Puerarin alleviates rheumatoid arthritis in rats by modulating TAK1-mediated TLR4/NF-κB signaling pathway

doi:10.12122/j.issn.1673-4254.2025.10.19

Abstract

Abstract:

Objective To explore the therapeutic mechanism of puerarin for alleviating synovitis in rats with collagen-induced arthritis (CIA). Methods In a SD rat model of CIA, we tested the effects of daily gavage of puerarin at low, moderate and high doses (10, 30, and 100 mg/kg, respectively) for 3 weeks, with tripterygium glycosides (GTW, 10 mg/kg) as the positive control, on swelling in the hind limb joints regions evaluated by arthritis index scoring. Mass fraction of the liver of the rats was calculated, and pathologies in joint synovial membrane were observed with HE staining. The expressions of transforming growth factor β‑activated kinase-1 (TAK1), Toll-like receptor 4 (TLR4), and nuclear factor kappa-Bp65 (NF‑κB p65) at the mRNA and protein levels in the synovial tissues were detected using Real-time PCR and Western blotting. Results Compared with those in the model group, the rats in GTW group and high-dose puerarin group showed significantly reduced mass fraction of the liver. Treatment with GTW and puerarin at the 3 doses all significantly alleviated plantar swelling, lowered arthritis index scores, and improved synovitis in CIA rats (P<0.05), and the effects of puerarin showed an obvious dose dependence. Both GTW and puerarin treatments significantly lowered TAK1, TLR4, and NF‑κB p65 mRNA and protein expressions in the synovium of CIA rats. Conclusion Puerarin alleviates synovium damages in CIA rats possibly by suppressing the TLR4/NF‑κB signaling pathway via downregulating TAK1 expression.

Key words: puerarin, rheumatoid arthritis, synovium, TAK1, Toll-like receptor 4, nuclear factor kappa-B p65

Maiyuan XU, Ni LI, Jiayi LI, Tao ZHANG, Liwen MA, Tao LIN, Haonan YU, Ning WU, Zunqiu WU, Li HUANG. Puerarin alleviates rheumatoid arthritis in rats by modulating TAK1-mediated TLR4/NF-κB signaling pathway[J]. Journal of Southern Medical University, 2025, 45(10): 2231-2239.

Figures/Tables 7

Tab.1 Primer sequences used for Real-time PCR

Primer	Sequences
GAPDH Forward: Reverse:
	5'-GAAGCTGGTCATCAACGGGA-3'
	5'-GGCGGAGATGATGACCCTTT-3'
TLR4 Forward: Reverse:
	5'-CCGCTCTGGCATCATCTTCATTG-3'
	5'-GCTTTTCCATCCAACAGGGCTTT-3'
TAK1 Forward: Reverse:
	5'-ATCAACAAGCACCACCGTAA-3'
	5'AATGTGGGAGTGGCTATCAGA-3'
NFκB-p65 Forward: Reverse:
	5'-GCTTGCTTTAGCCCTTTGGT-3'
	5'-AAGAGGGAAGAAGCCACCAG-3'

Tab.2 Effect of puerarin on swelling of bilateral hind limb joints in rats with collagen-induced arthritis (Mean±SD, n=10)

Group	1week before CIA modeling	1week before gavage	1 weeks of gavage	2 weeks of gavage	3 weeks of gavage
Normal	1.435±0.332	1.418±0.215	1.442±0.235	1.456±0.324	1.436±0.243
Model	1.465±0.351	1.892±0.416***	1.852±0.632***	1.853±0.347***	1.865±0.359**
GTW	1.441±0.236	1.798±0.342	1.633±0.285^###	1.536±0.245^###	1.425±0.257^###
Pur 10 mg/kg	1.425±0.317	1.865±0.416	1.831±0.375^###	1.768±0.447^###	1.703±0.654^##
Pur 30 mg/kg	1.437±0.341	1.843±0.324	1.736±0.458^#	1.563±0.152^###▲	1.478±0.139^##▲▲
Pur 100 mg/kg	1.427±0.387	1.824±0.332	1.715±0.519^▲▲	1.6145±0.614^##▲▲▲	1.444±0.335^#▲▲▲

Fig.1 Hind limb joint swelling of the rats in each group. A-F: Normal control group, CIA Model group, GTW group, Puerarin (Pur) 10 mg/kg group, Pur 30 mg/kg group, and Pur 100 mg/kg group, respectively. Joint swelling was measured using the same measurement standard and instrument calibration.

Tab.3 Effect of puerarin on arthritis index score in rats with collagen-induced arthritis (Mean±SD, n=10)

Group	1week before CIA modeling	1week before gavage	1 weeks of gavage	2 weeks of gavage	3 weeks of gavage
Normal	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
Model	6.52±1.22***	6.45±1.25***	6.44±1.34***	6.32±1.68***	6.37±1.39***
GTW	6.25±1.21	6.56±1.36	4.55±1.12^#	2.54±0.78^##	0.89±0.21^###
Pur 10 mg/kg	6.55±1.32	6.43±1.41	5.82±1.27	5.04±1.17^#	4.36±0.42^##
Pur 30 mg/kg	6.46±1.18	6.15±1.23	4.78±1.31	4.24±1.42	2.56±0.89
Pur 100 mg/kg	6.59±1.76	6.38±1.29	4.52±1.23	2.24±1.02	0.74±0.15

Fig.2 Effect of puerarin on mass fraction of the liver of the rats with collagen-induced arthritis (Mean±SD, n=10). *P<0.05 vs the Nor group; #P<0.05, ##P<0.01 vs the Mod group.

Fig.3 Pathological changes in the synovium of the rats. (HE staining, original magnification: ×100). A-F: Normal control group, CIA model group, GTW group, Puerarin(Pur) 10 mg/kg group, Pur 30 mg/kg group, and Pur 100 mg/kg group, respectively. Black arrows indicate synovial cell hyperplasia, yellow arrows denote shedding of synovial cells, green arrows indicate connective tissue hyperplasia, and purple arrows highlight widening of collagen fiber interstices.

Fig.5 Effect of puerarin on expression levels of TAK1, p-TAK1, NF-κB p65 and TLR4 proteins in the synovium of the rats with collagen-induced arthritis (n=10). A: Expression levels of TAK1 and p-TAK1 proteins in the synovium of the rats. B: Expression level of TLR4 protein in the synovium of the rats. C: Expression level of NF-κB p65 protein in the synovium of the rats. D: Western blotting for detecting expression levels of TAK1, p-TAK1, NF-κB p65 and TLR4 in each group. **P<0.01,***P<0.001 vs the Nor group; #P<0.05, ##P<0.01, ###P<0.001 vs the Mod group.

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