Ginkgetin delays the progression of osteoarthritis by inhibiting the NF-κB and MAPK signaling pathways.

doi:10.21203/rs.3.rs-5315063/v1

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Research Article

Ginkgetin delays the progression of osteoarthritis by inhibiting the NF-κB and MAPK signaling pathways.

https://doi.org/10.21203/rs.3.rs-5315063/v1

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Background

Osteoarthritis (OA) is considered an advancing chronic degenerative joint disease, leading to severe physical functional impairment of patients. Its development is closely related to increased inflammation and oxidative stress within the joint. Ginkgetin (GK), a natural non-toxic chemical, has proven anti-inflammatory, antioxidant, anti-tumor, and neuroprotective effects.

Methods

First, this study utilizes network pharmacology to explore the intrinsic connection between GK and OA. In vitro, SW1353 human cartilage cells were stimulated with Tert-butyl hydrogen peroxide (TBHP), and different GK concentrations were pre-treated to evaluate its protective effects. GK's anti-inflammatory and antioxidative effects were comprehensively assessed via MTT assay, western blot, cell immunofluorescence, ELISA, and transcriptome sequencing. Potential underlying mechanisms were also explored. In vivo, OA was induced in rats via anterior cruciate ligament transection (ACLT), and GK's impact on cartilage protection was further assessed via histological analysis and western blot.

Results

Network pharmacology has revealed that GK regulates OA via several key pathways, especially NF-κB, HIF-1, PI3K-AKT, and substances like reactive oxygen species. In vitro experiments showed GK effectively reverses oxidative stress damage from TBHP, inhibits inflammatory factor release, and protects cellular matrix (ECM) from degradation. These functions may be achieved via the NF-κB and MAPK signaling pathways. In vivo experiments showed GK significantly reduced proteoglycan loss from ACLT and inhibited matrix metalloproteinase 13 (MMP13) and glycan protease 5 (ADAMTS5) production, effectively preventing cartilage degeneration in rats.

Conclusion

The research findings indicate that GK is a novel approach for the treatment of OA.

Ginkgetin

Osteoarthritis

Network pharmacology

oxidative stress

inflammation

NF-κB

MAPK

Osteoarthritis (OA), a chronic degenerative joint disorder, can potentially lead to severe physical disability. Globally, OA has affected over 500 million people, posing a heavy socioeconomic burden[1]. OA is triggered by a combination of factors including excessive mechanical loading, inflammation, obesity, aging, and genetic factors[2–4]. Frontline research reveals that joint cartilage degradation and erosion, synovial membrane proliferation and inflammation, abnormal synovial joint angiogenesis, subchondral bone turmoil, and ligament/tendon instability can independently or collectively contribute to OA onset and progression[5]. Currently, no effective OA treatment exists. Non-surgical current clinical treatments are mostly palliative, aiming to alleviate symptoms but often accompanied by numerous side effects. Surgical treatment has some effects however, its risks and instability factors warrant concern. These treatments don't fundamentally reverse cartilage degradation or halt disease progression[5, 6]. Therefore, developing novel drugs with low toxicity and few side effects to intervene in OA processes is imperative.

Although the specific pathogenesis of OA remains elusive, recent research has revealed that inflammatory response and oxidative stress generation in joints are directly related to OA progression[7]. Under normal conditions, articular cartilage primarily comprises cartilage cells and their secreted extracellular matrix (ECM). These cells produce various ECM components, providing a conducive environment for cell survival. This mutually reinforcing mechanism maintains functional and structural balance in articular cartilage, but can be disrupted by oxidative stress and inflammation[8].

Chronic inflammation is one of the pathological processes of OA[9]. Chondrocytes, both sources of pro-inflammatory cytokines in OA and their target sites, are sensitive to environmental changes. Under unfavorable conditions, chondrocytes generate excessive pro-inflammatory cytokines and chemoattractants[10], which subsequently exert effects by inducing oxidative stress, promoting chondrocyte ECM transformation, and triggering apoptosis[11, 12]. These factors crucially influence osteoarthritis occurrence and progression. Generally, reactive oxygen species (ROS) exist in cells at low levels, playing vital roles in maintaining homeostasis and function. However, oxidative stress occurs due to excessive ROS generation, exceeding effective clearance by the cell's antioxidant defense system[13]. In chondrocytes, excessive ROS generation-induced oxidative stress is a major factor influencing osteoarthritis pathogenesis[14]. Numerous studies have shown that when chondrocytes are exposed to hydrogen peroxide (H2O2), Tert-butyl hydrogen peroxide (TBHP), or other pro-oxidants, there is increased inflammation and cell apoptosis, while also accelerating cartilage matrix degradation and inhibiting synthesis[15–17]. In conclusion, oxidative stress and inflammatory processes negatively affect joint health and function. Interventions targeting this mechanism could significantly impact OA treatment strategies.

In recent years, natural products have garnered significant attention in biomedical and pharmaceutical research due to their rich therapeutic potential and low toxicity[18–20]. Since the extraction of Ginkgetin (GK) from Ginkgo biloba leaves in 1929, its diverse biological activities have made it a hot research topic. These activities include anti-inflammatory, antioxidative, antimicrobial, anticancer, antiatherosclerotic, liver protective, and neuroprotective effects[21]. Studies by Li et al. revealed GK effectively protects neurons from cerebral ischemia/reperfusion injury by reducing inflammatory cytokines like iNOS and COX-2, inhibiting TLR4/NF-κB signaling[22]. Jeong et al.'s research showed GK clears reactive oxygen species, suppresses oxidative stress by maintaining antioxidant enzyme activity or inhibiting ERK1/2 activation induced by glutamate[23]. Tian et al. found GK inhibits PI3K/Akt-mediated apoptosis, reduces cleaved-caspase 3 levels, decreases Bax, and increases Bcl-2 expression, effectively inhibiting apoptosis and reducing infrared radiation-induced injury severity in rats[24]. However, GK's biological function in cartilage cells and mechanism in OA development remain unclear.

