n = 6; sham, n = 6) or 10-HDA (10 mg/kg, 8-week-old male C57BL/6 J mice, DMM, n = 6) through intra-articular injection 8 weeks after DMM surgery. p21 and p16 were stained green (middle boxes), DAPI (4,6-diamino-2-phenyl indole) was blue (upper boxes) and merged images were shown in the lower boxes. Scale bar = 50 μm. n = 6 mice. Data were shown as means ± SD. P values in (b, c, d) were performed by one-way ANOVA followed by Tukey’s HSD multiple comparison test. Source data are provided as a Source Data file.

Aging in mice promotes the progress of spontaneous OA, similar with what happens in human. Next, we explored the effect of 10-HDA on attenuating chondrocyte senescence and cartilage degeneration in naturally aged mice (Figs. 8a, 9a). Measurement of knee joint diameters revealed that compared to the young mice group (control group), transverse and anteroposterior diameters in the aging mice (vehicle group) were larger, and IA injection or oral delivery of 10-HDA both effectively decreased knee joint diameters, thereby preventing the joint swelling (Figs. 8b; 9b, c). Safranin O, toluidine blue staining, and OARSI score revealed 10-HDA partially alleviated aging-induced cartilage degeneration and improved the OARSI score, compared with the vehicle group (Figs. 8c, f; 9d, e). Immunohistochemistry (IHC) staining in knee joints further indicated that p16, p21, and Mmp13 protein levels were increased and Col2 was decreased in the aging mice (vehicle group); this effect was reversed partly after administration of 10-HDA (Figs. 8d, f; 9f). Immunofluorescence staining also showed Asph protein level was decreased in the aging mice, whereas IA injection of 10-HDA increased the expression of Asph (Fig. 8e, f).

Fig. 8 — a Young (8 weeks old, male, n = 6) or naturally aged (18 months old, male, n = 12) C57BL/6 J mice were administered after 1 week with vehicle (DMSO, 8 weeks old, male, n = 6; 18 months old, male, n = 6) or 10-HDA (10 mg/kg, 18 months old, male, n = 6) twice a week for 8 weeks through intra-articular injection and sacrificed at week 9 post-administration. The joints were collected 9 weeks post-administration. b Representative images, transverse and anteroposterior diameters (mm) of the knee joint were calculated by vernier caliper at week 9 post-administration. n = 6 mice. c Representative images of hematoxylin-eosin (HE), safranin O/fast green, and toluidine blue staining images in the knee joint at week 9 post-administration. Scale bar = 200 μm. d Representative images of p16, p21, type II collagen (Col2), matrix metallopeptidase 13 (Mmp13) immunohistochemical staining and quantification in the knee joint at week 9 post-administration. Scale bar = 50 μm. e Representative images of aspartyl β-hydroxylase (Asph) immunohistochemical in the knee joint at week 9 post-administration. Asph was stained red (middle boxes), DAPI (4,6-diamino-2-phenyl indole) was blue (upper boxes), and merged images were shown in the lower boxes. Scale bar = 50 μm. f OARSI score, quantification of p16, p21, Col2, Mmp13, Asph positive cells in the knee joint at week 9 post-administration. n = 6 mice. Data were shown as means ± SD. P values in (b), positive cells of p16, p21, Col2, Mmp13, Asph in (f) were determined using one-way ANOVA followed by Tukey’s HSD multiple comparison test. P values for the OARSI score in (f) were determined with the two-sided Kruskal–Wallis test followed by the Mann–Whitney U test. Experiments in (c, d, e) were repeated independently three times with similar results. Source data are provided as a Source Data file.

Fig. 9 — a Young (8 weeks old, male, n = 6) or naturally aged (18 months old, male, n = 12) C57BL/6 J mice were administered after 1 week with vehicle (CMC-Na, 8 weeks old, male, n = 6; 18 months old, male, n = 6) or 10-HDA (100 mg/kg, 18 months old, male, n = 6) three times a week for 8 weeks through oral administration and sacrificed at week 9 post-administration. The joints were collected 9 weeks post-administration. b Representative images of the knee joint in each group at week 9 post-administration. n = 6 mice. c Transverse and anteroposterior diameters (mm) of knee joint were calculated by vernier caliper at week 9 post-administration. n = 6 mice. d Representative images of hematoxylin-eosin (HE), safranin O/fast green, and toluidine blue staining in the knee joint at week 9 post-administration. Scale bar = 200 μm. e OA severity was analyzed by the OARSI system at week 9 post-administration. n = 6 mice. f Representative images of p16, p21, type II collagen (Col2), matrix metallopeptidase 13 (Mmp13) immunohistochemical staining and quantification of positive cells in the knee joint at week 9 post-administration. Scale bar = 50 μm. n = 6 mice. Data were shown as means ± SD. P values in (c, f) were determined using one-way ANOVA followed by Tukey’s HSD multiple comparison test. P values for the OARSI score in (e) were determined with two-sided Kruskal–Wallis test followed by the Mann–Whitney U test. Experiments in (d, f) were repeated independently three times with similar results. Source data are provided as a Source Data file.

In addition, we found high-dose of 10-HDA (200 mg/kg) for 4 weeks with IA injection in the wild-type 8 weeks old mice effectively promoted the Asph protein levels in the knee joints, but HE pathological results showed the cartilage structure remained normal in the 10-HDA group, compare to the normal control group, indicating there was no carcinogenicity to the cartilage (Supplementary Fig. 16). Further, IHC results revealed that in the human chondrosarcoma samples, there was no difference in the ASPH protein levels between the tumor and non-tumor area (Supplementary Fig. 17, Supplementary Data 3). We also detected some oncogene MET proto-oncogene, receptor tyrosine kinase (MET), epidermal growth factor receptor (EGFR), KRAS proto-oncogene, GTPase (KRAS), baculoviral IAP repeat containing 5 (BIRC5), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor C (VEGFC), B-Raf proto-oncogene, serine/threonine kinase (BRAF), erb-b2 receptor tyrosine kinase 2 (ERBB2) mRNA levels after ASPH over-expression in the human chondrocytes C28/I2 (Supplementary Fig. 18a), human normal bronchial epithelial cells BEAS-2B (Supplementary Fig. 18b) and human kidney epithelial cells HEK-293T (Supplementary Fig. 18c). These results suggested that over-expression of ASPH did not promote carcinogenesis in these normal cells and tissues, however, future studies need to further investigate the relationship between ASPH and OA.

