Ginkgetin ameliorates IL-1β-stimulated inflammation via modulating PI3K/AKT/NF-κB cascade in human osteoarthritis chondrocytes

Abstract

Osteoarthritis (OA) is a degenerative joint condition characterized by an increased density of the subchondral osseous tissue, cartilage degradation, and synovial membrane inflammation. Ginkgetin, a bioactive compound derived from Ginkgo biloba, exhibits notable anti-inflammatory properties in various disease models. In the current study, we aimed to determine the therapeutic potential of ginkgetin in mitigating IL-1β-induced inflammatory responses in human osteoarthritis chondrocytes. Our findings demonstrated that ginkgetin significantly inhibited the IL-1β-induced production of prostaglandin E2 (PGE2) and nitric oxide (NO). Additionally, ginkgetin downregulated the expression of IL-1β-induced proinflammatory mediators and cartilage-degrading enzymes. Furthermore, ginkgetin attenuated the degradation of key extracellular matrix components, including aggrecan and type II collagen. Mechanistically, ginkgetin exerted its protective effects by markedly suppressing the activation of the PI3K/AKT/NF-κB signaling pathway. Taken together, our findings demonstrated that ginkgetin ameliorates IL-1β-stimulated inflammation via modulation of the PI3K/AKT/NF-κB cascade, underscoring its viability as a promising therapeutic strategy for managing OA.

INTRODUCTION

Osteoarthritis (OA) is a chronic and degenerative joint disorder characterized by irreversible progression, predominantly affecting the elderly population globally, and is primarily attributed to pronounced joint instability (García-Muñoz et al., 2024; Yao et al., 2023). The major pathological features of OA include deterioration of hyaline cartilage, alteration of subchondral osseous structures, osteophyte development, and synovial membrane inflammation (Yao et al., 2023; Panikkar et al., 2021). Multiple risk factors, including age, sex, hereditary susceptibility, obesity, and prolonged inflammatory conditions, contribute to OA progression (Tong et al., 2022). Despite significant advancements in research, the underlying molecular and cellular mechanisms driving the OA pathogenesis remain incompletely elucidated. However, inflammation and proinflammatory cytokines play pivotal roles in disease progression. Oxidative stress disrupts the oxidant-antioxidant equilibrium in chondrocytes, leading to an increased production of inflammatory mediators (Nejadhosseinian et al., 2022). Additionally, IL-1β exacerbates extracellular matrix (ECM) disintegration by promoting the secretion of inflammatory modulators, including nitric oxide (NO), ADAMTS, prostaglandin E2 (PGE2), and matrix metalloproteinases (MMPs) (Ansari et al., 2020; Sanchez-Lopez et al., 2022). Furthermore, proinflammatory cytokines, including IL-6 as well as TNF-α, significantly contribute to the pathological development of OA (Molnar et al., 2021). Consequently, anti-inflammatory interventions are regarded as viable therapeutic approaches to decelerate OA progression and alleviate its deleterious effects.

The NF-κB signaling cascade plays a key role in orchestrating the catabolic and inflammatory responses triggered by IL-1β (Rigoglou and Papavassiliou, 2013). Upon stimulation by IL-1β, the NF-κB pathway is activated through a series of upstream signaling events, which ultimately results in the proteasomal degradation of IκBα, a key inhibitory regulator. (Choi et al., 2019). This degradation enables the nuclear translocation of the NF-κB p65 subunit, where it activates the transcription of genes associated with inflammation and matrix degradation, including PGE2, IL-6, TNF-α, MMPs, NO and ADAMTS, all of which collectively drive the pathological development of OA (Cho et al., 2021). Moreover, inhibition of the PI3K/AKT pathway may attenuate OA progression by suppressing NF-κB activity (Kang et al., 2024; Wan et al., 2023). Both the PI3K/AKT and NF-κB cascades are involved in ECM breakdown, underscoring their importance in OA pathology (Hsu et al., 2022). Thus, targeting the PI3K/AKT/NF-κB axis presents a potential therapeutic strategy for OA management.

Ginkgetin, a bioactive compound derived from Ginkgo biloba, exhibits a variety of biological activity, such as antibacterial, anti-atherosclerotic, anti-inflammatory, and neuroprotective effects (Tatlı Çankaya et al., 2023). Previous research has indicated that ginkgetin inhibits the EGFR/PI3K/AKT cascades in intestinal epithelial cells (Geng et al., 2024). Moreover, ginkgetin regulates the NK-κB signaling pathway to attenuate bone loss in mice (Wei et al., 2024). However, the precise mechanisms by which genkgetin protects against progression of OA remain need to be elucidated.

