| Objective:In this study,lipopolysaccharide（LPS）was used to induce acute lung injury（ALI）in mice,and the lung tissue of model mice was analyzed for nuclear factor kappa B（NF-κB）and endoplasmic reticulum stress（ERS）,and its branch sensor-activating transcription factor 6（ATF6）,apoptosis,necroptosis signal,and changes in pulmonary microvascular endothelial cell marker proteins and their regulatory relationship.Aiming to explore the lung microvascular endothelial cell NF-κB,ATF6,endoplasmic reticulum stress,apoptosis and necroptosis has regulation axis,and to explore its role in the pathogenesis of ALI,which provides a reference for clinical treatment.Materials and Methods:1.LPS-induced acute lung injury in miceMale BALB/c mice are grouped according to the random number table method,Different concentrations of LPS（2mg/kg,5mg/kg,10mg/kg,30mg/kg）were injected through the tail vein.Acting for 12h,24h,48h,72h and 96h,respectively.With the same dose of PBS buffer as a control,to explore the optimal concentration of LPS for establishing acute lung injury model in mice.Survival rate of mice in each group was calculated,and pathological changes of lung tissue of each group were observed.Lung injury score,wet to dry weight ratio（W/D）,bronchoalveolar lavage fluid（BALF）cell classification and total protein content were calculaded,arterial blood oxygenation index（Pa O2/Fi O2）were determined to comprehensicely evaluated the severity of lung injury.Detected the NF-κB signal pathway,endoplasmic reticulum stress,apoptosis,necropotosis and the expression of marker-related proteins in vascular endothelial cells in the lung tissue of each group of mice by Western blot（WB）.Terminal deoxynucleotidyl transferase-mediated DUTP-biotin Nick end labeling assay（TUNEL）was used to detect cell apoptosis rate of bronchial and lung tissue cells.Respectively from the system level,cell level,the organelles and molecular biological level analysis of LPS-induced mice ALI,and pulmonary microvascular endothelial cells apoptosis,necroptosis,ERS,the change of the NF-κB signaling pathway.2.4-PBA protected lung tissue endoplasmic reticulum stress from acute lung injuryIntraperitoneal injection of ERS inhibitor 4-phenylbutyric acid（4-PBA,100mg/kg）pre-intervention to reduce ERS.48 mice were randomly divided into 4groups（n=12）,solvent control group（PBS group）,model group（LPS group）,4-PBA intervention group（4-PBA+LPS group）and 4-PBA positive control group（4-PBA group）by random number table method.Samples were taken at 24h and 48h time points of LPS intervention.Comprehensive assessment of lung injury changes after 4-PBA intervention by detecting relevant indicators.NF-κB pathway,ERS,apoptosis and programmed cell death,and expression of related proteins in pulmonary microvascular endothelial cells were detected by WB.Cell apoptosis rate was detected by TUNEL assay.The expression changes of proteins in each pathway after 4-PBA intervention were analyzed.3.Short hairpin RNA interfered with the expression of p65 subunit（Rela）gene of NF-κB signaling,and explored the effect of Rela on LPS-induced ATF6expression in mouse lung tissue,and its effected on endoplasmic reticulum stress,apoptosis and necroptosis.Recombinant adeno-associated virus（r AAV）9 was transfected with Rela sh RNA for 6 weeks.Rela gene expression was knocked down in the lung of mice,and then induced by LPS for 24h.48 mice were randomly divided into 4 groups:the control group（Control sh RNA+PBS）,Rela sh RNA transfection group（Rela sh RNA+PBS）,transfection control+LPS group（Control sh RNA+LPS）,Rela sh RNA transfection+LPS group（Rela sh RNA+LPS）.WB and q RT-PCR were used to detect transfection efficacy.Survival rate of mice in each group was calculated,pathological changes and related indexes of lung were observed,cell classification and total protein in BALF were determined,and changes of lung injury after Rela gene knockdown were comprehensively evaluated.RELA pathway,ERS and its