ASPH was required for 10-HDA-mediated inhibition of chondrocyte senescence

Finally, we investigated the potential mechanisms by which 10-HDA regulates chondrocyte senescence, dependent on ASPH. Based on the literatures^45–47 and RNA-Seq results, we detected whether and how 10-HDA targeting ASPH in regulating chondrocyte senescence through the MAPK and p53 signaling pathways. We found that the increased levels of phosphorylation of extracellular signal-regulated kinase (ERK) and decreased phosphorylation of p53 induced by 10-HDA were abolished in ASPH-KO cells, thereby leading to the expression of senescence marker gene p21 was unaltered after treatment with 10-HDA. Nevertheless, transfection of ASPH-KO C28/I2 cells with a Δ2-over-expressing plasmid restored the effect of 10-HDA on promoting the phosphorylation of ERK (p-ERK) and inhibiting that of p53 and p21 (Fig. 10a, c, e).

*p21* (a) and *p16* (b) mRNA levels in the control and *ASPH* knockout (KO) C28/I2 cells with or without re-expression of ASPH-Δ2 treated with 10 nM 10-HDA and 10 ng/ml IL-1β for 48 h. n = 3 biologically independent replicates (a). n = 3 biologically independent replicates (b). c ASPH/ERK/p53/p21 pathway activation in the control and *ASPH-KO* C28/I2 cells with or without re-expression of ASPH-Δ2 treated with 10 nM 10-HDA and 10 ng/ml IL-1β for 48 h. d ASPH/GSK3β/p16 pathway activation in the control and *ASPH-KO* C28/I2 cells with or without re-expression of ASPH-Δ2 treated with 10 nM 10-HDA and 10 ng/ml IL-1β for 48 h. e Western blot quantification for Fig. 10c. n = 3 biologically independent replicates. f Western blot quantification for Fig. 10d. n = 3 biologically independent replicates. g A model used to explain the signal pathways of 10-HDA binding with ASPH, resulting in the inhibition of chondrocyte senescence and protection against OA. Data were shown as means ± SD. P values in (a, b, e, f) were determined using two-way ANOVA followed by Tukey’s HSD multiple comparison test. Source data and uncropped blots are provided as a Source Data file. ERK extracellular signaling-regulated kinases, p-ERK phospho-extracellular signaling-regulated kinases, GSK3β glycogen synthase kinase-3β, p-GSK3β phospho-glycogen synthase kinase-3β.

Previous studies showed that ASPH restrains senescence through inhibiting the phosphorylation of glycogen synthase kinase-3β (GSK3β) and p16 expression in hepatocellular carcinoma. Accordingly, we examined the role of the ASPH/GSK3β/p16 axis in chondrocytes treated with 10-HDA. We detected that the levels of phosphorylated GSK3β (p-GSK3β) and p16 were decreased with the increase of 10-HDA concentration in the C28/I2 cells (Supplementary Fig. 19a). In addition, we found that the 10-HDA-mediated inhibition of GSK3β phosphorylation and p16 protein levels were abolished by LiCl (a GSK3β phosphorylation agonist) (Supplementary Fig. 19b). Concomitantly, we observed that the increase of both phosphorylation of GSK3β and p16 protein levels in the ASPH-KO cells was diminished by LY294002 (a GSK3β phosphorylation inhibitor) (Supplementary Fig. 19c). The inhibition of GSK3β phosphorylation induced by 10-HDA was weaken in the ASPH-KO cells, thus leading to the unchanged p16 expression. However, expression of the ASPH Δ2 mutant regained the inhibitory effect on the GSK3β phosphorylation and p16 expression (Fig. 10b, d, f). Collectively, these results indicated that 10-HDA inhibits chondrocyte senescence through the ASPH/ERK/p53/p21 and ASPH/GSK3β/p16 axes (Fig. 10g).

Discussion

It was reported that oral administration of RJ daily can delayed osteoarthritis progress in OA mice, and RJ or 10-HDA could inhibit the inflammatory cytokine Il6 and ECM degrading enzymes Mmp3, Mmp13, Adamts5 mRNA levels in ATDC5 chondrocytes induced by TNF-α in vitro⁴⁸. To date, there is no direct evidence on the role of 10-HDA on OA in vivo. In the present study, we found that 10-HDA can increase anabolism, decrease catabolism, inhibit cartilage fibrosis and maintain the characteristics of hyaline cartilage, thus exhibiting protective effects in IL-1β-induced mouse primary chondrocytes and human cartilage explants in vitro (Figs. 1, 2; Supplementary Figs. 1–7), indicating that it might exert a therapeutic effect during the progression of OA. Subsequently, we used a DMM OA mouse model to investigate whether intra-articular injection or oral administration of 10-HDA can markedly attenuate surgical-induced osteoarthritis in vivo and found that it promoted anabolism, whereas inhibited catabolism in chondrocytes (Figs. 3, 4). We also found that 10-HDA was effective in relieving OA-related pain (Figs. 3, 4; Supplementary Fig. 8). Both DARTS and CETSA assays further demonstrated that ASPH is a binding target of 10-HDA, with Asn-706 identified as the critical amino acid participating in the interaction with 10-HDA (Fig. 5; Supplementary Figs. 11, 12). These protein stability assays have been widely used for drug-target detection^43,49,50. In addition, we determined that the regulation of chondrocyte metabolism by 10-HDA depends on ASPH (Fig. 6). Furthermore, we determined that 10-HDA can inhibit chondrocyte senescence and regulate chondrocyte homeostasis in the naturally aging mice (Figs. 7–9; Supplementary Fig. 14, 15). Finally, we verified that the ASPH/ERK/p53/p21 and ASPH/GSK3β/p16 signaling pathways are involved in the anti-senescent effect of 10-HDA (Fig. 10; Supplementary Fig. 19).

ASPH, a type II transmembrane protein, is a member of the α-ketoglutarate-dependent dioxygenase family⁵¹. Previously, ASPH was reported to participate in the regulation of oncogenesis in multiple tumors, including hepatic cellular cancer^51,52, pancreatic^52,53, and non-small cell lung cancer⁵⁴. ASPH has been shown to exert multiple biological functions, especially in promoting cell proliferation and colony formation^52,53. In this study, we found that ASPH was markedly down-regulated in the cartilage tissues of OA patients, DMM mice and naturally aged mice (Figs. 6a–d; 8e; Supplementary Fig. 13). Importantly, we confirmed that 10-HDA upregulate ASPH expression without inducing carcinogenic effects on chondrocytes (Figs. 6c, d; 8e; Supplementary Figs. 13, 16). Although ASPH isoform1 is reported to be associated with carcinogenesis, however, different isoforms of ASPH, including isoform1, exhibit functional variations in different cell types^55,56. Our research confirms that when 10-HDA exerts chondroprotective effects through binding with ASPH, ASPH is not implicated in cartilage-associated carcinogenesis (Fig. 6e, g, h; Supplementary Figs. 16–18). Targeting ASPH in chondrocytes may be anticipated to emerge as a therapeutic strategy for OA.