Recent studies have further investigated the biological functions and mechanisms of GK[25–27]. However, reports on its therapeutic benefits for degenerative OA remain scarce. This study utilizes network pharmacology and transcriptomics methods, using TBHP induced SW1353 human chondrocytes as an in vitro OA oxidative stress model[28]. The goal is to explore GK's potential to inhibit inflammation, protect against oxidative damage, and slow ECM degradation. Moreover, the study evaluates GK's protective effect in an ACLT (anterior cruciate ligament transection)-induced OA rat model after intra-articular administration[29]. Through this research series, the article further unveils GK's potential biological functions and mechanism in treating degenerative OA, seeking novel insights for OA treatment strategies.

2.1 Network pharmacology analysis

2.1.1 Ginkgetin target prediction

The two databases of SwissTargetPrediction (http://www.swisstargetprediction.ch/) and SuperPred (https://prediction.charite.de/) were used to search the complete target profile of Ginkgetin. The Uniprot (https://www.uniprot.org/)database was then used for calibration. During calibration, human-derived genes were removed and invalid duplicate targets were eliminated, resulting in standardized gene names.

2.2.2 Acquisition of osteoarthritis-related targets

By conducting a keyword search in the GeneCards (https://www.genecards.org/) and OMIM (https://www.omim.org/) databases using the term "osteoarthritis", we acquired associated target genes related to this disease. Subsequently, all target genes obtained from these databases were compiled into an Excel sheet, and redundant entries were removed. Finally, the UniProt database was used to verify and correct information on these disease-related target genes, ensuring accuracy and reliability of the data.

2.2.3 Drug-disease target prediction

By mapping the identified drug component targets with disease-related targets and drawing Venn diagrams, we subsequently constructed the "drug-target" network graph using Cytoscape 3.7.2 software.

2.2.4 Construction of target protein interaction network

In order to further study the protein-protein interaction of Ginkgetin in the treatment of osteoarthritis, the drug-intersection gene was uploaded to the protein interaction network construction (PPI) database String (https://string-db.org/) for protein interaction network construction. The species was set to "Homo sapiens" and the minimum interaction score was set to 0.4 to ensure confidence in the study. The rest of the parameters were kept as the default settings. The results were stored in TSV format and imported into Cytoscape 3.7.2. The network was analyzed using Cytoscape's Network Analyzer, and the analysis results were saved. The node size and color reflect the Degree size, with larger nodes corresponding to higher Degree values. The edge thickness reflects the Combinescore size, with thicker edges corresponding to higher Combinescore values. The core target was selected to create a protein interaction network diagram.

2.2.5 GO enrichment analysis and KEGG pathway analysis

The drug-disease intersection gene was uploaded to the DAVID database (https://david.ncifcrf.gov/summary.jsp), with the gene identifier set as OFFICIAL_GENE_SYMBOL and species as Homo Sapiens. Using DAVID 6.8, GO gene function was annotated to elucidate the role of Ginkgetin in osteoarthritis treatment from three aspects: biologicalProcess (BP), cellular component (CC), and molecular function (MF). To further clarify Ginkgetin's target in osteoarthritis treatment, KEGG pathway enrichment analysis was performed on the signaling pathway. The top 10 BP, CC, and MF pathways in GO function, and 20 pathways related to osteoarthritis were selected as the main gene function enrichment processes and signaling pathways of Ginkgetin in osteoarthritis treatment. The mechanism of action of Ginkgetin in osteoarthritis treatment was predicted.

2.2 Cell culture and reagents

The human chondrocyte line, SW1353, is a commonly used and mature cell line for replacing primary chondrocytes in research[30]. The cell line was provided by Wuhan Proxel Life Science and Technology Co., Ltd. and cultured in DMEM medium with 10% fetal bovine serum and 1% bispecific antibody at 37°C and 5% CO2.

Ginkgetin (GK, T4S2126) was purchased from TargetMol (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO; Solarbio, China) to prepare a 30 mM stock solution for subsequent cell experiments. A separate solution was prepared by dissolving GK in 40% PEG300 (10mg/mL) for intra-articular injection in anterior cruciate ligament transection (ACLT) rats. Tert-butyl hydrogen peroxide (TBHP, 416665), 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, D6883), and Dihydroethidium (DHE, 37291) were purchased from Sigma Aldrich (St. Louis, MO, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT, M158055) was purchased from Aladdin (Shanghai, China). JC-1 Mitochondrial Membrane Potential Assay Kit (40706ES60) and Hoechst 33342 (40732ES03) were purchased from Yeasen Biotechnology Co., Ltd (Shanghai, China). LDH (lactate dehydrogenase) Cytotoxicity Assay Kit (C0016), Griess assay kit (S0021S) were purchased from Beyotime Biotechnology (Shanghai, China). SOD (superoxide dismutase) activity assay kit (BC0175), CAT (catalase) activity assay kit (BC0205), MDA (malondialdehyde) content assay kit (BC0025) were purchased from Solarbio Science & Technology Co.,Ltd (Beijing, China).