The present study aimed to ascertain the ability of ginkgetin to impede IL-1β-stimulated inflammation in human osteoarthritis chondrocytes. Our findings demonstrate that ginkgetin exhibits significant therapeutic potential as viable pharmacological candidate drug for OA treatment.

MATERIALS AND METHODS

Primary human chondrocytes isolation and culture

Cartilage specimens from human joints were acquired based on the ethical guidelines of the Declaration of Helsinki and Tokyo, with the requisite authorization from the Medical Ethical Committee of Ganzhou People's Hospital. Samples were collected from eight individuals diagnosed with OA who underwent total knee arthroplasty at Ganzhou People's Hospital (All chondrocytes exhibited same phenotypes). The cartilage tissue was meticulously dissected into minute pieces, thoroughly washed with PBS for three times, and enzymatically digested at 37°C for 4 hr using a 2 mg/mL solution of collagenase II (From clostridium histolyticum, Thermo Fisher). Following digestion, the resultant cellular suspension was centrifugated (1,200 rpm for 3 min) to isolate the chondrocytes. The isolated cells were then subsequently propagated in DMEM/F12 culture medium with Fetal Bovine Serum (FBS, 10%) and P/S (Penicillin/Streptomycin, 1%). Cell cultures were maintained in a humidified incubator with 5% CO₂ at 37°C, and the growth medium was replenished at three-day intervals. The culture medium was refreshed every 48 hr, and second-passage chondrocytes were used for all subsequent experimental procedures.

CCK-8 assay

The influences of ginkgetin (HY-N0889, MCE) on human chondrocytes were analyzed using CCK-8 (Beyotime). Briefly, chondrocytes (1×10⁴ cells per well) were seeded into 96-well plates and allowed to adhere for 24 hr. The cells were treated with increasing concentration (0, 5, 10, 25, 50, 100 μM) of ginkgetin for 24 or 48 hr. Following treatment, cells were washed three times with PBS to remove residual media. Subsequently, CCK-8 reagents (10 μL) were added into 96-well plates, followed by incubation for 2 hr at 37°C. Absorbance was then measured at 450 nm using a microplate reader.

ELISA and Griess reaction

Chondrocytes (1×10⁶/well) were seeded in 6-well microplates and maintained for 24 hr. After the initial incubation, the cells were treated with IL-1β and ginkgetin for an additional 24 hr. Levels of PGE2 (#EHPGE2), TNF-α (#KHC3011), IL-6 (#EH2IL6), Aggrecan (#KAP1461), MMP-13 (#EHMMP13), MMP-3 (#BMS2014-3), ADAMTS (#EH14RB), MMP-9 (#BMS2016-2) in supernatant were quantified using commercially available ELISA kits (Invitrogen). The concentration of nitric oxide (NO) was quantified using the Griess assay system, which measures nitrite accumulation in culture supernatants as an indicator of NO production (#S0024, Beyotime).

qRT-PCR

Upon IL-1β stimulation and incremental treatment with ginkgetin, TRIzol reagent (Invitrogen) was used to isolate the total RNA from cultured chondrocytes. qRT-PCR was performed using a CFX96 system (Bio-Rad Laboratories). The thermal cycling protocol commenced at 98°C for 3 min, followed by 38 cycles at 95°C for 15 sec and annealing/extension (60 sec at 60°C). The cycle threshold (Ct) metrics were normalized against β-actin as the housekeeping gene. The primer sequences were as follows: iNOS F: 5’- GCTCTACACCTCCAATGTGACC-3’ and R: 5’-CTGCCGAGATTTGAGCCTCATG-3’, COX-2 F: 5’- CGGTGAAACTCTGGCTAGACAG-3’ and R: 5’-GCAAACCGTAGATGCTCAGGGA-3’, TNF-α F: 5’- CTCTTCTGCCTGCTGCACTTTG-3’ and R: 5’-ATGGGCTACAGGCTTGTCACTC-3’, IL-6 F: 5’- AGACAGCCACTCACCTCTTCAG-3’ and R: 5’-TTCTGCCAGTGCCTCTTTGCTG-3’.