mediated apoptosis and necroptosis in lung tissues were detected by WB,as well as the expression of vascular endothelial related proteins.Cell apoptosis rate was detected by TUNEL assay,the expression and distribution of phophorylated mixed lineage kinase domain-like pseudokinase（p-MLKL）in bronchial and pulmonary tissues were detected by immunohistochemistry.The effected of knocking down Rela gene in lung on each pathway protein and apoptosis and necroptosis of pulmonary microvascular endothelial cells were comprehensively analyzed.4.Knock down the expression of Atf6 gene in the lung,exploring the effect of ATF6 on the RELA signal,ERS,apoptosis and necroptosis in lung tissue.The expression of Atf6 gene in the lungs of mice was knocked down by the same method.To confirm the success of the knockdown of Atf6 gene.The changes of lung injury and the expression of related pathway protein were observed after knockdown,and the effects of ATF6 knockdown on lung injury,NF-κB pathway,ERS mediated apoptosis and necroptosis were analyzed.Results:1.There was no difference in 96h cumulative mortality between mice injected with LPS 2mg/kg through tail vein and those without injection.When the concentration of LPS was 5mg/kg,dyspnea,reduced activity and rough hair after injection,and the symptoms were obvious 24h-48h after injection,but there was no death.Mice injected with 10mg/kg LPS through the tail vein had significantly worse dyspnea,then some of them died within 48h.When the LPS concentration was increased to 30mg/kg,all mice died within 12h.Pathological results showed that LPS 2mg/kg had no obvious lung injury.After treatment with LPS 5mg/kg for 24h,obvious lung tissue damage was observed,which was manifested as inflammatory cell infiltration,perivascular edema and alveolar wall thickening,and lung injury was aggravated with time prolongation.Lung injury score increased,W/D ratio increased（P<0.05）,and Pa O2/Fi O2 in arterial blood decreased in a LPS dose-dependent way（P<0.05）.Biochemical results:the cell classification propotion and total protein content in BALF were increased.Phosphorylation levels of RELA and ERS related proteins and apoptosis related proteins caspase-12,cleaved caspase-3 and necroptosis associated protein MLKL phosphorylation level increased（P<0.05）,while the levels of endothelial marker molecules associtated proteins VE-cadherin and E-selectin were significantly decreased（P<0.05）.The result was the most significant change from 24h to 48h.Therefore,the LPS concentration of 5mg/kg was selected as the optimal intervention concentration for subsequent experiments.2.The intervention of 4-PBA alone had no noticeable effect on mice.Preintervention of 4-PBA can alleviated the degree of lung tissue destruction in mice with LPS-induced acute lung injury.Lung injury score and W/D ratio were significantly decreased（P<0.05）,oxygenation index was improved,the proportion of inflammatory cells and total protein level in BALF were decreased（P<0.05）.The phosphorylation levels of RELA and the protein expression of ERS,apoptosis and necroptosis related proteins in lung tissues were significantly decreased,and the expression levels of endothelial marker proteins were partially restored（P<0.05）.There was no significant difference in survival rate among groups（P>0.05）.3.After Rela sh RNA transfection in mice,the expression of Rela gene in the lungs of mice was significantly decreased in both protein and gene levels by WB and q RT-PCR（P<0.001）.After knocking down Rela gene,lung tissue injury was obvious,and lung injury score was increased.and W/D ratio increased,aerterial oxygenation index decreased.The total number of cells,neutrophils/lymphocytes ratio and total protein content in BALF were significantly increased（P<0.05）.However,there was no significant difference in survival rate among all groups（P>0.05）.At the same time,the expression of ATF6 protein,GRP78,GRP94 and endothelial cell marker proteins