To explore the underlying pathways and mechanism of 10-HDA in OA, we constructed ASPH-KO C28/I2 cells using CRISPR-Cas9 genome-editing technique and sent them for transcriptome sequencing. Sequencing results revealed that cellular senescence-related pathways were activated in the ASPH-KO cells, indicating that 10-HDA might regulate chondrocytes senescence through ASPH (Supplementary Fig. 14). Recent studies demonstrated that aging stands out as one of predominant risk factor for OA⁵⁷. Senescent chondrocytes are considered being responsible for cartilage injury, thereby participating in the occurrence or progression of OA. As cellular senescence may trigger metabolic reconfigurations that, over time, thereby promoting OA¹⁴. The major pathological feature in OA is usually the loss and damage of articular cartilage⁵⁸. Evidence suggests that the rise in age-related inflammation, termed “inflammation-aging,” triggered by stress-induced cellular senescence, may be one of the significant contributing factors to OA. Elevated levels of IL6 in the blood are associated with declining physiological functions and frailty in aging individuals, and higher IL6 levels are linked to an increased risk of knee osteoarthritis progression⁵⁹. With advancing age, the number of senescent cells in the joints were increased, the SASP, implicated in cartilage degradation and OA, was gradually enhanced. This entails an elevation in pro-inflammatory mediators like cytokines, chemokines, and matrix-degrading enzymes crucial for joint tissue degradation, further exacerbating OA¹⁴. Our study presents initial evidence that 10-HDA promotes chondrocyte anabolism (COL2, ACAN), inhibits cartilage degradation (MMP13), reduces the expression of pro-inflammatory factor IL6, and regulates SASP expression to mitigate the onset of OA (Figs. 1d; 2b–d; 3e, f; 4d; 6e, g, h; 8d, f; 9f; Supplementary Figs. 2b, 3b, 4b, 5b, 6, 7)^60,61. In the aging process, 10-HDA functions by restoring the appropriate cartilage metabolism balance, maintaining cartilage homeostasis, and slowing down the progression of age-related chronic conditions, including OA.

The upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus, which encodes p16^INK4A and p14^ARF, is a driving mechanism in cellular senescence. In aged human primary chondrocytes, the p16 level is significantly increased⁶². p21, encoded by cyclin-dependent kinase inhibitor 1A (CDKN1A), another important marker of senescence, has also been detected in senescent chondrocytes⁶³. SA-β-gal is a commonly used marker of senescence⁶⁴. Increased levels of SA-β-Gal in chondrocytes have been inherently associated with the severity of knee joint damage⁶⁵. Interestingly, IA injection of transplanted senescent chondrocytes in mice was reported to lead to OA-like articular cartilage damage⁶⁶. Currently, few small molecular compounds targeting senescent chondrocytes are known to be effective in the treatment of OA.

In our study, we found that 10-HDA exhibited anti-senescent effects on IL-1β-induced C28/I2 cells in vitro, DMM mice, and naturally aged mice in vivo (Figs. 7; 8d, f; 9f; 10; Supplementary Figs. 15, 19). During cellular senescence, cells are failed to proliferate⁶⁷. Cell cycle arrest is a major feature of senescent cells. As inhibitors of the cyclinE/CDK2 and cyclinD/CDK4, 6 complexes respectively, p21 and p16 are accumulated in senescent cells, eventually causing irreversible cell cycle arrest in G0/G1 and S phases, hence resulting in cellular senescence. Our study demonstrated that 10-HDA led to the upregulation in the levels of cell cycle maker genes cyclinD1, cyclinE1, CDK2, CDK4, and CDK6, whereas induced the down-regulation of cellular senescence maker genes p16 and p21 (Figs. 1c; 2a; 7c, d; 8d, f; 9f; 10; Supplementary Figs. 2a, 3a, 4a, 5a, 15, 19).

The role of ASPH in chondrocyte senescence has not yet been well elucidated. We thus attempted to clarify the underlying mechanism of the regulation of chondrocyte senescence by 10-HDA via ASPH. Previous studies indicated that ASPH directly binds to GSK3β, and inhibition of its activity induced the phosphorylation of GSK3β and expression of p16 in human hepatocellular carcinoma cells, thereby promoting cellular senescence⁵¹. In addition, the ratio of p-GSK3β to total Gsk3β was increased in bone marrow mesenchymal stem cells (BMSCs) transfected with ASPH siRNA⁶⁸. GSK3β is a serine/threonine kinase involved in numerous signaling pathways that possesses multiple critical functions, such as in apoptosis and cell division during embryonic development⁶⁹. Phosphorylated GSK3β is inactive after stimulation with different upstream kinases, including protein kinase B (AKT), MAPKs, and ribosomal protein S6 kinase (S6K)⁶⁹. ASPH binds physically with GSK3β by competing with AKT and p38, thereby blocking the GSK3β phosphorylation stimulated by these kinases⁵¹. Accordingly, inhibitors of GSK3β were reported to promote senescence⁷⁰. Firstly, we found that LiCl (a p-GSK3β agonist) promoted the expression of p-GSK3β and p16, thus reversing the 10-HDA-mediated inhibition of p-GSK3β and p16, indicating activation of phosphorylated GSK3β (p-GSK3β) reversed the anti-senescent role of 10-HDA in chondrocytes (Supplementary Fig. 19a, b). Conversely, LY294002 (a p-GSK3β inhibitor) suppressed the increase in the levels of p-GSK3β and p16 induced by ASPH knockout (Supplementary Fig. 19c). In line with the above findings, ASPH was identified as an upstream regulator of the GSK3β/p16 signaling pathway in OA. Additionally, we demonstrated that 10-HDA down-regulated the expression of p16 through the inhibitory effect of ASPH on the p-GSK3β (inactivation) (Fig. 10b, d, f).