Western blotting

Western blotting was performed as outlined in previous research (Chen et al., 2021). Briefly, cellular proteins were lysed using RIPA buffer and centrifugated at 13,000×g for 30 min at 4°C to collect the supernatant. The cytoplasmic and nuclear proteins were extracted using the extraction kit obtained from Beyotime according to the manufacturer’s instruction. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then the proteins were transferred onto PVDF membranes (Millipore) using electrotransfer. The PVDF membranes were then blocked with 5% skim milk blocking buffer for 1 hr to reduce nonspecific binding. The PVDF membranes with primary antibodies were stored at 4°C overnight. The following day, the membranes were incubated with secondary antibodies for 1 hr. After three washes with PBST, the results were visualized by Bio-Rad Imaging System using enhanced chemiluminescence reagent (ECL, #34578, Thermo Fisher). The primary antibodies were as follows: iNOS (#2982, Cell Signaling Technology), COX-2 (#4842, Cell Signaling Technology), Aggrecan (#28971, Cell Signaling Technology), Collagen (#ab270933, Abcam), ADAMTS5 (#12897, Cell Signaling Technology), MMP-13 (#67329, Cell Signaling Technology), p-p65 (#ab76320, Abcam), p65 (#ab32536, Abcam), p-PI3K (#4228, Cell Signaling Technology), PI3K (#4249, Cell Signaling Technology), p-AKT (#ab38449, Abcam), AKT (#ab8805, Abcam), Lamin A/C (#2032, Cell Signaling Technology), β-actin (#4967, Cell Signaling Technology). Secondary antibodies: Anti-mouse IgG, HRP-linked antibody (#7076, Cell Signaling Technology) and Anti-rabbit IgG, HRP-linked antibody (#7074, Cell Signaling Technology).

Statistical analysis

Statistical analyses were performed by GraphPad Prism software (version 6). Intergroup variances were analyzed using one-way ANOVA, followed by Tukey’s post-hoc analysis for pairwise comparisons. The results are presented as mean ± SD, and P-values < 0.05 was considered significant.

RESULTS

Minimal cytotoxic effects of ginkgetin on human chondrocytes

To evaluate the potential cytotoxicity of ginkgetin (Fig. 1A) on human chondrocytes, chondrocytes were exposed to increasing concentrations of ginkgetin for 24 and 48 hr, followed by cell viability assessment using CCK-8 assay. Our results demonstrated that 100 μM ginkgetin treatment for 24 hr showed no significant cytotoxic effects on human chondrocytes. However, extended exposure (48 hr) at the same concentration led to a marked reduction in cell viability (Fig. 1B and 1C). Consequently, based on these toxicity profiles, subsequent experiments investigating the modulatory effects of ginkgetin on IL-1β-stimulated human chondrocytes were conducted using lower concentrations of 10, 25, and 50 μM.

Fig. 1

The effect of ginkgetin on the viability of human chondrocyte. (A) Structure of Ginkgetin (CAS No. 481-46-9). (B) The human chondrocytes were cultured in increasing concentration of ginkgetin for 24 hr, cell viability was analyzed by CCK-8. (C) The human chondrocytes were cultured in increasing concentration of ginkgetin for 48 hr, cell viability was analyzed by CCK-8. The data in the figures represent the mean ± standard deviation (SD) of three independent experiments (n=3). Statistical significance between groups was shown as follows: *P < 0.05 compared with control group.

Ginkgetin attenuates IL-1β-stimulated proinflammatory biomarkers

Next, we investigated the inhibitory effects of ginkgetin on the IL-1β-stimulated upregulation of inflammatory mediators in human chondrocytes. As shown in Fig. 2A, 2C, and 2D, IL-1β stimulation markedly increased iNOS and COX-2 levels at both the protein and mRNA levels, an effect that was dose-dependently inhibited by ginkgetin. Furthermore, IL-1β significantly enhanced the production of PGE2 and NO, while ginkgetin incubation notably reduced their levels in a dose-dependent manner (Fig. 2E). Moreover, ginkgetin decreased the IL-1β-stimulated induction of TNF-α and IL-6 (Fig. 2B and 2F). Collectively, these findings demonstrate that ginkgetin markedly suppress IL-1β-induced inflammatory responses in human chondrocytes by modulating key inflammatory factors and cytokines at both transcriptional and translational levels.

Fig. 2

The effect of ginkgetin on IL-1β-induced inflammatory response in human chondrocyte. (A) The mRNA levels of iNOS and COX-2 were analyzed by qRT-PCR in human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (B) The mRNA levels of TNF-α and IL-6 were analyzed by qRT-PCR in human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (C) The protein levels of iNOS and COX-2 were analyzed by Western blotting in human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (D) Quantitative analysis of expression of iNOS and COX-2 in (C). (E) The levels of PGE2 and NO were analyzed by ELISA in the supernatant of human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (F) The protein levels of TNF-α and IL-6 were analyzed by ELISA in the supernatant of human chondrocytes treated with IL-1β with or without ginkgetin as indicated. The data in the figures represent the mean ± standard deviation (SD) of three independent experiments (n=3). Statistical significance between groups was shown as follows: ### P < 0.001 compared with control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with IL-1β treatment group.

