Skip to main content
NCBI home page
Chinese Medical Journal logoLink to Chinese Medical Journal
. 2025 Sep 17;138(21):2813–2823. doi: 10.1097/CM9.0000000000003762

Kaempferide inhibited progression of osteoarthritis by targeting the HIF-1 signaling pathway

Xianjie Wei 1,2, Hesuyuan Huang 1, Ping Yuan 3, Peisen Xie 1, Keshi Zhang 1,4,✉, Zhenpeng Guan 1,✉
Editor: Yanjie Yin
PMCID: PMC12574516  PMID: 40960306

Abstract

Background:

Osteoarthritis (OA) is a prevalent joint disorder that significantly impairs quality of life among elderly individuals because of chronic pain and physical disability. As the global burden of OA continues to rise, novel therapeutic strategies are urgently needed. Kaempferide (KA), a flavonoid derived from traditional Chinese herbal medicine, is known for its anti-inflammatory properties. However, the effect of KA on the progression of OA has not been well investigated. This study aimed to explore the therapeutic potential of KA in an OA model and investigate the underlying mechanisms via transcriptomic sequencing.

Methods:

An in vitro OA model was established using SW1353 cells treated with interleukin-1 beta (IL-1β) and different concentrations of KA (30, 60, or 90 μmol/L) for 24 h. The anti-inflammatory effects of KA were assessed using quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting. In vivo, a papain-induced OA rat model was used to evaluate the therapeutic effects of KA through histological and behavioral analyses. Transcriptomic sequencing was performed to explore the differentially expressed genes (DEGs) and related signaling pathways. Statistical analysis was conducted using one-way analysis of variance.

Results:

KA significantly increased cell viability in the OA chondrocyte model and downregulated the expression of inflammatory cytokines and cartilage degradation markers, with the greatest reduction observed at 90 μmol/L. In vivo, KA treatment mitigated cartilage degradation and improved gait behavior in OA rats. Transcriptomic analysis revealed substantial modulation of DEGs, implicating the hypoxia-inducible factor-1 (HIF-1) signaling pathway as a key mechanism. Further blocking and rescue experiments revealed that KA regulated key molecules within the HIF-1 pathway, specifically interferon-gamma (IFN-γ) and hypoxia-inducible factor 1-alpha (HIF-1α), confirming their critical roles in mediating the therapeutic effects of KA.

Conclusion:

KA inhibited the progression of OA by targeting the HIF-1 signaling pathway, reducing inflammation, and cartilage degradation.

Keywords: Kaempferide, Osteoarthritis, IL-1β, HIF-1 signaling pathway, NF-κB signaling pathway

Introduction

Osteoarthritis (OA) is a debilitating degenerative disease that impacts all joints of the human body and is characterized by cartilage degeneration, subchondral bone sclerosis, and synovial hypertrophy.[1] Its clinical manifestations primarily include pain, swelling, and restricted mobility. OA can be a major social burden, as it has a significantly negative impact on individuals and health care systems because of its high prevalence and associated high economic costs. Moreover, the prevalence of OA has increased in recent years, with the number of cases increasing from ~250 million in 2013 to 595 million in 2020, representing 7.6% of the global population. This number is projected to increase by 2050, with knee OA anticipated to increase by ~75%, hand OA by more than 48%, and hip OA by more than 76%.[2] Notably, OA ranks as the 15th leading cause of disability globally.[2–4] In addition, the economic impact of OA is substantial, with average medical costs reaching ~$15,000 per patient (discounted) and total potential wage losses for patients amounting to an astonishing $65 billion.[5,6]

Current treatments for early-stage OA are primarily palliative therapies, such as nonsteroidal anti-inflammatory drugs and intra-articular glucocorticoid injections, aimed at symptom relief and joint function improvement.[7] However, a noteworthy gap remains in the availability of disease-modifying osteoarthritis drugs (DMOADs) capable of halting or slowing OA progression,[8] which is affected by various risk factors, such as age, sex (with postmenopausal women being more susceptible), obesity, genetic predisposition, joint injuries, and repetitive stress from occupations or sports.[9]

The complexity of OA pathogenesis stems from intricate signaling pathways that govern joint health. Key pathways, such as Wnt canonical signaling, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR), are crucial for maintaining cartilage integrity and homeostasis.[10–14] These classic pathways are well studied and are known to regulate crucial processes in cartilage health, including cell proliferation, differentiation, and survival. Disruptions in these pathways can lead to increased cartilage degradation and impaired repair mechanisms. In addition to these well-established pathways, other significant signaling pathways involved in OA progression have been identified. These include the hypoxia-inducible factor-1 (HIF-1), focal adhesion, transforming growth factor-beta/bone morphogenetic protein (TGFβ/BMP), fibroblast growth factor (FGF) pathways, and Notch signaling also plays a role in OA progression.[15–19]

In addition to biochemical changes, OA also involves prominent biomechanical alterations in cartilage and chondrocytes, including increased tissue stiffness and altered stress–strain responses.[20] These changes disrupt the essential biomechanical stimuli required for cartilage homeostasis, leading to further degradation and OA development. In this context, the infrapatellar fat pad contributes to OA pathogenesis, as it secretes proinflammatory cytokines and adipokines that exacerbate joint inflammation and pain.[21]

Given the complex pathogenesis of OA, exploring new and effective drugs and uncovering their underlying mechanisms are of paramount importance. Flavonoids, a diverse group of naturally occurring compounds found in fruits, vegetables, and medicinal plants, have shown significant potential in managing inflammatory conditions, including OA.[22] Compounds, such as quercetin, kaempferide (KA), and epigallocatechin-3-gallate, possess potent anti-inflammatory, antioxidant, and cartilage-protective properties. They modulate the expression and activity of inflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), and inhibit key enzymes involved in inflammation and cartilage degradation, including cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs).[23–27] KA, a specific kind of flavanol, remains largely unexplored in the context of OA, making it a novel candidate for investigation.

For experimental purposes, SW1353 cells, a human chondrosarcoma cell line, serve as a robust model for studying chondrocyte biology and OA-related cartilage damage.[28,29] Furthermore, IL-1β-treated SW1353 cells simulate the inflammatory conditions of OA, providing a relevant in vitro system for investigating disease mechanisms and potential therapeutic interventions.[30] When exploring therapeutic interventions or simulating inflammatory OA models, a papain-induced OA model is frequently utilized. Papain, a proteolytic enzyme, breaks down proteoglycans, leading to joint inflammation. This model effectively mimics the early inflammatory processes of OA, making it ideal for testing potential treatments.[31]

Overall, this study aimed to evaluate the therapeutic potential of KA in an OA model by comparing its efficacy with that of dexamethasone (DXM), a standard anti-inflammatory agent for OA management.[32] Through transcriptomic sequencing analysis, this study sought to elucidate the specific mechanisms of action of KA by using IL-1β-treated SW1353 cells and a papain-induced OA rat model, potentially identifying KA as a new therapeutic option for OA.