Furthermore, KEGG analysis revealed the activation of the MAPK and p53 signaling pathways in the ASPH-KO chondrocytes (Supplementary Fig. 14e, f). It is becoming increasingly clear that MAPK cascades regulate senescence⁷¹, including pro-inflammatory factors, p16/Rb, and p53/p21 axes, thus leading to SASP and cell cycle arrest⁷². The major categories of MAPKs include ERKs (1 and 2), p38s (α, β, γ and δ), and JNKs (c-Jun N-terminal kinases, 1, 2 and 3), of which ERK and p38 MAPKs are most closely associated with senescence, directly modulating the abundance of CDK inhibitors, such as p16 (CDKN2A) and p21 (CDKN1A)^72,73. The ERK pathway plays a critical role in almost all cellular functions, especially proliferation and senescence, by promoting cell proliferation in dividing cells, whereas inducing cell cycle arrest in senescent cells^74,75. In senescent cells, DNA damage activates the p38 pathway, thereby triggering cellular senescence⁷⁶. p38 can directly phosphorylate p53 at Ser15, consequently stimulating the activation of p53, while phosphorylation of p53 at serine 15 is related to the induction of senescence^77,78.

Accumulating evidence supports the role of p53 in leading to senescence⁷⁹. Cell cycle arrest induced by senescence is mediated by two tumor suppressor pathways, p16/Rb and p53/p21⁷⁹. Recently, evidence has been presented that the phosphorylation of p53 at the Ser15 amino acid site promotes its recruitment on the p21 promoter, thus enhancing the expression of p21⁸⁰. Our study demonstrated that 10-HDA strongly up-regulated the ratio of phosphorylated ERK (p-ERK) to total ERK (t-ERK), whereas, the proportion of the phosphorylated p53 (p-p53) to total p53 (t-p53) was down-regulated (Fig. 10c, e). Particularly, the levels of p21, which is considered as a canonical target of p53⁷⁹ and a hallmark of senescence, were reduced after treatment with 10-HDA (Fig. 10a, c, e). These results indicated 10-HDA might exert anti-senescence effects in chondrocytes through regulating the ERK/p53/p21 axis. Furthermore, the reduction of p-ERK levels, accumulation of p-p53 and p21 in the ASPH-KO chondrocytes revealed the critical role of ASPH in inducing senescence of chondrocytes, suggesting it as a potential influencing factor for OA progression (Fig. 10a, c, e). Interestingly, the 10-HDA-mediated increase of p-ERK, decrease of p-p53 and p21 was simultaneously abolished by ASPH deficiency (Fig. 10a, c, e). Conversely, over-expression of an ASPH critical Δ2 mutant that binds to 10-HDA in ASPH-KO chondrocytes restored the 10-HDA-mediated regulation of ERK/p53/p21 pathway (Fig. 10a, c, e). Collectively, these results indicated that ASPH is a critical target for the anti-senescent effect of 10-HDA in chondrocytes through the ERK/p53/p21 and GSK3β/p16 axes, as shown in the proposed model presented in Fig. 10g.

In summary, this study proposes that 10-HDA, as a natural medicinal ingredient derived from food nutrition royal jelly, can effectively treat OA by inhibiting chondrocyte senescence, and identifies ASPH as a new target of 10-HDA, laying a certain foundation for future discoveries related to 10-HDA/ASPH interaction. Moreover, 10-HDA can modulate OA through ERK/p53/p21 and GSK3β/p16 pathways. Further research on the roles of 10-HDA in suppressing chondrocyte senescence and protecting articular cartilage is also necessary.

Methods

Ethics statement

The human chondrosarcoma and OA samples were randomly recruited from the first affiliated and the second affiliated hospital of Chongqing Medical University. Animal experiments, collection, and use of human samples were approved by the ethics committee of Chongqing Medical University (March 23, 2021). In accordance with the ethical guidelines for animal research, we strictly practice animal welfare monitoring and euthanasia in the study. All the animal experiments were compliant with the ARRIVE guidelines. Written informed consents were obtained from all the human donors.

The source of 10-HDA

(E)-10-Hydroxy-2-decenoic acid (10-HDA, C₁₀H₁₈O₃, Cat. HY-N1363) was obtained from the MedChemExpress (MCE) company.

Cell cultures and treatment

Human chondrocyte C28/I2 cells (gifted by Professor Chuanju Liu from New York University) and human normal bronchial epithelial cells BEAS-2B (gifted by Professor Mingsong Wu from Zunyi Medical University) were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Cat. 11965092) containing 10% fetal bovine serum (FBS, Gibco, Cat. 10099141C) and 50 μg/mL penicillin-streptomycin, incubated in 5% CO₂ and at 37 °C. Human embryonic kidney 293T (HEK-293T) cells (gifted by professor Mingsong Wu from Zunyi Medical University) were cultured in RPMI-1640 medium (Gibco, Cat. 11875119) with 10% FBS (FBS, Gibco, Cat. 10099141C). Mouse primary chondrocytes were isolated from 3–5 days old male C57BL/6 J mice. After the mice were sacrificed, we disinfected the surface of the body using 75% alcohol. Then opened the knee joint capsule and removed articular cartilage into 1 mg/ml collagenase II (Worthington Biochemical, Cat. WorthingtonLS004176) for overnight digestion, the next day cells were cultured in DMEM/F-12 (Gibco, Cat. 11320033) with 10% FBS (FBS, Gibco, Cat. 10099141C) after filtering out redundant tissues.

For IL-1β treatment, mouse primary chondrocytes or C28/I2 cells were seeded in 6-well cell plates at a density of 2 × 10⁶ cells/well and treated with or without 10 ng/ml IL-1β (MedChemExpress, mouse, Cat. HY-P7073; MedChemExpress, human, Cat. HY-P7028). For 10-HDA treatment, mouse primary chondrocytes or C28/I2 cells were treated with 0, 2, 5, 10 nM 10-HDA, dissolved in PBS. For agonist or inhibitor treatment, C28/I2 cells were treated with or without 10 mM Lithium chloride (LiCl) (Abcam, Cat. ab120853) and 30 μM LY294002 (Selleckchem, Cat. S1105). For over-expression ASPH (OE-ASPH) treatment, C28/I2, BEAS-2B or HEK-293T cells were transfected with pcDNA3.1/myc-His-ASPH full-length plasmid by Lipofectamine 2000 (ThermoFisher, Cat. 11668030) for 48 h.