Effects of ginkgetin on ECM of chondrocytes

Aggrecan and type II collagen (Col II) are the main components of ECM. Our findings demonstrated that ginkgetin enhanced the concentration of aggrecan and Col II, while decreasing the levels of ADAMTS-5, MMP-9, MMP-13, and MMP-3 in a dose-dependent manner (Fig. 3A-3F). Next, the effects of ginkgetin on the levels of MMP-13, aggrecan, collagen II, and ADAMTS-5 were investigated using western blotting. Consistent with the preceding results, IL-1β exposure markedly diminished the protein levels of type II collagen and aggrecan, whereas ginkgetin treatment restored their expression (Fig. 3G and 3H). Moreover, the elevated levels of ADAMTS-5 and MMP-13 stimulated by IL-1β were notably downregulated by ginkgetin in a concentration-dependent manner (Fig. 3G and 3H). Collectively, these findings indicate that ginkgetin could reduce the collapse of ECM in human chondrocytes.

Fig. 3

The effect of ginkgetin on IL-1β-induced degradation of ECM in human chondrocyte. (A)-(F) The protein levels of Aggrecan (A), Col II (B), MMP-13 (C), MMP-3 (D), ADMTS (E), MMP-9 (F) were analyzed by ELISA in the supernatant of human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (G) The protein levels of Aggrecan, Type II collagen, ADMTS5 and MMP-13 were analyzed by Western blotting in human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (H) Quantitative analysis of expression of Aggrecan, Type II collagen, ADMTS5 and MMP-13 in (G). The data in the figures represent the mean ± standard deviation (SD) of three independent experiments (n=3). Statistical significance between groups was shown as follows: ### P < 0.001 compared with control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with IL-1β treatment group.

Ginkgetin mitigates IL-1β-stimulated NF-κB signaling

To further elucidate the molecular mechanism underlying ginkgetin's anti-inflammatory effects, we analyzed the effects of ginkgetin on the NF-κB p65 cascade in human chondrocytes. As illustrated in Fig. 4A and 4B, IL-1β exposure led to increased p65 phosphorylation, a response that was effectively inhibited by ginkgetin treatment. Additionally, the levels of p65 in the nucleus protein extracts were detected in human OA chondrocytes by Western blotting. Our findings indicated that ginkgetin reduced the IL-1β-mediated nuclear translocation of p65 (Fig. 4C). Together, these data establish that ginkgetin exerts its protective effects, at least in part, through suppression of the NF-κB signaling pathway by preventing both p65 phosphorylation and subsequent nuclear translocation.

Fig. 4

The effect of ginkgetin on IL-1β-induced NF-κB pathway activation in human chondrocyte. (A) The protein levels of p-p65 and p65 were analyzed by Western blotting in human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (B) Quantitative analysis of expression of p-p65 in (A). (C) The protein levels of p65 in nucleus were analyzed by Western blotting in human chondrocytes treated with IL-1β with or without ginkgetin as indicated. The data in the figures represent the mean ± standard deviation (SD) of three independent experiments (n=3). Statistical significance between groups was shown as follows: ### P < 0.001 compared with control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with IL-1β treatment group.

Ginkgetin suppresses IL-1β-stimulated modulation of the PI3K/AKT cascade

Emerging evidence indicates that ginkgetin impedes the PI3K/AKT cascade (Geng et al., 2024), which functions as an upstream regulator of NF-κB signaling (Guo et al., 2024). Consistent with these reports, our findings demonstrated that ginkgetin administration significantly reduced the IL-1β-mediated of PI3K/AKT pathway activation (Fig. 5A and 5B). To confirm that the PI3K/AKT pathway acts upstream of NF-κB, chondrocytes were pretreated with the PI3K agonist 740 Y-P prior to ginkgetin exposure. The ginkgetin-mediated dephosphorylation of p65 was reversed by 740 Y-P treatment (Fig. 5C and 5D). These findings collectively suggest that ginkgetin diminishes IL-1β-stimulated upregulation of the PI3K/AKT/NF-κB signaling axis.

Fig. 5

The effect of ginkgetin on IL-1β-induced AKT pathway activation in human chondrocyte. (A) The protein levels of p-AKT, AKT, p-PI3K and PI3K were analyzed by Western blotting in human chondrocytes treated with IL-1β with or without ginkgetin as indicated. (B) Quantitative analysis of expression of p-AKT and p-PI3K in (A). The data in the figures represent the mean ± standard deviation (SD). Statistical significance between groups was shown as follows: ### P < 0.001 compared with control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with IL-1β treatment group. (C) The protein levels of p-p65 and p65 were analyzed by Western blotting in human chondrocytes treated with IL-1β, ginkgetin and 740 Y-P as indicated. (D) Quantitative analysis of expression of p-p65 in (C). The data in the figures represent the mean ± standard deviation (SD) of three independent experiments (n=3). Statistical significance between groups was shown as follows: ### P < 0.001 compared with IL-1β treatment group; *** P < 0.001 compared with IL-1β+ ginkgetin treatment group.

