Effect and its mechanism of emodin on the ferroptosis of chondrocytes in rats with knee osteoarthritis

doi:10.11958/20221976

Abstract

Abstract:

Objective To investigate the effect and its mechanism of emodin on the ferroptosis of chondrocytes in rats with knee osteoarthritis (KOA). Methods A total of 96 SD rats were divided into the sham operation (sham) group, the KOA group, the emodin (EMO) group and the emodin+Nrf2 inhibitor (EMO+ML385) group, with 24 rats in each group. Except for the sham group, KOA rat models were established by modified Hulth method in each group. The tumor necrosis factor-α (TNF-α), nitric oxide (NO) and prostaglandin E2 (PGE2) levels were detected by enzyme-linked immunosorbent assay (ELISA). General morphology of knee joint was observed with naked eyes. The pathological morphology of knee joint cartilage was observed with safranine O-solid green staining. Apoptosis rate of chondrocytes of knee joint was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Malondialdehyde (MDA), reactive oxygen species (ROS), glutathione (GSH) and ferrous ion (Fe²⁺) levels in knee joint cartilage were detected by biochemical reagent kit. Expression levels of matrix metalloproteinase (MMP)-3, MMP-13, collagen type II α1 (COL2A1), prostaglandin-endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long chain family member 4 (ACSL4), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) mRNA or protein in knee joint cartilage were detected by real-time fluorescence quantitative PCR (qRT-PCR), Western blot assay or immunohistochemistry. Results Compared with the sham group, the serum TNF-α, NO and PGE2 levels, Osteoarthritis Research Society International (OARSI) score, apoptosis rate, mRNA expression of MMP-3, MMP-13 and PTGS2, MDA, ROS, and Fe²⁺ levels, proportion of ACSL4 positive cells, and protein expression of ACSL4, nuclear Nrf2 and HO-1 increased in the KOA group. GSH levels, mRNA expression of COL2A1, proportion of GPX4 positive cells, and protein expression of GPX4 and cytoplasmic Nrf2 decreased (P<0.05). The articular cartilage was obviously damaged. EMO can improve the inflammatory reaction, knee joint cartilage tissue injury and chondrocyte ferroptosis of KOA rats, and further activate Nrf2/HO-1 signal pathway. ML385 not only inhibited the activation of Nrf2/HO-1 signal pathway, but also attenuated the improvement of EMO on knee joint cartilage tissue injury and chondrocyte ferroptosis of KOA rats. Conclusion Emodin can activate Nrf2/HO-1 signal pathway, inhibit chondrocyte ferroptosis and protect knee articular cartilage of KOA rats.

Key words: emodin, osteoarthritis, knee, arthritis, experimental, chondrocytes, ferroptosis, Nrf2/HO-1 signal pathway

CLC Number:

R684.3，R285.5

Figures/Tables 14

References 23

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基因名称	引物序列
MMP-3	上游：5′-TTTGGCCGTCTCTTCCATCC-3′
	下游：5′-GCATCGATCTTCTGGACGGT-3′
MMP-13	上游：5′-TTCTGGTCTTCTGGCACACG-3′
	下游：5′-TGGAGCTGCTTGTCCAGGT-3′
COL2A1	上游：5′-GGTAAGTGGGGCAAGACTGTTA-3′
	下游：5′-TGTTGTTTCTGGGTTCAGGTTT-3′
PTGS2	上游：5′-ATGTTCGCATTCTTTGCCCAG-3′
	下游：5′-TACACCTCTCCACCGATGAC-3′
GAPDH	上游：5′-ATGACAACTCCCTCAAGAT-3′
	下游：5′-GATCCACAACGGATACATT-3′

基因名称	引物序列
MMP-3	上游：5′-TTTGGCCGTCTCTTCCATCC-3′
	下游：5′-GCATCGATCTTCTGGACGGT-3′
MMP-13	上游：5′-TTCTGGTCTTCTGGCACACG-3′
	下游：5′-TGGAGCTGCTTGTCCAGGT-3′
COL2A1	上游：5′-GGTAAGTGGGGCAAGACTGTTA-3′
	下游：5′-TGTTGTTTCTGGGTTCAGGTTT-3′
PTGS2	上游：5′-ATGTTCGCATTCTTTGCCCAG-3′
	下游：5′-TACACCTCTCCACCGATGAC-3′
GAPDH	上游：5′-ATGACAACTCCCTCAAGAT-3′
	下游：5′-GATCCACAACGGATACATT-3′

组别	TNF-α/（ng/L）	NO/（μg/L）	PGE2/（μg/L）
Sham组	48.97±6.38	30.64±4.19	0.23±0.06
KOA组	117.54±8.67^a	58.27±5.63^a	0.52±0.08^a
EMO组	69.85±7.52^b	37.52±4.56^b	0.31±0.06^b
EMO+ML385组	102.71±8.35^c	51.38±5.32^c	0.46±0.07^c
F	127.745^＊＊	51.816^＊＊	30.789^＊＊

组别	TNF-α/（ng/L）	NO/（μg/L）	PGE2/（μg/L）
Sham组	48.97±6.38	30.64±4.19	0.23±0.06
KOA组	117.54±8.67^a	58.27±5.63^a	0.52±0.08^a
EMO组	69.85±7.52^b	37.52±4.56^b	0.31±0.06^b
EMO+ML385组	102.71±8.35^c	51.38±5.32^c	0.46±0.07^c
F	127.745^＊＊	51.816^＊＊	30.789^＊＊

组别	OARSI评分/分	凋亡率/%
Sham组	1.46±0.31	4.34±0.62
KOA组	6.50±0.69^a	21.17±3.74^a
EMO组	3.64±0.45^b	8.26±1.45^b
EMO+ML385组	5.78±0.52^c	17.58±2.67^c
F	158.438^＊＊	83.641^＊＊