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Objective: 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., Methods: 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., Results: 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., Conclusions: 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.

Abstract Targeting inflammation is considered a challenging pharmacological strategy to prevent or delay the development of inflammatory diseases, such as severe asthma, Crohn's disease, and rheumatoid arthritis. The angiotensin-(1–7) -Mas axis ((Ang-(1–7)-Mas axis) was confirmed to antagonize the effects of the Angiotensin II-AT 1 receptor axis and the latter is reported to regulate cardiovascular and renal function, as well as contribute to the inflammatory process. In this paper, we aim to explore the crucial effect of Ang-(1–7) in inflammation and disclose the mechanisms in lipopolysaccharide (LPS)-induced murine macrophages RAW264.7. We found that Ang-(1–7) inhibited the production and secretion of tumor necrosis factor-α and interleukin-6 in a concentration-dependent manner in LPS-induced macrophages. The overexpression of TLR4, phospho-JNK, and FoxO1 induced by LPS were also inhibited by incubation with Ang-(1–7). These inhibitory effects were reversed by A-779. Moreover, we also used a selective JNK inhibitor Sp600125 to further corroborate the involvement of TLR4, JNK, and FoxO1 in the anti-inflammatory action of Ang-(1–7). Our research reveals a new mechanism that Ang-(1–7) may drive anti-inflammatory effects via the Mas receptor through inhibition of the TLR4-mediated JNK/FoxO1 signaling pathway in LPS-induced macrophages. Our findings open new perspectives of Ang-(1–7)-Mas axis in local inflammation. Highlights • Ang-(1–7) inhibited inflammation in a dose-dependent manner in vitro. • Ang-(1–7) may exerts anti-inflammatory effects via TLR4/JNK/FoxO1 pathway. • The effect of Ang-(1–7) was mediated by the Mas receptor. [ABSTRACT FROM AUTHOR]