Electroacupuncture alleviates articular chondrocyte apoptosis in rats with knee osteoarthritis by regulating the TLR4/MyD88/NF-κB signaling pathway

doi:10.12122/j.issn.1673-4254.2026.08.16

Abstract

Abstract:

Objective To investigate the effect of electroacupuncture (EA) on chondrocyte apoptosis in rats with knee osteoarthritis (KOA) and the underlying mechanism. Methods Twenty-seven SD rats were randomly divided into blank group, KOA model group, and EA group (n=9). Rat models of KOA were established by intra-articular injection of monosodium iodoacetate, and EA treatment was administered at the acupoints "Neixiyan", "Dubi", "Xuehai", and "Yanglingquan" (dense-sparse wave, 2/100 Hz, 0.5-1.0 mA, 20 min/day for 14 days). Knee function of the rats was assessed using Lequesne MG scale, and cartilage pathologies and ultrastructural changes were observed with HE staining and transmission electron microscopy. The protein expressions of TLR4, MyD88, NF-κB p65, COL2, COL10, Bax, Bcl-2, and caspase-3 and mRNA levels of TLR4, MyD88, and NF‑κB p65 were detected using Western blotting and RT-qPCR; serum levels of IL-1β and TNF‑α were determined with ELISA. Another 15 rats were randomized into blank control, model, TLR4 inhibitor (TAK-242), EA, and sham-EA groups (n=3) for analysis of HMGB1 and key pathway protein expressions and NF‑κB p65 nuclear translocation. Results The KOA rats showed increased Lequesne MG scores, obvious chondrocyte injuries, upregulated TLR4, MyD88, NF‑κB p65, COL10, Bax, and caspase-3 expressions, increased serum IL-1β and TNF‑α levels with decreased COL2 and Bcl-2 expressions. EA significantly reduced Lequesne MG scores, improved cartilage pathologies and reversed the abnormal changes in protein expressions and inflammatory cytokines. The KOA rats showed increased HMGB1 expression and enhanced NF‑κB p65 nuclear translocation, and EA treatment effectively decreased HMGB1 expression, reduced nuclear NF‑κB p65, and increased cytoplasmic NF‑κB p65. The TLR4 inhibitor produced similar effects, while sham EA produced no significant effect. Conclusion EA alleviates cartilage injury in KOA rats possibly by inhibiting HMGB1-mediated activation of TLR4/MyD88/NF‑κB signaling and NF‑κB p65 nuclear translocation to regulate Bcl-2/Bax balance and suppress mitochondria-dependent chondrocyte apoptosis.

Key words: knee osteoarthritis, TLR4/MyD88/NF-κB signaling pathway, electroacupuncture, apoptosis, knee cartilage

Chunning LI, Qiqi YANG, Zhenfeng ZHANG, Liang BAO, Tianxin JIANG, Yu ZHANG, Fei LI. Electroacupuncture alleviates articular chondrocyte apoptosis in rats with knee osteoarthritis by regulating the TLR4/MyD88/NF-κB signaling pathway[J]. Journal of Southern Medical University, 2026, 46(8): 1886-1895.

Figures/Tables 9

Tab.1 Primer sequences for RT-qPCR

Gene	Amplicon size (bp)	Forward primer (5'→3')	Reverse primer (5'→3')
TLR4	188	CACCAGGAAGCTTGAATCCCT	ATCCAGCCACTGAAGTTCTGA
Myd88	178	CATACCCTTGGTCGCGCTTA	CCAGGCATCCAACAAACTGC
NF-κB p65	181	CATGGAGGTGGCGGTCTATC	CACAGCGTACATCAGGTGGT
GAPDH	78	TGGTGAAGCAGGCATCTGAG	TGAAGTCGCAGGAGACAACC

Fig.1 Comparison of LequesneMG scores of the rats in the 3 groups after modeling and intervention (Mean±SD, n=9). **P<0.01 vs Control group,##P<0.05 vs Model group.

Fig.2 Pathological changes of knee articular chondrocytes in rats from different groups (HE staining, Scale bar=20 μm). Arrows indicate nuclear pyknosis, disappearance, and cell swelling.

Fig.3 Transmission electron microscopy showing ultrastructural changes of the chondral nucleus membrane, chromatin, and mitochondria of the rats. The blue arrow indicates the nuclear envelope, the red arrow indicates the mitochondria, and the green arrow indicates the chromatin.

Fig.4 Western blotting for detecting protein expression levels of TLR4, MyD88, NF-κBp65, COL2, COL10, Bax, Bcl-2, and Caspase-3 in the knee joint cartilage of the rats (Mean±SD, n=3 ). **P<0.01 vs Control group, ##P<0.05 vs Model group.

Fig.5 Comparison of TLR4, MyD88, and NF-κB p65 mRNA expression levels in knee joint cartilage tissues among the 3 groups (Mean±SD, n=3). **P<0.01 vs Control group,##P<0.05 vs Model group.

Fig.6 Comparison of serum IL-1β and TNF-α levels among the 3 groups (Mean±SD, n=9). **P<0.01 vs Control group, ##P<0.05 vs Model group.

Fig.7 Western blotting for detecting expression levels of HMGB1, TLR4, and MyD88 proteins and nuclear translocation of NF‑κB p65 in knee articular cartilage tissues of the rats (Mean±SD, n=3). **P<0.01 vs Control group, ##P<0.05 vs Model group.

Fig.8 Comparison of TLR4, MyD88, and NF-κB p65 mRNA expression levels in knee joint cartilage tissue among five groups of rats (Mean±SD, n=3). **P<0.01 vs Control group, ##P<0.05 vs Model group.

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