The Regulatory Effect And Mechanism Of Albumin On The Neuthrophil Extracellular Traps(NETs)

Objective:The flow of blood in vessels is very important to keep the blood and oxygen supply.Usually,there is no blood clotting and thrombosis in vessels.Once there is thrombosis in vessel,it will lead to ischemia,hypoxia and even injury of tissues and organs.Neutrophil extracellular traps(NETs)are fibrous mesh-like structures that are released by neutrophil(PMN)cells into the extracellular environment.NETs are mainly composed of cellular DNA and cytoplasmic proteins released by PMN,and the most abundant protein is histone,followed by neutrophil elastase(NE)and myeloperoxidase(MPO).Recently,more and more research believe that NETs is involved in the formation of intravascular thrombosis,which is closely related to sepsis,autoimmune diseases,diabetes and even tumors.Therefore,it is of great significance to study the molecular mechanism of NETs in the occurrence of diseases and to find effective treatment methods.Albumin(ALB)is the most abundant protein in the blood vessels,playing an important role in maintaining plasma colloid osmotic pressure and blood volume,transporting and binds drugs or endogenous substances,and ACTS as an antioxidant.It is clinically used in hemorrhagic shock,liver cirrhosis with ascites edema,adult respiratory distress syndrome,malignant tumors and other treatments.We accidentally found that in serum-free medium,PMN could release NETs,but after adding serum,the formation of NETs was inhibited,indicating that there was some component in serum that inhibited the formation of NETs.As we know,there are a large number of PMN in blood vessels,but few NETs are generated in normal physiological state.If a large number of NETs are generated spontaneously,it may cause vascular embolism or even shock and DIC.Based on the above findings,we speculated that substances that have precise regulatory effects on the formation of NETs should be present in blood vessels,but the regulatory mechanism of such substances and their involvement is still unclearTo this end,we designed a series of experiments to research the substance basis and molecular mechanism of inhibiting the formation of NETs in plasma,so as to elucidate the substance basis and regulatory mechanism of inhibiting the formation of NETs in blood vesselsMethods:?.separation of human neutrophils and detection of NETs1 Isolation of neutrophils(PMN)in healthy people:peripheral venous blood of healthy people was taken,and neutrophils were separated by human neutrophil assay kit2 Neutrophil medium system:100%DMEM medium was used as the standard PMN medium system,and was added to the 35 mm laser confocal culture dish.The final concentration of PMN cells in the medium system was 5.0 105/ml.3 Identification of neutrophils:reye staining and trypan blue assay were used to identify the isolated cell types and the survival rate of PMN4 Detection of NETs:PMN on CO2 incubator(37 ?,5%CO2)in 2h after incubation,join DNA sytox green dye/sytox orange[sytox green/sytox orange,medium final concentration of 5 mM(The concentrations mentioned later are all final concentrations in the medium)].then green/red fluorescence was observed under the laser confocal microscope,and the mean fluorescence intensity(MFI)was determined by ZEN widefield software(the method was adopted for NETs detection in subsequent experiments,and it will not be repeated)?.Reconfirmation and component screening of the inhibitory effect of serum on the formation of NETs1 To confirm the effect of serum on NETs:the amount of NETs was detected in standard PMN medium system containing 10%fetal bovine serum(FBS)and 10%human serum(HBS)2 Ultrafiltration of plasma components:ultrafiltration centrifuge tubes with different molecular weights(3k Da,30k Da,50k Da,100k Da)were used to conduct ultrafiltration of human serum,and human serum components with different molecular weights were obtained3 Confirmation of the effect of plasma components in molecular weight segments on NETs:human plasma components in different molecular weight segments were added into the standard PMN medium system for 2 h to detect the production of NETs in each molecular weight plasma component in the experimental group,and the molecular weight range of plasma components that could inhibit NETs was preliminarily confirmed.?.Screening and identification of proteins in plasma that inhibit NETs formation1 Selection of proteins in blood:by reviewing