化痰祛湿和益气健运方药对巨噬细胞泡沫化的影响及自噬机制研究

Effects of“Huatan Qushi Formula”and“Yiqi Jianyun Formula”on the Foaming of Macrophages and the Autophagy Mechanism

目的:观察王琦教授自拟化痰祛湿方(薏脂颗粒)和益气健运方(茯元颗粒)对泡沫细胞脂质的影响,并探索其自噬机制。方法:培养RAW264.7细胞,用氧化低密度脂蛋白(ox-LDL)构建泡沫细胞模型。通过cell titer-blue法进行细胞活力测定,通过油红O染色法检测细胞脂质含量,通过蛋白质印迹法检测CD36和轻链3蛋白表达,用自噬蛋白芯片进一步研究两方对泡沫细胞自噬的影响。结果:油红O染色结果显示,ox-LDL模型组细胞脂质含量升高,泡沫细胞造模成功。两方干预后,细胞脂质含量降低。蛋白质印迹法结果显示:两方干预后CD36表达均升高,化痰祛湿方50 ng/μL组和益气健运方50 ng/μL、100 ng/μL组轻链3Ⅱ/轻链3Ⅰ水平升高。自噬芯片结果显示:与空白对照组比较,ox-LDL模型组P62升高。与ox-LDL模型组比较,化痰祛湿方组P62下降,自噬相关蛋白10上调,益气健运方组的自噬相关蛋白10、自噬相关蛋白12、自噬相关蛋白4A上调。结论:两方均可降低泡沫细胞脂质含量,可能是通过增强巨噬细胞的自噬作用从而促进脂质代谢,减少泡沫细胞的形成。

Objective:To observe the effects of Prof.WANG Qi′s self-prepared Huatan Qushi Formula(Yizhi Granules)and Yiqi Jianyun Formula(Fuyuan Granules)on foam cell lipids,and to explore the mechanism of autophagy.Methods:RAW264.7 cells were cultured,and a foam cell model was constructed with oxidized low-density lipoprotein(ox-LDL).Cell viability was determined by cell titer-blue method,the lipid content in cells was detected by oil red O staining method,CD36 and LC3 protein expressions were detected by Western Blot method,and the effects of the two formulas on foam cell autophagy were further studied using autophagy protein chips.Results:The Oil red O staining results showed that the lipid content in cells in the ox-LDL model group increased,suggesting that foam cell modeling was successful.After the intervention with the two formulas,the lipid content in cells decreased.The Western blot results showed that the CD36 expression increased after the intervention with the two formulas,and the LC3Ⅱ/LC3Ⅰexpression increased in the Huatan Qushi Formula(50 ng/μL)group and the Yiqi Jianyun Formula(50 ng/μL,100 ng/μL)group.The results of autophagy chips showed that compared with the control group,the P62 expression in the ox-LDL model group increased.Compared with the ox-LDL model group,the P62 expression decreased and the ATG10 expression was up-regulated in the Huatan Qushi Formula group,while the ATG10,ATG12,and ATG4A expression were up-regulated in the Yiqi Jianyun Formula group.Conclusion:Both formulas can reduce the lipid content in foam cells,and the possible mechanism is that they promote lipid metabolism and reduce the formation of foam cells by enhancing the autophagy of macrophages.