Methods

Reagents

KA, with a purity of at least 98%, was procured from Shanghai Yuanye Bio-Technology Co., Ltd. (B21132, Shanghai, China). DXM, also with a purity of at least 98%, was obtained from Solarbio (ID0170, Beijing, China). The experimental antibodies used included those against collagen II (ab307674), interferon-gamma (IFN-γ, ab171081), NF-κB (ab220803), hypoxia-inducible factor 1-alpha (HIF-1α, ab51608), vascular endothelial growth factor (VEGF, ab32152), inducible nitric oxide synthase (iNOS, ab178945), matrix metalloproteinase-2 (MMP-2, ab92536), matrix metalloproteinase-3 (MMP-3, ab52915), matrix metalloproteinase-13 (MMP-13, ab39012), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5, ab41037), and β-actin (ab6276) from Abcam (Cambridge, UK). Additional antibodies, such as those against tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2, AF8112), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3, AF7734), phospho-p65 (AF5875), and phospho-IκBα (AF5851), were obtained from Beyotime (Shanghai, China). The HIF-1α agonist dimethyloxallyl glycine (DMOG, HY-15893) and the HIF-1α inhibitor LW6 (HY-13671) were both supplied by MedChemExpress (Monmouth Junction, NJ, USA).

Cell viability assay

Before establishing the cell culture and treatment protocols, an initial KA concentration screening was performed. SW1353 cells, a human chondrosarcoma cell line, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were inoculated into 96-well plates at a density of 5 × 103 cells/well. After incubation with different concentrations of KA (1, 15, 30, 60, 90, or 120 µmol/L) at 37°C for 24 h, the absorbance of each well at 450 nm was measured. Then, the half-maximal inhibitory concentration (IC50) of KA for cell growth inhibition was calculated. In addition, to evaluate the protective effect of KA under inflammatory conditions, cell viability was also assessed in IL-1β (10 ng/mL)–treated cells following co-incubation with KA (0, 30, 60, or 90 µmol/L) for 24 h.

Cell culture and establishment of a chondrocyte OA model

Following Lin’s protocol,[33] cells were seeded in 6-well plates at a density of 1 × 105 cells per well and cultured in Dulbecco’s modified Eagle’s medium (DMEM, 11965092, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, 086-450, Wisent, St. Bruno, QC, Canada). The plates were then placed in a humidified incubator at 37°C with 5% CO2. To simulate conditions similar to those of human OA chondrocytes in vitro, the cells were treated with 10 ng/mL IL-1β for 24 h. The cells were subsequently allocated into five distinct groups: (1) a control group consisting solely of SW1353 cells without IL-1β treatment; (2) an OA group treated with IL-1β only; (3) an OA group treated with IL-1β plus a low dose (30 μmol/L) of KA; (4) an OA group treated with IL-1β plus a moderate dose (60 μmol/L) of KA; and (5) an OA group treated with IL-1β plus a high dose (90 μmol/L) of KA. After treatment, cells were further incubated with their respective conditions for an additional 24 h at 37°C.

Western blotting

Protein extraction was conducted following the protocol provided with the Total Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). The protein concentrations of each sample were subsequently determined using a BCA Assay Kit (Beyotime Biotechnology, Shanghai, China). Total protein samples were then subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk powder for 1 h and incubated overnight at 4°C with a panel of primary antibodies. These included rabbit anti-human IFN-γ (1:5000), anti-NF-κB (1:2000), anti-HIF-1α (1:1000), anti-VEGF (1:5000), anti-iNOS (1:1000), anti-MMP-2 (1:5000), anti-MMP-3 (1:2000), anti-MMP-13 (1:6000), anti-ADAMTS-5 (1:250), anti-Tie2 (1:2000), anti-PFKFB3 (1:1000), anti-phospho-p65 (1:2000), anti-phospho-IκBα (1:1000), and mouse anti-human β-actin (1:10000) antibodies. These antibodies were appropriately diluted and applied to the PVDF membranes containing the target protein bands, followed by overnight incubation at 4°C. The membranes were then incubated with the secondary antibodies horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:1000, A0216, Beyotime Biotechnology, Shanghai, China) and HRP-conjugated goat anti-rabbit IgG (1:1000, A0208, Beyotime Biotechnology) for 1 h at room temperature. β-actin was used as an internal control. The gray values of the protein bands were subsequently analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).

RNA extraction and quantitative real-time polymerase chain reactions (qRT-PCR) analysis

The expression of related genes was quantified via qRT-PCR, and RNA was isolated using a Total RNA Extraction Kit (1 μg) (Invitrogen, Carlsbad, CA, USA). In addition, complementary DNA (cDNA) and amplification products were synthesized from total RNA with a SuperScript III RT (ABI-Invitrogen, Foster City, CA, USA). The amplification conditions utilized were as follows: 35 s at 95°C followed by 40 cycles of 10 s at 95°C and 30 s at 60°C; 15 s at 95°C, 60 s at 60°C, and 15 s at 95°C for the melt curve. β-actin was used as the internal reference. Relative gene expression was calculated using the 2−ΔΔCt method. Each cDNA sample was tested in triplicate.

Transcriptome sequencing

Following 24 h of exposure to 90 μmol/L KA, total RNA was extracted for transcriptome sequencing in partnership with Nanjing Personaltech Genomics Technology Co., Ltd. (Nanjing, China). The RNA quality was evaluated using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Cluster generation and primer hybridization were executed on the cBot system (Illumina, San Diego, CA, USA) according to the manufacturer’s guidelines. Sequencing reagents were prepared according to the manufacturer’s protocols, and paired-end sequencing was carried out on an Illumina NextSeq 500 with the High Output v2 kit (Illumina Inc., San Diego, CA, USA). Image analysis, base calling, and sequence read generation were performed using the NextSeq Control Software v2.0 (Illumina Inc.) and real-time analysis Software v2 (RTA). The data were converted to FASTQ files using bcl2fastq2 v2.20 software (Illumina). The sequence data were aligned to the reference genome, and the fragments per kilobase of exon model per million mapped fragments (FPKM) values were calculated for gene expression estimation. Differential expression analysis between the two groups was conducted using DESeq2 software (Bioconductor, Fred Hutchinson Cancer Research Center, Seattle, WA, USA), with adjusted P values and log2-fold change thresholds for significance. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the differentially expressed genes (DEGs), with an adjusted P value of less than 0.05 as the threshold for significant enrichment. Gene set enrichment analysis (GSEA) was conducted on the GO and KEGG datasets.

Bioinformatics analysis

HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) was used to filter the original data to obtain clean reads for data analysis. Additionally, the spliced mapping algorithm HISAT2 was used for genome mapping of the preprocessed sequences. StringTie software (version 1.3.0, Johns Hopkins University, Baltimore, MD, USA) was used to count the number of fragments corresponding to each gene after the HISAT2 comparison, which were then normalized to the trimmed mean M values (TMM) of the FPKM for each gene.

