Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

Feixiang Wu,Yuming Sun,Xuerong Miao,Yantao Liu,Weifeng Yu,Xin Lv

doi:10.1155/2012/406435

Abstract

Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA) targeting Toll-like receptor 4 (TLR4) gene in ameliorating lipopolysaccharide- (LPS-) induced acute lung injury (ALI). Methods. In vitro, alveolar macrophages (AMs) were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL) for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group), 300 μL of Ad-EGFP (Ad-EGFP group), or 300 μL of Ad-siTLR4 (Ad-siTLR4 group) and then were intravenously treated with LPS (50 mg/kg) to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

Highlights

Pulmonary trauma, severe infection as well as hemorrhage can result in acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), which are common pulmonary diseases in humans, especially in patients undergoing surgery [1,2,3,4]
Real-time PCR showed Ad-siTLR4 significantly downregulated the mRNA expression of Toll-like receptor 4 (TLR4) at 12 h, 24 h, and 48 h after transfection when compared with the Ad-enhanced green fluorescent protein (EGFP) group (Figure 1(b))
Results revealed LPS could induce the production of these cytokines in vitro, which was associated with the upregulation of TLR4 expression (Figures 1(c) and 1(d))

Summary

Introduction

Severe infection as well as hemorrhage can result in acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), which are common pulmonary diseases in humans, especially in patients undergoing surgery [1,2,3,4]. Studies have confirmed that lipopolysaccharide (LPS) plays important roles in the development and progression of ALI or ARDS [5]. Increased serum endotoxin or LPS of Gram-negative bacteria may induce pulmonary inflammation leading to ALI and can be applied as a predictor of multiple organ failure (MOF), during sepsis. It is widely believed that Toll-like receptor 4 (TLR4) is required for the innate immune response to LPS of gram-negative bacteria [9, 10]. Some researchers speculate that it is LPS other than TLR4 that is directly involved in the induction of pulmonary immune response [11]. Jeyaseelan et al demonstrated that LPS-induced CD-14-dependent and -independent (CD11b-dependent) signaling pathways in the lung were entirely dependent on TLR4 and that blocking TLR4 might be beneficial for lung injury caused by LPS from gram-negative pathogens [12]

Methods

Results

Conclusion