2.3 Cell treatment and MTT assay

Cell viability was measured using the MTT assay. First, SW1353 human chondrocytes (cell density 5×10^4 cells/well) were seeded into 24-well plates, incubated for 24 hours, then treated with different concentrations (0, 1, 2, 5, 10, or 15 µM) of GK for 12 or 24 hours. Subsequently, 500 µL of MTT working solution diluted in serum-free medium was added per well and incubated for 2 hours to induce blue-purple formazan crystal formation. The crystals were then dissolved in DMSO. Finally, cell viability was evaluated by measuring absorbance at 490 nm. To determine appropriate TBHP stimulation conditions, cell viability was assessed under different TBHP concentrations: 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 400 µM, or 500 µM, with treatment duration of 4 hours. Unless otherwise specified, the routine practice in this experiment and subsequent studies is to pre-treat cells with 5 µM or 10 µM GK for 12 hours, followed by 300 µM TBHP stimulation for 4 hours.

2.4 Hoechst Staining AND PI Staining

Hoechst 33342 fluorescence dye was utilized to observe cells displaying apoptotic characteristics[31]. After processing the cells, 2–3 washes with phosphate-buffered saline (PBS) were performed, followed by the addition of 1 µg/mL Hoechst 33342 buffer prepared from serum-free media. The reaction lasted for 30 minutes. After washing again with PBS, the cells were observed using an inverted fluorescence microscope (Nikon, Japan) and images were captured. Since PI (Iodine-iodides) cannot penetrate the cell membrane of living cells, it can only enter the nucleus of apoptotic or dead cells, resulting in a red fluorescence[32, 33]. In this study, the concentration of the PI working solution was set to 50 µg/mL, and the staining process was carried out using the same steps as Hoechst 33342 staining.

2.5 ROS assay

H2DCFDA (10 mM) and DHE (10 mM) were diluted to a working concentration of 10 µM. After treatment, 300 µL of DCFH-DA or DHE working solution was added to each well and incubated at 37°C for 30 min. ROS levels were detected by fluorescence microscopy (Nikon, Japan), and relative fluorescence intensity was calculated using ImageJ-win64 software.

2.6 Mitochondrial menmbrane potential assay

JC-1 is widely considered an ideal fluorescent probe for assessing dynamic changes in mitochondrial membrane potential. Under normal physiological conditions, mitochondria have strong negative charge and JC-1 enters mitochondria as a polymer, resulting in strong red fluorescence. However, during apoptosis, mitochondrial depolarization reduces negative charge and JC-1 exists in the cytoplasm as a monomer, increasing green fluorescence[32]. Adhere strictly to the instructions provided in the reagent kit for each step. First, we wash each well with 1×JC-1 Assay Buffer, then add 250µL JC-1 working solution and incubate at 37℃ for 20 minutes. After incubation, we wash cells with 1×JC-1 Assay Buffer and observe under an inverted fluorescence microscope (Nikon, Japan).

2.7 Measurement of antioxidant enzyme activity and MDA content.

Using commercially available kits, the operation was carried out according to the manufacturer's instructions. First, cells were washed with PBS 1–2 times and then collected into an ice-centrifugation tube after centrifugation. After centrifugation, the supernatant was discarded, and a suitable amount of extraction solution was added. Subsequently, cells were disrupted using ultrasonic technology, and the supernatant was collected at 4°C under 8000g of centrifugation force for 10 minutes. The supernatant was then stored at -20°C for further testing. Strictly following the kit's usage instructions, the next steps were carried out, including measuring SOD and CAT activities, as well as MDA amount.

2.8 NO production assay and Enzyme-linked Immunosorbent Assay (ELISA)

By adopting a NO detection kit, we measured NO content in the culture medium supernatant via the Griess method. Collected supernatant from treated cells was combined with 100µL of reagent to generate a mixed reaction. Subsequently, absorbance at 540nm was measured and NO content was calculated based on the standard curve.

According to the manufacturer's operation guide, we used a conventional ELISA kit (BOSTER, Wuhan, China) to detect pro-inflammatory cytokines (IL-6 and TNF-α).

2.9 RNA-seq analysis and data processing

RNA-Seq was used to analyze cells in three groups: control (Con), TBHP, and TBHP + GK. Each group had three biological replicates. Total RNA was extracted with Trizol reagent (Seville Biotechnology, Wuhan, China) at ≥ 20 ng/µL per group. mRNA was isolated using the KAPA Stranded mRNA-Seq Kit, enriched with oligo-dT beads, randomly fragmented by heating, and reverse-transcribed into cDNA with strand synthesis master mix for high-throughput sequencing libraries. The library was tested and sequenced on the illumina NovaSeq 6000 platform. The raw data (rawreads) were obtained after RNA-seq sequencing, filtered, decontaminated, and mapped to the reference genome. High-quality mapped reads (MAPQ ≥ 30) were used for subsequent analyses.

2.10 Western blot analysis

Treated cells are lysed using IP buffer and then the supernatant is collected by centrifuge for further protein quantification. The leveled proteins were separated by one-step PAGE gel (Epizyme, Shanghai, China) electrophoresis and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Next, the PVDF membrane was blocked with 5% skim milk for 1 hour and then incubated with the primary antibody overnight at 4°C. Wash the membrane 3 times with TBST (10 min per wash) and then incubate with secondary antibody for 1 h at 37°C. Finally, the membrane was washed 3 times again with TBST (10 min per wash) and the images were captured on an ultra-sensitive multifunction imager (GE, Japan) and the grayscale values were analyzed with Image J software.

