Table 2.
Characteristics of the in vitro studies.
| Author (year, Country) | Cell type & source | Core study design (Duration of intervention) |
Outcome measures | Pivotal discovery |
|---|---|---|---|---|
| Gong et al. (27) (2023, China) |
Primary chondrocytes (healthy); SD rat (knee joint) | G1: control G2: IL-1β (10 ng/mL) G3: IL-1β (10 ng/mL) + CAD (10 μmol/L) (24 hours) |
Cell viability assay: CCK-8 kit Intracellular iron level: iron assay kit Intracellular GSH level: GSH test kit Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Malondialdehyde assay: MDA assay kit Mitochondrial membrane potential: JC-1 staining Mitochondrial morphology: Mito-Tracker Morphology: TB and Alcian Blue staining Protein expression: WB and IF Ultrastructure of mitochondria: TEM |
CAD ameliorated OA cartilage degradation by regulating ferroptosis via the p53/SLC7A11/GPx4 signalling pathway. |
| Guo et al. (29) (2022, China) |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: IL-1β (10 ng/mL) G3: IL-1β (10 ng/mL) + DFO (100 μmol/L) G4: IL-1β (10 ng/mL) + DFO (100 μmol/L) + si-Nrf2 (24 hours) |
Cell viability assay: CCK-8 kit Intracellular iron level: iron assay kit Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Malondialdehyde assay: MDA assay kit Morphology: TB staining Protein expression: WB Ultrastructure of mitochondria: TEM |
DFO alleviated the inflammatory response and ECM degradation in chondrocytes induced by IL-1β by inhibiting chondrocyte ferroptosis via Nrf2 signalling. |
| He et al. (24) (2023, China) |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: FAC (500 μmol/L) G3: FAC (500 μmol/L) + DFO (100 μmol/L) G4: FAC (500 μmol/L) + BCA (12 μmol/L) G5: FAC (500 μmol/L) + BCA (24 μmol/L) (48 hours) |
Apoptotic effect: annexin V-FITC/PI staining Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular iron level: calcein-AM Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Intracellular GSH: GSH test kit Mitochondrial membrane potential: JC-1 staining Morphology: TB staining Protein expression: WB |
Biochanin A protects against iron overload–associated knee osteoarthritis by regulating iron levels and the Nrf2 / System Xc- / GPx4 axis |
| Jing et al. (16) (2021, China) a |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: IL-1β (10 ng/mL) G3: TNF-α (10 ng/mL) G4: IL-1β (10 ng/mL) + si-DMT1 G5: TNF-α (10 ng/mL) + si-DMT1 (24 hours) |
Apoptotic effect: annexin V-FITC/PI staining and flow cytometry Gene quantification: PCR Intracellular iron level: calcein-AM Protein expression: WB |
DMT1 played a pivotal role in iron overload–induced OA progress. Inhibition of DMT1 suppressed IL-1β-induced inflammatory response and ECM degradation by blockading MAPK and PI3K/AKT pathway. |
| Jing et al. (30) (2021, China)b |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: FAC (100 μmol/L) G3: FAC (100 μmol/L) + IL-1β (10 ng/mL) G4: FAC (100 μmol/L) + DFO (100 μmol/L) G5: FAC (100 μmol/L) + NAC (100 μmol/L) (24 hours) |
Apoptotic effect: annexin V-FITC/PI staining Gene quantification: PCR Intracellular iron level: calcein-AM Intracellular ROS level: ROS assay kit Mitochondrial membrane potential: JC-1 staining and flow cytometer Mitochondrial morphology: Mito-Tracker Protein expression: WB |
The pro-inflammatory cytokines could disrupt chondrocytes’ iron homeostasis and promote iron influx; iron overload–induced oxidative stress and mitochondrial dysfunction play important roles in iron overload–induced cartilage degeneration. |
| Jing et al. (26) (2021 China)c |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: FAC (100 μmol/L) G3: FAC (100 μmol/L) + BA (10 μmol/L) (24 hours) |
Apoptotic effect: annexin V-FITC/PI staining Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular iron level: calcein-AM Intracellular ROS level: ROS assay kit Mitochondrial membrane potential: JC-1 staining Mitochondrial morphology: Mito-Tracker Protein expression: WB and IF |
Calcium chelators may be of value in the treatment of iron metabolism–related diseases and IOOA progression. |