CCK-8

Cell viability was measured by cell counting kit-8 (CCK-8) kit (MedChemExpress, Cat. HY-K0301) according to the manufacturer’s instructions. Mouse primary chondrocytes or C28/I2 Cells were seeded in 96-well microplates with a density of 5000 cells/well in 100 μl medium. After treatment for 48 h, added 10 μl CCK-8 reagent per well, then incubated for 2 h, finally detected the absorbance at 450 nm. All experiments were performed in five repeats.

qPCR

Total RNA from cells or tissues were lysed using Trizol reagent (Thermofisher, Cat.15596018) and cDNA was reversed by RT Master Mix for qPCR II kit (MedChemExpress, Cat. HY-K0511A). qPCR was performed using the chamQ universal SYBR qPCR master mix kit (Vazyme, Cat. Q711-02) on a RT-PCR machine (Bio-Rad CFX, ver.5.0.021.0616, Singapore). The gene expression analysis was carried out by the 2^−ΔΔCT system, and GAPDH was used as the internal reference. The human or mouse primer sequences for target genes were all listed in Supplementary Data 4 and 5.

Edu assay

The 5-ethynyl-2′-deoxyuridine (EdU) staining was performed according to the EdU kit (Beyotime, Cat. C0078). Incubated C28/I2 cells with working fluid and fixed for 15 min, and incubated by transparent fluid. Then added Click Additive Solution to incubate for 15 min darkly. Finally, dyed the cell nucleus with Hoechst 33342 solution for 10 min. The images were photographed with fluorescence microscope (Leica, DM6B, ver.3.7.22383.2, Germany) and the numbers of EdU-positive cells were analyzed by the ImageJ (ver. 1.51j8) software.

Tunel staining

The effect of 10-HDA on C28/I2 cells apoptosis was measured by one one-step tunel apoptosis assay kit (Beyotime, Cat. C1089). Fixed cells with 4% paraformaldehyde (Solarbio Life Sciences, Cat. P1110) for 30 min, and added 50 μl tunel reaction mixture to each well, then incubated darkly at 37 °C for 1 h. The images were photographed with fluorescence microscope (Leica, DM6B, ver.3.7.22383.2, Germany), and the numbers of tunel-positive cells were analyzed by the ImageJ (ver. 1.51j8) software.

Human samples

The human chondrosarcoma tissues were obtained from the right femur, sternum, basis cranii or right rib of chondrosarcoma patients (n = 5; aged 20–70 years; 3 male and 2 female; Supplementary Data 3). The human knee joint OA (damaged) and surrounding undamaged cartilage tissues were obtained from the patients undergoing total knee replacement surgery (n = 15; aged 50–80 years; 3 male and 12 female; Supplementary Data 6). Written informed consent was obtained from all subjects before human tissue samples were harvested. Human OA cartilage explants (about 4 × 4 mm) were placed in 6-well cell plates and cultured in DMEM/F-12 (Gibco, Cat. 11320033) with 10% FBS (Gibco, Cat. 10099141 C) in vitro. For IL-1β and 10-HDA treatment, the cartilage explants were treated with or without 10 ng/ml IL-1β (MedChemExpress, human, Cat. HY-P7028) and 0, 5, 10 nM 10-HDA, dissolved in PBS for one week. For 10-HDA treatment, the undamaged cartilage explants were treated with PBS, and the damaged cartilage explants were treated with 0, 5, 10 nM 10-HDA, dissolved in PBS for one week.

Drug administration in DMM mice

Induced OA in animals may also be more progressive in males such as following DMM in mice⁸¹, and as a result, the majority of murine DMM studies are done in males with direct comparison of OA outcome measures or interventions in females being uncommon⁸². Wild-type C57BL/6 J mice (7 weeks old, male, n = 45) were purchased from Ensiweier Inc. All the mice were housed in a standard specific pathogen-free (SPF) laboratory with controlled temperature (20–26 °C), humidity (ranging from 50% to 60 %), a 12 h light/dark cycle and fed chow (Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., Cat. 1010085), water ad libitum. The mice were randomly allocated to each experimental group. We performed destabilization of the medial meniscus (DMM) or sham surgery when mice were 8 weeks old. DMM surgery was performed on the right hind knee joint. For sham operation, exposed the knee joint cavity after the medial incision, and then close the surgical incision with suture. 10-HDA was administered 10 mg/kg and the vehicle group with 10 μl DMSO (Solarbio Life Sciences, Cat. D8371) through intra-articular injection (1 week after surgery), then conducted twice a week for 6 weeks. For oral administration, mice were fed orally with 100 mg/kg/d 10-HDA or a control diet with CMC-Na (Solarbio Life Sciences, Cat. IS9000) three times a week for 7 weeks. The weight of mice was measured every two days from day 0 to day 56 after surgery. The mice were euthanized by isoflurane (1.5–2% in O₂) 8 weeks after surgery. The weight of each organ was measured, and the coefficient of internal organs was calculated according to the following formula. Coefficient (%) = internal organ weight (g)/body weight (g) × 100.

Drug administration in aging mice

Wild-type C57BL/6 J mice (7 weeks, male, n = 12; 18 months old, male, n = 24) were purchased from Huachuang Sino Inc. All the mice were housed in a standard specific pathogen-free (SPF) laboratory with controlled temperature (20–26 °C), humidity (ranging from 50% to 60%), a 12 h light/dark cycle and fed chow (Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., Cat. 1010085), water ad libitum. The mice were randomly allocated to each experimental group. Performed drug administration when the mice were raised in the animal center for one week. For intra-articular injection administration, 10-HDA was administered 10 mg/kg, the vehicle group or control group (7 weeks old mice) with 10 μl DMSO (Solarbio Life Sciences, Cat. D8371) on the right hind knee joint, then conducted twice a week for 8 weeks. For oral administration, mice were fed orally with 100 mg/kg/d 10-HDA, or a control diet with CMC-Na (Solarbio Life Sciences, Cat. IS9000) in the vehicle group and control group (7 weeks old mice) three times a week for 8 weeks. The mice were euthanized by isoflurane (1.5–2% in O₂) 8 weeks after surgery.

Drug administration in normal mice

Wild-type C57BL/6 J mice (8 weeks old, male, n = 12) were purchased from Ensiweier Inc. All the mice were housed in a standard specific pathogen-free (SPF) laboratory with controlled temperature (20–26 °C), humidity (ranging from 50% to 60 %), a 12 h light/dark cycle and fed chow (Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., Cat. 1010085), water ad libitum. The mice were randomly allocated to each experimental group. Mice were administered DMSO (male, n = 6) or 200 mg/kg high-dose of 10-HDA (male, n = 6) twice a week for 4 weeks through intra-articular injection and sacrificed 4 weeks. The mice were euthanized by isoflurane (1.5–2% in O₂) and joints were collected 4 weeks after administration.

Joint swelling (edema)

Joint transverse and anteroposterior diameters of the knee in each group were measured with the animals in the supine position under anesthesia using a vernier caliper. Three measurements were performed in the same area for each joint, and the data were expressed in millimeters (mm)^83,84.