The antibodies used in this study are as follows: Bcl-2 (1:1000), Bax (1:1000), Bad (1:1000) Cleaved-Capase 3 (1:1000) were purchased from ABclonal Technology Co., Ltd (Wuhan, China); MMP3 (1:1000), MMP13 (1:1000), iNOS (1:1000), GAPDH (1:3000) were purchased from Abcam (Cambridge, MA, USA); COX-2(1:1000)、Collagen Ⅱ༈1:1000༉、ADAMTS-5༈1:1000༉、Aggrecan༈1:1000༉、p65༈1:1000༉、p-P65༈1:1000༉、IκBα༈1:1000༉、p- IκBα༈1:1000༉、p38༈1:1000༉、p-p38༈1:1000༉、JNK༈1:1000༉、p-JNK༈1:1000) purchased from Proteintech Group (Wuhan, China); Goat Anti-Rabbit IgG༈H + L༉was purchased from Elabscience Biotechnology Co., Ltd༈Wuhan, China༉。

2.11 Animals and in vivo experiment

In this study, 30 8-week-old Sprague-Dawley (SD) rats (weight range: 200 ~ 220g) were selected and purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. All animal procedures involved in the experiment have been reviewed and approved by the Laboratory Animal Welfare Committee of Qingdao University. To mitigate potential effects of experimental manipulation on animals, all SD rats underwent one-week environmental acclimatization prior to surgery. Afterwards, all experimental animals were randomly assigned to three groups: (1) Control group (n = 10): Only the right knee capsule was incised, but the anterior cruciate ligament was not severed; 10% chloral hydrate was injected intraperitoneally before surgery and the carrier (50 µL) dissolved GK was injected into the joint cavity twice weekly after surgery. (2) ACLT model group (n = 10): The right knee capsule was incised and the anterior cruciate ligament was cut simultaneously to induce knee instability in rats; the vehicle (50 µL) of dissolved GK was injected into the intra-articular cavity twice weekly after surgery. (3) ACLT + GK group (n = 10): Following joint capsule incision and anterior cruciate ligament shearing, GK (10mg/kg) was injected into the joint cavity twice weekly after surgery. In the eighth week of the experiment, rats were sacrificed and the right knee joint of three rats was randomly selected in each group for follow-up experiments. Joint samples were first fixed in 4% paraformaldehyde solution for 4 days, followed by 4 weeks of room temperature decalcification using 10% EDTA solution. After decalcification, the sample was further embedded. For staining and histological analysis of sections (5 µm thickness), hematoxylin-eosin (HE) staining method and modified saffron O-green cartilage staining kit (Solarbio) were used in this experiment. In another part of the experiment, protein samples were extracted from the knee joints of the remaining rats in each group for Western blot analysis.

2.12 Statistical analysis

The experiment was repeated at least three times, and results were presented as mean ± standard deviation (SD). Statistical analysis of multiple datasets was conducted using one-way ANOVA and Dunnett's multiple comparison test in GraphPad Prism version 9 software. Any statistical test with P value < 0.05 was considered statistically significant.

3.1 Results of network pharmacology analysis.

3.1.1 Prediction of Ginkgetin target in the treatment of OA

The flowchart of network pharmacology is shown in Fig. 1A, while the chemical structure of Ginkgetin (GK) is illustrated in Fig. 1B. We obtained 156 drug target points from related databases, 4547 target points related to osteoarthritis (OA), and intersected the drug target points with OA target genes to obtain 89 drug-target interaction genes for OA treatment. As depicted by the Venn diagram (Fig. 1C), the drug-target data was used to draw a graph with Cytoscape3.7.2, and the drug-target prediction result was obtained. The rectangle represents the target, while the circle represents the drug component (Fig. 1D). Subsequently, these 89 interaction target genes were imported into the String database to predict protein-protein interactions. The PPI network was drawn using Cytoscape3.7.2 software, with 82 nodes and 794 edges. As shown in Fig. 1E, the network was analyzed based on degree value, which reflects the size of the target and its color, and combinedscore value, which reflects the thickness of the edge. Among the core targets, HSP90AA1, HIF1A, PTGS2, NFKB1, BCL2, GSK3B, STAT1, CXCR4, BRD4, and BCL2L1 played a central role.

3.1.2 GO enrichment and KEGG pathway enrichment analysis results.

Based on drug-disease intersection genes, we performed gene function enrichment and KEGG pathway enrichment analyses using the DAVID database (Fig. 1F-G). In biological process (BP) analysis, key processes included positive regulation of signal transduction, cell proliferation, inflammation response, negative regulation of apoptosis, protein phosphorylation, angiogenesis, autophosphorylation, positive regulation of the ERK1-2 cascade, negative regulation of gene expression, and apoptosis. In cellular component (CC) analysis, key foci were cytoplasm, cytosol, plasma membrane, nucleus, nucleoplasm, nucleoplasm-cytoplasm, extracellular vesicles, extracellular space, extracellular region, membrane components, and high molecular weight complexes. In molecular function (MF) analysis, key processes were protein binding, homodimerization, ATP binding, enzyme binding, zinc ion binding, protein kinase activity, protein kinase binding, protein tyrosine kinase activity, and protein domain-specific binding.

KEGG analysis identified significant involvement of the NF-κB signaling pathway, HIF-1 signaling pathway, EGFR tyrosine kinase inhibitor resistance, PI3K-AKT signaling pathway, reactive oxygen species, RAS signaling pathway, p53 signaling pathway, Rap1 signaling pathway, and other related pathways.