were down-regulated by knocking down Rela gene（P<0.05）.The levels of apoptosis and necroptosis associated proteins were increased,and ERS was aggravated,and the expression levels of endothelial marker protein was decreased（P<0.05）.4.After transfected with Atf6 sh RNA into lung tissue of mice,the expression of ATF6 at protein and gene levels was decreased significantly（P<0.001）.The pathological injury of lung tissue was aggravated,and the lung injury score was increased（P<0.05）.Furthermore,the changes of oxygenation index,W/D ratio,cell proportion and total protein content in BALF,were consistent with the results of Rela gene knockdown.WB assay showed that after Atf6 sh RNA transfection,LPS-induced protein phosphorylation levels of RELA was significantly increased,the expression of GRP78,GRP94 protein were decreased,and the expression of apoptosis and necroptosis related proteins were significantly increased（P<0.05）.The expression level of endothelial marker protein was decreased（P<0.05）.Conclusion:1.Acute lung injury model of mice was successfully established by intravenous injection of LPS at a concentration of 5mg/kg,which was accompanied by NF-κB signal activation,ERS,apoptosis and necroptosis in lung tissue,and the decrease of pulmonary vascular endothelial cells.2.4-PBA partially alleviated ERS and improved LPS-induced lung tissue cell apoptosis,necroptosis,partially restored the number of pulmonary microvascular endothelial cells,and improved acute lung injury.3.The expression of Rela gene in the lung was knocked down,and the expression of ATF6 protein in the lung of mice induced by LPS was down-regulated.by increasing ERS,cell apoptosis and necroptosis,the number of pulmonary microvascular endothelial cells was reduced,and ALI was aggravated.4.Knockdown the expression of Atf6 gene in the lung could decrease GRP78,GRP94 protein levels,and up-regulates the RELA pathway protein,aggravates ERS,programmed cell death of lung tissue,the number of pulmonary microvascular endothelial cell was reduced,and ALI was aggravated.5.NF-κB,ATF6 signal,ERS,apoptosis and necroptosis of pulmonary microvascular endothelial cells may have axial regulationship and influence the pathogenesis of ALI.Objective: The role of NF-κB/ATF6-endoplasmic reticulum stress-apoptosis and necroptosis signal axis in human pulmonary microvascular endothelial cell（HPMVEC）was verified in vitro experiments.Materials and Methods: 1.TG interfered with HPMVECs to establish ERS model and detect related indicators.Culture HPMVECs in vitro,the concentration of TG on HPMVECs（0.25,0.5,1,and 2μmol/L）and time effects（12,24,36 and 48h）,CCK-8 assay detected cell proliferation activity,flow cytometry to detect cell apoptosis rate.The optimal concentration and action time of ERS in TG-induced HPMVECs were selected.WB detected the phosphorylation level of RELA and the expression of ERS pathway,apoptosis and necroptosis related protein.To evaluated the activation of various HPMVECs pathway proteins induced by TG.2.4-PBA inhibits ERS,to explore the changes of apoptosis and necroptosis of HPMVECs induced by TGThe HPMVECs were pre-intervention with 4-PBA 2mmol/L for 2h to inhibit ERS.Dimethyl sulfoxide（DMSO）was used as solvent control.The model group was treated with TG（0.5μmol/L）to induce HPMVECs for 24 h and 48 h,and divided into 4 groups,the control group（DMSO）,model group（TG）,4-PBA intervention group（4-PBA+TG）,4-PBA intervention control group（4-PBA+DMSO）.The expression of related pathway proteins was detected by WB,while flow cytometry examed the apoptosis rate.To evaluate the effected of 4-PBA intervention on HPMVECs.3.To investigate the effect of RELA on the expression of ATF6 in HPMVECs and its role in ERS,apoptosis and necroptosis by interfering with RELA gene expression.HPMVECs were transfected with RELA sh RNA or Control sh RNA by Lipofectamine 3000 plasmid.The optimal transfection rate was determined by green fluorescence,RELA knockdown