目的 探討藏藥肺熱普清散對斑馬魚炎癥模型的作用及機制。方法 挑選受精后 72 h 斑馬魚隨機移入 24 孔板中,分為對照組、模型組和肺熱普清散低、中、高質(zhì)量濃度(3、10、30 mg·L^-1)組,對照組與模型組不加藥,肺熱普清散與斑馬魚共孵育 1 h 后,除對照組外,其余各組用 20 μmol·L^-1硫酸銅處理 2 h 制備炎癥模型。在熒光顯微鏡下觀察綠色熒光標(biāo)記中性粒細(xì)胞的 Tg(Lyz:EGFP)系斑馬魚體內(nèi)炎癥細(xì)胞遷移情況;實時熒光定量 PCR(qRT-PCR)檢測野生型斑馬魚核因子κB2(NF-κB2)、白細(xì)胞介素(IL)-1b、腫瘤壞死因子(TNF)-α、IL-8 mRNA 水平;Western blotting 實驗檢測野生型斑馬魚 NF-κB、TNF-α 蛋白表達(dá)水平;免疫熒光法檢測各組魚尾 NF-κB 表達(dá)。結(jié)果 對照組斑馬魚熒光細(xì)胞主要分布于頭部和主動脈部,軀干部位主動脈以上熒光細(xì)胞稀少;模型組熒光細(xì)胞在各區(qū)域均增加,顯示出炎癥細(xì)胞向血管外遷移的明顯趨勢,軀干側(cè)線以上熒光細(xì)胞計數(shù)較對照組顯著增加(P<0.001);肺熱普清散 10、30 mg·L^-1組軀干部熒光細(xì)胞計數(shù)與模型組比較顯著降低(P<0.01、0.001)。與模型組比較,肺熱普清散 3、10、30 mg·L^-1 組 IL-1b、TNF-α、IL-8 的 mRNA 水平顯著下調(diào)(P<0.05、0.01),10、30 mg·L^-1 組 NF-κB2 mRNA 水平顯著下調(diào)(P<0.01);10、30 mg·L^-1 組 NF-κB、TNF-α 蛋白表達(dá)水平顯著降低(P<0.01、0.001)。免疫熒光結(jié)果顯示,模型組軀干部位和魚鰭 NF-κB 白色熒光點較對照組明顯增加,經(jīng) 10 mg·L^-1肺熱普清散處理后的相同部位熒光點減少。結(jié)論 肺熱普清散對硫酸銅誘導(dǎo)的斑馬魚炎癥模型具有抗炎作用,機制與抑制 NF-κB、TNF-α 表達(dá)有關(guān)。

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

西藏自治區(qū)科技廳中央引導(dǎo)地方科技發(fā)展項目(XZ202202YD0020C);濟南市"高校 20 條"資助項目引進(jìn)創(chuàng)新團隊項目(2020GXRC031);山東省自然科學(xué)基金重大基礎(chǔ)研究項目(ZR2021ZD29)