literatures,common and important proteins in blood were selected from the molecular weight range of plasma components with NETs inhibition,and a value within the physiological concentration range of this protein was selected as the experimental physiological concentration2 Effects of blood proteins on NETs generation:the above selected proteins were added into the standard PMN medium system at 10%of the experimental physiological concentration to detect the effects on NETs The most common and important proteins with NETs inhibitory effect were analyzed as the main research object?.Study on the effect of albumin on NETs after adding NETs key protease inhibitor1 After inhibition of MPO,albumin action on NETs:MPO inhibitor(final concentration 100 nM)was added to standard PMN medium containing BSA(0.5 mg/ml,1.5 mg/ml,4.5 mg/ml)to detect the changes in NETs production2 After inhibition of NE,albumin action on NETs:NE inhibitor(final concentration 100 nM)was added to standard PMN medium containing BSA(0.5 mg/ml,1.5 mg/ml,4.5 mg/ml)to detect the changes in NETs production.3 After inhibition of PAD4,albumin action on NETs:PAD4 inhibitor(final concentration 100 nM)was added to standard PMN medium containing BSA(0.5 mg/ml,1.5 mg/ml,4.5 mg/ml)to detect the changes in NETs production?.Effect of albumin antibody on the inhibition of NETs by albumin1 Antibodies against people albumin and albumin preliminary hatch configuration 200 mu g/ml HSA,adding albumin antibodies to 200 mu g/ml,HSA solution into the CO2 incubator(37?,5%CO2)for the night2 Detection of the effect of antibody incubation on albumin inhibition of NETs:4 kinds of PMN medium systems(no albumin,albumin antibody,200 g/ml HSA,200 g/ml conjugated antibody HSA)were tested for NETs?.Anti-nets effect of bovine serum albumin in oxidized or reduced state1 Preparation of BSA in oxidation state:BSA(40 mg/ml)and H2O2(45 mM)were incubated at room temperature for 1 h,and the incubated BSA was freeze-dried to obtain BSA in oxidation state.The concentration of oxidized BSA was determined by BCA protein concentration assay,and the concentration was adjusted to 40 mg/ml2 Reduction condition of the preparation of BSA,the BSA(40 mg/ml)and cystine(17 mM)at 37? for 24 h after incubation,the PD-10 desalination column off residual cystine;The column effluent was freeze-dried to obtain reduced BSA.BSA concentration in reduced state was determined by BCA protein concentration assay,and its concentration was adjusted to 40 mg/ml3 Influence of BSA in oxidation or reduction state on NETs:the amount of NETs was detected by adding BSA in oxidation or reduction state(4 mg/ml)into standard PMN medium system,and the results were compared with normal BSA results to determine the influence of BSA in oxidation or reduction state on NETs?.Effects of bovine serum albumin on NETs inhibition in the presence of LPS and escherichia coli1 Bovine serum albumin inhibits the action of NETs in the presence of LPS1.1 In the presence of LPS,he effects of different concentrations BSA inhibited NETs1.1.1 In the presence of low concentration LPS,the effect of the experimental physiological concentration BSA on inhibiting NETs:the LPS(final concentration:20 ng/ml,40 ng/ml,80 ng/ml,160 ng/ml)was added into the standard PMN culture medium system containing the physiological concentration BSA(40 mg/ml)to detect the amount of NETs production.1.1.2 In the presence of high concentration LPS,the effect of different concentrations of BSA on inhibiting NETs:the amount of NETs was detected by adding LPS(final concentration:160 ng/ml,320ng/ml,and 640ng/ml)into the standard PMN medium system containing BSA(40 mg/ml,5 mg/ml)1.2 The dose-effect relationship of BSA inhibiting NETs in the presence of LPS:LPS(final concentration:320 ng/ml)was added to the PMN culture medium containing BSA(0.5 mg/ml,1.5 mg/ml,4.5 mg/ml)to detect the production of NETs2 Effect of bovine serum albumin on NETs inhibition in the presence of escherichia coli2.1 In the presence of EC,the effect of different concentrations of BSA on inhibiting NETs2.1.1 In the presence of low concentration EC,the effect of different concentrations of BSA on inhibiting NETs was detected by adding EC(2.5105)into PMN culture medium containing two concentrations of BSA(40 mg/ml,0.5 mg/ml)2.1.2 The inhibitory effect of different concentrations of BSA on NETs in the presence of high concentration EC:the high concentration EC(2.9 107)was added to the PMN culture medium containing different