| Backgrounds:Systemic inflammatory response syndrome(SIRS),acute lung and intestinal injury can lead to the deterioration of severe acute pancreatitis(SAP).From Traditional Chinese medicine theory” Lung and large intestine are appearance and inside of each other”,it can be determined that there is a potential relationship and homology in large intestine-lung injury of SAP.Polymorphonuclear Neutrophil(PMN)is one of key cells of inflammatory injury in the whole progression of SAP.When PMN is specifically stimulated,it will release a DNA fiber structure decorated with PMN plasma protease called neutrophil extracellular traps(NETosis/NETs).NETosis can damage the whole body tissue in the state of non-infectious inflammation,so NETosis may be the key to pancreas-intestine-lung injury in SAP.However,previous studies have confirmed that Dachengqi decoction can improve gastrointestinal motility and reduce acute lung injury,but its therapeutic mechanism is unknown and the target is unknown.NETosis may be the therapeutic target of Dachengqi decoction and its monomer emodin on SAP.Objections:(1)to isolate and purify PMN and determine whether SAP-related inflammatory substances can induce NETosis;(2)to determine whether NETosis is the key point for PMN to aggravate the disease of SAP and lead to organ failure;(3)to determine whether emodin can inhibit the formation of NETosis and inhibition mechanism;(4)to determine whether emodin can protect pancreas-colon-lung cell from injury caused by NETosis;(5)Part five:to determine whether emodin can inhibit the formation of NETosis in SAP rats and reduce the injury of pancreas,colon,and lung in SAP rats.Methods:(1)Part one: SPF male SD rats and C57BL/6 male mice were killed,and the bone marrow PMN extraction methods of rats and mice were used to isolate and purify PMN.Phorbol 12-tetradecanoate 13-acetate(PMA),lipopolysaccharide(LPS)and the mixture of recombinant rat tumor necrosis factor α(TNF-α)/ recombinant rat interleukin 6(IL-6)were used to stimulate PMN.The activity of isolated PMN was detected by living cell nuclear fluorescent dye Draq5 and dead nuclear green cyanine dye(SYTOX green),and the purity of isolated PMN and the structure of NETosis were detected by co-staining of 2-(4-aminophenyl)-6-indole formamide dihydrochloride(DAPI)and anti-neutrophil elastase antibody(anti-NE).The images were collected by confocal microscope and image analysis software.(2)Part two: After the SPF male SD rats were killed,the femur was removed,and the rat bone marrow PMN extraction method was used to isolate and purify PMN.The isolated PMN were treated with emodin when induced by PMA,LPS and TNF-α / IL-6 mixture,respectively.The experimental methods were co-incubated with PMN+ stimulus+emodin at 37 ℃.The experimental data collection methods are as follows:(1)collecting cell supernatant to detect MPO and citrullinated histone,(2)dynamically observing cell morphological changes by confocal scanning,(3)detecting phosphorylation of protease protein kinase C(PKC),ERK and NADPH oxidase(P47)and release of reactive oxygen series(ROS)to clarify the mechanism of emodin intervention on NETosis.4.The death mode change of emodin-induced PMN was determined by specific apoptotic antibody Cleavage-capase3 / specific pyroptotic antibody GSDMD and confocal dynamic monitoring of cell morphology.(3)Part three:Purification of type I alveolar epithelial cells(AECI)and the method of coreaction of NETosis and AECI: two SPF-grade newborn SD rats were killed after severing the neck,and the lung tissues of them were washed and shredded.The small pieces of lung tissues were digested with 0.1% collagenase I and0.02%DNA hydrolase(DNase)solution at 37 ℃ for 50 rpm / min 30 min,and the digestive enzyme was diluted,centrifuged and then cells were resuspended with complete culture medium.Ig G-coated and rat tail collagencoated 6-well were used to purify and remove miscellaneous cells,respectively,and then the purified cells were planted into rat tail collagen-coated culture plates.The complete medium was cultured for 72 hours after AEC was completely adhered to the wall,then the glutamate-acidified culture medium was used to remove fibroblasts for about 7 days.Co-reaction method: method1: after AECI was cultured and matured,AECI was digested with trypsin without EDTA(that is calcium