The edgeR R package was used to compare the DEGs among the sample groups. After the P value was obtained, multiple testing correction was applied, and the P value threshold was determined by controlling the false discovery rate (FDR) and was expressed as a q value. Afterward, the fold change (FC) was calculated on the basis of the FPKM value. In addition, cutoffs of q value ≤0.05 and FC ≥|1.5| were applied to screen for DEGs. The DEGs were subsequently mapped to the GO database, and the number of genes corresponding to each entry was calculated. KEGG pathway enrichment analysis was performed similarly for the DEGs. The DEGs were sorted in descending order according to their |log2FC| values, and the top 20 dysregulated genes were identified. Eventually, KEGG pathways related to OA with a large number of DEGs were selected, and the genes enriched in these pathways were selected.

Animal experiments

Thirty male Sprague–Dawley rats (6 weeks old) were obtained from SpePharm Biotechnology Co., Ltd. (Beijing, China). The model was established according to earlier reports.[31] Briefly, a 4% papain solution (0.2 mL) and 0.03 mol/L L-cysteine (0.1 mL) were mixed and allowed to stand for 0.5 h. After that, the mixture (20 μL) was injected into the right knee joint of each rat. The dose of DXM administered was in accordance with the following empirical equation: Dose (rat, mg/kg) = Dose (cell, μg/mL) × Volume (mL/kg)/ Weight (kg).[34]

According to the report by Hu et al,[35] quercetin, a flavonoid compound, effectively improved cartilage damage in a rat meniscectomy model when it was administered at a dose of 8 μmol/L per 100 μL for 6 consecutive weeks. Based on this, for the present study, we selected concentrations of 1.5 and 3 mg/kg KA for the experiments.

Interventions were initiated beginning on the second day after model establishment. The OA + DXM group (n = 6) received intra-articular injections of DXM at a dosage of 1 mg/kg per week. The OA + KA (low dose, Lo) group (n = 6) received intra-articular injections of KA at 1.5 mg/kg per week, whereas the OA + KA (high dose, Hi) group (n = 6) was administered KA at 3 mg/kg per week. The control group and the OA group were treated with an equivalent volume of saline. The treatment period spanned 4 weeks. At the conclusion of the 4-week period, all of the rats were euthanized by cervical dislocation, and samples of articular cartilage were collected for subsequent analysis. All animal experiments adhered to current guidelines and were reviewed and approved by the Biomedical Ethics Committee of Peking University (No. LA2021238).

Gait behavior test

The gait behavior test evaluated the distance traveled by the rats within a 5-min time frame and their standing durations at various time points: Before the procedure and at 1, 2, 3, and 4 weeks postprocedure. At each assessment point, each animal was transferred to the testing area, and its genital region was marked with food-safe dye to prevent it from being analyzed twice. After the dye had dried, the rat was introduced into the testing chamber and given a brief period to acclimate to the new environment. During this time, the software was configured, and bumpers were adjusted to ensure that the animals remained within the field of view. The treadmill was initially set to a speed of 10 cm/s, allowing the animal to adapt to this pace. The speed was then incrementally increased to the testing speed of 20 cm/s, at which point recording commenced. The treadmill and recording were halted once 3–5 s of consistent stepping was captured, and the animal was returned to its home cage.

Safranin O–Fast Green staining

After the paraffin sections were dewaxed and hydrated with graded alcohol, they were stained with 0.5% fast green for 20 min and 0.5% safranin O for 5 min. The sections were then subjected to gradient alcohol dehydration, cleared in xylene, and sealed with neutral gum. The articular cartilage tissue was subsequently observed under a microscope (Leica Microsystems, Wetzlar, Germany).

Mankin scores

After safranin O–fast green staining was completed, the degree of the articular cartilage lesions was scored by two independent observers according to the modified Mankin scoring principle.[36] The score ranged from 0 to 14; the higher the score was, the more severe the joint degeneration was.

Immunohistochemical analysis

Paraffin sections with a thickness of 4 μm were hydrated in gradient alcohol. The sections were subsequently incubated in citrate buffer for 15 min at 95°C and then treated with a peroxidase blocker for 30 min. The sections were then incubated overnight at 4°C with primary antibodies specific for collagen II (1:100), MMP13 (1:2000), IFN-γ (1:200), HIF-1α (1:100), and VEGF (1:250). Sections incubated with phosphate-buffered saline (PBS) alone served as negative controls. The following day, a fluorescein isothiocyanate (FITC)-labeled secondary antibody was applied for 1 h. After being washed with PBS, the sections were developed with a 3,3′-diaminobenzidine (DAB) solution. The sections were then washed with tap water, counterstained with hematoxylin, dehydrated through a series of gradient alcohols, clarified with xylene, and finally sealed with neutral gum. Five sections from each sample were selected for photography. The mean optical density (MOD) of the immunohistochemical staining was quantified by two independent individuals following the double-blind principle to ensure unbiased assessment.

Enzyme-linked immunosorbent assay (ELISA)

The levels of COX-2, iNOS, lipoxygenase (LOX), leukotriene B4 (LTB4), and prostaglandin E2 (PGE2) in the OA cell model were assessed using ELISA. An absorbance microplate reader, SuPerMax 3000FA (Flash Co. Ltd., Shanghai, China), was used to measure the optical density (OD) values at a wavelength of 450 nm. The concentrations of the specific proteins were determined by comparing these OD values to a standard curve, which was generated using known concentrations of the respective proteins. This method allowed for the quantification of the levels of these inflammatory mediators in the OA cell model.

Statistical analyses

Statistical analyses were conducted using SPSS software (version 07 for Windows; SPSS Inc., Chicago, IL, USA). The results are presented as the mean ± standard deviation. One-way analysis of variance followed by Tukey’s honestly significant difference (HSD) test was employed for multigroup comparisons. A significance level of P <0.05 was used for all tests.

Results

Effects of KA on a chondrocyte OA model in vitro

The viability of SW1353 cells treated with various concentrations of KA alone (0, 1, 15, 30, 60, 90, or 120 μmol/L) was initially assessed. The cell viability significantly decreased at a concentration of 15 μmol/L [Figure 1A]. In the IL-1β-induced OA model, treatment with KA at concentrations of 30, 60, and 90 μmol/L significantly increased cell viability compared with that in the untreated OA group, with all P values less than 0.05 [Figure 1B]. The IC50 of KA in SW1353 cells was estimated to be 200.1 μmol/L.

Figure 1.

Figure 1

Open in a new tab

KA cytotoxicity and cell proliferation assays of KA. (A) Cytotoxicity and cell proliferation effects of different concentrations of KA on SW1353 cells. (B) Effects of different concentrations of KA on IL-1β-treated SW1353 cells (OA model). The data are shown as the mean ± SD. n = 3, *P <0.001; †P <0.0001, compared with the control group. ‡P <0.01; §P <0.0001, compared with the OA group. IL-1β: Interleukin-1 beta; KA: Kaempferide; ns: Not significant; SD: Standard deviation.

The levels of inflammatory cytokines (COX-2, iNOS, PGE2, LOX, and LTB4) in the supernatants of OA model cells were examined via ELISA after treatment with 0, 30, 60, and 90 μmol/L KA. The levels of these cytokines were significantly lower in the KA-treated groups than in the untreated OA group (P <0.01), with the 90 μmol/L KA group showing the most significant reduction [Figure 2A–E].

