Molecular Mechanism of Hydroxyl Safflower Yellow A in Knee Osteoarthritis through Modulation of Autophagy via Inhibition of the HIF-1α/BNIP3 Pathway

As a prevalent chronic orthopedic condition, knee osteoarthritis (KOA) affects middle-aged and elderly individuals, significantly impacting their quality of life^1,2. Despite its widespread occurrence, the precise etiology and pathophysiological mechanisms underlying KOA remain incompletely understood. Furthermore, for both prevention and curative treatment underscores the urgency to unravel the intricate pathological underpinnings of this condition. One area of emerging interest is the role of autophagy in cartilage homeostasis, with accumulating evidence suggesting that reduced autophagy within cartilage tissue contributes to its degeneration in KOA^3,4.

Autophagy, a cellular degradation process vital for maintaining cellular homeostasis, is of great importance in the maintenance and repair of cartilage. In the hypoxic microenvironment prevalent within articular cartilage, the hypoxia-inducible factor 1α (HIF-1α)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) signaling pathway emerges as a pivotal regulator of autophagy. Elevated HIF-1α expression has been observed in KOA patients, suggesting that dysregulation of this pathway may contribute to impaired autophagy and subsequent cartilage degeneration^5,6,7.

Acupuncture, moxibustion, herbs, and massage are four classic methods of traditional Chinese medicine for treating knee arthritis. The current treatments, such as non-steroidal anti-inflammatory drugs, hyaluronic acid injections, and arthroplasty, have been proven to be helpful but also associated with certain side effects⁸. Moreover, there is no recommended routine treatment for knee osteoarthritis. Over time, as a common complementary therapy for KOA, Traditional Chinese Medicine (TCM) has developed numerous herbal remedies that show promise in treating KOA⁹. Among these, Hydroxyl safflower yellow A has garnered significant attention due to its anti-inflammatory and autophagy-modulating properties^10,11. However, the exact mechanisms by which HSYA exerts its therapeutic influence on knee osteoarthritis (KOA), particularly in terms of regulating autophagy through the HIF-1α/BNIP3 pathway, remain uncertain. Therefore, we investigate the mechanism of HSYA's therapeutic effects in KOA by focusing on its impact on chondrocyte autophagy and its interaction with the HIF-1α/BNIP3 pathway. We hypothesize that HSYA mitigates knee osteoarthritis by enhancing chondrocyte autophagy and proliferation while suppressing apoptosis and inflammation via HIF-1α/BNIP3 pathway inhibition.

All procedures involving human samples were approved by the Ethics Committee of Zhanjiang Central People's Hospital (Approval No. KY-YS-2021-09). Written informed consent was obtained from all participants prior to sample collection. The reagents and the equipment used are listed in the Table of Materials.

1. Patients and study design

Thirty patients diagnosed with knee osteoarthritis (KOA) and undergoing total knee arthroplasty were recruited. The cohort included 14 males and 16 females, aged 52-75 years (mean ± SD: 64.3 ± 12.6 years), all fulfilling the American College of Rheumatology KOA criteria¹². Patients were excluded if they had received non-steroidal anti-inflammatory drugs or steroids within 2 weeks prior to surgery or intra-articular injections within 1 month.

2. Primary chondrocyte culture

Articular cartilage was collected under sterile conditions immediately after surgery and placed in cold PBS. Using sterile scalpels, the cartilage was cut into ~1 mm³ fragments on an ice-cold glass plate and transferred into a 50 mL centrifuge tube containing 10 mL of 0.25% trypsin-EDTA. The tube was incubated in a 37 °C shaking water bath at 80 rpm for 30 min and inverted gently every 5 min to facilitate digestion. The supernatant was aspirated, and the tissue pellet was washed once with PBS and centrifuged at 200 × g for 5 min. The pellet was then resuspended in 10 mL of 0.02% type II collagenase and digested at 37 °C for 4 h with shaking. The suspension was pipetted up and down every 30 min to promote dissociation, passed through a 70 µm strainer, and centrifuged again at 200 × g for 5 min. The resulting pellet was resuspended in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin and seeded into T25 flasks. Cells were incubated at 37 °C with 5% CO₂, the medium was replaced every 2-3 days, and cells from passages 1-2 were used for experiments. Experimental groups included normal control, blank model, HSYA (100 µmol/L), HIF-1α inhibitor YC-1 (10 µmol/L), rapamycin (10 nmol/L), HSYA + YC-1, and HSYA + rapamycin.

