叉头蛋白O3a在库普弗细胞内毒素耐受中的开关作用取决于PI3K/AKT信号通路。
The on-off action of Forkhead protein O3a in endotoxin tolerance of Kupffer cells depends on the PI3K/AKT pathway.
作者信息
Mu Di, Miao Chunmu, Cheng Yao, Li Peizhi, Gong Jianping, Zhang Wenfeng
机构信息
Key Laboratory of Laboratory Medical Diagnostics, Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
出版信息
Int Immunopharmacol. 2020 Mar 3;82:106342. doi: 10.1016/j.intimp.2020.106342.
BACKGROUND
The endotoxin tolerance (ET) of Kupffer cells (KCs) is an important protective mechanism for limiting endotoxin shock. As a key anti-inflammatory molecule, the roles and mechanism of Forkhead protein O3a (Foxo3a) in ET of KCs are not yet well understood.
METHODS
ET and nonendotoxin tolerance (NET) KCs models were established in vitro and in vivo. The levels of cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression and phosphorylation levels were detected by western blotting (WB). Changes in the localization of nuclear factor kappa B (NF-κB) and Foxo3a in KCs were detected by immunofluorescence assays. KCs apoptosis and survival rates were detected by flow cytometry and an automatic cell counter, respectively.
RESULTS
The activity of NF-κB and the levels of p-Foxo3a and tumor necrosis factor (TNF-α) in the ET group were significantly lower than those in the NET group, while the levels of Foxo3a and interleukin 10 (IL-10) in the ET group were significantly higher than those in the NET group. Overexpression of Foxo3a or the use of a phosphatidylinositol-3-hydroxykinase (PI3K) inhibitor suppressed the activation of NF-κB by decreasing the levels of p-Foxo3a by inhibiting the activity of PI3K/AKT, which improved the tolerance of KCs and mice to endotoxin. In contrast, silencing Foxo3a or the use of a PI3K agonist reduced the tolerance of KCs and mice to endotoxin. The PI3K agonist counteracted the inhibitory effects of Foxo3a overexpression on NF-κB, impairing the tolerance of KCs to endotoxin.
CONCLUSIONS
The on-off action of Foxo3a in the ET of KCs depends on the PI3K/AKT pathway.
背景
库普弗细胞（KCs）的内毒素耐受（ET）是限制内毒素休克的重要保护机制。作为一种关键的抗炎分子，叉头蛋白O3a（Foxo3a）在KCs的ET中的作用和机制尚未完全明确。
方法
在体外和体内建立ET和非内毒素耐受（NET）KCs模型。通过酶联免疫吸附测定（ELISA）检测细胞因子水平。通过蛋白质印迹法（WB）检测蛋白质表达和磷酸化水平。通过免疫荧光测定法检测KCs中核因子κB（NF-κB）和Foxo3a的定位变化。分别通过流式细胞术和自动细胞计数器检测KCs凋亡和存活率。
结果
ET组中NF-κB的活性、p-Foxo3a和肿瘤坏死因子（TNF-α）的水平显著低于NET组，而ET组中Foxo3a和白细胞介素10（IL-10）的水平显著高于NET组。Foxo3a的过表达或使用磷脂酰肌醇-3-激酶（PI3K）抑制剂通过抑制PI3K/AKT的活性降低p-Foxo3a的水平，从而抑制NF-κB的激活，这提高了KCs和小鼠对内毒素的耐受性。相反，沉默Foxo3a或使用PI3K激动剂降低了KCs和小鼠对内毒素的耐受性。PI3K激动剂抵消了Foxo3a过表达对NF-κB的抑制作用，损害了KCs对内毒素的耐受性。
结论
Foxo3a在KCs的ET中的开关作用取决于PI3K/AKT途径。
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