| Karim et al. (38) (2022, United Arab Emirates) |
Immortalised C-20/A4 cell line; human | G1: control G2: FAC (200 μmol/L) G3: FAC (300 μmol/L) (24/48 hours) |
Apoptotic effect: annexin V-FITC/PI staining Cell cycle progression analysis: flow cytometer Cell viability assay: MTT kit Intracellular iron level: calcein-AM and flow cytometer Intracellular ROS level: ROS assay kit Protein expression: WB and IF |
Iron overload disrupts cellular iron homeostasis, which compromises the functional integrity of chondrocytes and leads to oxidative stress and apoptosis. |
| Liu et al. (31) (2022, China) |
Primary chondrocytes (OA); human (articular cartilage) | G1: control G2: sh-SLC3A2-1 G3: sh-SLC3A2-2 G4: sh-SLC3A2-1 + Fer-1 (unknown drug concentration) G5: sh-SLC3A2-2 + Fer-1 (unknown drug concentration) (24/48/72 hours) |
Gene quantification: PCR Morphology: Alcian Blue, SOFG and TB staining Protein expression: WB Ultrastructure of mitochondria: TEM |
SLC3A2 inhibited ferroptosis and suppressed cartilage degeneration in OA. |
| Lv M et al. (33) (2022, China) |
Primary chondrocytes (healthy); mice (unknown race, knee joint) | G1: control G2: IL-1β (10 ng/mL) G3: IL-1β (10 ng/mL) + sh-SND1 G4: IL-1β (10 ng/mL) + sh-SND1 + sh-HSPA5 G5: IL-1β (10 ng/mL) + Ad-HSPA5 G6: IL-1β (10 ng/mL) + Ad-HSPA5 +sh-GPx4 (48 hours) |
Cell viability assay: MTT kit and EdU staining Intracellular iron level: ELISA kit Intracellular ROS and lipid ROS level: DCFH-DA Malondialdehyde assay: ELISA kit RNA-binding protein interaction: RIP Protein expression: CO-IP and WB |
The RNA-binding protein SND1 promotes the degradation of GPx4 by destabilising the HSPA5 mRNA and suppressing HSPA5 expression, promoting ferroptosis in osteoarthritis chondrocytes. |
| Lv Z et al. (35) (2022, China) |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: CPS (50 μmol/L) G3: JNJ (10 μmol/L) G4: TBHP (50 μmol/L) G5: CPS (50 μmol/L) + TBHP (50 μmol/L) G6: JNJ (50 μmol/L) +TBHP (50 μmol/L) (24 hours) |
Cell viability assay: CCK-8 kit and AM/PI staining Gene quantification: PCR Intracellular iron level: FerroOrange Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Protein expression: WB |
TRPV1 protects chondrocytes from ferroptosis. |
| Miao et al. (28) (2022, China) |
ATDC5 cell line; mouse | G1: sh-NC + IL-1β (10 ng/mL) G2: sh-GPx4-1 + IL-1β (10 ng/mL) G3: sh-GPx4-2 + IL-1β (10 ng/mL) (NR) |
Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular GSH: GSH test kit Intracellular iron level: FerroOrange Intracellular ROS and lipid ROS level: DCFH-DA and Liperfluo staining Malondialdehyde assay: MDA assay kit Mitochondrial membrane potential: JC-1 staining Protein expression: WB |
GPx4 downregulation could increase the sensitivity of chondrocytes to oxidative stress and aggravate ECM degradation through the MAPK/NF-κB pathway. |
| Mo et al. (36) (2021, China) |
ATDC5 cell line; mouse | G1: control G2: IL-1β (10 ng/mL) G3: IL-1β (10 ng/mL) + STM (20 μg/mL) G4: IL-1β (10 ng/mL) + STM (20 μg/mL) + Oe-NC G5: IL-1β (10 ng/mL) + STM (20 μg/mL) + Oe-SREBF2 (24 hours) |
Analysis of oxidative stress: GSH, MDA and SOD assay kit Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular iron level: iron colorimetric assay kit Protein expression: WB |
STM attenuated chondrocyte injury induced by IL-1β by regulating ferroptosis via down-regulation of SREBF2 and may have potential as a novel therapeutic method for knee osteoarthritis. |
| Pan et al. (21) (2022, China) |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: FAC (100 μmol/L) + IL-1β (10 ng/mL) G3: FAC (100 μmol/L) + IL-1β (10 ng/mL) + NAC (100 μmol/L) G4: FAC (100 μmol/L) + IL-1β (10 ng/mL) + NAR-L (10 μmol/L) G5: FAC (100 μmol/L) + IL-1β (10 ng/mL) + NAR-H (20 μmol/L) (48 hours) |
Apoptotic effect: annexin V-FITC/PI staining and flow cytometry Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular iron level: calcein-AM Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Malondialdehyde assay: MDA assay kit Mitochondrial membrane potential: JC-1 staining and flow cytometer Morphology: TB staining Protein expression: WB |