3.2 Ginkgetin reduced the decrease in cell viability caused by TBHP in SW1353 human chondrocytes.

The MTT method was used to detect the effect of GK (1 µM, 2 µM, 5 µM, 10 µM, and 15 µM) on the cell viability of SW1353 human chondrocytes for 12 hours and 24 hours. The results are shown in Fig. 2A. GK had no significant effect on the proliferation of SW1353 human chondrocytes at concentrations below 10 µM and slightly damaged SW1353 human chondrocytes at 15 µM. Therefore, GK at concentrations of 5 µM and 10 µM were used for follow-up experiments. In order to understand the effect of TBHP on SW1353 human chondrocytes and establish suitable TBHP induction conditions, different concentrations of TBHP (100–500 µM) were used to induce SW1353 human chondrocyte injury for 4 hours. The results are shown in Fig. 2B. Different concentrations of TBHP can reduce the cell viability of SW1353 human chondrocytes, and the damage increases with the concentration. Among them, 300 µM TBHP reduced the proliferation of SW1353 human chondrocytes by more than 50% after 4 hours, so 300 µM TBHP was selected to induce SW1353 human chondrocyte damage. Finally, the protective effect of GK on TBHP-induced SW1353 human chondrocytes was evaluated. The cell viability of SW1353 human chondrocytes was increased by 25.75% and 41.06% (Fig. 2C) after pretreatment with 5 µM and 10 µM Ginkgetin for 12 hours, then 300 µM TBHP was added for 4 hours, respectively. After 4 hours of TBHP treatment, SW1353 human chondrocytes showed morphological changes, the cells shrank and decreased, and the floating cells increased. The cell morphology recovered compared with the TBHP group after pretreatment with different concentrations of GK, and the floating cells decreased (Fig. 2D). The above results suggest that GK has a certain protective effect against damage induced by TBHP on SW1353 human chondrocytes in vitro.

3.3 Ginkgetin attenuates TBHP-induced oxidative stress in SW1353 human chondrocytes

To evaluate the ex vivo antioxidant capacity of GK, a skeletal OA oxidative stress model was induced in vitro by adding TBHP[8]. Subsequently, GK was administered, and corresponding oxidative stress indicators, including lactate dehydrogenase (LDH) release, superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), mitochondrial membrane potential, and intracellular ROS level, were measured.

The rupture of mitochondrial membrane potential is considered a significant indicator of mitochondrial dysfunction. It results in defects in the electron transport chain, decreased oxygen consumption during metabolism, and ATP consumption, leading to low energy metabolism and triggering oxidative stress response[34]. JC-1, an ideal fluorescent probe, can detect and evaluate changes in mitochondrial membrane potential in cells[32]. As shown in Fig. 3A and 3B, the green fluorescence intensity of JC-1 monomers increased after TBHP treatment, whereas the red fluorescence intensity of JC-1 oligomers decreased. This indicates that TBHP treatment significantly damages the mitochondrial membrane potential of SW1353 human chondrocytes. However, GK treatment can reverse the mitochondrial membrane potential damage induced by TBHP, and its effects are dose-dependent.

2',7'-Dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine (DHE) were used to assess intracellular ROS levels. Representative images are shown in Fig. 3C and fluorescence semi-quantitative analysis results (Figure D, E) indicate the ROS fluorescence intensity of the TBHP + GK group is significantly lower than the TBHP group, decreasing with increasing concentration.

SOD and CAT, as important antioxidants, play a key role in hydrogen peroxide decomposition. MDA is the final product of lipid peroxidation, reflecting tissue oxidative damage degree[13]. LDH, a stable cytoplasmic enzyme, is normally unable to pass through cell membranes. In damaged or dead cells, it can be released into the extracellular environment. By measuring LDH, SOD, CAT, and MDA levels using relevant kits, the results are shown in Figs. 3F-I. After TBHP treatment, SOD and CAT activity decreased compared to the control group, while MDA and LDH activity increased. With GK addition, a certain level of SOD and CAT activity was restored, and MDA and LDH levels were also reduced to some extent. The above results indicate that GK can alleviate mitochondrial damage induced by TBHP and attenuate oxidative stress damage caused by TBHP.

3.4 Ginkgetin reduces apoptosis in SW1353 human chondrocytes caused by TBHP.

The important role of oxidative stress-mediated chondrocyte apoptosis in OA occurrence and progression[35, 36]. We demonstrated GK's antioxidant effects in vitro in previous experiments. Next, we determined whether GK regulates TBHP-induced apoptosis of SW1353 human chondrocytes by Western blot and immunofluorescence staining. Propidium iodide (PI) cannot penetrate the cell membrane of living cells and can only enter the nucleus of apoptotic or dead cells, producing red fluorescence[33]. The results of Fig. 4A showed that the number of apoptotic or dead cells increased after TBHP treatment of SW1353 human chondrocytes, and GK attenuated this phenomenon. Subsequently, Hoechst 33342 fluorescent staining was used to visualize cells with apoptotic features. The results showed that SW1353 chondrocytes showed morphological characteristics consistent with apoptosis after TBHP treatment, including chromatin condensation and dense fluorescence as well as bright whitish nuclei (Fig. 4B). After GK intervention, cells with apoptotic characteristics gradually decreased with increasing GK concentration. Western blot results showed that the apoptosis-related proteins Bax, Bad, and Cleaved-capase 3 were up-regulated after TBHP treatment, and the anti-apoptotic protein BCL-2 was down-regulated. However, these conditions showed a reversal trend after GK intervention (Fig. 4C-G). These results suggest that GK can inhibit TBHP-induced apoptosis of SW1353 chondrocytes.

3.5 Ginkgetin inhibited TBHP-induced production of inflammatory mediators and ECM degradation in SW1353 human chondrocytes.

In chondrocytes, TBHP can induce ROS production, inhibit proteoglycan synthesis, and promote ECM degradation, causing expression of inflammatory cytokines and matrix metalloproteinases (MMPs)[16, 35]. Therefore, TBHP-induced SW1353 human chondrocytes are a representative in vitro OA model. An inflammatory microenvironment was induced by high oxidative stress with TBHP, then treated with GK to investigate effects on TBHP-induced inflammatory mediators and ECM degradation.