Figure 2.

Figure 2

Open in a new tab

KA inhibited IL-1β-induced inflammatory mediator expression in the SW1353 cell model. (A–E) ELISA analysis of the levels of COX-2, iNOS, PGE2, LOX, and LTB4 in IL-1β-treated SW1353 cells (OA model) treated with different concentrations of KA. The data are shown as the mean ± SD. n = 3, *P <0.0001; †P <0.001; ‡P <0.01; §P <0.05. (F) The mRNA levels of MMP-2, MMP-3, MMP-13, SOX9, ADAMTS-5, COL2A1, Aggrecan, and HAS2 in the SW1353 cell model were assayed via real-time polymerase chain reactions (RT-PCR). (G) Representative Western blotting results for MMP-2, MMP-3, MMP-13, and ADAMTS-5 in the SW1353 cell model. (H) Quantification of the Western blot data from (G). The data are shown as the means ± SDs. n = 3, *P <0.0001, compared with the control group. †P <0.001; ‡P <0.01; §P <0.05; ||P <0.0001, compared with the OA group. ADAMTS-5: A disintegrin and metalloproteinase with thrombospondin motifs 5; COL2A1: Collagen type II alpha 1 chain; COX-2: Cyclooxygenase-2; ELISA: Enzyme linked immunosorbent assay; HAS2: Hyaluronan synthase 2; IL-1β: Interleukin-1 beta; iNOS: Inducible nitric oxide synthase; KA: Kaempferide; LOX: Lipoxygenase; LTB4: Leukotriene B4; MMP-2: Matrix metalloproteinase-2; MMP-3: Matrix metalloproteinase-3; MMP-13: Matrix metalloproteinase-13; mRNA: Messenger RNA; MW: Molecular weight; PGE2: Prostaglandin E2; SD: Standard deviation; SOX9: SRY-box transcription factor 9.

Cartilage degradation-related markers (MMP-2, MMP-3, MMP-13, and ADAMTS-5) and cartilage matrix synthesis markers (COL2A1, aggrecan, SRY-box transcription factor 9 [SOX9], and hyaluronan synthase 2 [HAS2]) were quantified 24 h post-KA treatment via qRT-PCR. The levels of all cartilage degradation-related markers were lower in the KA-treated group than in the OA group [Figure 2F]. In contrast, the levels of markers of cartilage matrix synthesis were greater in the KA-treated group than in the control group [Figure 2F]. Compared with 30 μmol/L KA, a higher dose of KA (90 μmol/L) was more effective at reducing the expression of cartilage degradation markers [Figure 2F]. Western blotting analysis confirmed these results for MMP-2, MMP-3, MMP-13, and ADAMTS-5 [Figure 2G and H].

These results indicate that KA has no cytotoxicity within the appropriate concentration range and significantly reduces inflammatory cytokine levels in the chondrocyte OA model. These in vitro findings prompted further investigations into the effects of KA in an in vivo OA model to validate its therapeutic potential.

Effects of KA on a chondrocyte OA model in vivo

To validate the anti-inflammatory and chondroprotective effects of KA observed in vitro, a papain-induced OA rat model was used. Initial gait analysis revealed no significant differences in gait parameters among the surgical groups at baseline (0 w) [Figure 3A]. However, at 4 weeks posttreatment, compared with untreated OA rats, rats treated with DXM and KA presented enhanced gait performance, characterized by longer running times and increased standing times [Figure 3B]. Notably, the OA + KA(Hi) group demonstrated superior running performance and equivalent standing times compared with the OA + DXM group, indicating that KA treatment effectively improved gait behavior [Figure 3C].

Figure 3.

Figure 3

Open in a new tab

Gait behavior test in papain-induced rat OA. Gait behavior tests were performed from 0 to 4 weeks. (A) Gait behavior test results at 0 week. Data are shown as the mean ± SD. n = 6, ns, not significant, compared with the control group. (B) Gait behavior test results at 4 weeks. Data are shown as the mean ± SD. n = 6, P <0.0001, compared with the control group; †P <0.01, ‡P <0.05, ns, not significant, compared with the OA group. (C) Changes in gait behavior from 0 to 4 weeks. 0 w: 0 week; 1 w: 1 week; 2 w: 2 weeks; 3 w: 3 weeks; 4 w: 4 weeks; DXM: Dexamethasone; KA (Lo): Kaempferide low dose; KA (Hi): Kaempferide high dose; SD: Standard deviation; OA: Osteoarthritis.

After 4 weeks of treatment, the rats were euthanized, and knee joint cartilage samples were collected for examination [Figure 4A]. The control group displayed smooth, glossy cartilage surfaces, whereas the OA group presented significant cartilage damage. Compared with the OA group, the OA + DXM group presented substantial improvement in cartilage. The OA + KA(Lo) group also demonstrated some improvement, but noticeable cartilage damage persisted. In contrast, the OA + KA(Hi) group presented less pronounced damage than both the OA + KA(Lo) and OA groups did. Visual inspection revealed that the cartilage conditions in the control, OA + DXM, and OA + KA(Hi) groups were similar, suggesting a synergistic chondroprotective effect of KA.

Figure 4.

Figure 4

Open in a new tab

KA slowed the progression of OA in papain-induced rat OA. (A) Macroscopic images of the joints of the rats after 4 weeks of treatment. The control rats presented smooth, intact articular cartilage surfaces. Nontreated OA rats presented with cartilage loss, exposing pitting on the bone surface — KA and DXM ameliorated cartilage loss. DXM reduced erosion and cracking. KA joints presented intact cartilage with small fissures and chondral softening. (B) Histology images stained with Safranin O-Fast after 4 weeks of treatment (original magnification: ×10). Compared with the control, smooth articular cartilage surface with normal chondrocyte orientation, the lateral femoral cartilage of nontreated OA rats presented severe cartilage loss, exposing the calcified subchondral bone surface. DXM and KA treatments significantly reduced cartilage loss, as indicated by reduced Safranin O–Fast green intensity, condensed collagen fibers, fibrillation of the superficial layer, and cell death. (C) Mankin scores in different groups. The data are shown as the mean ± SD. n = 6, *P <0.0001, compared with the control group. †P <0.05, compared with the OA group. DXM: Dexamethasone; KA: Kaempferide; KA (Hi): Kaempferide high dose; KA (Lo): Kaempferide low dose; SD: Standard deviation; OA: Osteoarthritis.

The Mankin structure score, which is used to assess OA-related cartilage injuries, revealed significantly greater structural damage scores in the medial femoral condyle of the surgical knee than in the treated groups [Figure 4B and C]. Both the OA + KA(Lo) and OA + KA(Hi) groups presented lower Mankin scores than the OA + DXM group [Figure 4C]. Immunohistochemistry further demonstrated that KA treatment mitigated the loss of collagen II and reduced the levels of IFN-γ, HIF1α, VEGF, and MMP13 [Figure 5A and B].

Figure 5.