DISCUSSION

Knee osteoarthritis (OA) is the highly prevalent joint disorder and a leading cause of functional disability in elderly population (Latourte et al., 2020). Chronic low-grade inflammation plays a key role in the development of OA pathogenesis, contributing to cartilage degradation and disease progression (Doherty, 1999; Sanchez-Lopez et al., 2022). Therefore, anti-inflammatory agents, such as nonsteroidal anti-inflammatory drugs (NSAIDs), have been used to mitigate OA progression (Zhu et al., 2024). However, the development of effective therapeutic agents for OA remains an unmet clinical need. Ginkgetin, a bioactive compound derived from Ginkgo biloba, possesses significant medicinal properties, including anti-inflammatory effects. In this study, we investigated the therapeutic potential of ginkgetin in modulating IL-1β-induced inflammation in human OA chondrocytes, a well-established in vitro model of OA-related inflammation. Our findings demonstrate, for the first time, that ginkgetin effectively inhibits IL-1β-stimulated inflammatory responses and ECM degradation in human chondrocytes. In addition, the results suggest that ginkgetin exerts its anti-inflammatory effects by inhibiting the PI3K/AKT/NF-κB signaling cascade, highlighting its potential as a novel therapeutic candidate for OA management.

Inflammation plays a critical role in OA progression by disrupting cartilage homeostasis (Sanchez-Lopez et al., 2022). Secreted inflammatory mediators have a functional role on the dysregulation of processes underlying the OA pathophysiology (Rösch et al., 2022). Among these mediators, IL-1β serves as a pivotal proinflammatory cytokine in OA pathogenesis (Wang et al., 2024b), primarily through its stimulation of MMPs production that subsequently accelerates cartilage ECM (Chen et al., 2022). Similarly, PGE2, a well-documented inflammatory mediator, plays a key role on OA progression (Cui et al., 2021). Previous research has reported increased PGE2 levels in the synovial membranes of patients with OA, further linking these levels to disease progression (Cui et al., 2021). Nitric oxide (NO), another critical inflammatory mediator, promotes catabolic processes that exacerbate OA (Chen et al., 2022). Therefore, targeting or suppressing the production of inflammatory mediators is a promising therapeutic strategy for managing OA. In the current research, ginkgetin effectively suppressed IL-1β-stimulated PGE2 and NO production in chondrocytes. Additionally, MMPs facilitate cartilage matrix deterioration and are key contributors to cartilage deterioration. We found that ginkgetin significantly attenuates IL-1β-stimulated induction of MMP-13, MMP-9, and MMP-3. Collectively, these results suggest that ginkgetin may represents a promising therapeutic agent for OA progression.

The NF-κB signaling pathway, a well-established proinflammatory signaling cascade, serves as a pivotal transcriptional regulator in the initiation and progression of inflammatory responses (Rigoglou and Papavassiliou, 2013). The critical function of the NF-κB cascade in modulating proinflammatory factors is related to the OA pathophysiology (Rigoglou and Papavassiliou, 2013; Wang et al., 2024a). Suppressing NF-κB phosphorylation has emerged as a viable therapeutic avenue for managing OA (Ye et al., 2024). The NF-κB remains inactive in the cytoplasm but becomes phosphorylated and translocate to the nucleus upon IL-1β stimulation, where it activates target gene expression (Rigoglou and Papavassiliou, 2013). In this study, we investigated whether ginkgetin's anti-inflammatory effects in chondrocytes are mediated through the NF-κB pathway. Our findings revealed that ginkgetin effectively suppress the IL-1β-induced phosphorylation of p65 in human chondrocytes. Collectively, these findings suggest that NF-κB pathway is integral to ginkgetin’s anti-inflammatory activity in human OA chondrocytes.

The intracellular PI3K/AKT cascade plays a key role on the ECM degradation and cellular alterations associated with OA pathogenesis (Wang et al., 2024c). Previous studies have identified the PI3K/AKT cascade as a crucial upstream regulator of NF-κB activation (Fang et al., 2024). Our findings demonstrate that ginkgetin exerts anti-inflammatory properties by modulating IL-1β-stimulated inflammatory responses via inhibition of the PI3K/AKT/NF-κB signaling cascade in OA (Culley et al., 2015; Hu et al., 2022). In the previous study, Geng et al. shown that ginkgetin directly targets and activates EGFR, thereby interfering with PI3K/AKT signaling pathway (Geng et al., 2024). In addition, ginkgetin pretreatment has been shown to reduce inflammatory responses in DCD donor liver by modulating the JAK2/STAT3 signaling pathway (Liu et al., 2024). Furthermore, ginkgetin suppresses ovarian cancer growth through inhibition of JAK2/STAT3 and MAPK signaling pathways (Wu et al., 2023). It also exhibits neuroprotective effect in experimental cerebral ischemia/reperfusion, mediated through PI3K/Akt/mTOR signaling pathway (Tian et al., 2019).

