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

Summary

Hydroxyl Safflower Yellow A (HSYA) mitigates knee osteoarthritis by enhancing chondrocyte autophagy and proliferation while suppressing apoptosis and inflammation via HIF-1α/BNIP3 pathway inhibition.

Abstract

The reduction of autophagy in cartilage tissue is closely linked to the development of knee osteoarthritis (KOA), yet the mechanisms by which Hydroxyl safflower yellow A (HSYA) exerts protective effects remain incompletely understood. In this study, human KOA chondrocytes were isolated and assigned to different treatment groups, including normal control, blank model, HSYA, hypoxia-inducible factor-1α (HIF-1α) inhibitor, autophagy inducer, HSYA combined with HIF-1α inhibitor, and HSYA combined with autophagy inducer. Proinflammatory cytokines (IL-1β, TNF-α, IL-6) were quantified by ELISA, protein expression of HIF-1α, BNIP3, and autophagy-related markers was examined by Western blotting, and autophagic vesicles in mitochondria were evaluated using monodansylcadaverine staining and transmission electron microscopy. Compared with the normal control, the blank model group showed significantly increased levels of IL-1β, TNF-α, IL-6, and HIF-1α, accompanied by higher apoptosis rates, reduced proliferation, and downregulation of autophagy-related proteins (P < 0.05). Treatment with HSYA, HIF-1α inhibitor, or autophagy inducer significantly upregulated autophagy-related protein expression and reduced inflammatory cytokine levels. Moreover, HSYA combined with HIF-1α inhibitor or autophagy inducer produced the most pronounced effects, with marked reductions in cytokine release and apoptosis, along with enhanced chondrocyte proliferation and mitochondrial autophagic vesicle formation (P < 0.05). These findings demonstrate that HSYA exerts chondroprotective effects in KOA, at least in part, through inhibition of the HIF-1α/BNIP3 pathway, thereby promoting autophagy and attenuating inflammatory injury in chondrocytes.

Introduction

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.

Protocol

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.