and magnesium chelating agent),and then centrifuged and re-suspended into the petri dish of NETosis;method 2: after digesting NETosis with DNase,gently blowing it off the bottom plate,and then adding the suspension to the AECI culture hole;method 3: gently scraping off the NETosis with a cell scraper and adding it to the AECI culture hole together with the culture medium;(4)Part four: PMN was extracted and stimulated by PMA to produce NETosis and then incubated with pancreatic acinar cells,colonic epithelial cells(CEC)and AECI,which were divided into 5 groups:normal acinar / CEC/AECI cells,PMN+ acinar / CEC/AECI cells,NETs+acinar / CEC/AECI cells,NETs+DNase I+ acinar/ CEC/AECI cells,NETs+emodin + acinar / CEC/AECI cells.The experimental data collection methods are as follows: 1.the content of MPO and NF-kappa B in acinar / CEC/AECI cells,2.cell activity,3.E-cadherin detected by CEC/AECI,4.morphological changes of cells were dynamically observed by confocal microscope,5.NETs+AECI cell morphological change was observed in confocal living cell imaging system for 1 hour,and the incubation temperature was 36.8 ～37.3 ℃,6.aquaporin 5 and chemokine 5(CXCL5)were detected in ACEI.(5)Part five: male SD rats were randomly divided into sham operation group(n),severe acute pancreatitis model group(m)and emodin group(E),8 rats in each group.SAP model was established by retrograde injection of 3.5% sodium taurocholate into pancreaticobiliary tract in group M and E,and was replaced by normal saline in group n after laparotomy.Group E was given emodin(1mg/ ml)twice at 6h and 12 h after modeling,and the sham operation group and model group were given the same amount of normal saline.Blood samples,pancreas,colon and lung tissues were collected 24 hours after modeling.The contents of Amy and LPS in serum were detected by automatic biochemical analyzer;HE staining was used to evaluate the pathological injury of pancreas,colon and lung;The expression of MPO,Cit-Histone and E-cardherin in pancreas,colon and lung were detected by WB.Results:Part one: 1.PMN activity: the proportion of living PMN cells extracted from rat bone marrow is 86.3% The proportion of living PMN cells extracted from mouse bone marrow is82.1%.2.The purity of extracted rat bone marrow PMN is about 95.8%,and the purity of mouse bone marrow PMN is 87.9%.3.After PMN was stimulated by PMA for 4 hours,fixed staining and observed under confocal microscope,it was found that the nuclear lobular of most cells disappeared,the nuclear membrane was lysis,and cell membrane was broken.DNA fiber and cytoplasm melted together,and some cells had typical DNA(blue DAPI)filamentous structure overlapping with anti-NE(rose red).4.PMN was stimulated by high concentration of LPS(25ug/ml)for 4 hours,fixed staining,and observed under confocal microscope,it was found that the changes of most cells were the same as those stimulated by PMA,but the difference was that the filamentous structure of DNA(blue DAPI)overlapped with anti-NE(rose red)seems like tadpole.5.PMN was stimulated with TNF-α / IL-6 mixture for 3 hours,and NETosis changes occurred.After fixed staining,the morphological changes of cells were observed under confocal microscope,and it was found that the morphological changes of cells were consistent with those stimulated by PMA.Part two: 1.Emodin interfered with the content of MPO release induced by PMA in NETosis groups(high,middle and low concentration)was significantly lower than that in PMA stimulation group,and the inhibitory effect of emodin on MPO release was related to the concentration.Emodin can also effectively inhibit histone citrullination induced by PMA.Dynamic monitoring of cell morphology by confocal microscope showed that the morphology of emodin-interfered NETosis was completely different from that produced by simple PMA stimulation.PMN did not show nuclear lobule dissolution and nuclear membrane rupture,but the cell membrane swelled repeatedly after cultured 30 min,the cell membrane was intact,the nucleus was biased to one side and stained green,and the cell morphology was fixed after about 2 hours.2.Emodin significantly inhibited the release of cytoplasmic protein MPO and nuclear histone citrullination in NETosis group induced by LPS,but there was no significant difference between high and low concentrations of emodin.3.Emodin inhibited with the release of cytoplasmic protein MPO and nuclear histone citrullination in TNF-α / IL-6 complex induced NETosis.4.Explore the mechanism of emodin intervention on NETosis.Results: 1.the amount of ROS in normal PMN cells was the most,and the medium containing PMA also produced a large amount of ROS,when PMN was stimulated for half an hour,while both PMN intervened by emodin and pretreated with NADPH oxidase inhibitor DPI produced almost no ROS after half an hour of stimulation in the medium containing PMA.