While this study provides novel insights into ginkgetin's therapeutic potential for OA, several limitations should be acknowledged. First, our findings are restricted to in vitro experiments demonstrating ginkgetin's ability to mitigate IL-1β-induced inflammation in human OA chondrocytes. These results require validation through comprehensive in vivo studies to assess its therapeutic efficacy in established OA conditions. Second, the absence of direct comparisons with clinically approved anti-inflammatory drugs leaves ginkgetin's relative therapeutic advantages undefined. Third, although we have confirmed ginkgetin's protective effects on ECM integrity and inflammatory responses, its potential modulation of other critical OA pathological processes-including oxidative stress and chondrocyte apoptosis-remains to be investigated. Despite these limitations, our study provides compelling evidence that ginkgetin possesses both ECM-preserving and anti-inflammatory properties, highlighting its promise as a potential therapeutic candidate for OA management. Future studies should address these limitations to further elucidate ginkgetin's complete therapeutic profile and clinical potential.

In conclusion, our findings demonstrate that ginkgetin effectively mitigates IL-1β-stimulated inflammatory responses in human chondrocytes by modulating the PI3K/AKT/NF-κB signaling pathway. These results strongly suggest that ginkgetin represents a promising therapeutic candidate for OA intervention, warranting preclinical and clinical investigation.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

