目的 探讨藏药肺热普清散对斑马鱼炎症模型的作用及机制。方法 挑选受精后 72 h 斑马鱼随机移入 24 孔板中,分为对照组、模型组和肺热普清散低、中、高质量浓度(3、10、30 mg·L^-1)组,对照组与模型组不加药,肺热普清散与斑马鱼共孵育 1 h 后,除对照组外,其余各组用 20 μmol·L^-1硫酸铜处理 2 h 制备炎症模型。在荧光显微镜下观察绿色荧光标记中性粒细胞的 Tg(Lyz:EGFP)系斑马鱼体内炎症细胞迁移情况;实时荧光定量 PCR(qRT-PCR)检测野生型斑马鱼核因子κB2(NF-κB2)、白细胞介素(IL)-1b、肿瘤坏死因子(TNF)-α、IL-8 mRNA 水平;Western blotting 实验检测野生型斑马鱼 NF-κB、TNF-α 蛋白表达水平;免疫荧光法检测各组鱼尾 NF-κB 表达。结果 对照组斑马鱼荧光细胞主要分布于头部和主动脉部,躯干部位主动脉以上荧光细胞稀少;模型组荧光细胞在各区域均增加,显示出炎症细胞向血管外迁移的明显趋势,躯干侧线以上荧光细胞计数较对照组显著增加(P<0.001);肺热普清散 10、30 mg·L^-1组躯干部荧光细胞计数与模型组比较显著降低(P<0.01、0.001)。与模型组比较,肺热普清散 3、10、30 mg·L^-1 组 IL-1b、TNF-α、IL-8 的 mRNA 水平显著下调(P<0.05、0.01),10、30 mg·L^-1 组 NF-κB2 mRNA 水平显著下调(P<0.01);10、30 mg·L^-1 组 NF-κB、TNF-α 蛋白表达水平显著降低(P<0.01、0.001)。免疫荧光结果显示,模型组躯干部位和鱼鳍 NF-κB 白色荧光点较对照组明显增加,经 10 mg·L^-1肺热普清散处理后的相同部位荧光点减少。结论 肺热普清散对硫酸铜诱导的斑马鱼炎症模型具有抗炎作用,机制与抑制 NF-κB、TNF-α 表达有关。

Objective To explore the effects of Feire Puqing Powder (FPP) on inflammation and its mechanism on zebrafish model. Methods Zebrafish 72 h after fertilization were randomly transferred into 24-well plates and divided into control group, model group and FPP low, medium and high concentration (3, 10, and 30 mg·L^-1) groups. Control group and model group were not given any medicine. After FPP was co-incubated with zebrafish for one h, except the control group, the other groups were treated with 20 μmol·L^-1 copper sulfate for two h to prepare inflammation model. The number of inflammatory cells in the midline area of green fluorescence labeled neutrophils transgenic strain Tg (Lyz: EGFP) zebrafish was observed and recorded by fluorescence microscope. Quantitative real-time PCR (qRT-PCR) was used to detect mRNA levels of nuclear factor κB2 (NF-κB2), interleukin (IL-1b), tumor necrosis factor (TNF) -α and IL-8 in wild-type zebrafish. The expression levels of NF-κB and TNF-α in wild-type zebrafish were detected by Western blotting assay. The expression of NF-κB in fish tail of each group was detected by immunofluorescence method. Results In the control group, the fluorescent cells were mainly distributed in the head and the aorta, and the fluorescent cells above the aorta were rare in the trunk. The fluorescence cells in the model group were increased in all areas, showing an obvious trend of extravascular migration of inflammatory cells, and the fluorescence cell count above the lateral trunk line was significantly increased compared with the control group (P<0.001). Compared with model group, the fluorescent cell count of trunk cadre in 10 and 30 mg·L^-1 groups was significantly decreased (P<0.01, 0.001). Compared with model group, mRNA levels of IL-1b, TNF-α and IL-8 in FPP 3, 10 and 30 mg·L^-1 groups were significantly down-regulated (P<0.05, 0.01), and NF-κB2 mRNA levels in 10 and 30 mg·L^-1 groups were significantly down-regulated (P<0.01). The expression levels of NF-κB and TNF-α in 10 and 30 mg·L^-1 groups were significantly decreased (P<0.01, 0.001). The white fluorescence spots of NF-κB in the trunk and fin of the model group were significantly increased compared with the control group, and the fluorescence spots in the same parts of the model group were decreased after 10 mg· L^-1 FPP treatment. Conclusion Feirepuqing Powder had anti-inflammatory effect on zebrafish inflammation model induced by copper sulfate, and the mechanism was related to the inhibition of NF-κB and TNF-α expression.

R285.5

西藏自治区科技厅中央引导地方科技发展项目(XZ202202YD0020C);济南市"高校 20 条"资助项目引进创新团队项目(2020GXRC031);山东省自然科学基金重大基础研究项目(ZR2021ZD29)