Research Of Exosomal Cytokine Profile On Endotoxin-induced Acute Lung Injury In Mice

ALI(acute lung injury,ALI)is one of the common and difficult problem of Respiratory Diseases.There is no relevent reports of ALI/acute respiratory distress syndrome(ARDS)morbidity and mortality in China.However,in the United States,approximately150000 to 200000 cases are reported each year.The latest random survey showed that the mortality rate in ALI patients ranged from 35%-40%,and 25-30%patients died within 28 days.A variety of reasons can cause damage to the lungs and other organs,and can cause ALI,ARDS and/or multiple organ dysfunction syndrome(MODS).ALI/ARDS is often the first and the most common organ dysfunction in MODS,It plays an important or even an essential role in the pathogenesis of MODS development.Not only ALI/ARDS mortality is high,also its etiology and pathogenesis are complex,it has become a focused and difficult problem in clinical respiratory and intensive care research.Pothogensis of ALI/ARDS is not clear.There are many theories,such as pro-inflammatory/anti-inflammatory imbalance theory","coagulation/anticoagulation imbalance theory" and "oxidative stress theory" "apoptosis disorder theory" and so on.Among them,the inflammatory response is the body’s physiological response to various pathological damage and stimuli.Most scholars believe that uncontrolled lung or systemic inflammatory response is a major pathogenesis of ALI/ARDS.Cytokines and inflammatory mediators constitute the basis for ALI/ARDS inflammation and immune regulation,and through different signal transduction pathways regulating the immune response and relating to uncontrolled inflammatory response.Cytokines and inflammatory mediators play a key role in pathogenesis of ALI/ARDS.In China,it has been recognized that imbalance of cytokine plays a very important role in the regulation of inflammatory mediators ALI/ARDS development and progression of the disease in the early 1990s.Over the past 10 years,the study of ALI/ARDS in the regulation of cytokines and inflammatory mediators imbalance made some progress,but also exposed the study of defects and shortcomings.Fundamental aspects progresses:ALI reveal activative cell signaling pathways:NF-κB system,activates myosin light chain kinase(MLCK)and the MAPK pathway.Clinical studies aim to finding specific protein marker of ALI/ARDS,like troponin in acute myocardial infarction.On the other hand,searching for effective anti-inflammatory therapy.Review of basic and clinical research in the treatment of acute lung injury,we find that inhibiting the release of pro-inflammatory mediators in the initial stage of inflammation is the main theme.However,separately inhibit only one cytokine of proinflammatory mediators(such as application-specific TNF-α or IL-1β antagonist)by clinical studies can not improve patient prognosis.Studies show that inflammation subsides,like inflammation,also is a process of active and sequencing,controlled by endogenous lipid/chemical medium.Timely resolve Inflammation is a key step to prevent Inflammation to chronic disease.Therefore the mechanism of endogenous Inflammation subsides become a new hotspot in the research of Inflammation in recent years.Due to the inhibition effect of a certain kind of inflammatory medium alone is not good,we expect to find more comprehensive entrance to explain ALI/ARDS disease pathophysiology in the research of inflammatory response.Therefore the research of exosome was born.Exosomes can be formed through inward budding of endosomal membranes,giving rise to intracellular multivesicular bodies(MVBs)that later fuse with the plasma membrane,releasing the exosomes to the exterior.The study found that exosome is a diameter of 30-100 nm lipid molecular structure of double vesicles.A variety of cells have been shown to release exosomes,including epithelial cells,mast cells,platelets,antigen-presenting cells(APCs)and tumor cells.Exosomes can also be isolated from various bodily fluids,such as blood,urine,the BALF,amniotic fluid,breast milk,semen,saliva,joint synovial fluid,ascites tumor and malignant effusions,and it can be found in the cell conditioned medium in vitro.Exosome contains the miRNA,mRNA,protein and lipid and other different kinds of biological molecules Although exosomes component altered from the tissue source,but they have common protein components,including tetraspanins,heat shock protein,the plasma membrane and fusion related proteins,polycystic bubble produce related intracellular protein,these ingredients reflects its biological origin.In addition,exosomes also contains the cytokines,growth factor receptors,transcription factor receptor and other biologically active molecules.Under normal and disease states,these substances play a corresponding role in biological fluids that expressed in cells and secreted and loaded by the exosomes.Moreover,exosomes have a lot of physiological functions,such as mediating cell communication,signal transduction,genetic material transportation and regulating