Mechanism of Fuyuan Huoxue decoction in inhibiting chondrocyte ferroptosis of rats with knee osteoarthritis via the STING/ACSL4 pathway

doi:10.11958/20261146

Abstract

Abstract:

Objective To investigate the mechanism by which Fuyuan Huoxue decoction (FHD) inhibiting chondrocyte ferroptosis in a rat model of knee osteoarthritis (KOA) via the stimulator of interferon genes (STING)/acyl-CoA synthetase long-chain family member 4 (ACSL4) pathway. Methods SD rats were randomly divided into six groups: the sham operation (Sham) group, the model (KOA) group, the low-dose FHD (FHD-L) group, the high-dose FHD (FHD-H) group, the positive control (PC, celecoxib) group and the FHD-H+STING activator (FHD-H+DMXAA) group, with 12 rats in each group. The KOA model was established using the modified Hulth method. After successful modeling, each group received corresponding interventions by intragastric administration or tail vein injection for 8 consecutive weeks. Knee joint function was assessed using the Lequesne MG score method. Cartilage morphology was observed by HE staining. Cartilage degeneration was evaluated by Safranin O-fast green staining combined with the OARSI score. Levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-13 (MMP-13) and the disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) in cartilage tissue were measured by ELISA. Levels of Fe²⁺, malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) in cartilage tissue were determined using biochemical kits. The level of reactive oxygen species (ROS) in cartilage tissue was detected by DCFH-DA probe method. Iron deposition was observed by Prussian blue staining. Mitochondrial ultrastructure of chondrocytes was observed by transmission electron microscopy. The protein expressions of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and cyclooxygenase-2 (COX-2) in cartilage tissue were detected by immunohistochemistry. The protein expressions of STING and ACSL4 were detected by Western blot assay. Results Compared with the Sham group, Lequesne MG score, OARSI score, cartilage inflammatory and matrix degradation factors (IL-1β, TNF-α, MMP-13 and ADAMTS-5), ferroptosis-related indicators (Fe²⁺, MDA, ROS), Prussian blue staining positive area ratio, COX-2 positive expression area and STING and ACSL4 protein expression were significantly increased in the KOA group (P<0.05), while the levels of GSH and SOD, and the positive expression areas of GPX4 and SLC7A11 were significantly decreased (P<0.05). HE staining, Safranin O-fast green staining and transmission electron microscopy revealed severe cartilage structural damage, matrix loss, mitochondrial atrophy and cristae rupture in the KOA group. Compared with the KOA group, all the above indicators were improved to varying degrees in the FHD-L group, the FHD-H group and the PC group (P<0.05), with the FHD-H group showing better efficacy than the FHD-L group (P<0.05). Among them, the protective effects on the above indicators of the FHD-H group was better than that of the FHD-L group (P<0.05). Compared with the FHD-H group, the improvement effects of the above indicators in the FHD-H +DMXAA group were significantly reversed (P<0.05). Conclusion FHD may improve joint function and cartilage damage in KOA rats by inhibiting chondrocyte ferroptosis via suppressing the STING/ACSL4 pathway.

Key words: Fu Yuan Huo Xue Tang, osteoarthritis, knee, chondrocytes, ferroptosis, STING/ACSL4 pathway

CLC Number:

R684.3

Figures/Tables 12

References 32

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组别	Lequesne MG评分（n=12）		OARSI评分（n=6）
组别	给药前	给药后	OARSI评分（n=6）
Sham组	0.08±0.02	0.22±0.08	1.03±0.52
KOA组	8.81±1.34^a	8.67±1.23^a	14.67±2.16^a
FHD-L组	8.69±1.22^a	5.92±0.71^b	10.83±1.94^b
FHD-H组	8.75±1.18^a	2.67±0.59^bc	5.17±1.47^bc
PC组	8.78±1.25^a	2.83±0.44^bc	5.83±1.60^bc
FHD-H+DMXAA组	8.72±1.16^a	7.08±0.86^d	12.50±2.07^d
F	118.941^＊＊	220.018^＊＊	54.232^＊＊

