枫杨总黄酮通过抑制中性粒细胞胞外陷阱网的释放减轻大鼠类风湿关节炎

doi:10.12122/j.issn.1673-4254.2024.09.03

摘要/Abstract

摘要：

目的探讨枫杨总黄酮（PHSTF）干预类风湿关节炎（RA）的机制，为治疗类风湿关节炎提供新的理论基础。方法将25只雄性SD大鼠随机分为正常组、模型组（CIA组）、枫杨总黄酮低剂量组［CIA+PHSTF （45 mg/kg）］、枫杨总黄酮高剂量组［CIA+PHSTF （90 mg/kg）］、雷公藤多苷片组［CIA+ TPG（10 mg/kg）］，5只/组。除正常组外，对其余大鼠采用二次免疫法建立CIA大鼠模型。二次免疫结束后，枫杨总黄酮组和雷公藤多苷片组按相应剂量灌胃给药，1次/d，连续4周。采用ELISA检测血清中细胞因子（TNF-α、IL-1β）的水平；HE染色检测踝关节组织损伤情况；免疫组化（IHC）检测踝关节中性粒细胞胞外陷阱网（NETs）标志物瓜氨酸化组蛋白3（Cit-H3）的表达水平。建立细胞模型，设立枫杨总黄酮不同剂量组（0、12.5、25、50、100、200 μg/mL），利用CCK-8探索PHSTF用药浓度，采用迪夫快速染色法观察中性粒细胞的形态；设立对照组（Control），佛波酯（PMA）刺激中性粒细胞模拟NETs形成建立模型组（PMA），枫杨总黄酮低剂量组［CIA+PHSTF （100 μg/mL）］，枫杨总黄酮高剂量组［CIA+PHSTF （200 μg/mL）］。Western blotting检测枫杨总黄酮干预后NETs标志性蛋白Cit-H3，肽酰基精氨酸脱亚胺酶4（PADI4），中性粒细胞弹性蛋白酶（NE）以及髓过氧化物酶（MPO）的表达，免疫荧光检测Cit-H3的表达观察NETs形成。结果体内实验中，与模型组相比较枫杨总黄酮可明显改善CIA大鼠踝关节的肿胀程度（P<0.05），降低血清中炎症因子水平（P<0.05），显著改善踝关节组织病理程度，降低血清以及踝关节软骨中性粒细胞胞外诱捕网浓度，抑制中性粒细胞胞外诱捕网的释放（P<0.05）。体外实验中，枫杨总黄酮并未对中性粒细胞活性有明显影响（P<0.01），此外枫杨总黄酮显著抑制PMA诱导的中性粒细胞胞外诱捕网的释放（P<0.05）。结论枫杨总黄酮可以通过抑制NETs的产生，达到干预类风湿关节炎的目的。

关键词: 枫杨总黄酮, 中性粒细胞胞外陷阱网, 类风湿关节炎

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

杨锐, 舒翊秦, 文晖杰, 蔡熹, 王震, 张棽, 向阳, 吴昊. 枫杨总黄酮通过抑制中性粒细胞胞外陷阱网的释放减轻大鼠类风湿关节炎[J]. 南方医科大学学报, 2024, 44(9): 1645-1652.

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.

图/表 6

图1 枫杨总黄酮减轻CIA大鼠踝关节肿胀程度

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.

图2 枫杨总黄酮对CIA大鼠血清中炎症因子变化的影响

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.

图3 枫杨总黄酮改善CIA大鼠踝关节组织病理情况

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

图4 枫杨总黄酮抑制CIA大鼠中性粒细胞胞外陷阱的释放

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.

图5 枫杨总黄酮对正常中性粒细胞胞活性及形态无显著影响

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 ).

图6 枫杨总黄酮抑制NETs相关蛋白的表达

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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