• Ansari, M.Y., Ahmad, N. and Haqqi, T.M. (2020): Oxidative stress and inflammation in osteoarthritis pathogenesis: Role of polyphenols. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 129, 110452.
•  Chen,  J.,  Liu,  J.,  Chen,  S.,  Lai,  R.,  Zheng,  C.,  Lu,  J.,  Jiang,  X.,  He,  F.,  Yang,  C.,  Li,  K.,  Xie,  K.,  Tang,  Y. and  Wang,  L. (2022): Salinomycin alleviates osteoarthritis progression via inhibiting Wnt/β-catenin signaling. Int. Immunopharmacol., 112, 109225.
•  Chen,  Y.L.,  Yan,  D.Y.,  Wu,  C.Y.,  Xuan,  J.W.,  Jin,  C.Q.,  Hu,  X.L.,  Bao,  G.D.,  Bian,  Y.J.,  Hu,  Z.C.,  Shen,  Z.H. and  Ni,  W.F. (2021): Maslinic acid prevents IL-1β-induced inflammatory response in osteoarthritis via PI3K/AKT/NF-κB pathways. J. Cell. Physiol., 236, 1939-1949.
•  Cho,  Y.,  Jeong,  S.,  Kim,  H.,  Kang,  D.,  Lee,  J.,  Kang,  S.B. and  Kim,  J.H. (2021): Disease-modifying therapeutic strategies in osteoarthritis: current status and future directions. Exp. Mol. Med., 53, 1689-1696.
•  Choi,  M.C.,  Jo,  J.,  Park,  J.,  Kang,  H.K. and  Park,  Y. (2019): NF-κB Signaling Pathways in Osteoarthritic Cartilage Destruction. Cells, 8, 734.
•  Cui,  S.B.,  Wang,  T.X.,  Liu,  Z.W.,  Yan,  J.Y. and  Zhang,  K. (2021): Zinc finger protein A20 regulates the development and progression of osteoarthritis by affecting the activity of NF-κB p65. Immunopharmacol. Immunotoxicol., 43, 713-723.
•  Culley,  K.L.,  Dragomir,  C.L.,  Chang,  J.,  Wondimu,  E.B.,  Coico,  J.,  Plumb,  D.A.,  Otero,  M. and  Goldring,  M.B. (2015): Mouse models of osteoarthritis: surgical model of posttraumatic osteoarthritis induced by destabilization of the medial meniscus. Methods Mol. Biol., 1226, 143-173.
•  Doherty,  M. (1999): Synovial inflammation and osteoarthritis progression: effects of nonsteroidal antiinflammatory drugs. Osteoarthritis Cartilage, 7, 319-320.
•  Fang,  Y.,  Lou,  C.,  Lv,  J.,  Zhang,  C.,  Zhu,  Z.,  Hu,  W.,  Chen,  H.,  Sun,  L. and  Zheng,  W. (2024): Sipeimine ameliorates osteoarthritis progression by suppression of NLRP3 inflammasome-mediated pyroptosis through inhibition of PI3K/AKT/NF-κB pathway: an in vitro and in vivo study. J. Orthop. Translat., 46, 1-17.
•  Guo,  Q.,  Jin,  Y.,  Chen,  X.,  Ye,  X.,  Shen,  X.,  Lin,  M.,  Zeng,  C.,  Zhou,  T. and  Zhang,  J. (2024): NF-κB in biology and targeted therapy: new insights and translational implications. Signal Transduct. Target. Ther., 9, 53.
•  García-Muñoz,  A.M.,  Abellán-Ruiz,  M.S.,  García-Guillén,  A.I. and  Victoria-Montesinos,  D. (2024): Efficacy of Eggshell Membrane in Knee Osteoarthritis: A Systematic Review and Meta-Analysis. Nutrients, 16, 2640.
•  Geng,  Z.,  Zuo,  L.,  Li,  J.,  Yin,  L.,  Yang,  J.,  Duan,  T.,  Wang,  L.,  Zhang,  X.,  Song,  X.,  Wang,  Y. and  Hu,  J. (2024): Ginkgetin improved experimental colitis by inhibiting intestinal epithelial cell apoptosis through EGFR/PI3K/AKT signaling. FASEB J., 38, e23817.
•  Hsu,  H.C.,  Ke,  Y.L.,  Lai,  Y.H.,  Hsieh,  W.C.,  Lin,  C.H.,  Huang,  S.S.,  Peng,  J.Y. and  Chen,  C.H. (2022): Chondroitin Sulfate Enhances Proliferation and Migration via Inducing β-Catenin and Intracellular ROS as Well as Suppressing Metalloproteinases through Akt/NF-κB Pathway Inhibition in Human Chondrocytes. J. Nutr. Health Aging, 26, 307-313.
•  Hu,  W.,  Lin,  J.,  Wei,  J.,  Yang,  Y.,  Fu,  K.,  Zhu,  T.,  Zhu,  H. and  Zheng,  X. (2022): Modelling osteoarthritis in mice via surgical destabilization of the medial meniscus with or without a stereomicroscope. Bone Joint Res., 11, 518-527.
•  Kang,  W.,  Xu,  Q.,  Dong,  H.,  Wang,  W.,  Huang,  G. and  Zhang,  J. (2024): Eriodictyol attenuates osteoarthritis progression through inhibiting inflammation via the PI3K/AKT/NF-κB signaling pathway. Sci. Rep., 14, 18853.
•  Latourte,  A.,  Kloppenburg,  M. and  Richette,  P. (2020): Emerging pharmaceutical therapies for osteoarthritis. Nat. Rev. Rheumatol., 16, 673-688.
•  Liu,  J.,  Xiao,  J.,  Deng,  Q.,  Fu,  Z. and  Xiao,  Q. (2024): Ginkgetin Pretreatment Reduces Inflammatory Response in DCD Donor Liver via JAK2/STAT3 Signaling Pathway. Ann. Transplant., 29, e944153.