3. siRNA-Mediated HIF-lα, BNIP3 Knockdown

Primary chondrocytes were seeded in 6-well plates at 2-5 × 10⁵ cells/well and cultured at 37 °C until they reached 30%-50% confluency. For each well, 5 µL of 20 µM siRNA was diluted in 250 µL Opti-MEM, and 5 µL of lipid-based transfection reagent was diluted separately in 250 µL Opti-MEM. The two solutions were gently mixed and incubated at room temperature for 15-20 min to form complexes. The culture medium was replaced with 1.5 mL fresh DMEM with 10% FBS, and the siRNA-lipid complex (500 µL) was added dropwise along the well wall with gentle swirling. Cells were incubated for 6 h, the medium was replaced with complete medium, and the culture was continued for 48-72 h. Knockdown efficiency was verified by qRT-PCR and Western blot before further treatments.

4. ELISA

Cell culture supernatants were collected, centrifuged at 200 × g for 5 min, and diluted 1:2 in assay buffer when necessary. ELISA kits for IL-1β, TNF-α, and IL-6 were used according to the manufacturer's protocol. Standards, blanks, and samples were run in triplicate. Following substrate reaction, absorbance was measured at 450 nm using a microplate reader within 15 min.

5. MTT assay

Chondrocytes were seeded into 96-well plates at 5,000 cells/well in 100 µL of complete medium and incubated for 0 h, 24 h, 48 h, or 72 h. At each time point, 20 µL of MTT solution (5 mg/mL in PBS) was added directly to each well, and the plates were incubated at 37 °C for 30 min until purple formazan crystals became visible at the bottom of the wells. The supernatant was aspirated carefully without disturbing the crystals, 150 µL of DMSO was added to each well, and the plates were placed on a shaker at 100 rpm for 10 min to dissolve the crystals completely. Absorbance was measured at 490 nm using a microplate reader. The short incubation time was selected to avoid signal saturation in highly metabolically active cells.

6. Flow cytometry

Cells were harvested by trypsinization, centrifuged at 200 × g for 5 min, and washed twice with cold PBS. They were resuspended in 500 µL of binding buffer at ~1 × 10⁶ cells/mL, and 5 µL Annexin V-FITC and 5 µL PI were added. After gentle mixing, cells were incubated for 15 min at room temperature in the dark. Samples were analyzed on a flow cytometer, with at least 10,000 events collected per sample (excitation 488 nm, emission FITC 530/30 nm, PI > 600 nm). Apoptotic cells were quantified using standard software.

7. Western blotting

Cells were rinsed twice with cold PBS and lysed in 200 µL RIPA buffer containing protease inhibitors on ice for 30 min. Cells were scraped with a plastic scraper, pipetted up and down to reduce viscosity, and centrifuged at 12,000 × g for 10 min at 4 °C. Supernatants were collected, and protein concentrations were measured using the BCA assay. Equal amounts of protein (30 µg) were mixed with 5× loading buffer, heated at 95 °C for 5 min, and separated on 10%-12% SDS-PAGE gels. Electrophoresis was run at 80 V for stacking and 120 V for separation, and proteins were transferred onto PVDF membranes at 300 mA for 90 min. Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies (1:1,000). After three washes with TBST, membranes were incubated with HRP-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. Protein bands were visualized with ECL substrate and imaged using a chemiluminescence detection system with 30-120 s exposure. Band intensities were quantified using ImageJ, and GAPDH was used as the loading control.

8. Monodansylcadaverine (MDC) staining

A 50 µM MDC working solution was prepared from the stock solution immediately before use. Cells were fixed with 1 mL of 4% paraformaldehyde for 10 min at room temperature, washed twice with PBS, and incubated with 500 µL MDC working solution for 30 min at 37 °C in the dark. After two washes with PBS, coverslips were mounted with antifade mounting medium and observed under a fluorescence microscope (excitation 355 nm, emission 512 nm).