(2)emodin alone had no significant inhibitory effect on the activation of PKC gamma site,but had a significant inhibitory effect on the activation of betta site.Pretreatment with PKC inhibitor G?6976,emodin intervention could completely block the activation of PKC gamma site and effectively inhibit the activation of betta site.(3)emodin could inhibit the activation of ERK.(4)emodin alone enhanced activation of p47,but the simultaneous use of G?6976could reduce the activation of emodin on p47.5.The effect of emodin on the mode of cell death in NETosis: i.confocal fluorescence observation showed that the apoptosis marker Annexin V began to be stained gradually after 5min PMN was co-cultured with PMA and emodin,and the PMN stimulated by the complex of LPS,TNF-α and IL-6 interfered with emodin showed the same change.ii.Detection of death mode marker protein: the expression of GSDMD,a marker of pyroptosis,and Cleavage-capase3,a marker of apoptosis,was significantly increased in emodin intervention group.Part three: purity of AECI obtained from the improved method and cultured in selective acidification medium was above 99%.AEC cells can be completely transformed into AECI on the 10 th day of culture.The growth cycle of AECI is longer than that of normal medium.Method 3was used in the co-incubation reaction of NETosis and AECI observed under microscope using.The results showed that after the co-cultured,the gap between AECI cells enlarged,part of the cell membrane contracted like a cord,and some cells even detached from the bottom plate of the petri dish;Use method 1 for specimen collection and detection.Part four:1.The results of pancreatic acinar cell experiment: there was no expression of MPO in normal acinar(NY)and almost no expression of inflammatory phenotypic protein p P65;in PMN+ acinar(PY)group,a small amount of MPO entered acinar cells and increase of NF-κ B;there were a large number of MPO and NF-κ B expression in acinar cells in NETs+ acinar(APY)group,which were higher than those in the other three groups;MPO and NF-κ B in acinar cells in NETs+DNase I+ acinar(APDY)group were significantly lower than those in APY group.The contents of MPO and NF-κB in(APEY)acinar cells in NETs+ emodin + acinar group were significantly lower than those in APY group.2.Colonic epithelial cell(CEC)test: there was a small amount of NF-κ B expression in normal CEC(NC),there was no significant difference in NF-κ B expression between PMN+CEC(PC)group and NC group,while a large amount of NF-κ B expression in NETs+CEC(APC)group and was significantly higher than that in the other three groups,and NF-κ B expression in NETs+DNase I+CEC(APDC)group was significantly lower than that in APC group.The content of NF-κ B in NETs+emodin + CEC(APEC)group was significantly lower than that in APC group and APDC group.3.Results of AECI cell experiment: 1.AQP5 results: there was a small amount of expression of AQP5 in normal AECI(NA),and no significant difference in AQP5 expression between PMN+AECI(PA)group and NA group.AQP5 expression in NETs+AECI(APA)group increased,which was significantly higher than that in NA group.There was no significant change in AQP5 in NETs+DNase I+AECI(APDA)group compared with APA group,but the content of AQP5 in NETs+ emodin + AECI(APEA)group was significantly higher than that in APA group and APDA group.2.CXCL5results:there was no significant difference in the expression of CXCL5 between NA and PA groups;the expression of CXCL5 in APA group was significantly lower than that in NA group;the expression of CXCL5 in APDA group was significantly higher than that in APA group;the content of CXCL5 in APEA group was significantly higher than that in APA group and APDA group.3.The morphological changes of AECI in each group: the intercellular space of PA group