The Regulatory Function Of RIP140 On PPARγ During Septic Acute Lung Injury

Acute lung injury(ALI) and acute respiratory distress syndrome are an emergent and severe case in clinic. In ALI/ARDS, the integrity of the separation between the alveolus and the pulmonary circulation is compromised either by endothelial orepithelial injury or more commonly both. Despite many years of clinical researches in ARDS, it is still a life-threatening lung disease and the overall mortality of ARDS is more than 40%[1]. Besides, effective pharmacotherapy currently available to prevent the lung from over-inflammation or improve tissue repair are still rare. Thereby, exploration of new pathways to promote the resolution of pulmonary inflammation and alleviation of lung injury has been considered as a promising approach to develop novel therapies for ALI, which are urgently needed.Peroxisome proliferator–activated receptor(PPARγ), belongs to nuclear hormone receptor super family[2]. Based on its potent anti-inflammatory effect, many studies indicated that PPARγ might be a potential therapeutic target for acute lung injury[3~6]. However, sepsis and other inflammatory states are always accompanied with suppressed PPARγ activity and expression and the mechanism involved in this process remains unclear[6,7]. Receptor interacting protein(RIP140) was found to function as a corepressor for PPARγ by repressing its transcription activity[8,10]. The roles of RIP140 and the correlation between RIP140 and PPARγ during acute lung injury remain elusive.In our study, we firstly detected the expression of RIP140 in lung tissues and alveolar macrophage of rats with septic acute lung injury. To investigate the regulatory function of RIP140 on PPARγ, we constructed an adenoviruses expressing RIP140-specific short hairpin RNAs(sh RNAs) to silence the expression of RIP140 in macrophage and lung, then PPARγ expression and activation were observed.ObjectiveThis study was aimed to investigate the regulatory function of RIP140 on PPARγ during septic acute lung injury.Methods1.Expression and distribution of RIP140 in lung tissues during septic acute lung injury.CLP(cecal ligation and puncture) was performed to induce septic acute lung injury. Western blot, immunohistochemistry, immunofluorescence technique were performed to observd the expression and distribution of RIP140 during septic acute lung injury.2.Effect of down-regulating RIP140 on the expression of PPARγ and proinflammatory gene in macrophagesRIP140 and PPARγ protein expression was determined by Western blot in macrophages after LPS stimulation. Macrophages were exposed to adenoviruses at a multiplicity of infection of 100 for 48 hours, treated with LPS(1 μg/m L) or LPS plus GW9662(10 μmol/l) for 12 hours, or left untreated. Western blot and ELISA were performed to detected the expression and activation of PPARγ. Proinflammatory gene expression in macrophages after down-regulation of RIP140 was detected by RT-qPCR、ELISA.3.Effect of down-regulating RIP140 on the expression of PPARγ and proinflammatory gene in lung of mices during ALI.The activity of RIP140 in lung of wild-type C57Bl6 mice was disrupted by delivering an adenovirus expressing a RIP140-specific shRNA or control sh RNA via inhalation. Then ALI was induced by intraperitoneal injection of 20 mg lipopolysaccharide/kg body weight. PPARγ expression was determined by western-blot, the proinflammatory gene expression in lung of mices with ALI was detected by quantitative PCR analysis, HE staining was performed to observ the pathological changes.Results1.Six houres after CLP operation, the expression of RIP140 was started to elevate(P<0.05) in alveolar macrophages from rats with septic and was mainly expressed in the nuclei.2.Compared with that of the control shRNA-infected cells, remarkably increased PPARγ expression was found in RIP140 knockdown cells after LPS administration(P<0.05). In addition, an increase of PPARγ expression was correlated with a significant increase of PPAR DNA binding activity(P<0.05). However, administration of PPARγ antagonist GW9662 before LPS administration reversed the RIP140 deficiancy induced increase in PPARγ expression and activity(P<0.05).3.In comparision to control-shRNA mices,we found that down-regulating RIP140 can restore PPARγ expression in lung of mices during ALI(P<0.05).. However, this effect could be reversed by administration of PPARγ antagonist GW9662 before LPS administration(P<0.05). RIP140 deficiency can significantly inhabit the gene expressions of TNF-α,IL-1β, IL-6 as compared with the control sh RNA transfected mice(P<0.05), these proinflammatory gene expression levels can be partially reversed by the PPARγ antagonist GW9662(P<0.05). In comparision with control shRNA transfected mice, pathological changes were attenuated in lung of RIP140 deficiency mice and a significantly lower lung injury score was observed. However, when the PPARγ activity was blocked, these beneficial effects were partially reversed.Conclusions1. During septic ALI, the expression of RIP140 was elevated in in alveolar macrophages, and was mainly expressed in the nuclei.2.Down-regulation of RIP140 could suppress proinflammatory gene expression and cytokines release in macrophages via elevating PPARγ expression and activation.3.Silencing of RIP140 can significantly restore PPARγ expression in lung of mices with ALI. This restoration of PPARγ in turn could suppress the proinflammatory gene expression in lung and alleviate the LPS induced acute lung injury.