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 tiss....

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 Col....

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 si.......

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