concentrations of BSA(0.16 mg/ml,0.5 mg/ml,1.5 mg/ml,4.5 mg/ml,40 mg/ml)to detect the changes in the amount of NETs generated2.1.3 The inhibitory effect of different concentrations of BSA on NETs in the presence of low concentrations of EC:as the concentration of EC in previous experiments was too high,the concentration of EC was reduced for the experiment,and EC(2.5 105)was added to the PMN culture medium containing different concentrations of BSA(0.16 mg/ml,0.5 mg/ml,1.5 mg/ml,4.5 mg/ml,40 mg/ml)to detect the changes in the amount of NETs generated2.2 Dynamic observation of BSA inhibiting NETs generation in the presence of EC:EC(5.0×106)was added to a standard PMN medium containing BSA(1.5 mg/ml)and NETs were observed at three time points(30 min,60 min,120 min)to detect whether EC would affect the growth rate of NETs.?.Effects of Ca2+concentration and Ca2+channel blocker on the inhibitory effect of albumin on NETs1 The effect of Ca2+ and Ca2+ free medium on the formation of NETs PMN was incubated in the medium system with and without calcium PMN respectively,and the formation of NETs was observed2 Effects of different Ca2+concentrations on the formation of NETs PMN culture medium systems containing different Ca2+concentrations(0%,0.625%,1.25%,2.5%,5%,10%,20%)were prepared with Ca2+and Ca2+free DMEM as basic nutrients.PMN was incubated in the above concentration medium for 2 h to observe the differences in the formation of NETs.3 Effect of calcium channel blocker on NETs formation3.1 Effects of 2-ammoethoxy diphenyl ester boric acid(2-apb)on the inhibition of NETs by HSA:the amount of NETs was detected by adding 2-apb(final concentration 5 ?M)into the standard PMN medium containing HSA(1.5 mg/ml,4.5 mg/ml).3.2 The effect of verapamil on the production of NETs:the production of NETs was detected by adding verapamil(final concentration 10 ?M)into the standard PMN medium system.?.Effects of bovine serum albumin on intracellular total reactive oxygen species(ROS)and mitochondrial ROS levels in neutrophils1 Effect of BSA on intracellular total reactive oxygen species(ROS)level of PMN1.1 PMN ROS levels of detection:add the PMN standard PMN culture medium containing 10%BSA system,and in the CO2 incubator(37°C,5%CO2)incubation 20 min,in culture system to join DCFH-DA ROS fluorescence probe(10 microns)and after incubation for 30 min,gently beat off that stick a wall of PMN,collect the dark to 1.5 ml EP tube,using flow cytometry instrument detection of fluorescence intensity of ROS active oxygen probe.1.2 Effects of different kinds of proteins on ROS in PMN:BSA(4 mg/ml),HSA(4 mg/ml),apo A1(0.14 mg/ml)and HDL(0.3 mg/ml)were added into the standard PMN medium system,and the fluorescence intensity of ROS ROS probe was detected by flow cytometry.2 Effect of BSA on the level of mitochondrial reactive oxygen species(mitoROS)in PMN2.1 PMN mitoROS detection:the level of the standard system of PMN medium containing 10%FBS,and in the CO2 incubator(37 ?,5%CO2)incubation for 20 min,in culture system to join mitoROS fluorescent probe MitoSOX Red and take out after incubation for 30 min,gently beat off that stick a wall of PMN,collect the dark to 1.5 ml EP tube,using flow cytometry instrument testing mitoROS active oxygen probe fluorescence intensity.2.2 Effects of mitochondrial respiratory chain disruptors on the inhibition of NETs formation by albumin:antimycin A and rotenone are the inhibitors of mitoROS formation.In the standard PMN medium system,anti-mycin(final concentration of 20 ?M)and rotenone(final concentration of 10 ?M)were added,and the changes in the amount of mitoROS fluorescence probe and NETs generation were detected to further clarify the role of mitoROS in the generation of NETs2.3 Effects of different kinds of proteins on mitoROS level in PMN BSA(final concentration of 4 mg/ml),HSA(final concentration of 4 mg/ml),apo A1(final concentration of 0.14 mg/ml)and HDL(final concentration of 0.3 mg/ml)were added into the standard PMN medium system,and the fluorescence intensity of the fluorescence probe was detected by flow cytometry.?.effect of autophagy interfering agents on the inhibition of NETs by albumin1 Effect of autophagy inhibitors on the inhibition of NETs by albumin add 3-ma(final concentration is 1 mM)into the standard PMN medium system containing BSA(0.5 mg/ml,1.5 mg/ml,4.5 mg/ml)and observe the formation of NETs.2 Effect of autophagy agonists on the inhibition of NETs by albumin:the formation of NETs was observed by adding