Atorvastatin increases lipopolysaccharide-induced expression of tumour necrosis factor-α-induced protein 8-like 2 in RAW264.7 cells.

Wei Zhang,Li Wang,Chuan-Yun Qian,Mei-Xian Su,Ming-Wei Liu

doi:10.3892/etm.2014.1722

Abstract

RAW264.7 cells are one of the major sources of productive inflammatory biomediators, including tumour necrosis factor-α (TNF-α) and interleukin (IL)-6. TNF-α-induced protein 8-like 2 (TIPE2) is an essential negative regulator of Toll-like and T-cell receptors, and the selective expression in the immune system prevents hyper-responsiveness and maintains immune homeostasis. The aim of the present study was to investigate whether atorvastatin upregulates the expression of TIPE2 and further regulates the inflammatory response and oxidation emergency response in RAW264.7 cells. RAW264.7 cells were incubated in Dulbecco’s modified Eagle’s medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. Following incubation, the medium was collected and the levels of TNF-α and IL-6 were measured using an enzyme-linked immunosorbent assay. The cells were harvested, and the mRNA and protein expression levels of TIPE2, macrophage migration inhibitory factor (MIF), IκB and nuclear factor (NF-κB)-κB were analysed using quantitative polymerase chain reaction and western blotting analysis, respectively, the expression of NOS, COX-2 and HO-1 protein were essayed by western blotting analysis, NO and ROS activities were determined. The results revealed that LPS increased the mRNA and protein expression levels of TIPE2, MIF and NF-κB, as well as the production of IL-6 and TNF-α, in a dose and time dependent manner in RAW264.7 cells. Meanwhile, LPS enhanced the expression of NOS and COX-2 protein, blocked HO-1 protein expression, increased NO and PGE2 production and ROS activity in a dose dependent manner in RAW264.7 cells. Atorvastatin significantly increased LPS induced expression of TIPE2, downregulated the expression of NOS, COX-2, MIF and NF-κB and the production of PGE2, NO, IL-6 and TNF-α in a time and dose dependent manner, and increased HO-1 protein expression, reduced ROS production in a dose dependent manner. The observations indicated that atorvastatin upregulated LPS induced expression of TIPE2 and consequently inhibited MIF, NF-κB, NOS and COX-2 expression and the production of NO, PGE2, TNF-α and IL-6, increased HO-1 expression, and inhibited ROS activity in cultured RAW264.7 cells.

Highlights

Sepsis, which results in multiple organ failure, remains a leading cause of mortality and morbidity in intensive care units [1,2]
The expression of nuclear factor‐κB (NF‐κB) p65 and the secretion of IL‐6 and tumour necrosis factor‐α (TNF‐α) in the two groups increased with LPS at each time point; when compared with the RAW264.7 macrophages, the NF‐κB p65 expression and secretion of IL‐6 and TNF‐α were significantly elevated in the TNF‐α‐induced protein 8‐like 2 (TIPE2)‐siRNA RAW264.7 macrophages at 6, 9 and 12 h (Figs. 1B and 2C and D)
When compared with the RAW264.7 macrophages, the NF‐κB p65 expression and secretion of IL‐6 and TNF‐α were significantly elevated in the TIPE2‐siRNA RAW264.7 macrophages treated with 5, 10 and 15 ng/ml LPS

Summary

Introduction

Sepsis, which results in multiple organ failure, remains a leading cause of mortality and morbidity in intensive care units [1,2]. Uncontrolled hyperinflammatory and inappropriate cytokine responses during early sepsis are hypothesised to be the cause of multiple organ dysfunction syndrome (MODS) during sepsis. Controlling inflammation during early sepsis may reduce organ injury and prevent mortality following septic insult. Sepsis is the leading cause of mortality in critically ill patients, and the incidence of sepsis is increasing [3,4]. The mortality rate of severe sepsis is very high (up to 70%) and the calculated costs exceed $15 billion per year in the United States [3].

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