Department of Nephrology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China,
Inflamm Res. 2015 Jun;64(6):453-62. doi: 10.1007/s00011-015-0825-x. Epub 2015 May 1.
Augmenter of liver regeneration (ALR) is a growth factor that is ubiquitously expressed in multiple forms among eukaryotes. The present study focused on the role of endogenous ALR on the hypoxia/reoxygenation (H/R)-induced inflammatory response in human kidney 2 (HK-2) cells, and the underlying molecular mechanisms.
To determine the relationship between exogenous and endogenous ALR, exogenous ALR was administrated to HK-2 cells, and endogenous ALR protein and mRNA expression was examined by Western blotting and quantitative real-time polymerase chain reaction (qPCR), respectively. In order to knockdown endogenous ALR expression, HK-2 cells were infected with lentiviral shRNA/ALR, after which cell viability was determined by the MTS cell viability assay. Cells were subjected to hypoxia for 6 h and reoxygenation for 12 h. Levels of monocyte chemotactic protein (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR. Cells were harvested, and nuclear and phosphorylated protein extracts were prepared from the HK-2 cell lysates. Nuclear factor κB (NF-κB), and phosphorylated extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were analyzed by Western blotting. The translocation of NF-κB was detected by immunofluorescence.
Exogenous ALR inhibited the expression of endogenous ALR. Lentiviral shRNA/ALR markedly downregulated endogenous ALR expression, whereas there were no changes in ALR expression in lentiviral shRNA/control HK-2 cells. The results of the MTS assay showed that silencing ALR expression did not influence cell viability. H/R led to increased production of MCP-1, IL-6, and TNF-α. However, knockdown of ALR attenuated the inflammatory response via inhibition of ERK, p38, and JNK phosphorylation. The translocation of NF-κB into the nucleus was also decreased.
These results suggest that there is a negative feedback loop involving ALR in HK-2 cells. Knockdown of ALR exerts anti-inflammatory actions via suppression of the mitogen-activated protein kinase signaling pathway.
肝再生增强因子（ALR）是一种在真核生物中以多种形式广泛表达的生长因子。本研究旨在探讨内源性 ALR 在人肾 2 细胞（HK-2 细胞）缺氧/复氧（H/R）诱导的炎症反应中的作用及其潜在的分子机制。
为了确定外源性和内源性 ALR 之间的关系，我们向 HK-2 细胞中添加外源性 ALR，然后通过 Western blot 和实时定量聚合酶链反应（qPCR）分别检测内源性 ALR 蛋白和 mRNA 的表达。为了敲低内源性 ALR 的表达，我们用慢病毒 shRNA/ALR 感染 HK-2 细胞，然后通过 MTS 细胞活力测定法检测细胞活力。将细胞置于缺氧 6 小时，复氧 12 小时。通过酶联免疫吸附试验（ELISA）和 qPCR 测定单核细胞趋化蛋白（MCP-1）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的水平。从 HK-2 细胞裂解物中提取核蛋白和磷酸化蛋白提取物，通过 Western blot 分析核因子 κB（NF-κB）和磷酸化细胞外信号调节激酶（ERK）、p38 和 c-Jun N-末端激酶（JNK）。通过免疫荧光检测 NF-κB 的易位。
外源性 ALR 抑制内源性 ALR 的表达。慢病毒 shRNA/ALR 显著下调内源性 ALR 的表达，而慢病毒 shRNA/对照 HK-2 细胞中 ALR 的表达没有变化。MTS 测定结果表明，沉默 ALR 表达不会影响细胞活力。H/R 导致 MCP-1、IL-6 和 TNF-α 的产生增加。然而，通过抑制 ERK、p38 和 JNK 磷酸化，敲低 ALR 可减轻炎症反应。NF-κB 向核内的易位也减少。
这些结果表明，在 HK-2 细胞中存在涉及 ALR 的负反馈回路。敲低 ALR 通过抑制丝裂原活化蛋白激酶信号通路发挥抗炎作用。