•  Molnar,  V.,  Matišić,  V.,  Kodvanj,  I.,  Bjelica,  R.,  Jeleč,  Ž.,  Hudetz,  D.,  Rod,  E.,  Čukelj,  F.,  Vrdoljak,  T.,  Vidović,  D.,  Starešinić,  M.,  Sabalić,  S.,  Dobričić,  B.,  Petrović,  T.,  Antičević,  D.,  Borić,  I.,  Košir,  R.,  Zmrzljak,  U.P. and  Primorac,  D. (2021): Cytokines and Chemokines Involved in Osteoarthritis Pathogenesis. Int. J. Mol. Sci., 22, 9208.
•  Nejadhosseinian,  M.,  Djalalinia,  S.,  Haerian,  H.,  Alikhani,  M.,  Mansour,  A.,  Mousavian,  A.H.,  Mardani-Fard,  H.A.,  Kasaeian,  A. and  Faezi,  S.T. (2022): The effects of antioxidants on knee osteoarthritis: A systematic review and meta-analysis. Front. Nutr., 9, 1026450.
•  Panikkar,  M.,  Attia,  E. and  Dardak,  S. (2021): Osteoarthritis: A Review of Novel Treatments and Drug Targets. Cureus, 13, e20026.
•  Rigoglou,  S. and  Papavassiliou,  A.G. (2013): The NF-κB signalling pathway in osteoarthritis. Int. J. Biochem. Cell Biol., 45, 2580-2584.
•  Rösch,  G.,  El Bagdadi,  K.,  Muschter,  D.,  Taheri,  S.,  Dorn,  C.,  Meurer,  A.,  Straub,  R.H.,  Zaucke,  F.,  Schilling,  A.F.,  Grässel,  S. and  Jenei-Lanzl,  Z. (2022): Sympathectomy aggravates subchondral bone changes during osteoarthritis progression in mice without affecting cartilage degeneration or synovial inflammation. Osteoarthritis Cartilage, 30, 461-474.
•  Sanchez-Lopez,  E.,  Coras,  R.,  Torres,  A.,  Lane,  N.E. and  Guma,  M. (2022): Synovial inflammation in osteoarthritis progression. Nat. Rev. Rheumatol., 18, 258-275.
•  Tatlı Çankaya,  I.İ.,  Devkota,  H.P.,  Zengin,  G. and  Šamec,  D. (2023): Neuroprotective Potential of Biflavone Ginkgetin: A Review. Life (Basel), 13, 562.
•  Tian,  Z.,  Tang,  C. and  Wang,  Z. (2019): Neuroprotective effect of ginkgetin in experimental cerebral ischemia/reperfusion via apoptosis inhibition and PI3K/Akt/mTOR signaling pathway activation. J. Cell. Biochem., 120, 18487-18495.
•  Tong,  L.,  Yu,  H.,  Huang,  X.,  Shen,  J.,  Xiao,  G.,  Chen,  L.,  Wang,  H.,  Xing,  L. and  Chen,  D. (2022): Current understanding of osteoarthritis pathogenesis and relevant new approaches. Bone Res., 10, 60.
•  Wan,  J.,  Li,  M.,  Yuan,  X.,  Yu,  X.,  Chen,  A.,  Shao,  M.,  Kang,  H. and  Cheng,  P. (2023): Rutaecarpine ameliorates osteoarthritis by inhibiting PI3K/AKT/NF-κB and MAPK signalling transduction through integrin αVβ3. Int. J. Mol. Med., 52, 97.
•  Wang,  F.,  Xiao,  J.,  Li,  M.,  He,  Q.,  Wang,  X.,  Pan,  Z.,  Li,  S.,  Wang,  H. and  Zhou,  C. (2024a): Picroside II suppresses chondrocyte pyroptosis through MAPK/NF-κB/NLRP3 signaling pathway alleviates osteoarthritis. PLoS One, 19, e0308731.
•  Wang,  L.,  Shao,  T.,  Liu,  C.,  Han,  Z.,  Zhang,  S.,  Dong,  Y.,  Han,  T.,  Cheng,  B. and  Ren,  W. (2024b): Liensinine inhibits IL-1β-stimulated inflammatory response in chondrocytes and attenuates papain-induced osteoarthritis in rats. Int. Immunopharmacol., 138, 112601.
•  Wang,  Z.,  Xu,  H.,  Wang,  Z.,  Wang,  Y.,  Diao,  J.,  Chen,  J.,  Xie,  Y.,  Zhang,  L.,  Li,  M.,  Bian,  Y. and  Zhou,  Y. (2024c): Traditional Chinese Manual Therapy (Tuina) Improves Knee Osteoarthritis by Regulating Chondrocyte Autophagy and Apoptosis via the PI3K/AKT/mTOR Pathway: An in vivo Rat Experiment and Machine Learning Study. J. Inflamm. Res., 17, 6501-6519.
•  Wei,  G.,  Liang,  X.,  Wu,  F.,  Cheng,  C.,  Huang,  S. and  Zeng,  Y. (2024): Ginkgetin attenuates bone loss in OVX mice by inhibiting the NF-κB/IκBα signaling pathway. PeerJ, 12, e17722.
•  Wu,  L.,  Qian,  C.,  Zhang,  W.,  Shi,  M.,  Chen,  X.,  Wang,  Y. and  Lin,  F. (2023): Ginkgetin suppresses ovarian cancer growth through inhibition of JAK2/STAT3 and MAPKs signaling pathways. Phytomedicine, 116, 154846.
•  Yao,  Q.,  Wu,  X.,  Tao,  C.,  Gong,  W.,  Chen,  M.,  Qu,  M.,  Zhong,  Y.,  He,  T.,  Chen,  S. and  Xiao,  G. (2023): Osteoarthritis: pathogenic signaling pathways and therapeutic targets. Signal Transduct. Target. Ther., 8, 56.
•  Ye,  H.,  Cai,  T.,  Shen,  Y.,  Zhao,  L.,  Zhang,  H.,  Yang,  J.,  Li,  F.,  Chen,  J. and  Shui,  X. (2024): MST1 knockdown inhibits osteoarthritis progression through Parkin-mediated mitophagy and Nrf2/NF-κB signalling pathway. J. Cell. Mol. Med., 28, e18476.
• Zhu, R., Fang, H., Wang, J., Ge, L., Zhang, X., Aitken, D., Cai, G. (2024): Inflammation as a therapeutic target for osteoarthritis: A literature review of clinical trials, 43, 2417-2433.