HE, safranin O/fast green, and toluidine blue staining

The human cartilage explants, heart, liver, spleen, lung, kidney, brain or knee joint of mice were fixed in 4% paraformaldehyde (Solarbio Life Sciences, Cat. P1110) at 4 °C for 48 h, decalcified using EDTA decalcifying solution (Solarbio Life Sciences, Cat. E1171) for 2 weeks at 37 °C, dehydrated and embedded in paraffin, sliced into 5 μm thick sections, then stained with hematoxylin and eosin dye (Solarbio Life Sciences, Cat. G1120) for 10 min and 2 min respectively, modified safranin O-fast green stain kit (Solarbio Life Sciences, Cat. G1371) or toluidine blue dye (Solarbio Life Sciences, Cat. G3668) according to the manufacturer’s instructions. Finally, mounted sections with optical resin. To evaluate the severity of osteoarthritis in mice, we employed the OARSI scoring system and guidelines.

Von frey test

Mechanical allodynia test was measured by a set of von Frey filaments (North Coast Medical Inc., CA, USA, EXACTA, Cat. NC12775-99). Prior to test the mechanical withdrawal threshold, the mice were acclimated to the testing environment on a wire mesh grid for 30 min. Then we measured the withdrawal threshold of the paw using different forces of filaments, performed on the plantar surface center of the hind paws. A positive response was recorded when the animal withdrew paw quickly, licked or shock its paw. The von Frey filaments experiments were performed by the same investigator blinded to mice groups.

Gait analysis

Gait patterns and relative parameters of mice were measured by the Catwalk® gait analysis system (Noldus Inc.). In brief, put the mice on a glass platform in a dark room and let them move freely. The beam from the fluorescent lamp illuminates the platform. When the paw of mice touches the glass, the camera below will record the image of paw print and captured gait and parameters from the Catwalk® software (CatWalk XT 10.6).

DARTS

DARTS assay is used to measure the interaction stability of small molecule drugs with targeting proteins and performed according to previously reported protocol⁴¹. Briefly, C28/I2 or HEK-293T cells were lysed and centrifuged, then incubated with 10-HDA (200 µM) or DMSO (Solarbio Life Sciences, Cat. D8371) for 1 h at room temperature. Each lysate mixture containing equal proteins was digested by thermolysin (Sigma, Cat. T7902) at room temperature for 10 min. To stop the proteolysis, PMSF (Solarbio Life Sciences, Cat. IP0280) was added on ice for 5 min. Western blot was performed to test the targeting protein levels.

CETSA

To further confirm that 10-HDA targeted ASPH, CETSA-western blot experiment was carried out. CETSA is used to test the binding efficacy of drug and protein. Briefly, harvest C28/I2 cells were lysed with RIPA-containing protease inhibitor cocktail and centrifuged at 14,000 × g at 4 °C for 20 min. The protein lysate of cells was equally divided into PCR tubes and each was incubated with 10-HDA (200 μM) or DMSO (Solarbio Life Sciences, Cat. D8371) under room temperature for 1 h. Then samples were heated under different temperatures (40, 43, 46, 49, 52, 55, 58, 61, and 64 °C) for 3 min, followed by centrifugation for 20 min (20,000 × g, 4 °C), and the soluble supernatant was collected to detect the targeted proteins by western blot assay.

pcDNA3.1/myc-His-ASPH plasmids construction, validation and transfection

cDNA from human Hela cells was used as template for PCR amplification using PowerPol 2 × PCR Mix with Dye (Abclonal, Cat. RK20719). Primers sequences of ASPH full-length (FL), domain deletion mutants (Δ1, Δ2, Δ3, Δ4), and amino acid site deletion mutants were shown in Supplementary Data 7. We transformed PCR products by ligation with the double-cleaved pcDNA3.1 vector, picked the single clones, and sent to Tsingke Biotechnology for sequencing. Validation of PCR products or over-expression for ASPH FL, domain deletion mutants, and amino acid site deletion mutants was in Supplementary Fig. 10a, b. Uncropped gels and blots in Supplementary Fig. 10a, b are provided in Supplementary Fig. 20. C28/I2 or HEK-293T cells were transfected with pcDNA3.1/myc-His-ASPH plasmids by Lipofectamine 2000 (ThermoFisher, Cat. 11668030) for 48 h.

Establish of ASPH knockout chondrocytes by CRISPR-Cas9

Briefly, ASPH sgRNA was inserted into the lentiCRISPRv2 vector. Lentiviral CRISPR, VSVG, and psPAX2 plasmids were co-transfected into HEK-293T cells for 48 h to obtain the lentivirus. C28/I2 cells were infected with lentivirus and 1 mg/ml polybrene (Sigma, Cat. H9268) for 2 d, selected with 0.25 μg/ml puromycin (Abcam, Cat. Ab141453) for 2 d, then performed with monoclonal screening. ASPH sgRNA primers sequences were shown in Supplementary Data 8. LentiCRISPRv2 vector, VSVG, and psPAX2 plasmids were gifted by Professor Chuanju Liu from New York University.

RNA-seq analysis for ASPH-KO vs normal control C28/I2 cells

Total RNA of ASPH-KO or normal control (NC) group C28/I2 cells was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in the First-Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After the adenylation of 3′ ends of DNA fragments, Adapter with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing (Novogene Experimental Department). Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.

Differentially expressed genes (DEGs) are screened and subjected to hierarchical cluster analysis. Differential expression analysis of two groups was performed by the DESeq2 R package (1.20.0). Genes with an adjusted p value ≤ 0.05 found by DESeq2 were assigned as differentially expressed. GO (Gene Ontology) enrichment analysis of DEGs was performed using clusterProfiler (3.8.1) software, in which gene length bias was corrected. GO terms with corrected P value less than 0.05 were considered significantly enriched by differential expressed genes (Supplementary Data 9). We used clusterProfiler (3.8.1) software to test the statistical enrichment of differential expression genes in KEGG pathways. For Gene Set Enrichment Analysis (GSEA), the genes were ranked according to the degree of differential expression in the two groups (ASPH KO and NC group), and then the predefined Gene Set were tested to see if they were enriched at the top or bottom of the list. GSEA was performed with the list of DEGs. We used the local version of the GSEA analysis tool (http://www.broadinstitute.org/gsea/index.jsp), then GO and KEGG data sets were used for GSEA independently. The p-values of differential expression, GO and KEGG enrichment analysis were adjusted using Benjamini and Hochberg’s approach for multiple test correction. The multiple correction for GSEA is the false discovery rate (FDR). The raw data of RNA-seq analysis for differential gene expression profiles between ASPH KO and NC C28/I2 cells are deposited in the Sequence Read Archive (SRA) database under accession number PRJNA1105433.