To evaluate the regulatory effect of GK on inflammation in an in vitro OA model induced by TBHP, the Griess method was used to detect the reactive nitrogen radical NO, the expression of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) proteins was detected by Western Blot, and the release of inflammatory mediators TNF-α and IL-6 was detected by ELISA technology. As shown in Fig. 5A, only a small amount of NO was produced in the Con group, while the content of NO in the cell culture supernatant was significantly increased in SW1353 human chondrocytes induced by TBHP stimulation, and the release of NO was limited in a dose-dependent manner after the addition of GK pretreatment. The results of ELISA showed that GK could reduce the production of TNF-α and TL-6, inflammatory mediators of SW1353 human chondrocytes induced by TBHP (Figs. 5B, 5C). Western blot results (Fig. 5D-F) showed that TBHP successfully activated the expression of iNOS and COX-2 proteins, and the pretreatment of GK inhibited the expression of iNOS and COX-2 proteins, with a more significant effect at higher GK concentrations. The above results suggest that GK can protect SW1353 chondrocytes from inflammation caused by oxidative stress.

Western blot analysis revealed expression of ECM degradation enzymes, including ADAMTS5, MMP3, and MMP13, along with Collagen II, under influence of TBHP. Results indicate TBHP induces decrease in Collagen II protein expression while ECM degradation enzyme levels significantly increase. Additionally, our experiments demonstrated Ginkgetin inhibits expression of ADAMTS5, MMP3, and MMP13 proteins induced by TBHP, thereby promoting increase in Collagen II protein expression (Fig. 5G-K). These findings suggest GK inhibits ECM degradation induced by TBHP.

3.6 Ginkgetin was shown to influence mRNA expression in TBHP-induced SW1353 chondrocytes using RNA-seq.

To study the molecular mechanisms of GK in improving an in vitro model of TBHP-induced osteoarthritis, transcriptome sequencing was performed. First, the correlation between samples calculated by the Pearson correlation coefficient is shown in the heatmap (Fig. 6A). Redder colors indicate better sample correlation, indicating reasonable samples and reliable RNA-seq results. The PCA (Principal Component Analysis) score plot (Fig. 6B) showed clear separation between the Con and TBHP groups and between the TBHP and TBHP + GK groups (with good reproducibility within each group). These findings suggest significant alteration of SW1353 human chondrocytes (mRNA expression) and demonstrate the regulatory effect of GK on TBHP-induced OA models in vitro (mRNA expression). Differential gene (DEG) analysis was performed using a difference analysis tool, and the P-value, Q-value, and FC of each gene were calculated. The volcano map of DEGs was drawn according to the analysis, and the results are shown in the figure (Fig. 6C-D). Compared to the Con group, the TBHP group showed 4229 DEGs (including 1143 up-regulated and 3068 down-regulated). In addition, 4691 DEGs were exhibited in the TBHP + GK group (2679 up-regulated and 2012 down-regulated) compared to the TBHP group. Figure 6E shows 1626 common DEGs in the TBHP + GK vs. TBHP and TBHP vs. Con groups. Subsequently, all differentially migrated genes were used for functional enrichment, and the GO enrichment results (Fig. 6F-G) visually showed the top ten significantly enriched BP, CC, and MF items. Moreover, the enrichment results of KEGG pathway revealed multiple enrichment pathways such as inflammation, cell cycle, and apoptosis (Fig. 6H-I), providing a hint for further exploring the mechanism of GK in OA treatment.

3.7 Effect of Ginkgetin on TBHP-induced NF-κB and MAPK signaling pathways in SW1353 human chondrocytes.

The NF-κB and MAPK signaling pathways play a crucial role in regulating various aspects of chondrocyte function, such as proliferation, differentiation, apoptosis, and bone matrix metabolism[5, 37]. The transcriptional specificity of NF-κB is achieved through its interaction with the MAPK pathway[38].

Based on network pharmacology and RNA-Seq results and the crucial roles of NF-κB and MAPK signaling in OA pathogenesis and treatment, we further verified whether Ginkgetin regulates NF-κB and MAPK pathways in TBHP-induced OA models in vitro. Western Bolt assay detected NF-κB pathway-related and MAPK signaling pathway-related proteins. The results showed TBHP treatment increased phosphorylation expression of p65 and IκBα in SW1353 human chondrocytes, down-regulated IκBα, and activated the NF-κB signaling pathway in cells. Ginkgetin decreased phosphorylation expression of p65 and IκBα and increased IκBα expression (Fig. 7A-B). Similarly, TBHP stimulation significantly increased p38, JNK, and ERK phosphorylation. Ginkgetin inhibited p38, JNK, and ERK phosphorylation at different concentrations (Fig. 7C-D). These results suggest Ginkgetin inhibits the NF-κB and MAPK pathways activated by TBHP.

3.8 Ginkgetin delays the progression of OA after ACLT trauma in rats.

To explore Ginkgetin's effect on osteoarthritis (OA) in vivo, an in vivo OA model of anterior cruciate ligament transection (ACLT) in rats was constructed, similar to human OA in pathogenesis and progression. Rats were divided into sham operation, ACLT, and ACLT + GK groups based on different injection drugs. Figure 8A shows the flow chart of animal experiments by Figdraw. Histological assessment via H&E and saffron green staining revealed (Fig. 8B) relatively intact articular surfaces in the sham group with rich, evenly distributed cartilage tissue. The ACLT group exhibited more cartilage erosion and proteoglycan loss than the sham group, indicating extensive cartilage damage. However, the ACLT + GK group showed some cartilage protection compared to the ACLT group. To further clarify Ginkgetin's effect on extracellular matrix (ECM) degradation in the OA model in vivo, Western Blot assay detected protein expressions of MMP3, MMP13, and ADAMTS-5 in each group. Compared to the sham group, cartilage ECM-degrading enzymes MMP3, MMP13 and ADAMTS-5 increased in the ACLT group. However, intra-articular injection of Ginkgetin reduced MMP3, MMP13 and ADAMTS-5 protein levels (Fig. 8C-F). These results indicate Ginkgetin has potential for cartilage protection and ECM homeostasis maintenance in vivo, delaying OA progression post-ACLT trauma in rats.

