Liu Jiao, Li Guang, Xie Wen-Jie, Wang Lu, Zhang Rui, Huang Ke-Sheng, Zhou Qing-Shan, Chen De-Chang
Department of Critical Care Medicine, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China.
Department of Critical Care Medicine, Ruijin Hospital, Shanghai Jiaotong University, Shanghai 200025, China.
Chin Med J (Engl). 2017 May 20;130(10):1236-1243. doi: 10.4103/0366-6999.205853.
Surfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.
Tetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.
HK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.
The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.
表面活性蛋白A（SP-A）有助于调节脓毒症诱导的急性肾损伤。在先前的一项研究中，我们证明脂多糖（LPS）可刺激人肾小管上皮（HK-2）细胞中SP-A的表达。本研究评估了LPS诱导HK-2细胞中SP-A生物合成的可能信号转导机制。
采用四氮唑盐比色法（MTT）检测LPS刺激不同时间点后HK-2细胞的活力。用100 ng/ml的LPS刺激HK-2细胞不同时间，以确定LPS对SP-A和Toll样受体4（TLR4）信使核糖核酸（mRNA）表达以及丝裂原活化/细胞外信号调节激酶（MEK）1、细胞外信号调节激酶1/2（ERK1/2）、p38丝裂原活化蛋白激酶（p38MAPK）和核因子-κB（NF-κB）抑制因子α（IkB-α）磷酸化的影响。然后，用TLR4抑制剂CLI-095预处理HK-2细胞，以分析LPS暴露前HK-2细胞中SP-A和TLR4的mRNA和蛋白水平以及细胞质和细胞核中NF-κB的表达。
暴露于100 ng/ml LPS 1、6和24小时的HK-2细胞活力未受影响，表明100 ng/ml LPS对细胞无毒性作用（P = 0.16）；然而，HK-2细胞中SP-A mRNA和蛋白的生物合成显著增加（P = 0.02）。至于机制，LPS增强跨膜受体TLR4蛋白表达。随后，LPS时间依赖性地增加MEK1、ERK1/2和p38MAPK的磷酸化。此外，LPS暴露2小时后，磷酸化IκB-α和核NF-κB水平增加。CLI-095预处理可显著抑制LPS诱导的SP-A和TLR4 mRNA以及NF-κB表达。
本研究表明，LPS可通过依次激活TLR4相关的MEK1-ERK1/2-NF-κB依赖性途径增加人肾上皮细胞中SP-A的合成。
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