小檗碱和大黄酸对LPS诱导巨噬细胞炎症反应及TLR2/NF-κB信号通路的影响

Effects of berberine and rhubarb acid on LPS-induced macrophage inflammatory reaction and TLR2/NF-κB signaling pathway

目的观察小檗碱和大黄酸对脂多糖(LPS)诱导巨噬细胞炎症反应及炎性相关信号通路Toll样受体2(TLR2)/核转录因子κB(NF-κB)的影响。方法将巨噬细胞RAW264. 7随机分为4组,空白组以完全培养基常规培养,LPS诱导组加入10 ng/m L LPS诱导4 h,小檗碱组和大黄酸组分别用20μmol/L小檗碱和30μmol/L大黄酸预处理2 h后加入10 ng/m L LPS诱导4 h。ELISA法检测细胞上清液炎性因子IL-1β、IL-6水平,RT-qPCR和Western blotting法分别检测细胞中的TLR2、白细胞介素4受体相关激酶(IRAK4)、NF-κΒp65 mRNA和蛋白。结果与空白组比较,LPS诱导组、小檗碱组和大黄酸组细胞上清液IL-1β、IL-6水平升高(P均<0. 05),细胞中TLR2、IRAK4及NF-κΒp65 mRNA和蛋白表达增加(P均<0. 05);与LPS诱导组比较,小檗碱组和大黄酸组细胞上清液IL-1β、IL-6水平降低(P均<0. 05),细胞中TLR2、NF-κΒp65 mRNA和蛋白及IRAK4 mRNA表达下降(P均<0. 05),小檗碱组IRAK4蛋白表达下降(P <0. 05)。结论小檗碱和大黄酸可通过下调TLR2/NF-κB信号通路分子的表达,从而抑制LPS诱导巨噬细胞炎性因子的产生和释放,以发挥其抗炎作用。

Objective To observe the effects of berberine and rhubarb acid on lipopolysaccharide(LPS)-induced macrophage inflammatory response and inflammatory-associated Toll-like receptor 2(TLR2)/nuclear transcription factorκB(NF-κB)signaling pathway.Methods Macrophages RAW264.7 were randomly divided into four groups.The cells in the blank group were cultured in complete medium.The cells in the LPS induction group were induced with 10 ng/mL LPS for 4 h,and the berberine group and the rhubarb acid group were pretreated with 20μmol/L berberine and 30μmol/L rhubarb acid for 2 h and then induced by 10 ng/mL LPS for 4 h.The levels of inflammatory factors IL-1βand IL-6 in the cell supernatant were detected by ELISA.The mRNA and protein expression levels of TLR2,interleukin-4,and receptor-associated kinase(IRAK4)and NF-κΒp65 were detected by RT-qPCR and Western blotting,respectively.Results Compared with the blank group,the levels of IL-1βand IL-6 in the supernatant increased,and the mRNA and protein expression levels of TLR2,IRAK4,and NF-κΒp65 increased in the LPS induction group,berberine group,and rhubarb acid group(all P<0.05).Compared with the LPS induction group,the levels of IL-1βand IL-6 in the supernatant of the berberine and rhubarb acid group decreased(both P<0.05),and the expression of TLR2 and NF-κΒp65 mRNA and protein and IRAK4 mRNA in the cells decreased(all P<0.05);and IRAK4 protein expression in the berberine group decreased(P<0.05).Conclusion Berberine and rhubarb acid can inhibit the production and release of macrophage inflammatory factors induced by LPS and thus play its anti-inflammatory effect by down-regulating the expression of inflammatory-associated TLR2/NF-κB pathway signaling molecules.