Osteoarthritis (OA) is currently the most common degenerative joint disease globally and a leading cause of disability among middle-aged and elderly individuals[5]. Current treatment for OA centers on alleviating pain and other uncomfortable symptoms, but does little to halt or slow disease progression[5, 6]. Consequently, developing new OA drugs is urgently needed. Based on Ginkgetin (GK)'s beneficial properties in other diseases, we investigated its role in OA treatment. In this study, we first used network pharmacology for in-depth analysis of GK and OA, revealing potential mechanisms. Then, we utilized tert-butyl hydrogen peroxide (TBHP) to stimulate SW1353 human chondrocytes for an in vitro OA model, and concurrently, anterior cruciate ligament transection (ACLT) established a rat OA model. We further explored GK's therapeutic effects in OA at both in vitro and in vivo levels.

Ginkgetin (GK), a natural and non-toxic diflavonoid, exhibits various biological activities including anti-inflammatory and antioxidant effects [21]. Network pharmacology is a research method using bioinformatics to integrate drug action networks with biological networks, enabling systematic analysis of complex drug-organism interactions through constructing a "drug-target-pathway-disease" model[39]. In this study, we first used network pharmacology to construct a potential target network of GK and OA, and further explored related target genes. The results showed GK negatively regulates OA's inflammatory response and apoptosis, and KEGG pathway analysis revealed key roles of NF-κB and reactive oxygen species in GK-OA interactions. These findings provide important theoretical support for subsequent in vitro and in vivo experiments.

In articular cartilage, chondrocytes are the only cell type[40, 41]. Numerous studies have demonstrated a positive correlation between the degree of chondrocyte apoptosis and the severity of OA. OA-related factors, including various mechanical stresses, pro-inflammatory cytokines, and oxidative stress, can trigger chondrocyte apoptosis, resulting in a decrease in chondrocyte number. At the same time, these factors may also disrupt the balance between anabolism and catabolism in chondrocytes, such as increasing matrix metalloproteinases (MMPs) and glycanases (ADAMTS), further weakening cartilage homeostasis and exacerbating the OA process[42, 43]. The results of this study show that: TBHP exhibits higher stability than hydrogen peroxide(H2O2), so it was selected as an exogenous reactive oxygen species (ROS) donor to stimulate SW1353 human chondrocyte death and mimic the oxidative stress-induced inflammatory microenvironment in vitro. TBHP can induce apoptosis by increasing ROS levels and disrupting mitochondrial membrane potential. It can also activate the apoptosis-promoting proteins Bax and Bad, while inhibiting the activity of the anti-apoptotic protein Bcl-2, and triggering activation of the apoptosis effector molecule caspase-3. The experimental results of this study revealed that GK effectively inhibited TBHP-induced apoptosis of SW1353 human chondrocytes by reducing the protein levels of Bax, Bad, and cleaved-caspase-3, and increasing the expression of Bcl-2 protein.

Oxidative stress refers to an imbalance of oxidative and antioxidant activity in the body, resulting in the production of a large amount of intracellular ROS, considered an important factor in aging and disease[13]. Studies have shown that ROS-induced apoptosis is considered the predominant form of chondrocyte death in OA[44], particularly after joint injury, while post-traumatic ROS accumulation is primarily caused by mitochondrial dysfunction[45]. Superoxide dismutase (SOD) and catalase (CAT), as the main antioxidant enzymes in living organisms, play a vital role in maintaining the balance between oxidation and antioxidation. In chondrocytes, SOD and CAT regulate ROS levels, forming the first line of protection against oxidative damage to chondrocytes, and their activity can reflect the degree of cellular oxidative damage[8, 13]. Malondialdehyde (MDA) is a marker of lipid peroxidation. When exposed to an oxidizing agent like hydrogen peroxide, oxidative stress is induced in normal cells and lipid peroxidation occurs[46]. It has been found that the stimulation effect of TBHP can destroy the mitochondrial membrane potential, increase ROS levels and lipid peroxidation in cells, and inhibit SOD and CAT activities, thereby successfully inducing oxidative damage in cells. However, this phenomenon was reversed when GK was introduced, suggesting it is possible to protect cells from oxidative stress by increasing antioxidant enzyme activity.

This study evaluated the inflammatory response induced by oxidative stress, another important pathogenic factor in OA. Excess ROS can trigger inflammation in chondrocytes and lead to synthesis and secretion of pro-inflammatory cytokines, such as activation of nuclear factor-κB (NF-κB) and production of tumor necrosis factor-α (TNF-α)[47]. TBHP, a ROS source, induces production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in human chondrocytes[48], demonstrated in this study. iNOS and COX-2 can promote proteoglycan degradation and inhibit type II collagen synthesis by activating MMPs and ADAMTS, disrupting chondrocyte-extracellular matrix (ECM) balance and exacerbating OA development[49]. Among them, iNOS is a key enzyme in nitric oxide (NO) synthesis, and NO, as a highly reactive cytotoxic free radical, can not only inhibit type II collagen synthesis and promote its degradation, but also inhibit chondrocyte ECM synthesis[50]. In addition, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), as highly expressed inflammatory cytokines in osteoarthritic joints, positively correlate with joint inflammation severity, so their expression levels can be important reference indicators for measuring OA progression[51]. We found SW1353 human chondrocytes produced more inflammatory mediators and led to ECM degradation under TBHP stimulation, while GK intervention reduced inflammation and ECM degradation. In the in vivo model, we used the ACLT model, commonly used to induce OA in rats, to accelerate knee degeneration by artificially altering knee stability and accelerating cartilage wear. Histological analysis and Western Blot revealed GK also inhibited ECM degradation in vivo.