Figure 5

Open in a new tab

Immunohistochemical staining of papain-induced rat OA. Immunohistochemical staining of the collagen II, IFN-γ, HIF1α, VEGF, and MMP13 proteins in the cartilage tissue with (A) representative and (B) quantified data. Scale bar = 100 μm. The data are shown as the mean ± SD. n = 6, *P <0.0001, compared with the control group. †P <0.05; ‡P <0.01, compared with the OA group. Collagen II: Collagen type II; DXM: Dexamethasone; HIF-1α: Hypoxia-inducible factor 1-alpha; IFN-γ: Interferon-gamma; KA (Hi): Kaempferide high dose; KA (Lo): Kaempferide low dose; MMP-13: Matrix metalloproteinase-13; OA: Osteoarthritis; SD: Standard deviation; VEGF: Vascular endothelial growth factor.

These in vivo findings confirm the anti-inflammatory and chondroprotective effects of KA observed in vitro. To elucidate the molecular mechanisms underlying these effects, a transcriptomic analysis was conducted.

Transcriptomic analysis of the effects of KA on OA

To elucidate the molecular mechanisms underlying the effects of KA in IL-1β-treated SW1353 cells (chondrocyte OA model), RNA sequencing was conducted to identify DEGs among samples from the normal group, the OA group, and the OA group treated with KA. As depicted in Supplementary Figure 1A, http://links.lww.com/CM9/C566, compared with the control group, the OA group had 1415 DEGs. KA treatment in the OA group modulated the expression of 230 genes relative to the OA group. In total, 1397 DEGs were identified when comparing the OA + KA group with the control group.

GO enrichment analysis of the 230 DEGs regulated by KA revealed the top 20 GO terms at a significance level of P <0.05 [Supplementary Figure 1B, http://links.lww.com/CM9/C566]. These included 14 biological processes, such as the cellular response to oxygen levels, the cellular response to hypoxia, and the response to decreased oxygen levels, among others. Additionally, six molecular functions were highlighted, primarily involving cytokine activity, cytokine receptor binding, and growth factor receptor binding, among others [Supplementary Figure 1B, http://links.lww.com/CM9/C566].

Further KEGG pathway enrichment analysis of the KA-regulated DEGs revealed the top 20 signaling pathways at a significance level of P <0.05, with a strong emphasis on the HIF-1 signaling pathway [Supplementary Figure 1C, http://links.lww.com/CM9/C566]. Notably, the DEGs included down-regulated genes, such as SLC2A1, ENO2, PDK1, EGLN3, IL6, PFKFB3, and Tie-2 [Supplementary Figure 1D, http://links.lww.com/CM9/C566]. GSEA of the HIF-1 signaling pathway indicated that 28 genes were significantly enriched at the leading edge of HSA04066 (NES = −2.0483, P <0.01) [Supplementary Figure 1E, http://links.lww.com/CM9/C566].

Given the critical role of the HIF-1 signaling pathway, subsequent RT-PCR validation confirmed the observed changes in messenger RNA (mRNA) expression levels. Specifically, SLC2A1, ENO2, PDK1, Egl-9 family hypoxia inducible factor 3 (EGLN3), and IL6 expression were significantly lower in the OA group following KA treatment than in the untreated OA group (P <0.05) [Supplementary Figure 1F, http://links.lww.com/CM9/C566].

These transcriptomic findings provide insights into the molecular pathways through which KA exerts its anti-inflammatory and chondroprotective effects in the context of OA, highlighting the potential of KA as a therapeutic agent for OA.

Changes in HIF-1 signaling pathway expression in OA with KA treatment and validation

The mRNA expression levels of key inflammatory and hypoxia-related factors, such as IFN-γ, NF-κB, HIF-1α, VEGF, iNOS, Tie2, and PFKFB3, were significantly lower in the OA group treated with KA than in the untreated OA group (P <0.05) [Figure 6A and B]. These findings suggest that the protective mechanism of KA in OA may be associated with the modulation of these targets and signaling pathways.

Figure 6.

Figure 6

Open in a new tab

Validation of the HIF-1 and NF-κB signaling pathways in the KA-treated SW1353 cell model. (A) The mRNA levels of IFN-γ, NF-κB, HIF-1α, VEGF, and iNOS in the SW1353 cell model were assayed by reverse transcription-polymerase chain reaction. (B) The mRNA levels of Tie2 and PFKFB3 in the SW1353 cell model. (C) Representative Western blotting results for IFN-γ, NF-κB, HIF-1α, VEGF, and iNOS in the SW1353 cell model. (D) Quantification of the Western blotting data from (C). (E) Representative Western blot results for Tie2 and PFKFB3 in the SW1353 cell model. (F) Quantification of the Western blotting data from (E). (G) Representative immunofluorescence results for IFN-γ, NF-κB, HIF-1α, VEGF, and iNOS in the SW1353 cell model. (H) Quantification of the immunofluorescence data from (G). (I) Representative Western blotting results for p-p65, p65, p-IκBα, and IκBα in the SW1353 cell model. (J) Quantification of the Western blotting data from (I). The data are shown as the mean ± SD. n = 3, *P <0.0001; †P <0.05; ‡P <0.01; §P <0.001. HIF-1α: Hypoxia-inducible factor 1-alpha; IFN-γ: Interferon-gamma; IL-1β: Interleukin-1 beta; iNOS: Inducible nitric oxide synthase; KA: Kaempferide; mRNA: Messenger RNA; MW: Molecular weight; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; OA: Osteoarthritis; PFKFB3: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3; p-IκBα: Phosphorylated IBα; p-p65: Phosphorylated p65; SD: Standard deviation; Tie2: Tyrosine kinase with immunoglobulin-like and EGF-like domains 2; VEGF: Vascular endothelial growth factor.

The protein expression levels of these same key target genes in the HIF-1 signaling pathway were found to be elevated in the OA group relative to those in the control group and were subsequently reduced upon KA treatment (P <0.05) [Figure 6C–F]. Immunofluorescence analysis further supported these findings, revealing an increase in key proteins in the HIF-1 signaling pathway in the OA group, which was mitigated by KA treatment [Figure 6G and H]. These results collectively confirm that the mechanism by which KA reduces OA damage may indeed be linked to the HIF-1 signaling pathway.

To further explore the role of NF-κB in this context, the expression of p65, phosphorylated p65 (p-p65), IκBα, and phosphorylated IκBα (p-IκBα) was examined. The results indicated that KA may attenuate the phosphorylation of p65 and IκBα, suggesting a reduction in NF-κB activation [Figure 6I and J].

To validate the involvement of the HIF-1 signaling pathway, exogenous IFN-γ, and emapalumab, an anti-IFNγ antibody that blocks its function, were employed. Treatment with exogenous IFN-γ led to increases in the mRNA and protein expression levels of IFN-γ, NF-κB, HIF-1α, VEGF, and iNOS (all P <0.05), which were subsequently decreased by KA or emapalumab treatment (all P <0.05) [Figure 7A–C]. Moreover, the combination of KA and emapalumab treatment resulted in a more pronounced reduction in the expression of these genes (all P <0.001) [Figure 7A–C].

Figure 7.

