Protective Effect And Mechanism Of Mitochondrial Division Inhibitor 1 In A Rat Model Of Acute Lung Injury

[Background]:Clinical acute lung injury(ALI)is a common complication that occurs following sepsis among ICU patients and is associated with a high morbidity and mortality,imposing a significant burden on society.Death resulting from sepsis occurs due to lung failure and multiple organ dysfunctions,for which there are currently no biological therapies.Lipopolysaccharide(LPS)is a major endotoxin component of gram-negative bacteria and plays an essential role in the development of ALI.Currently,it is thought that the multiple organ dysfunctions associated with sepsis can be attributed to a pathochemical and pathophysiological injury cascade,including the inflammatory response,macroautophagy,mitochondria dysfunction,and apoptosis,moreover,oxidative stress is also involved in the pathogenesis of ALI.The lysosomal machinery is used to remove dysfunctional mitochondria through selective degradation via autophagy,a process termed mitophagy.However,the role of mitophagy in the development of disease remains controversial.While some studies have confirmed that excessive mitophagy promotes mitochondrial damage in chronic obstructive pulmonary disease,and excessive mitochondrial fission is related to neurodegeneration and other mitochondriopathies,its role in ALI remains unknown.It has been demonstrated that mitochondrial dynamics,which are primarily regulated by the GTPase dynamin-related protein,play a crucial part in regulating mitophagy.Mitophagy can be blocked using the mitochondrial division inhibitor 1(Mdivi-1).Thus,previous studies have shown that excessive mitophagy can cause mitochondrial damage and that Mdivi-1 can provide protection against various pathological conditions.In this study,we hypothesize that mitophagy contributes to the development of ALI,whereas treatment with Mdivi-1 may prevent LPS-induced mitophagy and alleviate lung injury in rats.[Objective]:This experiment intends to verify the protective effects of Mdivi-1 on LPS-induced acute lung injury(ALI),mitophagy,apoptosis,oxidative stress and inflammation response in vivo and mitochondrial dysfunction in vitro.To contribute insight into the clinical practice and the study of potential therapy for the treatment of ALI.[Method]:1.A rat model of ALI was induced by an intratracheal administration of LPS(5 mg/kg body weight).The animals were randomly divided into 4 groups using a random number table:1)control group(pretreated with 0.5 mL DMSO for 60 min via the caudal vein and given 0.5 mL intratracheal normal saline(NS));2)Mdivi-1 control group(rats pretreated with a dose of Mdivi-1(3 mg/kg)dissolved in 0.5 mL DMSO for 60 min and given 0.5 mL intratracheal NS);3)LPS group(rats pretreated with 0.5 mL DMSO for 60 min via the caudal vein followed by LPS instillation);4)Mdivi-1 group(rats pretreated with a dose of Mdivi-1(3 mg/kg)dissolved in 0.5 mL DMSO for 60 min via the caudal vein followed by LPS instillation).2.The A549 cells were stimulated in 10 μg/mL LPS for 6 h to establish LPS-induced ALI in vitro.The cell were randomly divided into 4 groups(n = 6 per group)using a random number table:1)control group(cells treated with vehicle and without LPS stimulation);2)Mdivi-1 control group(cells treated with 10μM of Mdivi-1 and without LPS stimulation);3)LPS group(cells pretreated with vehicle 60 min,followed by LPS stimulation);4)Mdivi-1 group(cells pretreated with 10μM of Mdivi-1 60 min before LPS stimulation).DMSO was used as vehicle.3.Pathological changes in acute lung injury were assessed by HE staining,measurement of wet/dry weight(W/D)ratio,oxygenation index(PaO2/FiO2)analysis and lung microvascular permeability detection.Then,the mitophagy related proteins were evaluated by western blot.In order to evaluate the mitochondrial function,A549 cells were stained with JC-1 and the mitochondrial membrane potential(△Ψm)was observed using laser confocal-scanning microscopy;furthermore,intracellular ATP was determined by a luciferase-based assay.Oxidative stress was assessed by the levels of superoxide dismutase(SOD),malondialdehyde(MDA)and lipid peroxides(LPO)in lung tissues.The level of Caspase-3 activity was determined in the lung tissue homogenates,pulmonary cell apoptosis was determined by TUNEL staining,and Bax&Bcl-2 protein expression was determined by immunohistochemistry.The level of inflammatory mediators in the lung and BALF were measured to reflecting the lung inflammation response.[Result]:1.In rat model,as expected,remarkable lung injury and dysfunction was observed following LPS administration,as evidenced by the level of histopathologic deterioration,elevated W/D weight ratio,and decreased oxygenation index,which is similar to that reported in previous studies,Then,treatment with the moderate and high dose of Mdivi-1 was associated with beneficial effects in LPS-induced ALI;2.Mitophagy leads to the phagocytosis of mitochondria,thus decreasing the intracellular mitochondrial mass when it is enhanced.TOM20 and TIM23 levels were decreased in rats with ALI,indicating that mitophagy was up-regulated.Mdivi-1 treatment inhibited the down-regulation of TOM20 and TIM23 expression,and the increased LC3 II/LC3 I ratio and decreased p62 expression induced by LPS were not reversed following Mdivi-1 treatment.Moreover,expression of the mitochondrial biogenesis markers,PGC-la and mt-TFA,were not significantly down-regulated following LPS or Mdivi-1 treatment.These findings indicted that Mdivi-1 had no significant effect on mitochondrial production or general autophagy,suggesting that Mdivi-1 affects mitochondrial mass by modulating mitophagy.3.LPO levels,MDA accumulation and SOD consumption were reversed by Mdivi-1 treatment,suggesting an attenuation of oxidative stress;furthermore,Mdivi-1 inhibited LPS-induced apoptosis,Bad up-regulation,Bcl-2 down-regulation and Caspase-3 activation.These findings indicate that Mdivi-1 pretreatment plays an anti-apoptotic role in LPS-induced ALI.Moreover,Mdivi-1 treatment dramatically prevented the increase in proinflammatory cytokines(TNF-a,IL-1β,IL-6)in lungs and BALF of LPS-challenged rats,and prevented the increase in lung MPO activity and F4/80 expression level.These results demonstrate that Mdivi-1 treatment ameliorates LPS-induced lung neutrophil infltration and inflammation.[Conclusion]:The present study demonstrates that Mdivi-1 inhibits mitophagy and protects ALI-challenged rats from LPS-induced apoptosis,oxidative stress and inflammation.Furthermore,in this study,the wider protection and inhibition of mitophagy provided by Mdivi-1(3 or 5 mg/kg)may represent a promising target for the treatment of ALI in the future.