NAR can reduce oxidative stress through the Nrf2/HO-1 pathway and alleviate cartilage damage under iron overload and has the potential to treat IOOA. |
| Wan et al. (25) (2023, China) |
1. Primary chondrocytes (healthy); human (articular cartilage) 2. Primary chondrocytes (healthy); mice (unknown race, knee joint) 3. AMPKα-knockout mice |
G1: control G2: IL-1β (10 ng/mL) G3: AICAR (10 mmol/L) + IL-1β (10 ng/mL) G4: IL-1β (10 ng/mL) + baicalein (5 μmol/L) G5: AICAR (10 mmol/L) + IL-1β (10 ng/mL) + baicalein (5 μmol/L) (36 hours) |
Apoptotic effect: annexin V-FITC/PI staining and flow cytometry Cell proliferation assay: EdU staining Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular iron level: Phen green SK diacetate Intracellular lipid ROS level: C11-BODIPY Protein expression: WB and Co-IP Ultrastructure of mitochondria: TEM |
Baicalein suppresses ferroptosis by inducing AMPK phosphorylation and facilitating AMPK holoenzyme assembly, stability and activity. |
| Wang S et al. (23) (2022, China) |
Primary chondrocytes; Col2a1-CreERT GPx4flox/flox mice (knee joint) | G1: control G2: 1 MPa (1 Hz) G3: 1 MPa (1 Hz) + GsMTx4 (10 nmol/L) (24 hours) |
Cell live/dead assay: calcein/PI cell viability/cytotoxicity assay kit Gene quantification: PCR Intracellular GSH level: GSH assay kit Intracellular ROS level: DCFH-DA Mitochondrial membrane potential: JC-1 staining Mitochondrial morphology: Mito-Tracker Protein expression: WB Ultrastructure of mitochondria: TEM |
Mechanical overloading induced ferroptosis in chondrocytes through the Piezo 1 ion channel. |
| Wang X et al. (22) (2022, China) |
Primary chondrocytes (healthy); SD rat (knee joint) | G1: control G2: IL-1β (10 ng/mL) G3: IL-1β (10 ng/mL) + Fer-1 (1 μmol/L) G4: IL-1β (10 ng/mL) + ATX (10 μmol/L) (24 hours) |
Cell viability assay: CCK-8 kit Intracellular GSH level: GSH test kit Intracellular iron level: colorimetric assay Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Malondialdehyde assay: MDA assay kit Mitochondrial iron level: Mito-FerroGreen Mitochondrial membrane potential: JC-1 staining Mitochondrial morphology: Mito-Tracker Morphology: TB staining Protein expression level: WB and IF Ultrastructure of mitochondria: TEM |
Both Fer-1 and ATX are able to mitigate chondrocyte injury and osteoarthritis progression by inhibiting ferroptosis and the regulation of mitochondrial function. |
| Wen et al. (34) (2023, China) |
Primary chondrocytes (OA); human (articular cartilage) | G1: control G2: UCPH-101 (20 μmol/L) G3: siRNA EAAT1 G4: siRNA control (24 hours) |
Apoptotic effect: PI/Hoechst Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular GSH and GSSG level: GSH and GSSG test kit Intracellular iron level: iron assay kit Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Malondialdehyde assay: MDA assay kit Ratio of senescent: β-galactosidase staining Protein expression: WB |
The EAAT1-glutamate-GPx4 anti-ferroptosis axis is a key survival mechanism for SenChos, and EAAT1 is an effective and specific target for anti-senescence therapy in osteoarthritis. |
| Xu et al. (37) (2022, China) |
Primary chondrocytes (OA); human (articular cartilage) | G1: si-NC G2: erastin (5 μmol/L) + si-NC G3: erastin (5 μmol/L) + si-NC + TF3 (30 μmol/L) G4: erastin (5 μmol/L) + si-Nrf2 + TF3 (30 μmol/L) (24 hours) |
Apoptotic effect: PI staining Cytotoxicity assays: MTS assay kit Gene quantification: PCR Intracellular ROS level: DCFH-DA Mitochondrial iron level: Mito-FerroGreen Protein expression level: WB |
TF3 significantly inhibits chondrocyte ferroptosis by activating the Nrf2/GPx4 signalling pathway, suggesting that TF3 serves as a potential therapeutic supplement for OA. |
| Yao et al. (15) (2021, China) |
Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: control G2: IL-1β (10 ng/mL) G3: IL-1β (10 ng/mL) + Fer-1 (1 μmol/L) G4: FAC (100 μmol/L) G5: FAC (100 μmol/L) + Fer-1 (1 μmol/L) (48 hours) |
