Chen Muhu, Zhong Wu, Hu Yingchun, Liu Jitao, Cai Xianfu
Department of Emergency, The First Affiliated Hospital of Sichuan Medical University Luzhou 646000, Sichuan, P. R. China.
Int J Clin Exp Med. 2015 Oct 15;8(10):17824-9. eCollection 2015.
This study aims to investigate the effect of 5(S), 6(R)-7-trihydroxymethyl heptanoate (BML-111) on the Wnt5a/frizzled-5 (FZD5)/calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathway in septic mice, and to explore whether this pathway mediates the effect of BML-111 on inflammatory response in lipopolysaccharide (LPS)-induced RAW 264.7 cells.
The cecal ligation and puncture-induced mouse model of sepsis was constructed, and the mice were pretreated with BML-111. In vitro, LPS-induced RAW 264.7 cells were incubated with various concentrations of BML-111. Activation of Wnt5a/FZD5/CaMKII signaling pathway was achieved by transfection of the Wnt5a overexpression plasmid. The levels of interleukin-1 beta (IL-1β), IL-6 and IL-8 in the mouse serum and cell supernatant were determined by ELISA assay. The expression of Wnt5a, FZD5 and CaMKIIδ was examined by western blot analysis.
The results from the in vivo studies revealed that BML-111 shows inhibitory effect on IL-1β, IL-6 and IL-8 expression in the serum of septic mice, and suppresses the expression of Wnt5a, FZD5 and CaMKIIδ protein. The in vitro studies demonstrated that BML-111 inhibits Wnt5a, FZD5 and CaMKIIδ proteins in a dose-dependent manner. BML-111 suppressed the levels of IL-1β, IL-6 and IL-8 in LPS-induced RAW 264.7 cells; however, this effect could be attenuated by transfection of the Wnt5a overexpression plasmid.
This study firstly demonstrated that BML-111 suppresses Wnt5a/FZD5/CaMKII signaling pathway in sepsis, and Wnt5a/FZD5/CaMKII signaling pathway mediates the effect of BML-111 on inflammatory reactions. These findings provided a novel molecular basis for the potential effect of BML-111 in sepsis.
本研究旨在探讨5(S),6(R)-7-三羟甲基庚酸酯（BML-111）对脓毒症小鼠Wnt5a/卷曲蛋白5（FZD5）/钙/钙调蛋白依赖性蛋白激酶II（CaMKII）信号通路的影响，并探究该通路是否介导BML-111对脂多糖（LPS）诱导的RAW 264.7细胞炎症反应的作用。
构建盲肠结扎穿刺诱导的小鼠脓毒症模型，并用BML-111对小鼠进行预处理。在体外，将LPS诱导的RAW 264.7细胞与不同浓度的BML-111孵育。通过转染Wnt5a过表达质粒来激活Wnt5a/FZD5/CaMKII信号通路。采用酶联免疫吸附测定（ELISA）法测定小鼠血清和细胞上清液中白细胞介素-1β（IL-1β）、IL-6和IL-8的水平。通过蛋白质印迹分析检测Wnt5a、FZD5和CaMKIIδ的表达。
体内研究结果显示，BML-111对脓毒症小鼠血清中IL-1β、IL-6和IL-8的表达具有抑制作用，并抑制Wnt5a、FZD5和CaMKIIδ蛋白的表达。体外研究表明，BML-111以剂量依赖性方式抑制Wnt5a、FZD5和CaMKIIδ蛋白。BML-111抑制LPS诱导的RAW 264.7细胞中IL-1β、IL-6和IL-8的水平；然而，转染Wnt5a过表达质粒可减弱这种作用。
本研究首次证明BML-111在脓毒症中抑制Wnt5a/FZD5/CaMKII信号通路，且Wnt5a/FZD5/CaMKII信号通路介导BML-111对炎症反应的作用。这些发现为BML-111在脓毒症中的潜在作用提供了新的分子基础。