Dual-luciferase reporter assay

HEK-293T Cells were seeded into 24-well plates and then transfected with the luciferase reporter plasmids, including vector of pGL3-basic, positive control of pGL3-control, pGL3-Col2 or pGL3-MMP13, siASPH (5′-TGGGAACAAAGAGGCATATAA-3′) and (5′-TTATATGCCTCTTTGTTCCCA-3′) and 10 nM 10-HDA for 48 h. The luciferase activity was measured for duplicate repeats and analyzed by the dual-Luciferase reporter assay system (Promega, Cat. E1910) according to the manufacturer’s instructions.

Western blot

Cells or human cartilage tissue were lysed with RIPA lysis solution (Beyotime, Cat. P0013B), which contains 1% protease inhibitor PMSF (Solarbio Life Sciences, Cat. IP0280) and 1% phosphatase inhibitor (MedChemExpress, Cat. HY-K0021), and denatured via boiling. Next, the samples were separated by SDS-PAGE gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Cat. IPVH00010), blocked with tri-buffered saline tween (TBST) solution containing 5% bovine serum albumin for 2 h at room temperature, incubated overnight at 4 °C with the corresponding primary antibodies. Next day, washed the PVDF membranes three times using TBST buffer and incubated the corresponding secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat. SA00001-1, 1:5000 dilution; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat. SA00001-2, 1:5000 dilution) for 2 h at room temperature. Protein signal was detected by chemiluminescent (ECL) (Affinity, Cat. KF005) solution. The following primary antibodies were used: COL2 (Proteintech, Cat. 28459-1-AP, 1:1000 dilution), ACAN (Abcam, Cat. ab3778, 1:1000 dilution), MMP13 (Proteintech, Cat. 18165-1-AP, 1:1000 dilution), ASPH (Abcam, Cat. ab172475, 1:20000 dilution), ERK (Proteintech, Cat. 11257-1-AP, 1:1000 dilution), p-ERK (Proteintech, Cat. 28733-1-AP, 1:1000 dilution), p53 (Proteintech, Cat. 60283-2-Ig, 1:1000 dilution), p-p53 (Proteintech, Cat. 28961-1-AP, 1:1000 dilution), GSK3β (Abcam, Cat. ab280376, 1:1000 dilution), p-GSK3β (Abcam, Cat. ab75814, 1:10000 dilution), p16 (Proteintech, Cat. 10883-1-AP, 1:1000 dilution), p21 (Proteintech, Cat. 10355-1-AP, 1:1000 dilution), C-Myc-Tag (Affinity, Cat. T0052, 1:1000 dilution). The membranes were washed with TBST buffer three times and then exposed to ECL chemiluminescent kit (Affinity, Cat. KF8003). Antibody against GAPDH (Proteintech, Cat. 10494-1-AP, 1:5000 dilution) served as loading control. Uncropped scans of all blots are provided in the Source Data or Supplementary Fig. 20.

SA-β-gal

Fixed C28/I2 cells for 15 min, then incubated with staining reagents of SA-β-galactosidase (SA-β-gal) assay kit (Beyotime, Cat. C0602) overnight at 37 °C. The images were photographed with optical microscope, and the numbers of SA-β-gal positive cells were analyzed by the ImageJ (ver. 1.51j8) software.

Immunofluorescence

Fixed paraffin section of human cartilage tissue or mice knee joints with 4% paraformaldehyde (Solarbio Life Sciences, Cat. P1110), permeated with 0.5% TritonX-100 (Solarbio Life Sciences, Cat. IT9100), and incubated with the primary antibody: COL2 (Proteintech, Cat. 28459-1-AP, 1:100 dilution), ACAN (Proteintech, Cat. 13880-1-AP, 1:100 dilution), MMP13 (Proteintech, Cat. 18165-1-AP, 1:100 dilution), ASPH (Santa Cruz Biotechnology, Cat. sc-271391, 1:100 dilution), p16 (Proteintech, Cat. 10883-1-AP, 1:100 dilution), p21 (Proteintech, Cat. 10355-1-AP, 1:100 dilution) at 4 °C overnight. Incubated the secondary antibody (CoraLite488-conjugated Goat Anti-Mouse IgG(H + L), Proteintech, Cat. SA00013-1, 1:200 dilution; CoraLite488-conjugated Goat Anti-Rabbit IgG (H + L), Proteintech, Cat. SA00013-2, 1:200 dilution; Proteintech, CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L), Cat. SA00013-4, 1:200 dilution) for 4 h and DAPI (Solarbio Life Sciences, Cat. C0065) for 5 min at room temperature. The images were photographed with fluorescence microscope (Leica, DM6B, ver.3.7.22383.2, Germany). Statistical analyses of immunofluorescence were performed with ImageJ software. Target protein-positive cells (%) = Target protein-positive cell numbers/DAPI-positive cell numbers.

Immunohistochemistry

Paraffin sections of human cartilage tissue or mice knee joints were performed antigen retrieval, endogenous peroxidase blocking and incubated with the primary antibody: COL2 (Proteintech, Cat. 28459-1-AP, 1:100 dilution), MMP13 (Proteintech, Cat. 18165-1-AP, 1:100 dilution), p16 (Proteintech, Cat. 10883-1-AP, 1:100 dilution), p21 (Proteintech, Cat. 10355-1-AP, 1:100 dilution), ASPH (Santa Cruz Biotechnology, Cat. sc-271391, 1:100 dilution; Invitrogen, Cat. PA5-78827, 1:100 dilution) at 4 °C overnight. Then sections were incubated with secondary antibody (goat anti-rabbit secondary antibody, Servicebio, Cat. G1213-100UL, 1:200 dilution; goat anti-mouse secondary antibody, Servicebio, Cat. G1214-100UL, 1:100 dilution) for 50 min, performed with DAB (ZSGB-BIO, Cat. ZLI-9018) solution for a few minutes, and dyed with hematoxylin (Solarbio Life Sciences, Cat. G1080) for 8 min at room temperature. The images were photographed with optical microscope. Statistical analyses of immunohistochemical were performed with ImageJ software. Target protein-positive cells (%) = Target protein-positive cell numbers/hematoxylin-positive cell numbers.