组别	Lequesne MG评分（n=12）		OARSI评分（n=6）
组别	给药前	给药后	OARSI评分（n=6）
Sham组	0.08±0.02	0.22±0.08	1.03±0.52
KOA组	8.81±1.34^a	8.67±1.23^a	14.67±2.16^a
FHD-L组	8.69±1.22^a	5.92±0.71^b	10.83±1.94^b
FHD-H组	8.75±1.18^a	2.67±0.59^bc	5.17±1.47^bc
PC组	8.78±1.25^a	2.83±0.44^bc	5.83±1.60^bc
FHD-H+DMXAA组	8.72±1.16^a	7.08±0.86^d	12.50±2.07^d
F	118.941^＊＊	220.018^＊＊	54.232^＊＊

组别	IL-1β/ （pg/mg）	TNF-α/ （pg/mg）	MMP-13/ （ng/mg）	ADAMTS-5/（ng/mg）
Sham组	12.47±2.15	8.23±1.56	3.42±0.58	2.15±0.42
KOA组	86.34±7.82^a	72.56±6.93^a	28.67±3.14^a	18.93±2.08^a
FHD-L组	58.26±6.14^b	48.37±5.25^b	18.42±2.56^b	12.67±1.85^b
FHD-H组	28.53±3.67^bc	22.18±3.42^bc	8.35±1.27^bc	5.28±0.96^bc
PC组	31.42±4.05^bc	24.56±3.78^bc	9.64±1.48^bc	6.12±1.03^bc
FHD-H+DMXAA组	67.38±6.52^d	56.29±5.84^d	22.53±2.87^d	15.47±1.92^d
F	158.243^＊＊	153.825^＊＊	115.575^＊＊	114.235^＊＊

组别	IL-1β/ （pg/mg）	TNF-α/ （pg/mg）	MMP-13/ （ng/mg）	ADAMTS-5/（ng/mg）
Sham组	12.47±2.15	8.23±1.56	3.42±0.58	2.15±0.42
KOA组	86.34±7.82^a	72.56±6.93^a	28.67±3.14^a	18.93±2.08^a
FHD-L组	58.26±6.14^b	48.37±5.25^b	18.42±2.56^b	12.67±1.85^b
FHD-H组	28.53±3.67^bc	22.18±3.42^bc	8.35±1.27^bc	5.28±0.96^bc
PC组	31.42±4.05^bc	24.56±3.78^bc	9.64±1.48^bc	6.12±1.03^bc
FHD-H+DMXAA组	67.38±6.52^d	56.29±5.84^d	22.53±2.87^d	15.47±1.92^d
F	158.243^＊＊	153.825^＊＊	115.575^＊＊	114.235^＊＊

组别		Fe²⁺/（μmol/g）	MDA/（nmol/g）		ROS（荧光强度）
Sham组		0.28±0.05	1.32±0.21		100.00±8.56
KOA组		1.86±0.17^a	5.78±0.52^a		286.54±18.63^a
FHD-L组		1.28±0.14^b	4.12±0.43^b		218.36±15.42^b
FHD-H组		0.58±0.08^bc	2.18±0.26^bc		138.27±11.35^bc
PC组		0.65±0.09^bc	2.35±0.28^bc		145.38±12.17^bc
FHD-H+DMXAA组		1.52±0.15^d	4.86±0.47^d		248.65±16.28^d
F		155.527^＊＊	126.533^＊＊		157.966^＊＊
组别	GSH/ （μmol/g）		SOD/ （U/mg）	普鲁士蓝染色阳性面积比/%
Sham组	8.64±0.87		42.38±3.86	0.42±0.11
KOA组	2.35±0.31^a		15.28±1.94^a	8.76±0.94^a
FHD-L组	3.86±0.42^b		24.63±2.35^b	5.83±0.72^b
FHD-H组	6.72±0.58^bc		36.47±2.86^bc	1.85±0.34^bc
PC组	6.48±0.62^bc		35.26±2.74^bc	2.12±0.41^bc
FHD-H+DMXAA组	3.12±0.38^d		20.18±2.13^d	6.94±0.85^d
F	114.110^＊＊		90.650^＊＊	163.643^＊＊