基于RNA-Seq技术探讨洋参芎芍方改善RAW264.7巨噬细胞代谢及炎症反应的机制

Effeve of Yangshen Xiongshao Formula on the Metabolism and Inflammatory Response of RAW264.7 Macrophages Based on RNA-Seq Technology

目的:探讨洋参芎芍方对RAW264.7巨噬细胞代谢、炎症反应的影响及其可能的机制。方法:应用细胞增殖与活性检测(CCK-8)法检测RAW264.7巨噬细胞活性;分别使用葡萄糖、总胆固醇(TC)、游离胆固醇(FC)试剂盒检测巨噬细胞葡萄糖摄取率、TC、FC;酶联免疫吸附法(ELISA)检测RAW264.7巨噬细胞肿瘤坏死因子-α(TNF-α)水平;油红O染色法检测RAW264.7巨噬细胞脂质积聚;真核RNA测序(RNA-seq)技术检测RAW264.7巨噬细胞基因表达,通过蛋白免疫印迹法(Westren Blot)检测洋参芎芍方对RAW264.7巨噬细胞过氧化物酶体增殖物激活受体-gamma(PPAR-γ)蛋白表达的影响。结果:与空白组比较,洋参芎芍方在25~6400 mg/L梯度范围内均促进RAW264.7巨噬细胞的活性(P<0.01);与空白组比较,模型组可增加RAW264.7巨噬细胞的葡萄糖摄取率、TC与FC浓度、TNF-α水平及脂质积聚(P<0.05),与模型组比较,洋参芎芍中剂量组、高剂量组可减少RAW264.7巨噬细胞葡萄糖摄取率、TC与FC浓度、TNF-α水平及脂质积聚(P<0.05);RNA测序结果表明过PPAR-γ信号通路是调控巨噬细胞代谢的关键通路,洋参芎芍方(400 mg/mL)可降低PPAR-γ蛋白表达,Westren Blot结果显示模型组PPAR-γ蛋白表达降低,洋参芎芍方可增加PPAR-γ蛋白表达(P<0.05)。结论:洋参芎芍方可能通过增加PPAR-γ蛋白表达改善巨噬细胞代谢及炎症反应。

Objective:To explore the effect of the Yangshen Xiongshao Formula on the metabolism and inflammatory response of RAW264.7 macrophages.Methods:The Cell Proliferation and Cytotoxicity Assay Kit(CCK-8)was used to detect the activity of RAW264.7 macrophages;the glucose,total cholesterol(TC),and free cholesterol(FC)kits were used to detect the glucose uptake rate,TC,and FC of macrophages,respectively;enzyme-linked immunosorbent assay(ELISA)was used to detect the level of tumor necrosis factor-α(TNF-α)in RAW264.7 macrophages;Oil Red O staining was used to detect lipid accumulation in RAW264.7 macrophages;RNA sequencing(RNA-seq)technique was used to detect gene expression in RAW264.7 macrophages;Western Blot was used to observe the effect of Yangshen Xiongshao formula on RAW264.7 macrophages.Results:Compared with the blank group,the Yangshen Xiongshao formula promoted the activity of RAW264.7 macrophages in the gradient range of 25 to 6400 mg/L(P<0.01);In the model group increased the macrophage glucose uptake rate of RAW264.7 macrophages,TC and FC concentration,TNF-αlevel,and lipid accumulation increased(P<0.05).Compared with the model group,the medium-and high-dose Yangshen Xiongshao formula groups the glucose uptake rate of RAW264.7 macrophages,TC and FC concentration,TNF-αlevel,and lipid accumulation of RAW264.7 macrophages(P<0.05).RNA-seq results showed that the peroxisome proliferator-activated receptor-gamma(PPAR-γ)signaling pathway was the key to regulating of macrophages.Yangshen Xiongshao formula(400 mg/mL)could reduce the expression of PPAR-γmRNA.Western Blot results showed that the expression of PPAR-γprotein in the model group decreased,and Yangshen Xiongshao formula could increase the expression of PPAR-γprotein(P<0.05).Conclusion:Yangshen Xiongshao formula may improve macrophage metabolism and inflammatory response by increasing the expression of PPAR-γprotein.