The Role Of MtDNA And NETs In The Seawater Inhalation Induced Acute Lung Injury

Background: Drowning is a serious social security problem,and it causes a large number of casualties every day.As one of the most serious complication of drowning,ALI/ARDS occurs in about 1/3 of the drowning victims,and it becomes the leading cause of death by drowning.Among the drowning casued by water,seawater drowning is the most serious and it is difficult to treat.Because of lacking of basic researchs,the pathogenesis and causes of seawater inhalation induced ALI/ARDS are still not clear and need further research.Seawter is alkaline liquid with low temperature,high osmotic pressure,high concentration of sodium and potassium,and containing a variety of bacteria.Its concentration of various ions are significantly different from fresh water or body fluids,and its osmotic pressure is about three times of human plasma(942vs.300 m Osm/kg).The low temperature and high osmotic pressure of seawater can produce a strong damage to lung tissue and cells together.Moreover,because the osmotic pressure of seawater is higher than that of the plasma,the fluid in the interstitium and capillaries of the lung will seep into the alveolar cavity,and finially leading to pulmonary edema and hypoxemia.mt DAMP is a kind of substance derived from mitochondria when the body is injured.It mainly includes mtDNA and formyl peptide.Clinical studies have shown that the level of free mtDNA in the blood of patients died from sepsis and ARDS in ICU is significantly higher than that in patients who have improved,and the risk of death in patients with high concentration of mtDNA is higher than that in other patients.mtDNA can also play a role in promoting inflammation.mtDNA is different from the nuclear DNA which has a lot of methylated Cp G motifs,and is similar to bacterial DNA.The immune system can recognize this difference and be activated.mtDNA can be recognition by immune cells,vascular endothelial cells and smooth muscle cells by TLR 9,and then promotes the activation of MAPK pathway and releasing of proinflammatory cytokines.In animal experiments,mtDNA intravenous injection is sufficient to cause acute lung injury.It plays an important role in the initiation and early development of acute lung injury by activating neutrophils through TLR 9 receptor.NETs is a bactericidal mechanism of neutrophils,and it is mainly composed of DNA,MPO,and histone.When stimulated by bacteria or other things,neutrophils are activated and may form NETs.NETs can provide high concentration of immune factors in the local lesions,and thus plays a role in killing bacteria.NETs has a smooth filamentous structure,and numbers of filaments can be prepared into bundles.Then these bundles can be compiled into a network which is similar to the cloud when observed under a microscope.The presence of NETs facilitates the presence of high concentrations of antimicrobial substances in the infection site,and it also can increase their antimicrobial activity.However,NETs may also has a negative impact.NETs is also involved in the development of a number of non infectious diseases,such as gestational hypertension(pre-eclampsia)and systemic lupus erythematosus.Excessive neutrophil infiltration and NETs formation are also involved in the progression of ALI/ARDS.In an animal experiment about transfusion related lung injury,the formation of NETs was detected in the microcirculation of the lung.Addition of NETs into culture lung epithelial cells can lead to the death of the cells.Another experiment also showed that NETs was also toxic to lung endothelial cells.JNK belongs to MAPK pathway,and it is phosphorylated and activated when the cell is stimulated.It can transfer to the mitochondria or the nucleus where it plays a role in regulating cell function.JNK-mitochondria pathway is involved in the production of ROS and the regulation of apoptosis.The experiments showed that part of p-JNK was transferred to the nucleus,and there was also part of them were localized to the mitochondria by bingding to Sab.The localization of JNK to mitochondria can lead to the disorder of mitochondrial function,resulting into the decrease of energy supply,the decrease of mitochondrial membrane potential,the increase of ROS production and the occurrence of apoptosis.Through the combination of Tat-Sab KIM1 and Sab,the combination of Sab and JNK can be blocked and JNK mitochondrial localization also be inhibited.Methods: Part 1: In this part of the experiment,we established the rat model of seawater inhalation induced acute lung injury,and do some test.First,we measured the wet to dry ratio of lung tissue,which represented the degree of pulmonary edema.Second,we also fixed lung tissue,did