Figure 7

Open in a new tab

Validation of the HIF-1 signaling pathway in the KA-treated SW1353 cell model. (A) The mRNA levels of IFN-γ, NF-κB, HIF-1α, VEGF, and iNOS in the SW1353 cell model treated with exogenous IFN-γ, emapalumab, IFN-γ+KA, or emapalumab+KA. (B) Western blotting data for IFN-γ, NF-κB, HIF-1α, VEGF, and iNOS in the SW1353 cell model treated with exogenous IFN-γ, emapalumab, IFN-γ+KA, or emapalumab+KA. (C) Quantification of the Western blotting data from (B). (D) The mRNA levels of HIF-1α, VEGF, iNOS, Tie2, and PFKFB3 in SW1353 cells treated with DMOG, LW6, DMOG+KA, or LW6+KA. (E) Western blotting data for HIF-1α, VEGF, iNOS, Tie2, and PFKFB3 in SW1353 cells treated with DMOG, LW6, DMOG+KA, or LW6+KA. (F) Quantification of the Western blotting data from (E). The data are shown as the mean ± SD. n = 3, *P <0.01; †P <0.0001; ‡P <0.05; §P <0.001. The concentrations utilized in the experiments were as follows: KA (90 μmol/L), emapalumab (2 pmol/L), IFN-γ (1 ng/mL), IL-1β (10 ng/mL), LW6 (10 μmol/L), and DMOG (90 μmol/L). DMOG: Dimethyloxalylglycine; HIF-1α: Hypoxia-inducible factor 1-alpha; IFN-γ: Interferon-gamma; IL-1β: Interleukin-1 beta; iNOS: Inducible nitric oxide synthase; KA: Kaempferide; mRNA: Messenger RNA; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; PFKFB3: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3; SD: Standard deviation; Tie2: Tyrosine kinase with immunoglobulin-like and EGF-like domains 2; VEGF: Vascular endothelial growth factor.

Additionally, to specifically target HIF-1α, DMOG, an agonist of HIF-1α, and LW6, an inhibitor of HIF-1α, were used. DMOG treatment increased the expression of mRNAs and proteins in the HIF-1α pathway, whereas KA treatment had the opposite effect [Figure 7D–F]. LW6 treatment also decreased the expression of mRNAs and proteins in the HIF-1α pathway, and this effect was further enhanced by the addition of KA [Figure 7D–F].

These comprehensive findings strongly support the conclusion that HIF-1 is the primary signaling pathway through which KA exerts its therapeutic effects in OA, highlighting the potential of targeting this pathway for the treatment of OA.

Discussion

KA, a multifaceted natural flavanol compound, has significant potential in the treatment of OA because of its diverse pharmacological properties.[37] This study aimed to elucidate the core targets and mechanisms by which KA ameliorates OA. Our investigations revealed that KA has the potential to alleviate OA injury and enhance gait behavior in model rats by significantly downregulating the expression of inflammatory cytokines, particularly MMP-2 and MMP-3.[38–41]

MMPs, a class of proteolytic enzymes pivotal in OA progression, promote the degradation of cartilage matrix components, such as collagen II, a critical factor in balancing the synthesis and degradation of the extracellular matrix (ECM) in articular cartilage. We observed that KA effectively counters IL-1β-induced MMPs and other inflammatory cytokines, including LOX, iNOS, PGE2, LTB4, and COX-2, ultimately inhibiting collagen degradation, a clear indication of its anticatabolic effects through inflammatory cytokine suppression. These findings underscore the potential of KA in mitigating the chondrocyte dysfunction associated with OA.

Although the results of the in vitro experiments were informative, our study also utilized an in vivo OA animal model to thoroughly investigate the protective effects of KA. Morphological and ultrastructural observations revealed that KA significantly reduced cartilage degradation and lowered Mankin’s histological score in papain-induced OA rats. Additionally, the results indicated that KA substantially improved gait behavior in rats. These findings suggest the potential utility of KA for OA treatment.

DXM, renowned for its anti-inflammatory properties, is commonly used for intra-articular injection in OA treatment. It effectively reduces OA-related inflammation and slows cartilage degradation, making it a standard positive control in OA treatment studies. Our findings revealed that KA has comparable, if not superior, anti-inflammatory and chondroprotective effects compared with DXM, underscoring its potential as an alternative therapeutic agent for OA.

Transcriptomic analysis was employed to explore the mechanism of KA in treating OA. We detected differential expression of genes, such as SLC2A1, ENO2, PDK1, EGLN3, IL6, Tie2, and PFKFB3 in the KA-treated OA group compared with the OA group alone. These findings suggest that the mechanism by which KA reduces OA damage may be related to the HIF-1 signaling pathway. The mRNA or protein expression of key target genes (IFN-γ, NF-κB, HIF-1α, VEGF, iNOS, Tie2, and PFKFB3) in the HIF-1 signaling pathway was significantly increased in the OA group but decreased with KA treatment. Previous evidence indicates that HIF-1α in the HIF-1 signaling pathway is extensively localized in early chondrogenesis, is expressed primarily in dedifferentiated chondrocytes,[42–44] and influences SOX9 expression during skeletogenesis.[45] Posttreatment in the OA model resulted in reduced levels of these genes and increased expression of SOX9, COL2A1, and ACAN, suggesting the potential importance of the HIF-1 signaling pathway in the therapeutic effects of KA on OA.

Furthermore, our study demonstrated that KA significantly decreased the expression levels of Tie2 and PFKFB3 in the SW1353 cell model, suggesting that KA may affect OA treatment by affecting angiogenesis and glucose metabolism pathways.[46,47]

Our investigation also focused on the role of IFN-γ as a pivotal mediator within the HIF-1 signaling pathway. IFN-γ serves as a crucial inflammatory mediator and plays roles in immune regulation, antiviral defense, and antitumor activities.[48] Our results revealed that IFN-γ has an important role in regulating the expression of key genes within the HIF-1 signaling pathway, demonstrating its potential as an upstream factor in KA-mediated OA treatment.

In addition to the HIF-1 signaling pathway, we explored the involvement of the NF-κB pathway. The Western blotting data highlighted the capacity of KA to significantly inhibit the phosphorylation of p65 and IκBα, which aligns with the observed reduction in the synthesis of inflammatory factors.

Despite all of the above, our study has limitations. First, no apparent pathways were associated with OA in the KEGG of the network pharmacology process, possibly due to outdated information in the databases used. Second, the in vitro study utilized SW1353 cells (a human chondrosarcoma cell line), which may not fully represent normal chondrocytes. Future studies should focus on utilizing various chondrocyte models, including primary chondrocytes from animals or other relevant models, to more accurately elucidate the mechanisms and roles of KA in OA progression.

In conclusion, our study demonstrated the anti-inflammatory and disease-modifying effects of KA in chondrocytes and rats, suggesting its potential as a beneficial therapeutic agent for patients with OA. However, further research is needed to address challenges, such as drug stability, optimal dosage determination, and long-term safety, to ensure its safe and effective utilization in clinical practice.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 82102531 and 82172410).

Conflicts of interest

None.