RAPA(5 mM)into the PMN culture medium containing 3 different concentrations of BSA(0.5 mg/ml,1.5 mg/ml,4.5 mg/ml)3 Effects of CaMKK inhibitor on NETs generation:STO609(concentration)was added into the standard PMN medium system to detect the amount of NETs productionResults:?.separation of human neutrophils and detection of NETs1 Isolation,culture and identification of neutrophils(PMN)in healthy people:the target cells were isolated by the PMN isolation solution kit of human peripheral blood according to the instructions.The cell nucleus was observed to be rod-shaped and 4-lobed by Swiss staining,and the cytoplasm contained purpse-red particles,which confirmed that the isolated cells were PMN.The survival rate was higher than 93%by trypan blue rejection test.2 Detection of NETs:green/red reticular or filamentous NETs were generated around PMN in the confocal dish under the laser confocal microscope?.Reconfirmation and component screening of the inhibitory effect of serum on the formation of NETs1 A large number of NETs can be observed in the standard PMN medium system,but NETs can hardly be observed in the PMN medium system containing 10%FBS.The above results show that 10%FBS has a significant inhibitory effect on the formation of NETs2 Ultrafiltration of plasma components:the lower filtrate in the ultrafiltration centrifuge tube was obtained by ultrafiltration centrifuge,and the lower filtrate was plasma components with different molecular weight specifications3 With different molecular weight specifications of the components of the plasma NETs observation:3 kDa centrifugal tube,30 kDa ultrafiltration of plasma components to the NETs have no obvious effect,and more than 50 kDa ultrafiltration centrifugal tube of the plasma components has inhibitory effect on NETs,of which 100 kDa ultrafiltration centrifugal tube of the plasma components the strongest inhibitory effect of NETs.The above results indicate that plasma contains components that inhibit the formation of NETs and may be of high molecular weight?.Identification of protein components in blood that inhibit the formation of NETs1 Selection of proteins in blood:Through literature review,the molecular weight in the blood of the common and most important protein human serum albumin(human serum albumin,HSA),high density lipoprotein cholesterol(ldl-c),high density lipoprotein,HDL),lipoprotein A1(apolipoprotein A1,apo A1),fibrinolytic enzyme(fibrinogen,FIB),gamma globulin(gamma globulin),transferrin(transferrin,agency),IgG In view of the protein structure and conservativeness and accessibility,the bovine serum albumin(BSA)was used to replace HSA in this experiment In the physiological concentration range of the above proteins,the median value was selected as the experimental concentration,and the concentration values were 40 mg/ml(HSA/BSA,3 mg/ml(HDL),1.4 mg/ml(apo A1),22 mg/ml(FIB),17 mg/ml(y-globulin),3 mg/ml(TRF),and 50 mg/ml(Ig G),respectively2 Observation of the effect of important and common proteins in blood on NETs:the final concentration of the experimental protein was set as 10%of the physiological concentration,i.e.,4 mg/ml HSA/BSA,0.3 mg/ml HDL,0.14 mg/ml apo A1,2.2 mg/ml FIB,1.7 mg/ml y-globulin,0.3 mg/ml TRF,5 mg/ml Ig G),and the amount of NETs was detected.The results showed that 10%of the experimental physiological concentration of HSA/BSA,HDL,FIB had a strong inhibitory effect on NETs,while ?-globulin,apo A1,TRF,Ig G had no significant inhibitory effect on NETs3 Selection of main research subjects:since HSA is the highest in blood and has important physiological significance,HSAis selected as the main research object.However,because HSAis expensive,BSAis selected to replace it for subsequent relevant experiments.?.Research on the effect of albumin on NETs after inhibiting the key protease of NETs1 Studies on the effect of albumin on NETs after inhibiting the key MPO protease:(1)NETs can be generated in large quantities in the standard PMN medium system.(2)NETs could still be generated in the medium system with low concentration BSA(0.5 mg/ml),and there was no significant change in the production of NETs in the medium system with low concentration BSA(0.5 mg/ml)when MPO inhibitor was added.(3)there was still a small amount of NETs generated in the PMN medium system with a lower concentration of BSA(1.5 mg/ml),and there was no significant change in the amount of NETs generated after adding MPO inhibitor into the PMN medium system with a lower concentration of BSA(1.5 mg/ml).