was larger than that of normal AECI,but no cell membrane contraction showed strip shape and cell exfoliation,while in APA group,a large number of cell membrane contracted and even exfoliated.In APDA group,the contraction edge of cell membrane showed zigzag change,a small amount of cell membrane showed strip shape change,and no cell exfoliation was found.In APEA group,the intercellular space enlarged and the cell membrane contracted,but there was no cord change and cell exfoliation.Part five:The pathological changes of pancreas,colon and lung,such as edema,inflammatory cell infiltration and necrosis,were more severe than those in sham operation group(M=22.38±3.74 vs N=5.50±2.38,P<0.05).Emodin intervention could significantly reduce the above markers(M=22.38±3.74 vs E=12.88±2.23,P<0.05);2.MPO expression in pancreas,colon and lung of SAP rats was significantly higher than that in sham operation group and Emodin intervention group(Pancreas : M= 2.35±1.16 vs N=0.34±0.03,M=2.35±1.16 vs E=1.65±0.98,P<0.05;Colon:M=2.24±0.78 vs N=0.33±0.1,M=2.24±0.78 vs E=1.05±0.56,P<0.05;Lung:M=1.83±0.54 vs N=0.45±0.24,M=1.83±0.54 vs E=1.39±0.73,P<0.05);3.The content of CitH expression in colon of SAP rats was significantly higher than that of sham operation group and Emodin intervention group(M= 1.85±0.64 vs N=0.51±0.03,M= 1.85±0.64 vs E= 1.03±0.18,P<0.05);4.The expression of E-cadherin in colon and lung tissue of SAP rats was significantly lower than that of sham operation group(Lung:M= 0.18±0.04 vs N= 1.02±0.16,P<0.05;Colon:M= 0.41±0.26 vs N= 0.82±0.1,P<0.05).Emodin could significantly increase the expression of E-cadherin in lung tissue(Lung:E=0.68±0.21 vs M=0.18±0.04,P<0.05).Conlusions:1.According to the characteristics of neutrophils in mice,neutrophils with high purity were isolated from bone marrow,and neutrophils were successfully induced to form NETosis,by using PMA and SAP-related inflammatory mediators(LPS,the mixture of TNF-α and IL-6).It was confirmed that NETosis was involved in the pathophysiological process of SAP.2.Emodin was used to interfere with the induction of NETosis induced by PMA.It was found that emodin could inhibit NETosis induced by PMA,LPS,and the mixture of TNF-α and IL-6,and the inhibitory effect of emodin on NETosis formation was concentration-dependent.3.Through the dynamic monitoring of cell changes by using living cell culture and confocal delay photography system,the mechanism of emodin interfering with NETosis formation was explored,and it was found that emodin inhibited NETosis by inducing neutrophils to transform into another death mode with less inflammatory damage to the body.4.PKC inhibitor G?6976 was used to interact with emodin,it was found that G?6976 and emodin had synergistic effect,which could enhance the inhibitory effect of emodin on NETosis.It can be inferred that there may be a synergistic relationship among the monomers of Da-cheng-qi decoction.A selective medium for alveolar epithelial cells was developed,and an improved scheme for isolation,purification and culture of alveolar epithelial cells was established,which made it easier to obtain alveolar epithelial cells and higher cell activity.5.By co-incubating NETosis with pancreatic acinar cells,AECI and CEC,it was found that NETosis could damage pancreatic acinar cells,AECI and CEC,destroy the integrity of AECI,cause disturbance of water metabolism of AECI and decrease AECI ability of chemotactic immune cells.It is confirmed that NETosis can indeed cause pancreatic-intestinal-lung cell injury in SAP.The use of DNase I,an intervention drug for NETosis,can reduce the damage of three kinds of cells,so the target role of NETosis in pancreas-intestine-lung injury can be determined.Emodin can reduce the inflammatory injury of these cells by inhibiting the entry of MPO into cells,inhibit the damage of NETosis to the integrity of alveolar epithelial cells,and increase the ability of water metabolism and chemotaxis of immune cells.6.SAP model rats had severe inflammatory injury in pancreas,colon and lung tissue caused by inflammatory cell infiltration and release of a large amount of MPO;Emodin can reduce the pancreas,intestine,and lung injury of SAP by reducing the infiltration of inflammatory cells,inhibiting the production of Cit-H and reducing the formation of NETosis. |
PDF Full Text Request