Reserarch On The Mechanisms Of 14-3-3 Proteins In TLR4 Signaling Transduction And Lipopolysaccharide-induced Acute Lung Injury

Acute lung injury(ALI)and its most severe form acute respiratory distress syndrome(ARDS)are common causes of clinical acute respiratory failure,which results in a significant number of deaths worldwide with a fatality rate of 35%to 60%.Clinically,its manifestations are dyspnea and distress,radiographic manifestations show bilateral pulmonary exudative lesions or consolidation,and histological manifestations are severe acute inflammation response in the lungs and neutrophil alveolitis.Bacterial or viral pneumonia are the most common cause of ALI and ARDS,the pathogenesis involves a variety of factors,such as the imbalance of inflammatory response,the disturbance of alveolar capillary barrier regulation,and the imbalance of redox.Previous studies have established a critical role for TLR4 in the pathogenesis of acute lung injury,and the modification of TLR4 signaling will alleviate LPS induced ALI in animal models.During the study,it was found that the recruitment of TICAM-2 into Toll-like receptor 4(TLR4)plays a key role in the initiation of TLR4 signal transduction,but the specific mechanism of action is still unclear.Therefore,more comprehensive and deeper research is needed to provide a more theoretical basis for the clinical treatment of acute lung injury.14-3-3 family proteins are highly conserved acidic proteins with seven isotypes(β,ε,γ,η,σ,τ/θ and ζ)encoded by separate genes in mammalian cells.They function as intracellular adaptors in a variety of biological processes,such as signal transduction,cell cycle control,and apoptosis.Recent studies have revealed that 14-3-3 proteins exist in macrophage and participate in micropinocytosis process,polarization and inflammatory responses.Studies have shown a possible interaction between 14-3-3 and TLR2,thus revealed 14-3-3 as novel intermediates within TLR2 signaling pathway.14-3-3ζ is also involved in TIC AM-1 speckle-like signalosomes formation thus regulates TLR3 mediated signal transduction and cytokine production.However,whether and how 14-3-3 proteins participate in TLR4 signalosome complex and LPS induced ALI needs to be further investigated.Our experiment found that TLR4 activation would form a complex of 14-3-3 and TLR4.Knocking down 14-3-3 or interfering with 14-3-3 interaction with TLR4 can significantly inhibit the transduction of TLR4 signaling pathways NF-κB and MAPK and the expression of inflammatory cytokines in MyD88-dependent(IL-6 and TNF-α)and non-dependent(IP-10 and RANTES).Further studies showed that the recombinant 14-3-3θprotein could bind directly to the intracellular domain of TLR4 and act as a bridge to recruit TICAM-2 to TLR4 and form the TLR4-14-3-3-TICAM-2 complex.Interfering TLR4-14-3-3-TICAM-2 complex formation by myrsR18 inhibited lipopolysaccharide(LPS)-induced respiratory epithelia apoptosis when co-cultured with THP-1 macrophage via TNF-α dependent pathway in vitro.In the LPS-induced acute lung injury model in mice,myrsR18 significantly reduce the expression of inflammatory factors in bronchopulmonary lavage fluid and the infiltration of leukocytes in lung tissues,and alleviated LPS-induced acute lung injury in mice.This experiment proves for the first time that 14-3-3 can directly bind to TLR4 and act as a connector to promote TICAM-2 into TLR4,further forming the ternary complex of TLR4-14-3-3-TICAM-2,thus promoting the transduction of the TLR4 signaling.Knocking down or interfering with 14-3-3 inhibits the formation of the above complex,inhibits MyD8 8-dependent and non-independent TLR4 signaling transduction,and inhibits the expression of inflammatory cytokine,thereby alleviating LPS-induced acute lung injury.14-3-3 protein plays an important role in TLR4 signaling and is a potential target for the treatment of ALI.