(4)only a very small amount of NETs was generated in the PMN medium system containing BSA(4.5 mg/ml),and the formation of NETs could hardly be observed when MPO inhibitor was added into the standard PMN medium system with high concentration of BSA(4.5 mg/ml).The results showed that the addition of MPO inhibitor can reduce the amount of NETs,and the addition of BSA can strengthen the inhibition of NETs,and the inhibition is enhanced with the increase of BSA concentration.2 Studies on the effect of albumin on NETs after inhibiting NE key protease:(2)when NE inhibitor was added into the medium system containing low concentration BSA(0.5 mg/ml),NETs were generated,but the production was reduced.(3)only filamentous NETs were observed after adding NE inhibitor into the medium system with a lower concentration of BSA(1.5 mg/ml).(3)the formation of NETs could hardly be observed in the standard PMN medium system with the addition of high concentration BSA(4.5 mg/ml)NE inhibitor.The results showed that the inhibition of NETs by NE inhibitor itself was very low,but the inhibition of NETs by BSA could be enhanced.The above experiments indicated that the addition of NE inhibitor could reduce the amount of NETs,and the addition of BSA could strengthen the inhibition of NETs,and the inhibition was strengthened with the increase of BSA concentration3 Studies on the effect of albumin on NETs after PAD4 key protease inhibition:(2)when PAD4 inhibitor was added to the medium containing low concentration BSA(0.5 mg/ml),the production of NETs decreased despite the presence of the inhibitor.(3)when PAD4 inhibitor was added to the medium with a low concentration of BSA(1.5 mg/ml),only filamentous NETs could be observed;(4)the generation of NETs could hardly be observed when PAD4 inhibitor was added to the standard PMN medium system with high concentration BSA(4.5 mg/ml).The results show that PAD4 inhibitor has a low inhibitory effect on NETs by itself,but it can enhance the inhibitory effect of BSA on NETs.The above experiments show that the addition of PAD4 inhibitor can reduce the amount of NETs,and the inhibition of NETs can be enhanced after the addition of BSA,and the inhibition is enhanced with the increase of BSA concentration?.Effect of albumin antibody on albumin inhibition of NETs1 Pre-incubation of albumin antibody and albumin:HSA binding antibody was successfully obtained2 Effect of albumin antibody on albumin inhibition of NETs:after the BSA(BSA of conjugated antibody)co-incubated with antibody was added to the PMN culture system,the generation of reticular NETs could still be observed,indicating that the inhibitory function of the BSA of conjugated antibody on NETs decreased?.Anti-nets effect of bovine serum albumin in oxidized or reduced state1 Preparation of oxidized BSA:obtain oxidized BSA2 Preparation of reduced albumin:obtain oxidized BSA3 Effects of BSA in oxidation or reduction state on NETs:in the standard PMN medium system containing albumin in oxidation state or reduction state(4 mg/ml),the production of NETs was significantly higher than that in the standard PMN medium system containing normal BSA(4 mg/ml).The results showed that the inhibitory effect of albumin on NETs could be affected by changing the state of albumin oxidation and reduction?.Effects of bovine serum albumin on NETs inhibition in the presence of LPS and escherichia coli1 In the presence of LPS,BSA inhibits the effect of NETs1.1 In the presence of low concentration LPS,BSA of different concentrations inhibited the effects of NETs1.1.1 In the presence of low concentration LPS,the effect of experimental physiological concentration BSA on inhibiting NETs:the results showed that in the environment of experimental physiological concentration BSA,LPS stimulated PMN to generate NETs was significantly inhibited,and the increase of LPS concentration would lead to a large number of PMN deaths and the generation of very few filamentous NETs.1.1.2 In the presence of high concentration LPS,the effects of different concentrations of BSA on inhibiting NETs:the experimental results show that in the environment of high concentration BSA,even with extremely high concentration LPS stimulation,PMN is difficult to generate NETs;In low concentration BSA environment,extremely high concentration LPS can cause a large number of NETs1.2 The dose-effect relationship between the generation of BSA and NETs in the presence of LPS:the generation of NETs decreased