Cell viability assay: CCK-8 kit Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Protein expression level: WB and IF |
Chondrocyte ferroptosis contributes to the progression of osteoarthritis. |
| Zhou et al. (20) (2021,China) | Primary chondrocytes (healthy); C57BL/6J mice (knee joint) | G1: mock G2: mock + IL-1β (10 ng/mL) G3: mock + IL-1β (10 ng/mL) + D-mannose G4: Ad-Epas1 + IL-1β (10 ng/mL) G5: Ad-Epas1 + IL-1β (10 ng/mL) + D-mannose G6: Ad-Epas1 + IL-1β (10 ng/mL) + D-mannose + Fer-1 (24 hours) |
Analysis of oxidative stress: GSH, MDA and SOD assay kit Cell viability assay: CCK-8 kit Gene quantification: PCR Intracellular ROS and lipid ROS level: DCFH-DA and C11-BODIPY Mitochondrial morphology: Mito-Tracker Morphology: TB and SOFG staining Protein expression: WB and IF |
D-mannose decreases chondrocyte ferroptosis sensitivity by inhibiting HIF-2α expression. |
Ad-Epas1, adenovirus vector-endothelial PAS domain-containing protein 1; Ad-HSPA5, adenovirus vector-heat shock protein family a member 5; Ad-SND1, adenovirus vector-staphylococcal nuclease domain containing 1; AICAR, acadesine; AKT, protein kinase B; AMPKα, adenosine 5’monophosphate-activated protein kinase alpha; Annexin V-FITC/PI, annexin V‐fluorescein isothiocyanate/propidium iodide; ATX, astaxanthin; BA, BAPTA acetoxymethyl ester; BCA, biochanin A; CAD, cardamonin; Calcein-AM, calcein acetoxymethyl ester; CCK-8, cell counting kit-8; Co-IP, co-immunoprecipitation; Col2a1-CreERT GPx4flox/flox, tamoxifen-inducible chondrocyte-specific homozygous GPx4 conditional knockout; CPS, capsaicin; DCFH-DA, dichlorodihydrofluorescein diacetate assay; DFO, deferoxamine; DMT1, divalent metal transporter 1; EAAT1, excitatory amino acid transporter protein 1; ECM, extracellular matrix; EdU, 5-ethynyl-2’-deoxyuridine; ELISA, enzyme linked immunosorbent assay; EPAS1, endothelial PAS domain-containing protein 1; FAC, ferric ammonium citrate; Fer-1, ferrostatin-1; GPx, glutathione peroxidase; GSH, glutathione; GsMTx-4, M-theraphotoxin-Gr1a; GSSG, oxidised glutathione; HIF-2α, hypoxia-inducible factor 2 alpha; HO-1, heme oxygenase 1; HSPA5, heat shock protein family A member 5; IL-1β, interleukin-1β; IOOA, iron overload-induced osteoarthritis; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; MTT, methylthiazolyldiphenyl-tetrazolium bromide; NAC, N-acetyl-cysteine; NAR-L, naringenin low concentration; NAR-H, naringenin high concentration; NF-κB, nuclear factor kappa-B; NR, not reported; Nrf2, nuclear factor–erythroid factor 2; OA, osteoarthritis; Oe-NC, negative control overexpressing vector; Oe-SREBF2, sterol regulatory element binding transcription factor 2 overexpressing vector; PCR, polymerase chain reaction; PI, propidium iodide; PI3K, phosphoinositide 3-kinase; RIP, RNA immunoprecipitation; ROS, reactive oxygen species; sh-GPx4, short hairpin RNA-glutathione peroxidase 4; sh-NC, short hairpin RNA-negative control; sh-SLC3A1, short hairpin RNA-resolute carrier family 3 member 1; sh-SLC3A2, short hairpin RNA-solute carrier family 3 member 2; sh-SND1, short hairpin RNA-staphylococcal nuclease domain containing 1; sh-HSPA5, short hairpin RNA-heat shock protein family A member 5; si-DMT1, small interfering RNA-divalent metal transporter 1; si-NC, small interfering negative control; si-Nrf2, small interfering RNA-nuclear factor-erythroid factor 2; siRNA, small interfering ribonucleic acid; SLC3A2-1, solute carrier family 3 member 2; SLC3A2, solute carrier family 3 member 2; SLC7A11, solute carrier family 7 member 11; SND1, staphylococcal nuclease domain containing 1; SOD, superoxide dismutase; SOFG, safranin O-Fast Green; System Xc-, cystine-glutamate antiporter; SREBF2, sterol regulatory element binding transcription factor 2; STM, stigmasterol; TB, Toluidine blue; TBHP, tert-butyl hydroperoxide. TEM, transmission electron microscopy; TF3, theaflavin-3,3’-digallate; TNF-α, tumour necrosis factor alpha; TRPV1, transient receptor potential vanilloid 1; WB, Western blot.