H-E staining,and observed under the microscope.Third,we measured the content of protein and the number of neutrophils in the rat alveolar lavage fluid.We also detected the content of mtDNA in the alveolar lavage fluid by q PCR method.Fourth,in order to further explore the effect of mtDNA on lung tissue and cell injury,we isolated the mitochondria of rat cardiac myocytes,extracted the mtDNA,and driped into the lung of rats.In addition,we cultured HUVEC and A549 cells and explore the release of mtDNA and its effects on cell injury and monolayer permeability.Fifth,we isolated neutrophils from healthy volunteers and examined the effect of mtDNA on neutrophil adhesion and chemotaxis.In addition,we also detected the release of cytokines in neutrophils stimulated by mtDNA.Part 2: In this part,we examined the effects of seawater and mtDNA stimulation on NETs formation.We detected NETs formation by three methods.First,we detected NETs by H-E staining and observed the cell free DNA in the alveolar cavity and lung tissue.Second,we detected cell free DNA content in bronchoalveolar lavage by fluorescent staining.Third,we detected the content of Neutrophil elastase-DNA composites by ELISA.We also induced and isolated NETs by PMA,and cultured HUVEC and A549 cells with NETs.The cell injury and permeability of monolayer were detected.Finally,we used NAC to inhibit the production of ROS and tested the effect of antioxidants on NETs formation.Part 3: In this part,we isolated lung mitochondrial protein and cytoplasmic/nuclear protein,and detected the expression of p-JNK and JNK by western blots,and examing the transfer of JNK after seawater stimulation.We also examined the effects of Tat-Sab KIM1 on JNK-mitochondrial pathway,ROS production,and lung injury.Results: Part 1: 1.The presence of mtDNA could be detected in bronchoalveolar lavage fluid of the rat model of seawater inhalation induced ALI/ARDS.2.The presence of mtDNA could be detected in the culture supernatants of HUVEC and A549 cells challenged by seawater exposure.3.Intratracheal instillation mtDNA could induce the occurrence of acute lung injury,and inhibition of mtDNA production could play an important role in improving acute lung injury.4.mtDNA could cause the injury of HUVEC and A549 cells and change the permeability of monolayer cells.5.mtDNA could activate neutrophils,promote neutrophil adhesion,exudation and migration chemotaxis,and promote the release of cytokines.6.mtDNA stimulation could induce the lung of SD rats and lung cells to produce a large amount of ROS.Part 2: 1.Seawater or mtDNA inhalation could cause the lung of SD rats to produce sterile NETs.2.Co-culture of neutrophils and HUVEC cells could induce the production of NETs.3.NETs could induce the change of permeability of HUVEC cells and A549 monolayer cells.4.The production of NETs was dependent on ROS,and the formation of NETs was significantly decreased after antioxidant treatment.Part 3: 1.Inhibition of the transfer of JNK to mitochondria could improve lung injury by reducing the production of ROS.2.There is a phenomenon of JNK transfer to mitochondria in seawater inhalation induced acute lung injury.3.Application of Tat-Sab KIM1 peptide could inhibit the mitochondrial transfer of JNK.4.Application of Tat-Sab KIM1 could inhibit the production of ROS,play a role in improving lung injury.Conclusion: The results showed that seawater stimulation could disturb the normal structure of the lung tissue and the function of cells and mitochondria,resulting in the release of a large amount of mtDNA into the alveolar space.The release of mtDNA could cause the lung cell injury,damage of blood gas barrier,and also could activate the immune system because of its immunogenicity.And then gave rise to neutrophil adhesion,exudation and releasing of inflammatory factors.The excessive activation of the immune system caused by mtDNA,especially the activation of neutrophils,further aggravated the extent of lung injury.NETs is a kind of bactericidal extracellular substances released by neutrophils.Rat lung would form amount of NETs when stimulated by seawater or mtDNA.The NETs had high concentrations of inflammatory cytokines and cell killing substances,and could cause damage to the lung tissue.The results showed that the formation and damage of mtDNA and NETs were dependent on ROS,and it was more easily to be damaged by mtDNA and NETs when the lung tissue cells were exposed to oxidative stress.The activation of JNK-mitochondria pathway could induce mitochondrial dysfunction,resulting in the accumulation of ROS in cells.Inhibited JNK-mitochondria location by specific Tat-Sab KIM1 peptide,the mitochondrial stability improved,autophagy and apoptosis inhibited and lung injury reduced.