as the concentration of BSA increased,and the generation of NETs could hardly be observed in the medium containing BSA(4.5mg/ml)PMN.The amount of NETs was increased in PMN culture system with LPS.However,as the concentration of BSA increased,the amount of NETs induced by LPS decreased gradually.The results showed that there was a significant dose-effect relationship between BSA and LPS-induced NETs inhibition2 In the presence of EC,BSA inhibits the effect of NETs2.1 In the presence of EC,different concentrations of BSA inhibit the effect of NETs2.1.1 The effect of BSA on inhibiting NETs in the presence of low concentration EC:the results showed that no NETs were generated in the presence of EC in the environment of experimental physiological concentration BSA,while the changes of NETs in the environment of low concentration BSA were not obvious2.1.2 The inhibitory effect of different concentrations of BSA on NETs in the presence of high concentration EC:the results showed that in the PMN culture system without EC,the production of NETs decreased as the concentration of BSA increased.When high concentration of EC was added into PMN culture system,PMN died directly without the formation of NETs.The above results show that in high concentration EC environment,PMN does not release NETs in direct death2.2 Dynamic observation of NETs inhibition by BSA in the presence of EC:the results showed that non-ec induced NETs in the low concentration BSA(4.5 mg/ml)environment gradually showed reticular opening over time.The growth rate of NETs induced by EC did not change significantly,and the wider fibrous structure of NETs could be observed at 60 min,but the local part of the ec-induced fibrous structure of NETs could be observed as a cluster at 120 min.The above results show that EC has no obvious effect on the formation rate and production amount of NETs,but it may affect the morphology of the fibrous structure of NETs,which may be related to the need of morphological changes when NETs catch bacteria?.effects of Ca2+ concentration and Ca2+channel blocker on albumin inhibition of NETs1 Effect of Ca2+concentration on the inhibition of NETs by albumin1.1 The influence of Ca2+and Ca2+free medium on the generation of NETs:large amount of NETs were generated in the standard PMN medium system containing Ca2+,while few in the standard PMN medium system without Ca2+.The results show that PMN can not generate NETs in Ca2+free environment,indicating that the generation of NETs is closely related to Ca2+.1.2 Effects of different Ca2+concentrations on NETs generation:the concentration of Ca2+in standard PMN medium system was 265 mg/dl.The production of NETs was significantly reduced in the Cat+free and low-concentration Ca2+(1.66 mg/dl?3.31 mg/dl)medium system,and NETs could be generated in large quantities in the PMN medium system with Ca2+concentration higher than 6.62 mg/dl.The results show that within a certain range of Ca2+concentration,the formation of NETs is positively correlated with the Ca2+concentration.2 Effect of Ca2+channel blocker on the formation of NETs2.1 The influence of verapamil on the generation of NETs:NETs can be generated in large quantities in the standard PMN medium system;In the standard medium system with a low concentration of HSA(1.5 mg/ml),there were more NETs generated.The addition of verapamil into the standard medium system reduced the generation of NETs,but there were still NETs generated.The results show that verapamil can slightly reduce the production of NETs,which may be related to calcium channels.2.2 Effects of 2-ammoxyl diphenyl ester boric acid(2-apb)on the inhibition of NETs by albumin:NETs can be generated in large quantities in standard PMN medium system;In the standard medium system with low concentration of HSA(1.5 mg/ml),more NETs were generated.The amount of NETs in the standard medium system containing low concentration HSA(1.5 mg/ml)was not significantly changed by adding 2-apb.No NETs were generated in the standard medium system containing HSA(4.5 mg/ml),and there was no significant change in the production of NETs in the standard medium system containing HSA(4.5 mg/ml)when 2-apb was added.The results showed that 2-apb had no significant effect on the formation of NETs,nor on the inhibition of NETs by albumin.?.Effects of plasma proteins on levels of total reactive oxygen species(ROS)and mitochondrial ROS in neutrophils1 Effect of BSA on intracellular total reactive oxygen species(ROS)level