| Backgroud and ObjectiveThe nechanism of acute respiratory distress syndrome(ARDS)is complicated.As research progresses,people gradually realized that apoptosis of lung tissue plays a key role in the progression and aggravation of ARDS.In recent years,High mobility group protein B1(HMGB1)binds to receptor for advanced glycation end-products(RAGE)to form the HMGB1/RAGE axis,which plays an important role in the inflammation,oxidative stress and apoptosis.However,the pro-apoptosis mechanisim of the axis in the pathogenesis of ARDS remains unclear.In many pathological settings,persistent stress states cause endoplasmic reticulum stress,(ERs)and unfolded protein response(UPR).Under these circumstances,binding immunoglobulin protein(BiP)dissociates from Protein kinase R-like ER kinase(PERK),leading to its phosphorylation.Activated PERK phosphorylates eukaryotic translation-initiation factor 2?(eIF2?),which shuts down cap-dependent translation and facilitates the production of activating transcription factor 4(ATF4).ATF4 is a transcription factor,induces another transcription factor,C/EBP homologous protein(CHOP),which involved in the induction of apoptosis.Therefore,we aimed to explore the mechanisim of the HMGB1-RAGE axis induces apoptosis via triggering ER stress in the pathogenesis of ARDS.Section 1(In vivo)Methods1.A total of 152 male C57BL/6 mice were randomly divided into eight experimental groups(18 mice per group):The CLP group;the Sham group;the HMGB1 inhibitor groups(CLP+GA group and Sham+GA group);the RAGE inhibitor groups(CLP+FPS-ZM1 group and Sham+FPS-ZM1 group);the ERs inhibitor groups(CLP+4-PBA group and Sham+4-PBA group).2.The survivor rate in each group was observed at 96h after CLP operation.3.Histology of lung was observed in light microscope and the lung injury scores were determined by a semi-quantitative measure;the wet to dry ratio4(W/D)was measured in the lung tissue and the PaO2/FiO2 were calculated in the artery blood gas of mice in different groups;to count the classification and count the immune cell in the bronchoalveolar lavage fluid(BALF)in different groups;to detect the tumor necrosis factor-a(TNF-a)and interleukin 6(IL-6)levels in BALF by ELISA;4.To detect the protein levels of HMGB1?RAGE?Bip?IRE la?PERK?p-eIF2??eIF2? ATF4?CHOP and Caspase-3 in the lung tissue by Western blot;5.To calculate the apoptosis rate of lung tissue by TUNEL.Result1.Mice in CLP group developed severe lung injury.A more severe lung injury score,a higher W/D ratio and lower PaO2/FiO2 ratio in CLPs compared to that observed in shams.Then the GA?FPS-ZM1 and 4-PBA injection resulted in a significant attenuation of these pathological changes,improvement of lung injury score,W/D ratio and PaO2/FiO2 ratio.2.The CLP operation in mice resulted in immune cell significant infiltration compared to that observed in shams(P<0.05).Then,the neutrophils rate in BALF of the CLP+4-PBA group were significantly reduced by 4-PBA treatment(P<0.05)and the lymphocyte rate in BALF of the CLP+GA and CLP+4-PBA group were significantly reduced by GA and 4-PBA treatment(P<0.05).The CLP operation in mice resulted in inflammatory cytokines into the blood compared to that observed in Shams(P<0.05).Then,the IL-6 levels of the CLP+FPS-ZM1 and CLP+4-PBA group were notably lower in blood compared with the CLP group(P<0.05)and the TNF-a levels of the three groups were notably lower in blood compared with the CLP group(P<0.05).3.The HMGB1,RAGE,IREla,Bip,PERK,p-eIF2a,eIF2a,ATF4,CHOP and Caspase-3 protein expression in the CLP group was markedly greater than that in shams(P<0.05);and then,compared with the CLP group,the GA,FPS-ZMland 4-PBA treatment resulted in a significant decrease of the these protein expression(P<0.05).4.Mice in CLP group had significantly more apoptotic rates of lung tissue than sham mice(P<0.05).And then,compared with the CLP group,the GA,FPS-ZMland 4-PBA treatment resulted in a significant decrease of the apoptotic rates(P<0.05).5.The mice in the CLP groups revealed the acute respiratory