Results

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 r...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% Osmium Tetroxide	Electron Microscopy Sciences	19150	Further fixes and stains samples for TEM analysis
2% Glutaraldehyde	Sigma-Aldrich	G5882	Fixes cells for TEM sample preparation
4% Paraformaldehyde	Servicebio	G1101	Fixes cells for MDC staining and TEM sample preparation
AG1478	Selleck Corporation	S1005	20 mg/kg intravenous injection, ErbB receptor kinase inhibitor in rat model
Annexin V-FITC/PI Apoptosis Detection Kit	Bioswamp	BS3030	Stains apoptotic cells for flow cytometry detection
Autophagy inducer (RAPA)	Sigma Corporation	R0395	Rapamycin, autophagy inducer, used at 10 nmol/L in chondrocyte experiments
Beclin1 Antibody	Santa Cruz Biotechnology	sc-48341	Primary antibody for Western blot detection of autophagy-related protein Beclin1
BNIP3 Antibody	Santa Cruz Biotechnology	sc-517348	Primary antibody for Western blot detection of BNIP3
Carbon dioxide incubator	Yiheng	BPN-150CW	for cell culture
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	D2650	Dissolves formazan crystals in MTT assay
DMEM	Beyotime	C0001	Cell culture medium, supplemented with 10% FBS for chondrocyte culture
ELISA Microplate Reader	Thermo Fisher Scientific	Multiskan FC	Measures OD values in ELISA and MTT assays
Enhanced Chemiluminescence (ECL) Reagent	Thermo Fisher Scientific	32106	Visualizes protein bands in Western blot
Epoxy Resin (EPON812)	Electron Microscopy Sciences	14120	Embeds samples for TEM sectioning
FBS	Beyotime	C0205	Fetal bovine serum, added to DMEM for chondrocyte nutrition
Flow Cytometer	Beckman (mentioned for apoptosis)	CytoFLEX S	Detects chondrocyte apoptosis rate and cellular markers
Fluorescence Microscope	Olympus	IX73	Observes MDC-stained autophagic vesicles
Gel Imaging System	Bio-Rad	ChemiDoc XRS+	Captures and quantifies protein bands from Western blot
HIF-1α Antibody	Santa Cruz Biotechnology	sc-13515	Primary antibody for Western blot detection of HIF-1α
HIF-1α inhibitor (YC-1)	Selleck Corporation	S1047	HIF-1α pathway inhibitor, used at 10 μmol/L in cell experiments
High-Speed Refrigerated Centrifuge	Eppendorf	5430R	used for separating components
HRP-conjugated Secondary Antibody	Jackson ImmunoResearch	115-035-003	Binds primary antibodies for signal amplification in Western blot
IL-1β ELISA Kit	Shenggong Biology	C5236	Quantifies IL-1β levels in cell supernatants via ELISA
IL-6 ELISA Kit	Shenggong Biology	C5237	Quantifies IL-6 levels in cell supernatants via ELISA
LC3 Antibody	Santa Cruz Biotechnology	sc-398822	Primary antibody for Western blot detection of autophagy-related protein LC3
Lipofectamine 3000 reagent	Thermo Fisher Scientific	L3000001	lipid-based transfection reagent for tansfection
Monodansylcadaverine (MDC)	Sigma-Aldrich	D4008	0.05 mol/L, stains autophagic vesicles for fluorescence microscopy
MTT Reagent	Beyotime	C0009	0.5 mg/mL, used in MTT assay to assess chondrocyte proliferation
NanoDrop 2000	Thermo Fisher Scientific	ND-1000	determine the concentration of RNA
NRG1 ELISA Kit	Abcam (UK)	ab213961	Measures NRG1 concentration in heart tissues via ELISA
P62 Antibody	Santa Cruz Biotechnology	sc-28359	Primary antibody for Western blot detection of autophagy-related protein P62
Phosphorylated ErbB4 (p-ErbB4) Antibody	Affinity	AF3445	Primary antibody for Western blot detection of activated ErbB4
PrimeScript RT Reagent Kit	TaKaRa	RR047A	RNA reverse transcription to provide templates for qPCR experiment
Real-time quantitative PCR system	Bio-Rad	CFX96	for RT-PCR detect
Recombinant human NRG1 (rh-NRG1)	R&D Systems	396-HB	5 mg/kg intravenous injection to activate NRG1/ErbB4 pathway in rat model
RIPA Buffer	Meilunbio	MA0151	Lysis buffer with protease/phosphatase inhibitors for protein extraction
Safflower Yellow A (HSYA)	Chengdu Mansit Biotechnology Co., Ltd.	Must-160601	Bioactive component from Carthamus tinctorius L., used at 100 μmol/L for chondrocyte treatment
SDS-PAGE Equipment	Bio-Rad	Mini-PROTEAN Tetra	Separates proteins by molecular weight in Western blot
TB Green Premix Ex Taq II	TaKaRa	RR820A	for qPCR experiment
TNF-α ELISA Kit	Shenggong Biology	C5238	Quantifies TNF-α levels in cell supernatants via ELISA
Total ErbB4 Antibody	Proteintech	19943-1-AP	Primary antibody for Western blot detection of total ErbB4
Transmission Electron Microscope (TEM)	Hitachi (mentioned for autophagic vesicles)	H-7650	Observes autophagic vesicles in mitochondria at ultrastructural level
Trizol Reagent	Thermo Fisher Scientific	15596026	for RNA extraction
Trypsin-EDTA	Gibco (Thermo Fisher)	25200056	Used for enzymatic digestion of articular cartilage to isolate chondrocytes
Type II Collagenase (0.02%)	Worthington Biochemical	CLS-2	Digests cartilage tissue for chondrocyte isolation
β-catenin Antibody	Santa Cruz Biotechnology	sc-7963	Internal reference antibody for Western blot normalization