of PMN1.1 Determination of intracellular ROS level in PMN:PMN in standard PMN medium system generates a large amount of ROS(1183429±136026),while PMN in standard PMN medium system containing 10%BSA has a low intracellular ROS level(368608±12145).1.2 Effects of different proteins on intracellular ROS level of PMN:PMN in the medium system containing HDL and apoAl produced a large amount of ROS(HDL,1107035±179906).Apo Al,1178918±114703).The above results showed that BSA/HSA could significantly inhibit the generation of PMN intracellular ROS,while HDL and apoAl had no significant effect on PMN intracellular ROS.2 Effect of BSA on the level of mitochondrial active oxygen(mitoROS)in PMN cells2.1 Detection of intracellular mitoROS level in PMN:a large number of mitoROS were generated in PMN in the standard PMN medium system(20878±8270),and the intracellular ROS level of PMN in the standard PMN medium system containing 10%BSA was low(6977±139).2.2 Effects of different proteins on the intracellular mitoROS level of PMN:PMN culture medium system with BSA and HDL showed different degrees of decrease in the intracellular mitoROS level of PMN.2.3 Effects of mitochondrial respiratory chain disruptors on albumin inhibition of NETs formation2.3.1 Influence of mitochondrial respiratory chain disruptor on intracellular mitoROS level of PMN:anti-my cin A can significantly inhibit the intracellular mitoROS level of PMN,and rotenone can significantly increase the intracellular mitoROS level of PMN2.3.2 Effects of mitochondrial respiratory chain disruptors on the inhibitory effect of albumin on the formation of NETs:Anti-mycin A and rotenone had no significant effect on the formation of NETs,indicating that the changes in the intracellular mitoROS level of neutrophils had no significant relationship with the formation of NETs?.the effect of autophagy interfering agents on the inhibition of the formation of NETs by albumin1 The effect of autophagy agonists on the inhibition of NETs by albumin:the autophagy agonist RAPA was added to the standard PMN medium system without BSA and containing BSA(4.5 mg/ml).The results show that BSA can inhibit the generation of NETs,but RAPA can significantly increase the generation of NETs,and RAPA can eliminate the inhibitory effect of BSA on the generation of NETs.The results showed that the inhibition effect of BSA on the formation of NETs was related to the inhibition of autophagy,and enhanced autophagy could eliminate the inhibition effect of BSA on the formation of NETs2 Effect of autophagy inhibitors on the inhibition of NETs by albumin autophagy inhibitor 3-ma was added to the standard PMN medium system without BSA and containing BSA(4.5 mg/ml).The results showed that both 3-ma and BSA could significantly inhibit the formation of NETs in the above culture system,but the addition of 3-ma did not significantly change the inhibitory effect of BSA on the formation of NETs.The results showed again that the inhibition of NETs generation by BSA was related to the inhibition of autophagy,so the inhibition of autophagy did not affect the inhibition of NETs generation by BSA.3 effects of CaMKK inhibitor on NETs generation:NETs can be generated in large quantities in standard PMN medium system;The amount of NETs was significantly reduced in the PMN medium containing BSA(4 mg/ml).When the CaMKK inhibitor STO 609 was added to the standard PMN medium system,NETs could still be generated in large quantities.The results showed that STO609 had no significant effect on the formation of NETs,nor on the inhibition of NETs by albuminConclusion:1 Normal people have substances in plasma that inhibit the formation of NETs,among which albumin,a common and important protein,has a significant inhibitory effect on the formation of NETs,while Ig,-globulin,TRF and apo A1 have no significant effect on NETs2 LPS can induce a large number of NETs,and EC can change the morphology of NETs.In the presence of LPS and EC,albumin can effectively inhibit the formation of NETs3 The extracellular Ca2+concentration is closely related to the generation of NETs.Reducing the extracellular Ca2+concentration can effectively inhibit the generation of NETs,and the non-selective Ca2+channel inhibitor verapamil can reduce the generation of NETs.4 The mechanism by which albumin inhibits the formation of NETs may be related to the reduction of PMN intracellular ROS and mitoROS levels and the inhibition of autophagy,but not related to the CaMKK pathway.