Based on network pharmacology and RNA-seq analysis, we focused on GK's effects on the NF-κB and MAPK signaling pathways. In the pathogenesis of OA, signaling pathways of the mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NF-κB) play crucial roles in regulating release of inflammatory mediators and changes in the ECM of cartilage cells[5, 37, 38]. Excess ROS disrupts the MAPK pathway, causing p38, JNK, and ERK phosphorylation-activation, continuously upregulating downstream MMPs, ADAMTS, and inflammatory mediators, ultimately leading to ECM degradation and inflammation[52]. The NF-κB family contains five members: RelA/p65, RelB, c-Rel, NF-κB1/p50 (p105), and NF-κB2/p52 (p100). NF-κB is a homologous/heterodimer complex, the most common and specific intracellular being p50 and Rel (p65). In steady state, nuclear transcription factors κB and κB inhibitory protein (IκB) in the cytoplasm are stimulated by IL-1β, TNF-α, LPS, H2O2, etc., transmitting upstream signals to IκB kinases (IKKs), which phosphorylate IκB in chondrocytes, activating NF-κB[5, 38]. The activated NF-κB transfers from cytoplasm to nucleus, inducing degrading enzyme secretion (e.g., MMPs and ADAMTS5), resulting in articular cartilage degradation[53]. NF-κB can also induce PGE2 (prostaglandin E2), iNOS, NO, and COX-2 to enhance joint damage, promoting tissue inflammation, catabolic factor synthesis, and chondrocyte apoptosis[10, 12]. We confirmed MAPK and NF-κB activation under TBHP stimulation; phosphorylation expression of p65, IκBα, p38, JNK, and ERK reduced in a dose-dependent manner with GK intervention, preliminarily demonstrating GK inhibits overactivation of MAPK and NF-κB signaling during cartilage degeneration, providing some protection. However, it's unclear whether GK directly inhibits phosphorylation or reduces ROS production to inhibit phosphorylation.

However, there are many limitations to this study. OA is a complex pathophysiological process, and various inflammatory mediators, such as IL-1β and TNF-α, are involved in the degenerative process of OA. These mediators are also used to mimic the in vitro environment of OA. The application of TBHP focuses on stimulation-induced oxidative stress microenvironment, and the use of inflammatory mediators and TBHP will be further considered to better mimic the degenerative process of OA. In addition, in vivo experiments, we did not set up a drug concentration gradient, nor did we take into account the toxic effect of ginkgo in vivo experiments, and further verification is needed in vivo experiments.

Based on the above results, this study demonstrated that Ginkgetin can effectively inhibit TBHP-induced oxidative stress, apoptosis, and inflammatory response in chondrocytes. Through in vitro and in vivo experiments, Ginkgetin exhibited significant protective effects on cartilage tissue. These protective effects may be primarily achieved by inhibiting overactivation of the NF-κB and MAPK signaling pathways. Therefore, this study provides a scientific basis for Ginkgetin as a potential drug for treating osteoarthritis (OA).

Ethics approval and consent to participate

All animal procedures involved in the experiment have been reviewed and approved by the Laboratory Animal Welfare Committee of Qingdao University.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests" in this section.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82472431, 82401552) and Shandong Provincial Natural Science Foundation (No. ZR2024QH590)

Authors’ contributions

Liang Zhu and TengBo Yu conceived the study. Liang Zhu and Xiao Xiao designed the research; Liang Zhu, Yanchi Bi, and Ting Liang performed the experiments and contributed essential reagents or tools. Liang Zhu and Yanchi Bi analyzed the data. Liang Zhu and Xiao Xiao wrote the paper. TengBo Yu and Xiao Xiao provided suggestions. All authors reviewed the manuscript.

Acknowledgements

Not applicable

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Ginkgetin delays the progression of osteoarthritis by inhibiting the NF-κB and MAPK signaling pathways.

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Network pharmacology analysis

2.1.1 Ginkgetin target prediction

2.2.2 Acquisition of osteoarthritis-related targets

2.2.3 Drug-disease target prediction

2.2.4 Construction of target protein interaction network

2.2.5 GO enrichment analysis and KEGG pathway analysis

2.2 Cell culture and reagents

2.3 Cell treatment and MTT assay

2.4 Hoechst Staining AND PI Staining

2.5 ROS assay

2.6 Mitochondrial menmbrane potential assay

2.7 Measurement of antioxidant enzyme activity and MDA content.

2.8 NO production assay and Enzyme-linked Immunosorbent Assay (ELISA)

2.9 RNA-seq analysis and data processing

2.10 Western blot analysis

2.11 Animals and in vivo experiment

2.12 Statistical analysis

3. Results

3.1 Results of network pharmacology analysis.

3.1.1 Prediction of Ginkgetin target in the treatment of OA

3.1.2 GO enrichment and KEGG pathway enrichment analysis results.

3.2 Ginkgetin reduced the decrease in cell viability caused by TBHP in SW1353 human chondrocytes.

3.3 Ginkgetin attenuates TBHP-induced oxidative stress in SW1353 human chondrocytes

3.4 Ginkgetin reduces apoptosis in SW1353 human chondrocytes caused by TBHP.

3.5 Ginkgetin inhibited TBHP-induced production of inflammatory mediators and ECM degradation in SW1353 human chondrocytes.

3.6 Ginkgetin was shown to influence mRNA expression in TBHP-induced SW1353 chondrocytes using RNA-seq.

3.7 Effect of Ginkgetin on TBHP-induced NF-κB and MAPK signaling pathways in SW1353 human chondrocytes.

3.8 Ginkgetin delays the progression of OA after ACLT trauma in rats.

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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