Trimetazidine Protects Against LPS-induced Acute Lung Injury Through MTORC₂/SGK₁ Pathway

Research backgroundALI/ARDS is a clinical syndrome characterized of diffusive pulmonary infiltration of inflammatory cells, lung circulation, pulmonary capillary permeability and hypoxemia, which is a still majority and difficulty of Respiration and Critical Care Medicine studies the prognosis is poor, It can rapidly progress a dangerous disease to multiple organ dysfunction and have important clinical significance for prevention of the diseases. While it has done a lot of experimental and clinical research at home and abroad, has not clarified the pathogenesis of ALI/ARDS yet. Researchers found that if it can effectively remove excessive alveolar edema fluid, to maintain gas exchange and improve oxygenation in patients with essential, can significantly reduce the mortality of these patients. Alveolar epithelial sodium channel（ENaC） is the alveolar sodium and water transport and promotes an important part of edema fluid removal,which is completed alveolar membrane transport of sodium ions and the rate-limiting step of providing AFC source of power, while synergies sodium potassium ATP enzyme together to complete the process of transport of sodium and water, play a very important role in leading the AFC.Initially, the related ALI/ARDS is mainly concentrated in the control of inflammation, such as free radical scavengers, corticosteroids, anti-oxidants and pulmonary surfactant, etc.But there are not satisfied with the efficacy or serious adverse reactions and so on. Thus, one aspect of the problem is difficult to play a fundamental role in the treatment of diseases. Important step treatment of ALI/ARDS is to exclude excessive edema fluid to the outside of the alveolar space, and promote lung edema clearance process, which is to maintain gas exchange and oxygenation in patients with restoration of positive significance. Alveolar epithelial cells have the ability to remove excess liquid, lipopolysaccharide endotoxin-induced injury is a determined factors of ALI/ARDS edema fluid accumulation. This study by mTORC₂/SGK₁ way while protecting the alveolar epithelial cells may enhance alveolar edema fluid clearance（AFC）, it has an important value for the study of the treatment of ALI / ARDS, to explore the mechanism of ENaC in the AFC more strong the clinical value, is also looking for an effective regulation of ENaC and upstream signaling pathways drugs provide new therapeutic targets.Trimetazidine [1-（2,3,4-trimethoxybenzyl） piperazine; TMZ] is an anti-ischemic and myocardial metabolism drug that modifies metabolic function[9], promote the and myocardial metabolism drug that modifies metabolic function[8].It has also been found TMZ is cardiovascular effectiveness to heart disease mainly through the inhibition of fatty acid beta oxidation[9],promote the oxidation of glucose[10], thereby reducing generation of ATP required oxygen consumption. Interestingly, Akan et al. reported that in acute pancreatitis, the IL-1a, IL-6 and TNF-á levels were significantly lowered by TMZ treatment, TMZ was considered to protect pancreatic tissue[21]. Recently, TMZ was found to protect the energy status after ischemia and reduces reperfusion injury in a rat single lung transplant model[11]. However, the effect of TMZ on acute pulmonary edema, has not been studied yet.Mammalian target of rapamycin（mTOR）, a multifunctional kinase plays an important role in cell proliferation, and metabolic reprogramming by regulating nutrient availability, cellular energy levels, oxygen levels, and mitogenic signal[12]. Serum and glucocorticoid-regulated kinase 1（SGK₁） is a downstream of mTOR pathway. It plays a fundamental role in ion and solute transport processes in epithelia[13]. SGK₁ is essential for normal sodium（Na+） and potassium homeostasis in mice [14] and for Na+ transport in cultured cells[15]. Accumulating evidence has implied that mTOR and SGK₁ play important roles in ALI pathogenesis in inflammation and autophagy[16-17]. In addition to reduction of inflammation, the rate of alveolar fluid clearance（AFC） is also a crucial prognostic factor for ALI/ ARDS patients since a lower AFC rate is relative with higher mortality in ARDS patients[18]. AFC is mediated by ion transporters, such as ENaC[19]. ENaC is a heteromultimeric protein composed of á, a and ? subunits. It has been found that ENaC plays an important role in the transepithelial absorption of sodium and fluid from alveolar spaces[17-18].ObjectiveThe aim of the present study is to explore the effects of TMZ on LPS-induced ALI/ADRS model in vitro and in vivo. We investigated the role of TMZ on ALI-associated pulmonary edema, ENaC expression, and the regulation of the mTORC₂/SGK₁ signaling pathway in ALI/ADRS model.Methods: this experiment is made up of three partsthe first one:To investigate the effect of mTORC₂ / SGK₁ signaling pathway in ENaC in vitro experiments. using ELISA, WB, immunofluorescence method described on ENaC expression of this pathway.The second one:To detect the mTORC₂/SGK₁ signaling pathway Movies on ENaC activity in vivo;assay the ENaC effect of mTORC₂/SGK₁ signaling pathway in vivo; to detect the expression of ENaC channels by using ELISA,WB,immunohistochemical and dry / wet weight and other means.The three one:Trimetazidine protects against LPS-induced acute lung injury through mTORC₂/SGK₁ pathwayTo observe the expression of ENaC subunits by trimetazidine protecting against LPS-induced acute lung injury through mTORC₂/SGK₁ pathway becoming a new avenue to improve the treatment of ALI/ARDS.Results1. To investigate the effect of mTORC₂/SGK₁ signaling pathway in ENaC in vitro.We used pro-inflammatory mediators IL-6, IL-8 and TNF-á as indicator of inflammation of ALI/ARDS malignancy. Cell supernatant was collected, IL-6, IL-8 and TNF-á were measured by ELISA assay. We found that the IL-6 IL8 and TNF-á were highly increased in LPS induction indicated that our model was well established.2、To detect the mTORC₂/SGK₁ signaling pathway Movies on ENaC activity in vivo;Since ENaC was the downstream gene of mTORC₂/SGK₁ pathway, 24 it was reported that mTOR plays an important role in ALI /ARDS[16-17]. This evidence promoted us to hypothesize that whether TMZ regulates ENaC through mTOR/SGK₁ pathway. We applied western blotting to measure the mTOR and SGK₁ protein levels to test this hypothesis.3. Trimetazidine protects against LPS-induced acute lung injury through mTOR/SGK₁ pathwayall of α-, β-, andγ-ENaC protein in the plasma membrane were decreased in LPS induced group and were recovered by TMZ treatment, indicating that TMZ could not only alleviate the inflammation but also increase Na+ transportation by increasing ENaC family; TMZ could down-regulate mTORC₂ in LPS induced cells,whereas SGK₁ was increased by TMZ, suggesting that TMZ could regulate the mTORC₂/SGK₁ pathway;the lung W/D ratio of LPS treated rat was significant increased, however, it could be reduced by TMZ treatment in dose-dependent manner.Conclusion:1.TMZ could alleviate pulmonary injury and edema in ALI/ARDS in vitro and in vivo model, providing a promising treatment for ALI/ARDS therapy.2.TMZ could alleviate ALI by decreasing the proinflammatory cytokines secretion in both LPS induced cells and rats model.3.TMZ also promoted AFC by increasing expression of ENaC, a mechanism independent of the alleviation of inflammation but dependent of ENaC.4.TMZ decreased mTORC₂ protein and increased SGK₁ protein expression in LPS induced model.our study provide evidence to show that TMZ may be a promising drug therary which could decrease the inflammation and effectively clear alveolar edema fluid leading to effective gas exchange which are extremely very important for the outcome of ALI/ARDS. In addition, our study also suggested that mTORC₂/SGK₁ might be a novel target of curing ALI/ARDS.