Statistical and reproducibility

Statistical analyses were performed by IBM SPSS 19.0 software. The data are presented as mean ± SD. Parametric test based on unpaired two-tailed Student’s t-test for comparisons between two groups or one-way ANOVA followed by post hoc test for multigroup comparisons. For nonparametric test of multigroup comparisons, the Kruskal-Wallis test followed by the Mann–Whitney U test was used. In all experiments, p value < 0.05 was statistically significant. n indicates the number of biologically independent samples, mice per group or human specimens. ImageJ (ver. 1.51j8) software was used to analyze the positive cell numbers of immunohistochemical or immunofluorescence and protein expression levels of western blot. All graphs were made using GraphPad Prism 9.0 software. All experiments were repeated at least three times. The sample size for each experiment was not predetermined by statistical method. No data were excluded from the analyses. The experiments were randomized in this study. The experiments were performed in a blind manner that the investigators were not aware of the identification of animals as well as the study groups.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information^{(4.6MB, pdf)}

Peer Review File^{(10.4MB, pdf)}

41467_2024_51746_MOESM3_ESM.pdf^{(112.4KB, pdf)}

Description Of Additional Supplementary File

Supplementary Data 1^{(10.3KB, xlsx)}

Supplementary Data 2^{(10KB, xlsx)}

Supplementary Data 3^{(9.1KB, xlsx)}

Supplementary Data 4^{(10.3KB, xlsx)}

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Supplementary Data 9^{(582.5KB, xlsx)}

Reporting summary^{(9.1MB, pdf)}

Source data

Source Data^{(4MB, zip)}

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81871769 and 82272550 to F.J.G.), Chongqing Science and Technology Bureau Foundation (China) (CSTB2024NSCQ-MSX0243 to F.J.G.), Distinguished Professor Foundation of Chongqing Medical University (202097 to F.J.G.), CQMU Program for Youth Innovation in Future Medicine (W0146 to F.J.G. and Y.D.), China Postdoctoral Science Foundation (2021M700632 to B.K.), Chongqing Science and Technology Bureau Foundation (China) (cstc2021jcyj-bshX0214 to M.T.F.).

Author contributions

Conception and design: F.J.G. Data curation: N.N.G., M.T.F., Y.M.P. Methodology: B.K., N.B.F., X.L.L., N.N.G., M.T.F., F.M.Z. Validation: N.N.G., M.L.X., L.D., C.C., Y.D., L.L., Y.L.Y., K.W.L, J.Q.C. Drafting of the article: F.J.G., N.N.G. Writing-review & editing: F.J.G., N.N.G. Final approval of the article: F.J.G. All authors contributed to the article and approved the submitted version.

Peer review

Peer review information

Nature Communications thanks Maria Mayan, Suzanne de la Monte, Jean-Marc Brondello, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The raw data of RNA-seq analysis for differential gene expression profiles between ASPH knockout (KO) and normal control (NC) C28/I2 cells have been deposited in the Sequence Read Archive (SRA) under accession number PRJNA1105433. All data that support the findings of this study are available within the article and its Supplementary Information files. The source data for Figs. 1–10 and Supplementary Figs. 1–20 generated in this study are provided in the Source Data file. Source data are provided with this paper. A reporting summary for this article is available as a Supplementary Information file. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Nana Geng, Mengtian Fan.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-51746-3.

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PERMALINK

10-hydroxy-2-decenoic acid prevents osteoarthritis by targeting aspartyl β hydroxylase and inhibiting chondrocyte senescence in male mice preclinically

Nana Geng

Mengtian Fan

Biao Kuang

Fengmei Zhang

Menglin Xian

Lin Deng

Cheng Chen

Yiming Pan

Jianqiang Chen

Naibo Feng

Li Liang

Yuanlan Ye

Kaiwen Liu

Xiaoli Li

Yu Du

Fengjin Guo

Abstract

Introduction

Results

10-HDA boosted proliferation and anabolism, whereas inhibited apoptosis and catabolism in chondrocytes

Fig. 1. Effects of 10-HDA on the chondrocyte proliferation, apoptosis, anabolism, catabolism, and inflammation in vitro.

10-HDA enhanced anabolism and repressed catabolism of chondrocytes in human cartilage explants

Fig. 2. Effects of 10-HDA on the anabolism and catabolism of human cartilage tissues.

Intra-articular injection of 10-HDA relieved OA and pain in a surgically induced DMM model in vivo

Fig. 3. Local delivery of 10-HDA attenuated OA development and pain-related behaviors in a surgically induced model in vivo.

Oral administration of 10-HDA protected against OA in a DMM model in vivo

Fig. 4. Oral delivery of 10-HDA relieved OA development and pain-related behaviors in a DMM model in vivo.

ASPH is a unique target of 10-HDA

Fig. 5. ASPH, a novel target of 10-HDA, and its functional domains were identified through DARTS assay.

10-HDA regulated chondrocyte metabolism dependent on the Asp-Arg-Hydrox domain of ASPH

Fig. 6. ASPH was down-regulated in OA cartilage and Asp-Arg-Hydrox domain of ASPH was required for 10-HDA regulation of chondrocyte anabolism and catabolism.

10-HDA alleviates OA by inhibiting chondrocytes senescence in vitro and in vivo

Fig. 7. 10-HDA inhibited chondrocyte senescence in OA.

Fig. 8. Local delivery of 10-HDA attenuated chondrocyte senescence and cartilage degeneration in naturally aged mice in vivo.

Fig. 9. Oral delivery of 10-HDA relieved chondrocyte senescence and cartilage degeneration in naturally aged mice in vivo.

ASPH was required for 10-HDA-mediated inhibition of chondrocyte senescence

Fig. 10. 10-HDA inhibited chondrocyte senescence by regulating the ASPH/ERK/p53/p21 and ASPH/GSK3β/p16 signaling pathways.

Discussion

Methods

Ethics statement

The source of 10-HDA

Cell cultures and treatment

CCK-8

qPCR

Edu assay

Tunel staining

Human samples

Drug administration in DMM mice

Drug administration in aging mice

Drug administration in normal mice

Joint swelling (edema)

HE, safranin O/fast green, and toluidine blue staining

Von frey test

Gait analysis

DARTS

CETSA

pcDNA3.1/myc-His-ASPH plasmids construction, validation and transfection

Establish of ASPH knockout chondrocytes by CRISPR-Cas9

RNA-seq analysis for ASPH-KO vs normal control C28/I2 cells

Dual-luciferase reporter assay

Western blot

SA-β-gal

Immunofluorescence

Immunohistochemistry

Statistical and reproducibility

Reporting summary

Supplementary information

Source data

Acknowledgements

Author contributions

Peer review

Peer review information

Data availability

Competing interests

Footnotes

Supplementary information

References

Associated Data