immune response,etc.For example:the study found that exosomes can release internal signal molecular bond on the cell membrane surface,to complete the communication between cells.Such as exosomes derived from the tumor cells secrete a large number of cell growth factors,such as fibroblast growth factor,vascular endothelial growth factor,vascular auxin,etc.these components can be released from the cell under acid condition,act on endothelial cell membrane surface receptors to promote cancer growth.Exosomes from isolated BALF of asthma patients compared with the normal person’s,they can induce more IL-8 and leukotriene secreted from alveolar epithelial cell.Other studies confirmed that exosomes related microRNAs involved in regulating the pathophysiological process of asthma disease,and so on.Exosomes contains the miRNA,mRNA,protein and lipid and other different kinds of biological molecules.Also includes cytokines,growth factor receptors,transcription factor receptor and other biologically active molecules.Under normal and disease states,these substances play a corresponding role in biological fluids that expressed in cells and secreted and loaded by the exosomes.Exosomes can be stable in the body fluids,because its surface express CD55 and CD59 to avoid activating by the opsonin,alexin and the clotting factor;At the same time because of its small size(<100 nm),which can effectively avoid the mononuclear-macrophage phagocytosis,and can be freely pass through the vessel wall and extracellular matrix,thus can be widely distributed in the body fluids.So cytokines loading by exosomes can be stable and be found widely in body fluids,which play a rich physiological functions,such as mediating cell communication and regulation of immune response.Given the exosome’ characteristics of stability and widely distributed in the body fluids,Does this help us to further awareness of the pathogenesis of ALI?Cytokines are small molecule protein with a broad range of biological activities,It can be synthesised and secreted by stimulated inflammatory cells.As an intercellular signal transduction molecules,mainly regulate immune response,involved in immune cell differentiation development,mediated inflammatory reaction to stimulate hematopoietic function and participated in tissue repairment,etc.Now there has been many studies on cytokine levels in serum of ALI/ARDS,but in view of cytokines in exosomes has not been reported.Moreover,and existing detection index is less lead to differences of results.For the understanding of cytokines in the blood is incomplete and uncomprehensive.The research significance of this experiment is to explore the role of exosomes in acute lung injury(ALI),from a new perspective to know disease development process,to describe more comprehensive cytokine expression profile of acute lung injury(ALI)to provide a new opportunity for explain the happening of the disease development mechanism.Not only that,to lay the foundation for the further study of exosomes’s proteomics and the miRNA in acute lung injury,to attempt to finding diagnostic marker of ALI.This topic employs classic model to duplicate the ALI in mice induced by intraperitoneal injection of LPS,the model was one of the first animal model of ALI.It can imitate clinical lung injury caused by Gram-negative bacteria,it can be copied easily.It has been reported LPS through toll-like receptor 4 pathway to activate innate immune response,can be well researched the host inflammatory response.After the success of the molding,collected specimens of different time points for the experimental group and control group,observe lung tissue pathology change,ratio of dry weight/wet of lung tissue in the early acute lung injury induced by LPS.Using the optimized ExoQuick Exosome Precipitation Solution(Cat#:EXOQ5A-1)kit method to extract the serum’s exosomes,obtain a higher purity of exosomes for future research.Using the multi-factor detection technology(multi-analyse profiling,xMAP)to research the difference of serum and serum exosomes cytokines profile.Methods:18 healthy 6-8 weeks C57/BL6 mice,male,weight(20±2)g,were randomly divided into three groups:control group(n=6),2h ALI group(n=6),8h ALI group(n=6),all experimental animals hunger 8 hours before stimulation,only drink without eating.Experimental mice intraperitoneal injection of 10 mg/kg of LPS,duplicate the model of endotaxin-induced acute lung injury in mice respectively in 2 h and 8 h.Control group intraperitoneal injection normal saline that with same amount of LPS.AIl mice open chest and extract blood specimens from left ventricle after anesthetizing fully,take the supernatant after centrifugation,-80℃cryopreserved.Using optimized ExoQuick Exosome Precipitation Solution(Cat#:EXOQ5A-1)kit method of extracting high purity of serum exosomes and proving the exosome by transmission electron microscopy and Western Blot;XMAP was used to study the expression of cytokines of the control group and experimental group in serum and serum exosomes.Obtain the lung tissue specimens,measure