distress symdrome.Although our study showed that a notably high mortality of 80%in the CLP group compared with the shams(P<0.05)?which was reduced to 70-60%by the GA,FPS-ZMland 4-PBA treatment.Section 2(In vitro)Method1.The A549 cell viability in different HMBG1 concentrations groups(0.1,1?10 and 100 ug/mL)were examined by CCK-8 assay at 12,24,and 48h.The apoptotic rate was detected by flow cytometer and the protein levels of PERX/eIF2a/ATF4 signalling by western blot at 24h in different HMBG1 concentrations groups(0.1,1 and lOug/mL).2.To determine the best multiplicity of infection(MOI)value of shRNA-PERK and shRNA-Negative lentivirus transfected A549 cell.And then,the PERK protein in A549 cell was detected by western blot after transfection with lentivirus with best MOI.3.The A549 cell was transfected by shRNA-PERK and shRNA-Negative lentivirus with best MOI and then was stimulated by HMBG1(lug/mL)for 24h.The A459 cell was divided into six groups:the Control group;the HMGB1 group;the lentivirus groups(shRNA-PERK group and shRNA-Negative group);the HMGB1+lentivirus groups(HMGB1+shRNA-PERK group and HMGBl+shRNA-Negative group).We detected the protein of PERK/eIF2a/ATF4 signaling and apoptotic biomarker by western blot and calculated the apoptotic rate of A549 cell by TUNEL.4.The A549 cell was pre-incubated by RAGE inhibitor(anti-RAGE antibody,20ug/mL)and negative antibody IgG2b(60ug/mL)and then was stimulated by HMBG1(lug/mL)for 24h.The A459 cell was divided into six groups:the Control group;the HMGB1 group;the anti-RAGE group;the IgG2b group;the HMGB1+anti-RAGE group;the HMGBl+IgG2b group.We detected the protein of PERX/eIF2a/ATF4 signaling and apoptotic biomarker by western blot and calculated the apoptotic rate of A549 cell by TUNEL.Result1.Compared with control group,the cells viability significantly decreased after stimulation with different HMBG1 concentrations at 24h and 48h(P<0.05).Moreover,the apoptotic rate significantly increased gradually with the concentration of HMGB 1 at 24h compared with control group(P<0.05).2.Compared with control group,the RAGE,Bip and p-eIF2a protein expression increased gradually with the concentration of HMGB 1 for 24h.However,PERK and ATF4 protein expression decreased after 10)j.g/mL HMBG1 stimulation for 24h.3.The best MOI value is 10 and the transfection efficiency of shRNA-PERK and shRNA-Negative lentivirus is 77.8%and 72.1%,respectively.Compared with control group,the PERX protein expression in A549 cell significantly decreased after transfection with shRNA-PERK(P<0.05).4.Compared with control group,the PERK,p-eIF2a/eIF2a,ATF4,CHOP and Caspase-3 proteins expression significantly increased in HMBG1 group(P<0.05);and then,compared with HMGB1 group,the shRNA-PERK transfection resulted in a significant decrease of these protein expression in the(HMGB1+shRNA-PERK)group(P<0.05).The apoptotic rate of A549 cell significantly increased in HMGB1 groups compared with that in control group(P<0.05);and then,compared with HMGB1 group,the shRNA-PERK transfection resulted in a significant decrease of apoptotic rate in the(HMGB1+shRNA-PERK)group(P<0.05)5.Compared with control group,the RAGE,Bip,PERX,p-eIF2a/eIF2a,ATF4,CHOP and Caspase-3 proteins expression significantly increased in HMBG1 group(P<0.05);and then,compared with HMGB1 group,the RAGE inhibitor pre-incubation resulted in a significant decrease of these protein expression in(HMGB1+anti-RAGE)group(P<0.05).The apoptotic rate of A549 cell significantly increased in HMBG1 group compared with that in control group(P<0.05);and then,compared with HMGB1 group,the RAGE inhibitor pre-incubation resulted in a significant decrease of apoptotic rate in the(HMGB1+anti-RAGE)group(P<0.05)Conclusion1.HMGB1,RAGE expression and ERs activation play a pro-apoptosis role in the process of ARDS caused by CLP2.HMGB1 activates ERs via RAGE receptor to induce apoptosis.The main mechanism is the activation of PERKX/eIF2a/ATF4 signalling pathway in ERs. |
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