参附黄方对斑马鱼炎症、巨噬细胞吞噬功能的影响

Effects of Shenfuhuang Fomula(参附黄方)on Inflammation and Phagocytic Function of Macrophages in Zebrafish

目的探讨参附黄方对斑马鱼炎症、巨噬细胞吞噬功能的作用及其机制。方法以转基因中性粒细胞荧光斑马鱼、野生型AB品系斑马鱼为实验对象,分别建立斑马鱼炎症模型和巨噬细胞吞噬模型。采用死亡率和心血管毒性评价筛选参附黄方的药用浓度;采用中性粒细胞计数评价参附黄方对斑马鱼炎症模型的作用,并以吲哚美辛为对照;检测斑马鱼白细胞介素10(IL-10)、肿瘤坏死因子α(TNF-α)、诱导型一氧化氮合酶(iNOS)、重组人精氨酸酶1(ARG1)、白细胞介素1β(IL-1β)mRNA表达评价参附黄方对斑马鱼吞噬细胞吞噬功能的影响。结果参附黄方对斑马鱼的最大安全浓度为10 mg/ml,本研究选择参附黄低、中和高剂量浓度分别为1.1、3.3和10 mg/ml。在炎症模型中,模型对照组斑马鱼炎症部位中性粒细胞个数高于正常对照组(P<0.01);与模型对照组比较,吲哚美辛组和参附黄中、高剂量组斑马鱼炎症部位中性粒细胞个数降低(P<0.01);参附黄中剂量组炎症消退作用最强为44.1%。在巨噬细胞吞噬模型中,与正常对照组比较,参附黄中、高剂量组TNF-α、IL-10 mRNA表达升高,参附黄高剂量组IL-1βmRNA表达升高、ARG1 mRNA表达降低(P<0.05或P<0.01)。结论参附黄方具有很好的免疫激活作用,其机制可能与降低中性粒细胞数量和调节巨噬细胞分型有关。

Objective To explore the effects of Shenfuhuang Fomula(参附黄方)on inflammation and phagocytosis of macrophages in zebrafish and its mechanism.Methods Transgenic neutrophil fluorescent zebrafish and wild-type AB strain zebrafish were used as experimental subjects to establish a zebrafish inflammation model and a macrophage phagocytosis model,respectively.Mortality and cardiovascular toxicity were applied to screen the medicinal concentration of Shenfuhuang Fomula;neutrophil count was used to evaluate the effect of Shenfuhuang Fomula on zebrafish inflammation model,and indomethacin was set as a control;zebrafish white blood cells interleukin 10(IL-10),tumor necrosis factor alpha(TNF-α),inducible nitric oxide synthase(iNOS),recombinant human arginase 1(ARG1),interleukin 1β(IL-1β)mRNA expression were detected to evaluate the effect of Shenfuhuang Fomula on the phagocytic function of zebrafish phagocytes.Results The maximum safe concentration of Shenfuhuang Fomula for zebrafish was 10 mg/ml.In this study,the low,medium and high dose concentrations of Shenfuhuang Fomula were 1.1,3.3 and 10 mg/ml,respectively.In the inflammation model,the number of neutrophils in the inflammation site of the model control group was higher than that of the normal control group(P<0.01);compared with the model control group,in the indomethacin group and in the Shenfuhuang Fomula middle and high dose groups,the number of neutrophils in the inflammation site of zebrafish decreased(P<0.01);the inflammation subsided in the Shenfuhuang Fomula medium dose group was 44.1%.In the macrophage phagocytic model,compared with the normal control group,the expression of TNF-αand IL-10 mRNA in the Shenfuhuang Fomula medium and high dose groups increased,and the expression of IL-1βmRNA in the Shenfuhuang Fomula high dose group increased,and the expression of ARG1 mRNA decreased(P<0.05 or P<0.01).Conclusion Shenfuhuang Fomula has a good immune activation effect,and its mechanism may be related to reducing the number of neutrophils and regulating the typing of macrophages.