the wet weight of the right lung and weighing again after drying,calculation of wet/dry weight ratio(W/D).Lavage after harvesting the lung tissue,put in the ice precooling of PBS rinsed three times to remove residual blood adequately,add 4%paraformaldehyde fixed,conventional paraffin embedding slices,after HE dyeing observation lung tissue pathology change.And scratches experiment:using the PBS,LPS,serum exosomes extracted from the control group and experimental group stimulate(RAW264.7)24 h to verify the function of serum exosomes in ALI.Results:1.The W/D of the control group in the lung tissue of mice was 4.037 ±0.130,the experimental group 2 hours was 4.607±0.201,the experimental group 8 hours was 5.109±0.158.Compared with the control group,experimental group 2 hours W/D was significantly increased(P<0.001),Compared with the control group,the experimental group 8 hours W/D was significantly increased(P<0.001);the experimental group in different time point W/D value also have significant difference(P<0.001).2.lung tissue pathology change:compared with the control group,the experimental group alveolar walls broadening,alveolar cavity inflammation visible and pulmonary interstitial hemorrhage and exudation were observed,over time,more and more serious,lung tissue of LPS stimulation 8h,in a large number of inflammatory cells and red blood cells and plasma protein exudation,alveolar structure damage,acute lung injury(ALI)building success.3.using transmission electron microscopy(TEM)and Western blot test to verify exosomes extraction successfully.The microscopic observation of the diameter of exosome is between 30-100 nm,assumes the circular or elliptic;the Western blot tested exosomes CD63 expressions,and serum supernatant after extracting exosomes without CD63 expressions.4.On the one hand,ALI group serum has 18 kinds of cytokines in mice compared with control group with significant difference(P<0.05),respectively,IL-6,TNF-alpha,IFN-gamma,IL-1alpha,IL-1 beta,IL-17,the MIP-1 alpha,MIP-1beta,MIP-2,KC,IP-10,MCP-1,Eotaxin,RANTES,MIG,G-CSF,IL-10 and LIF.There are 6 kinds of proinflammatory factor,is on the rise after stimulated,IL-6 and TNF-alpha on peak at 2h,IFN-gamma,IL-1 alpha,IL-1 beta,IL-17 on peak at 8h.IL-6,IL-1alpha,IL-lbeta 2 h and 8 h experimental group compared to control group with significant difference(P<0.05).IFN-gamma(P=0.969),IL-17(P=0.823)2 h group had no significant difference compared with control group.Nine kinds of chemokines after stimulated is on the rise,the MIP-1 alpha,MIP-1beta,MIP-2,KC,MCP-1 on peak at 2h,IP-10,Eotaxin,RANTES,MIG on peak at 8h.MCP-1,IP-10,Eotaxin,KC 2 h and 8 h experimental group compared to control group had significant difference(P<0.05).MIP-1 alpha(P=0.629),the MIP-1 beta(P=0.354),the MIP-2(P=0.920)in 8 h group had no significant difference compared with control group.RANTES(P=0.434),MIG(P=0.532)2 h experimental group had no significant difference compared with control group.G-CSF is on the rise after modeling,G-CSF at 8 h on peak,compared with the control group with significant difference(P<0.05).There are also 2 kinds of anti-flammatory factor is on the rise,IL-10 is on peak at 2h,LIF is at 8 h,IL-10 2h and 8h experimental group compared to control group with significant difference(P<0.05).LIF(P=0.121)2h group had no significant difference compared with control group.On the other hand,ALI group mice serum exosomes have 11 kinds of cytokines compared with the control group with significant difference(P<0.05),respectively,IL-6,MIP-1beta,MCP-1,IP-10,Eotaxin,RANTES,KC,MIG,G-CSF,MIP-2 and LIX.After stimulated,only one kind of proinflammatory factor,IL-6 is on the rise,and on peak at 8h,2 h and 8 h of experimental group compared to control group with significant difference(P<0.05).But 2h compared with 8 h,there was no significant difference(P=0.615);There are 9 kinds of chemokines is on the rise after modeling,MIP-1beta,MIP-2,KC is on peak at 2h,MCP-1,IP-10,Eotaxin,RANTES,MIG,LIX is at 8h,MCP-1and IP-10 2h and 8 h experimental group compared to the control group had significant difference(P<0.05).Eotaxin(P=0.138),RANTES(P=0.575),MIG(P=0.716)2 h group there was no significant difference compared with control group.MIP-1 beta(P=0.141),the MIP-2(P=0.780)8 h group were no significant difference compared with control group.LIX(P=0.557),KC(P=0.051)2 h had no significant difference compared with 8 h group.G-CSF is on the rise,peaked at 8h,2 h and 8 h experimental group compared to control group had significant difference(P<0.05).5.Scratches experiment:using the PBS,LPS,serum exosomes extracted from the control group and experimental group stimulate(RAW264.7)24h to valify serum exosomes in ALI can promote macrophage migration to the scratch parts like LPS.Conclusion:LPS-induced acute lung injury,pro-inflammatory and anti-inflammation factor mostly free form present in serum,The cytokines of exosomes are mainly chemokines,exosomes from serum of LPS induced acute lung injury can presenting role in promoting the migration of macrophages.