9. Transmission Electron Microscope (TEM)

Cells were harvested and pelleted by centrifugation at 200 × g for 5 min, then fixed in 2% glutaraldehyde at 4 °C overnight. Samples were washed three times with PBS for 5 min each and post-fixed in 1% osmium tetroxide for 1 h at 4 °C in a fume hood. Dehydration was performed through a graded acetone series of 30%, 50%, 70%, 90%, and 100% for 10 min each. Samples were infiltrated with 1:1 acetone/epoxy resin for 1 h and then with pure resin overnight. Embedding was performed in fresh resin and polymerized at 60 °C for 48 h. Ultrathin sections of 50-70 nm were cut with an ultramicrotome, mounted on 200-mesh copper grids, stained with 2% uranyl acetate for 30 min followed by lead citrate for 15 min, washed with distilled water, air dried, and examined under a transmission electron microscope at 80 kV. As a safety note, glutaraldehyde and osmium tetroxide are toxic and volatile and must be handled only in a fume hood with gloves and protective eyewear. Waste should be disposed of in designated hazardous containers according to institutional safety protocols.

10. Statistical analysis

All experiments were performed in triplicate. Data are expressed as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by LSD post-hoc test, with P < 0.05 considered significant.

HSYA's effect on chondrocyte pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) expression
Compared with the normal control group, all other groups showed significantly elevated levels of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) (P < 0.05, Table 1, Figure 1A-G). Treatment with HSYA, HIF-1α inhibitor, autophagy inducer, HSYA + HIF-1α inhibitor, or HSYA + autophagy inducer significantly reduced cytokine levels relative to the blank model group (P < 0.05). Among these, the combined treatments (HSYA + HIF-1α inhibitor or HSYA + autophagy inducer) further reduced cytokine expression, with decreases of approximately 35%-40% compared with the model group (P < 0.05). In the gene interference experiments (Figure 1H-J), siRNA knockdown of HIF-1α or BNIP3 led to reductions of ~25%-30% in cytokine levels compared with the model + blank group. HSYA treatment further enhanced these effects. Specifically, cytokine levels in the siHIF-1α + HSYA group were ~20% lower than in the siHIF-1α + blank group, and levels in the siBNIP3 + HSYA group were ~22% lower than in the siBNIP3 + blank group (P < 0.05).

Effect of HSYA on chondrocyte proliferation
The normal control group exhibited baseline proliferation activity, whereas the blank model group showed a significant reduction (P < 0.05, Figure 2A). Proliferation was partially restored following treatment with HSYA, HIF-1α inhibitor, or autophagy inducer, and markedly enhanced by combined HSYA + HIF-1α inhibitor or HSYA + autophagy inducer treatments (P < 0.05). In the siRNA experiments (Figure 2B), knockdown of HIF-1α or BNIP3 promoted proliferation by ~25% compared with the model control group. HSYA treatment further enhanced proliferation, resulting in ~40% higher proliferation compared with the model group (P < 0.05).

Effect of HSYA on apoptosis and autophagy-related protein expression in chondrocytes
Western blot analysis revealed significant differences in protein expression among the groups (Table 2). The blank model group showed marked upregulation of HIF-1α and reduced expression of LC3, P62, Beclin1, and BNIP3 compared with controls (P < 0.05). Treatment with HSYA, HIF-1α inhibitor, or autophagy inducer increased LC3, P62, Beclin1, and BNIP3 expression and decreased HIF-1α levels (P < 0.05). The combined treatments with HSYA + HIF-1α inhibitor or HSYA + autophagy inducer produced the largest changes, with LC3 and Beclin1 levels nearly doubled compared with the blank model group. Although P62 is typically reduced when autophagy is activated, here its levels increased following HSYA treatment. This may indicate enhanced autophagosome accumulation or incomplete flux, a phenomenon also reported in previous studies of chondrocyte autophagy.

