Pterocarya hupehensis Skan total flavones ameliorate rheumatoid arthritis in rats by suppressing formation of neutrophil extracellular traps

doi:10.12122/j.issn.1673-4254.2024.09.03

Abstract

Abstract:

Objective To investigate the therapeutic mechanism of Pterocarya hupehensis Skan total flavonoids (PHSTF) for rheumatoid arthritis (RA). Methods Twenty-five male SD rats were randomly divided into normal control group, RA model group, PHSTF treatment (45 and 90 mg/kg) groups, and Tripterygium glycosides (TPG) tablet (10 mg/kg) group (n=5). Except for those in the normal control group, all the rats were subjected to collagen-induced arthritis (CIA) modeling using a secondary immunization method, after which PHSTF and TPG were administered via gavage once daily for 4 weeks. After the treatments, serum levels of TNF-α and IL-1β were measured using ELISA, and ankle joint pathologies were assessed with HE staining; the expression of citrullinated histone H3 (Cit-H3), a neutrophil extracellular trap (NET) marker, in the ankle joints was evaluated with immunohistochemistry. In primary cultures of rat peripheral blood neutrophils stimulated with phorbol ester (PMA), the effects of PHSTF (100 and 200 μg/mL) on the expressions of Cit-H3, peptidylarginine deiminase 4 (PADI4), neutrophil elastase (NE), and myeloperoxidase (MPO) were examined with Western blotting; immunofluorescence assay was used to observe Cit-H3 expression and NET formation in the cells. Results In the CIA rat models, PHSTF significantly alleviated ankle swelling, decreased serum levels of TNF-α and IL-1β, improved histopathological changes in the ankle joints, and reduced Cit-H3 expression in both the serum and ankle joint cartilage. In the isolated rat neutrophils, PHSTF showed no significant effect on cell viability but strongly inhibited PMA-induced NET release. Conclusion PHSTF can alleviate RA by inhibiting the formation of NETs.

Key words: Pterocarya hupehensis Skan total flavonoids, neutrophil extracellular traps, rheumatoid arthritis

Rui YANG, Yuqin SHU, Huijie WEN, Xi CAI, Zhen WANG, Chen ZHANG, Yang XIANG, Hao WU. Pterocarya hupehensis Skan total flavones ameliorate rheumatoid arthritis in rats by suppressing formation of neutrophil extracellular traps[J]. Journal of Southern Medical University, 2024, 44(9): 1645-1652.

Figures/Tables 6

Fig.1 Pterocarya hupehensis Skan total flavonoids (PHSTF) alleviates ankle joint swelling in rat models with college-induced arthritis (CIA). A: Timeline of modeling and treatment of the rats. B: Observation of the ankle joints of the rats in different groups. C: Comparison of severity of arthritis among the 5 groups (Mean±SD, n=5). D: Comparison of ankle joint diameter for assessing joint swelling (Mean±SD, n=5). CIA: Collagen-induced arthritis; TPG: Tripterygium glycosides; PHSTF: Pterocarya hupehensis Skan total flavonoids. *P<0.05, **P<0.01 vs control group.

Fig.2 Effect of PHSTF on serum levels of TNF-α (A) and IL-1β (B) in CIA rats (Mean±SD, n=5). **P<0.01 vs control group; #P<0.05, ##P<0.01 vs CIA group.

Fig. 3 PHSTF treatment for 7 weeks improves ankle joint histopathology in CIA rats (HS staining, original magnification: ×100).

Fig.4 PHSTF inhibits neutrophil extracellular trap (NET) release in CIA rats. A, B: Immunohistochemical detection of citrullinated histone 3 (CitH3) expression in the ankle joint tissue in each group (n=5) at week 7 (×200). C: Comparison of serum levels of NETs release among control, CIA, and PHSTF (45 and 90 mg/mL) groups, presented as myeloperoxidase MPO/DNA complex levels (Mean±SD, n=4). **P<0.01 vs control group; #P<0.05, ##P<0.01 vs CIA group.

Fig.5 PHSTF does not affect normal rat neutrophil viability or morphology. A: Survival rates of neutrophils treated with 12.5, 25, 50, 100, and 200 μmol/L PHSTF for 4 h determined by CCK-8 assay. B: Effect of different concentrations of PHSTF on neutrophil morphology observed with Diff-Quick stain Kit (×200 ).

Fig. 6 PHSTF inhibits expressions of NETs-related proteins in PMA-induced rat neutrophils. A: Western blotting for citrullinated histone H (Cit-H3), neutrophil elastase (NE), myeloperoxidase (MPO), and peptidyl arginine deiminase (PADI4) in the 3 groups. β-actin was used as an internal reference (Mean±SD, n=3. **P<0.01 vs control group; ##P<0.01 vs PMA group). B: Immunofluorescence staining of cell crawls in different groups (scale bar=20 μm).

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