Supplementary Material

cm9-138-2813-s001.pdf (297.4KB, pdf)
Open in a new tab

Footnotes

Xianjie Wei, Hesuyuan Huang, and Ping Yuan contributed equally to this work.

How to cite this article: Wei XJ, Huang HSY, Yuan P, Xie PS, Zhang KS, Guan ZP. Kaempferide inhibited progression of osteoarthritis by targeting the HIF-1 signaling pathway. Chin Med J 2025;138:2813–2823. doi: 10.1097/CM9.0000000000003762

References

  • 1.Katz JN, Arant KR, Loeser RF. Diagnosis and treatment of hip and knee osteoarthritis: A review. JAMA 2021;325:568–578. doi: 10.1001/jama.2020.22171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Collaborators GBDO . Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: A systematic analysis for the Global Burden of Disease Study 2021. Lancet Rheumatol 2023;5:e508–e522. doi: 10.1016/S2665-9913(23)00163-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Diseases GBD, Injuries C. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020;396:1204–1222. doi: 10.1016/S0140-6736(20)30925-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Litwic A, Edwards MH, Dennison EM, Cooper C. Epidemiology and burden of osteoarthritis. Br Med Bull 2013;105:185–199. doi: 10.1093/bmb/lds038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Losina E Michl G Collins JE Hunter DJ Jordan JM Yelin E, et al. Model-based evaluation of cost-effectiveness of nerve growth factor inhibitors in knee osteoarthritis: Impact of drug cost, toxicity, and means of administration. Osteoarthritis Cartilage 2016;24:776–785. doi: 10.1016/j.joca.2015.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Long H, Zeng C, Shi Y, Wang H, Xie D, Lei G. Length of stay and inpatient charges of total knee arthroplasty in China: Analysis of a national database. Chin Med J 2023;136:2050–2057. doi: 10.1097/CM9.0000000000002220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Sharma L. Osteoarthritis of the knee. N Engl J Med 2021;384:51–59. doi: 10.1056/NEJMcp1903768. [DOI] [PubMed] [Google Scholar]
  • 8.Grandi FC, Bhutani N. Epigenetic Therapies for osteoarthritis. Trends Pharmacol Sci 2020;41:557–569. doi: 10.1016/j.tips.2020.05.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Perruccio AV, Young JJ, Wilfong JM, Denise Power J, Canizares M, Badley EM. Osteoarthritis year in review 2023: Epidemiology & therapy. Osteoarthritis Cartilage 2024;32:159–165. doi: 10.1016/j.joca.2023.11.012. [DOI] [PubMed] [Google Scholar]
  • 10.Zhu M Chen M Zuscik M Wu Q Wang YJ Rosier RN, et al. Inhibition of beta-catenin signaling in articular chondrocytes results in articular cartilage destruction. Arthritis Rheum 2008;58:2053–2064. doi: 10.1002/art.23614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ulivi V, Giannoni P, Gentili C, Cancedda R, Descalzi F. p38/NF-kB-dependent expression of COX-2 during differentiation and inflammatory response of chondrocytes. J Cell Biochem 2008;104:1393–1406. doi: 10.1002/jcb.21717. [DOI] [PubMed] [Google Scholar]
  • 12.Terkeltaub R, Yang B, Lotz M, Liu-Bryan R. Chondrocyte AMP-activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha. Arthritis Rheum 2011;63:1928–1937. doi: 10.1002/art.30333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ge Y Zhou S Li Y Wang Z Chen S Xia T, et al. Estrogen prevents articular cartilage destruction in a mouse model of AMPK deficiency via ERK-mTOR pathway. Ann Transl Med 2019;7:336. doi: 10.21037/atm.2019.06.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Pal B, Endisha H, Zhang Y, Kapoor M. mTOR: A potential therapeutic target in osteoarthritis? Drugs R D 2015;15:27–36. doi: 10.1007/s40268-015-0082-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Bouaziz W Sigaux J Modrowski D Devignes CS Funck-Brentano T Richette P, et al. Interaction of HIF1alpha and beta-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice. Proc Natl Acad Sci U S A 2016;113:5453–5458. doi: 10.1073/pnas.1514854113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Lian C Wang X Qiu X Wu Z Gao B Liu L, et al. Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin beta1-SMAD1 interaction. Bone Res 2019;7:8. doi: 10.1038/s41413-019-0046-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Thielen NGM, van der Kraan PM, van Caam APM. TGFbeta/BMP signaling pathway in cartilage homeostasis. Cells 2019;8:969. doi: 10.3390/cells8090969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Im HJ Muddasani P Natarajan V Schmid TM Block JA Davis F, et al. Basic fibroblast growth factor stimulates matrix metalloproteinase-13 via the molecular cross-talk between the mitogen-activated protein kinases and protein kinase Cdelta pathways in human adult articular chondrocytes. J Biol Chem 2007;282:11110–11121. doi: 10.1074/jbc.M609040200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Liu Z Chen J Mirando AJ Wang C Zuscik MJ O’Keefe RJ, et al. A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis. Sci Signal 2015;8:ra71. doi: 10.1126/scisignal.aaa3792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Pettenuzzo S Arduino A Belluzzi E Pozzuoli A Fontanella CG Ruggieri P, et al. Biomechanics of chondrocytes and chondrons in healthy conditions and osteoarthritis: A review of the mechanical characterisations at the microscale. Biomedicines 2023;11:1942. doi: 10.3390/biomedicines11071942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wisniewska E Laue D Spinnen J Kuhrt L Kohl B Bussmann P, et al. Infrapatellar fat pad modulates osteoarthritis-associated cytokine and mmp expression in human articular chondrocytes. Cells 2023;12:2850. doi: 10.3390/cells12242850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Al-Khayri JM, Sahana GR, Nagella P, Joseph BV, Alessa FM, Al-Mssallem MQ. Flavonoids as potential anti-inflammatory molecules: A review. Molecules 2022;27:2901. doi: 10.3390/molecules27092901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yue ME, Jiang TF, Shi YP. Fast determination of flavonoids in Hippophae rhamnoides and its medicinal preparation by capillary zone electrophoresis using dimethyl-beta-cyclodextrin as modifier. Talanta 2004;62:695–699. doi: 10.1016/j.talanta.2003.09.024. [DOI] [PubMed] [Google Scholar]
  • 24.Zhang Q, Cui H. Simultaneous determination of quercetin, kaempferol, and isorhamnetin in phytopharmaceuticals of Hippophae rhamnoides L. by high-performance liquid chromatography with chemiluminescence detection. J Sep Sci 2005;28:1171–1178. doi: 10.1002/jssc.200500055. [DOI] [PubMed] [Google Scholar]