Effect of HSYA on apoptosis rates in chondrocytes
Flow cytometry analysis showed that the blank model group exhibited a significantly higher apoptosis rate than the normal control group (P < 0.05, Figure 3A,B). HSYA, HIF-1α inhibitor, and autophagy inducer each reduced apoptosis, while the combined treatments (HSYA + HIF-1α inhibitor or HSYA + autophagy inducer) produced the lowest apoptosis rates, with reductions of ~45%-50% compared with the blank model group (P < 0.05). In the siRNA experiments, apoptosis was also decreased by knockdown of HIF-1α or BNIP3, and further reduced by HSYA treatment.

Effect of HSYA on apoptosis rates and autophagic vesicle formation in chondrocytes
MDC staining and TEM imaging revealed clear differences in autophagic vesicle formation across groups (Figure 4A-D). The blank model group exhibited a sparse distribution of vesicles, whereas HSYA, HIF-1α inhibitor, or autophagy inducer treatments increased vesicle numbers. The combined treatments (HSYA + HIF-1α inhibitor or HSYA + autophagy inducer) produced the most marked enhancement, with approximately two-fold more vesicles observed by TEM compared with the model group. Consistent with these findings, apoptosis rates measured by flow cytometry (Figure 5A-E) were lowest in the combined treatment groups.

DATA AVAILABILITY:
All data generated in this study are provided in Supplementary File 1.

Figure 1: The expression of chondrocyte pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) in each group (n = 3). (A-G) The protein expression levels of IL-1β, TNF-α, and IL-6 were detected by Western blotting (WB). (H-J) The contents of IL-1β, TNF-α, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) (P < 0.05). Please click here to view a larger version of this figure.

Figure 2: Comparison of chondrocyte proliferation activity in each group (n = 3). (A) The treated groups, and (B) the HIF-1α and BNIP3 knockdown groups, P< 0.05. Please click here to view a larger version of this figure.

Figure 3: Comparison of the apoptosis rate of each group (n = 3). (A) 1, Normal group; 2, Model group; 3, HSYA group; 4, HIF-1α inhibitor group; 5, Autophagy group; 6, HSYA + HIF-1α inhibitor group; 7, HSYA + Autophagy inducer group. (B) The HIF-1α and BNIP3 gene knockdown groups. Compared to the normal control group, *P < 0.05. Compared to the blank model group, #P< 0.05. Compared to the HSYA + HIF-1α inhibitor group, & P< 0.05. Please click here to view a larger version of this figure.

Figure 4: TEM imaging of the autophagic vesicle formation in mitochondria. (A) Blank model. (B) HSYA group. (C) HIF-1α inhibitor group. (D) Autophagy inducer group. Scale bar = 1 µm. Please click here to view a larger version of this figure.

Figure 5:Effect of HSYA on cell apoptosis rates in each group (n = 3). (A) Blank model. (B) HSYA group. (C) HIF-1α inhibitor group. (D) Autophagy inducer group. (E) The total analysis of each group, compared to the normal control group, **P< 0.01. Please click here to view a larger version of this figure.

Table 1: Comparison of pro-Inflammatory cytokine levels in each group. Compared to the normal control group, *P < 0.05. Compared to the blank model group, #P< 0.05. Compared to groups treated with HASY, the HIF-1α inhibitor, and the autophagy inducer, & P< 0.05.

Table 2: Comparison of the proteins in the different groups. Compared to the normal control group, *P< 0.05. Compared to groups treated with HSYA, the HIF-1α inhibitor, and the autophagy inducer, #P< 0.05.

Supplementary File 1: The raw data generated during the study. Please click here to download this File.

Osteoarthritis (OA), a prevalent chronic disease, significantly impairs patients' quality of life. Among the various OA subtypes, KOA is the most common, contributing to the global disease burden. Despite its high incidence, the precise etiology and underlying pathophysiological mechanisms of KOA remain elusive. The intricate interplay between mechanical and biochemical processes within articular cartilage, particularly the regulation of chondrocyte function, is crucial in KOA pathogenesis. Notably, a marked reduction in autophagy activity has emerged as a pivotal factor in KOA initiation and progression^13,14. Hence, enhancing autophagy presents a promising therapeutic strategy for promoting chondrocyte survival and mitigating disease severity.

