蒺藜总皂苷对LPS诱导的巨噬细胞分泌IL-1β、IL-6、TNF-α、IL-2、NO的影响和机制

Effects and mechanism of gross saponins of tribulus terrestris on IL-1β,IL-6,TNF-α,IL-2 and NO secreted by LPS-induced macrophages

目的:探讨蒺藜总皂苷对脂多糖(LPS)诱导的巨噬细胞分泌IL-1β、IL-6、TNF-α、IL-2、NO的影响和机制。方法:以巨噬细胞RAW264.7作为实验对象,分成Control(空白对照)组、LPS(LPS处理)组和GSTT-L(蒺藜总皂苷低剂量)、GSTTM(蒺藜总皂苷中剂量)、GSTT-H(蒺藜总皂苷高剂量)组(分别以10、20、40μg/ml的蒺藜总皂苷和LPS处理)。CCK-8法检测细胞增殖变化,ELISA法检测培养液上清中IL-1β、IL-6、TNF-α、IL-2水平,Griess法检测培养液上清中NO含量,Western blot检测诱导型一氧化氮合酶(iNOS)、Toll样受体4(TLR4)蛋白表达。在巨噬细胞RAW264.7中转染pcDNA3.1-TLR4,然后给予蒺藜总皂苷和LPS处理,同样利用上述方法测定细胞增殖、培养液上清中IL-1β、IL-6、TNF-α、IL-2水平、NO含量以及iNOS、TLR4蛋白表达水平。结果:与Control组比较,LPS组巨噬细胞增殖活性下降,细胞分泌的IL-1β、IL-6、TNF-α、IL-2和NO增多,细胞中iNOS、TLR4蛋白表达水平升高。与LPS组比较,GSTT-L、GSTT-M、GSTT-H组巨噬细胞增殖活性依次升高,细胞分泌的IL-1β、IL-6、TNF-α、IL-2和NO依次减少,细胞中iNOS、TLR4蛋白表达水平依次降低。pcDNA3.1-TLR4可以逆转蒺藜总皂苷对LPS诱导的巨噬细胞增殖、分泌IL-1β、IL-6、TNF-α、IL-2、NO以及细胞中iNOS、TLR4蛋白表达作用。结论:蒺藜总皂苷抑制LPS诱导的巨噬细胞分泌IL-1β、IL-6、TNF-α、IL-2、NO,与下调TLR4表达有关。

Objective:To investigate the effect and mechanism of gross saponins of tribulus terrestris on IL-1β,IL-6,TNF-α,IL-2 and NO secreted by macrophages induced by lipopolysaccharide(LPS).Methods:Macrophage RAW264.7 was used as the experimental object,divided into Control(blank control)group,LPS(LPS treatment)group and GSTT-L(low dose of gross saponins of tribulus terrestris),GSTT-M(middle dose of gross saponins of tribulus terrestris),GSTT-H(high dose of gross saponins of tribulus terrestris)group(10,20,40 μg/ml gross saponins of tribulus terrestris and LPS treatment). CCK-8 method was used to detect the changes of cell proliferation,ELISA method was used to detect the levels of IL-1β,IL-6,TNF-α and IL-2 in the culture supernatant,Griess method was used to detect the NO content in the culture supernatant,and Western blot was used to detect inducible nitric oxide synthase(iNOS)and toll-like receptor-4(TLR4)protein expression. pcDNA3.1-TLR4 was transfected in macrophage RAW264.7,and then treated with gross saponins of tribulus terrestris and LPS,and the same method was used to determine cell proliferation,IL-1β,IL-6,TNF-α,IL-2 level and NO content as well as iNOS and TLR4 protein expression.Results:Compared with the Control group,the proliferation activity of macrophages in the LPS group decreased,IL-1β,IL-6,TNF-α,IL-2 and NO secreted by the cells increased,and the expression levels of iNOS and TLR4 protein in the cells increased. Compared with LPS group,GSTT-L,GSTT-M,GSTT-H group macrophage proliferation activity increased in sequence,IL-1β,IL-6,TNF-α,IL-2 and NO secreted by cells decreased in turn,the expression levels of iNOS and TLR4 decreased in turn. pcDNA3.1-TLR4 could reverse the expression of gross saponins of tribulus terrestris to the secretion of IL-1β,IL-6,TNF-α,IL-2 and NO by LPS-induced macrophages and iNOS and TLR4 protein expression in cells.Conclusion:Gross saponins of tribulus terrestris inhibit IL-1β,IL-6,TNF-α,IL-2 and NO secreted by LPS-induced macrophages,the mechanism of action is related to the down-regulation of TLR4 expression.