  • 25.Pundir S Garg P Dviwedi A Ali A Kapoor VK Kapoor D, et al. Ethnomedicinal uses, phytochemistry and dermatological effects of Hippophae rhamnoides L.: A review. J Ethnopharmacol 2021;266:113434. doi: 10.1016/j.jep.2020.113434. [DOI] [PubMed] [Google Scholar]
  • 26.Jiao Z Xu W Zheng J Shen P Qin A Zhang S, et al. Kaempferide prevents titanium particle induced osteolysis by suppressing JNK activation during osteoclast formation. Sci Rep 2017;7:16665. doi: 10.1038/s41598-017-16853-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Wang D Zhang X Li D Hao W Meng F Wang B, et al. Kaempferide protects against myocardial ischemia/reperfusion injury through activation of the PI3K/Akt/GSK-3beta pathway. Mediators Inflamm 2017;2017:5278218. doi: 10.1155/2017/5278218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Gebauer M Saas J Sohler F Haag J Soder S Pieper M, et al. Comparison of the chondrosarcoma cell line SW1353 with primary human adult articular chondrocytes with regard to their gene expression profile and reactivity to IL-1beta. Osteoarthritis Cartilage 2005;13:697–708. doi: 10.1016/j.joca.2005.04.004. [DOI] [PubMed] [Google Scholar]
  • 29.Xiao J Luo C Li A Cai F Wang Y Pan X, et al. Icariin inhibits chondrocyte ferroptosis and alleviates osteoarthritis by enhancing the SLC7A11/GPX4 signaling. Int Immunopharmacol 2024;133:112010. doi: 10.1016/j.intimp.2024.112010. [DOI] [PubMed] [Google Scholar]
  • 30.Pang KL Chow YY Leong LM Law JX Ghafar NA Soelaiman IN, et al. Establishing SW1353 chondrocytes as a cellular model of chondrolysis. Life (Basel) 2021;11:272. doi: 10.3390/life11040272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Murat N, Karadam B, Ozkal S, Karatosun V, Gidener S. Quantification of papain-induced rat osteoarthritis in relation to time with the Mankin score. Acta Orthop Traumatol Turc 2007;41:233–237. [PubMed] [Google Scholar]
  • 32.Mori A, Takagi A, Ikeda Y, Yamamoto A. Improved method for direct DNA sequencing of the human lipoprotein lipase gene using an auto DNA sequencer. Clin Biochem 2000;33:323–327. doi: 10.1016/s0009-9120(00)00073-4. [DOI] [PubMed] [Google Scholar]
  • 33.Zeng L Wang W Rong XF Zhong Y Jia P Zhou GQ, et al. Chondroprotective effects and multi-target mechanisms of Icariin in IL-1 beta-induced human SW 1353 chondrosarcoma cells and a rat osteoarthritis model. Int Immunopharmacol 2014;18:175–181. doi: 10.1016/j.intimp.2013.11.021. [DOI] [PubMed] [Google Scholar]
  • 34.Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016;7:27–31. doi: 10.4103/0976-0105.177703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Hu Y, Gui Z, Zhou Y, Xia L, Lin K, Xu Y. Quercetin alleviates rat osteoarthritis by inhibiting inflammation and apoptosis of chondrocytes, modulating synovial macrophages polarization to M2 macrophages. Free Radic Biol Med 2019;145:146–160. doi: 10.1016/j.freeradbiomed.2019.09.024. [DOI] [PubMed] [Google Scholar]
  • 36.Kawasaki K, Fushimi T, Nakamura J, Ota N. Guava leaf extract suppresses osteoarthritis progression in a rat anterior cruciate ligament transection model. Food Sci Nutr 2018;6:800–805. doi: 10.1002/fsn3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Choi JK, Kwon OY, Lee SH. Kaempferide prevents photoaging of ultraviolet-B irradiated NIH-3T3 cells and mouse skin via regulating the reactive oxygen species-mediated signalings. Antioxidants (Basel) 2022;12:11. doi: 10.3390/antiox12010011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mehana EE, Khafaga AF, El-Blehi SS. The role of matrix metalloproteinases in osteoarthritis pathogenesis: An updated review. Life Sci 2019;234:116786. doi: 10.1016/j.lfs.2019.116786. [DOI] [PubMed] [Google Scholar]
  • 39.Wang T, He C. Pro-inflammatory cytokines: The link between obesity and osteoarthritis. Cytokine Growth Factor Rev 2018;44:38–50. doi: 10.1016/j.cytogfr.2018.10.002. [DOI] [PubMed] [Google Scholar]
  • 40.Fei J, Liang B, Jiang C, Ni H, Wang L. Luteolin inhibits IL-1beta-induced in flammation in rat chondrocytes and attenuates osteoarthritis progression in a rat model. Biomed Pharmacother 2019;109:1586–1592. doi: 10.1016/j.biopha.2018.09.161. [DOI] [PubMed] [Google Scholar]
  • 41.Li Z Liu B Zhao D Wang B Liu Y Zhang Y, et al. Protective effects of Nebivolol against interleukin-1beta (IL-1beta)-induced type II collagen destruction mediated by matrix metalloproteinase-13 (MMP-13). Cell Stress Chaperones 2017;22:767–774. doi: 10.1007/s12192-017-0805-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Hu S Zhang C Ni L Huang C Chen D Shi K, et al. Stabilization of HIF-1alpha alleviates osteoarthritis via enhancing mitophagy. Cell Death Dis 2020;11:481. doi: 10.1038/s41419-020-2680-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Zhang FJ, Luo W, Lei GH. Role of HIF-1alpha and HIF-2alpha in osteoarthritis. Joint Bone Spine 2015;82:144–147. doi: 10.1016/j.jbspin.2014.10.003. [DOI] [PubMed] [Google Scholar]
  • 44.Yudoh K, Nakamura H, Masuko-Hongo K, Kato T, Nishioka K. Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1 alpha in articular chondrocytes: Involvement of HIF-1 alpha in the pathogenesis of osteoarthritis. Arthritis Res Ther 2005;7:R904. doi: 10.1186/ar1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Zhang C, Yang F, Cornelia R, Tang W, Swisher S, Kim H. Hypoxia-inducible factor-1 is a positive regulator of Sox9 activity in femoral head osteonecrosis. Bone 2011;48:507–513. doi: 10.1016/j.bone.2010.10.006. [DOI] [PubMed] [Google Scholar]
  • 46.Sarkar K Rey S Zhang X Sebastian R Marti GP Fox-Talbot K, et al. Tie2-dependent knockout of HIF-1 impairs burn wound vascularization and homing of bone marrow-derived angiogenic cells. Cardiovasc Res 2012;93:162–169. doi: 10.1093/cvr/cvr282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Xu C, Tang Y, Yang H, Jiang S, Peng W, Xie R. Harpagide inhibits the TNF-alpha-induced inflammatory response in rat articular chondrocytes by the glycolytic pathways for alleviating osteoarthritis. Int Immunopharmacol 2023;127:111406. doi: 10.1016/j.intimp.2023.111406. [DOI] [PubMed] [Google Scholar]
  • 48.Wang S, Umrath F, Cen W, Salgado AJ, Reinert S, Alexander D. Pre-conditioning with IFN-gamma and hypoxia enhances the angiogenic potential of iPSC-Derived MSC secretome. Cells 2022;11:988. doi: 10.3390/cells11060988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Chinese Medical Journal are provided here courtesy of Wolters Kluwer Health

RESOURCES