The management of knee osteoarthritis (KOA) follows a step-by-step principle. Non-pharmaceutical treatments (such as weight loss, exercise, and physical therapy) are the foundation. Although they have no side effects and can delay degeneration, they take effect slowly and are only suitable for mild symptoms. In drug treatment, topical non-steroidal anti-inflammatory drugs (NSAIDs) are safe but have limited efficacy. Oral NSAIDs relieve pain quickly but carry gastrointestinal and cardiovascular risks and do not protect cartilage. Cartilage protectants (such as glucosamine) are safe but take effect slowly, and their efficacy is questionable. Intra-articular injection of sodium hyaluronate provides long-lasting lubrication but requires multiple injections. Glucocorticoids have quick anti-inflammatory effects but are prone to damage cartilage. Surgical treatment (arthroscopy, unicompartmental/total knee replacement) is suitable for severe patients. Total knee replacement has definite therapeutic effects but causes significant trauma and complications. Arthroscopy is minimally invasive but has poor long-term outcomes.

In contrast, hydroxy safflower Yellow A (HSYA), as a natural active ingredient, shows significant advantages over conventional treatments: Not only can it alleviate synovial inflammation by inhibiting inflammatory pathways (comparable to the anti-inflammatory effect of NSAIDs but without gastrointestinal/cardiovascular risks), but it can also enhance chondrocyte autophagy and proliferation while suppressing apoptosis and inflammation via HIF-1α/BNIP3 pathway inhibition (making up for the shortcoming of NSAIDs that only relieve symptoms without protecting cartilage, and it is easier to observe the effect than glucosamine), with high safety and few side effects It can be developed into multiple dosage forms such as oral, injectable and topical, with flexible administration. Its multi-target effect of "anti-inflammatory + cartilage repair" provides a new direction for the treatment of KOA that is more balanced in terms of efficacy and safety.

Currently, definitive clinical treatments for KOA are limited. However, studies have highlighted the potential of TCM in inducing autophagy in chondrocytes, demonstrating promising efficacy¹⁵. HSYA exhibits diverse pharmacological effects. Its regulatory role in chondrocyte autophagy¹⁶, prompting an in-depth exploration of the mechanisms underlying HSYA's effects on chondrocyte behavior. In the hypoxic microenvironment within articular cartilage, elevated levels of HIF-1α impede autophagy¹⁷. Therefore, this study aims to elucidate how HSYA modulates autophagy.

Meanwhile, there are several important factors to be noted in this study: Firstly, sample heterogeneity (differences in baseline characteristics such as patient age and severity of KOA, as well as the delay in post-extraction processing); Secondly, variations in chondrocyte isolation and culture (gelatinase digestion conditions, cell passage and dedifferentiation, stability of the culture environment); Thirdly, technical deviations in detection (non-specific binding of ELISA antibodies, protein extraction and quantification errors in WB, non-standardized operation in apoptosis and proliferation detection). In the experiment, it is necessary to strictly standardize key steps: sample inclusion requires clear inclusion and exclusion criteria (such as controlling patient age and KOA classification) and timely digestion and processing; chondrocyte culture requires a standardized digestion process, using only 1-2 generations of cells and verifying the chondrocyte-specific phenotype; technical operations need to verify the specificity of reagents and add quality control samples; further improvements can be achieved by establishing a KOA sample library linked to clinical data, using 3D culture to simulate the in vivo microenvironment or inducing KOA-like phenotypes with IL-1β, introducing high-throughput detection technologies (such as multiplex cytokine chips), and combining animal models to verify the in vivo effects. Additionally, elucidating the molecular intricacies of HSYA's effects on articular cartilage will be imperative to ascertain its practical significance in KOA management¹⁸. The core advantage of this scheme lies in: using primary chondrocytes from clinical samples as a model to ensure the relevance of research results to clinical pathology; achieving multi-dimensional validation of efficacy and mechanism through the use of multiple technologies, avoiding the limitation of "observing phenomena without understanding the mechanism"; the used technologies are mature and easy to operate, facilitating replication by other teams, and laying an important foundation for the translational research of HSYA in the treatment of KOA.