was confirmed by WB and q RT-PCR.HPMVECs were induced by TG（0.5μmol/L）for 24 h,and were divided into 4 groups after transfection of RELA: Transfection control group（Control sh RNA + DMSO）,RELA sh RNA transfection group（RELA sh RNA + DMSO）,RELA sh RNA transfection +TG group（RELA sh RNA +TG）,Transfection control +TG group（Control sh RNA + TG）.WB detected the expression of ERS-related protein,apoptosis and necroptosis related protein.The apoptosis rate was detected by flow cytometry.To observe the effect of knocking down RELA gene on the expression of ATF6 and its role in ERS,apoptosis and necroptosis.4.By knocking down the expression of ATF6 gene,explore the effect of ATF6 on the RELA signal,ERS,apoptosis and necroptosis of HPMVECs.HPMVECs were transfected with ATF6 sh RNA or Control sh RNA,and verified the successful knockdown of ATF6.HPMVECs were induced by TG for 24 h.Cell were grouped: Transfection control group（Control sh RNA + DMSO）,ATF6 sh RNA transfection group（ATF6 sh RNA + DMSO）,ATF6 sh RNA transfection + TG group（ATF6 sh RNA + TG）,Transfection control +TG group（Control sh RNA + TG）.The RELA pathway activation,ERS,apoptosis,and necroptosis related proteins of the experimental cells were detected by WB after ATF6 knockdown.Cell apoptosis rate was detected by flow cytometry.Results: 1.In the first generation,HPMVECs grew like a cobblestone adherent to the wall,and the cell morphology changed gradually with the increase of passage times.The cells were treated with 0.25μmol/L TG for 48 h,the cells were in good condition under the microscope,and the difference between the mother cells and the control group could not be distinguished by the naked eye.The TG concentration is 0.5μmol/L,observed under a microscope,the peripheral morphology of the cells was slightly irregular at 24 h,and a few cells showed signs of shedding.When TG concentration was more than 1μmol/L,the phenomenon of cell shedding and death after 24 h was further aggravated.When TG concentration was less than 0.5μmol/L,and the 48 h cell proliferation curve did not move down significantly.When the TG concentration was more than 0.5μmol/L,the cell proliferation activity was concentration-dependent and time-dependent to an inevitable extent decline.RELA and ERS related proteins（ATF6,GRP78,GRP94）,apoptosis-related proteins（cleaved caspase-3）and necroptosis-related proteins（p-MLKL,MLKL）levels increased significantly（P < 0.05）.Therefore,0.5μmol/L TG intervention cells were selected in the following experiment to complete the subsequent experiment.2.After 4-PBA treatment,the cell proliferation activity was partially restored after down-regulation induced by TG（P < 0.05）,Decreased the expression levels of RELA,ERS-related proteins,apoptotic proteins,and the protein expression levels of necroptotic proteins（P < 0.05）,the damage of HPMVECs was reduced.3.The optimal transfection rate of HPMVECs transfected with RELA sh RNA was greater than 70%.Compared with the control group,the expression of sh RNA knockdown RELA at protein level and gene level was significantly decreased（P < 0.001）.WB assay showed that RELA sh RNA transfection down-regulated TG-induced ATF6,GRP78,GRP94 protein expression,and ERS was increaded.But cleaved caspase-3,MLKL,p-MLKL levels were significantly increased,and the apoptosis rate was increased by flow cytometry,and the apoptosis,necroptosis of HPMVECs were increased（P < 0.05）.4.The optimal transfection rate of ATF6 sh RNA transfected with HPMVECs was higher than 70%.Knockdown of ATF6 gene significantly increased RELA protein expression in TG-induced HPMVECs.The protein expression of GRP78,GRP94 decreased,and the protein expression of cleaved caspase-3 and p-MLKL were significantly increased（P < 0.05）.Flow cytometry showed that the apoptosis rate was increased（P < 0.05）.The damage of HPMVECs was aggravated.Conclusion: 1.The ERS cell model of HPMVECs was successfully established by TG 0.5μmol/L.2.4-PBA reduced ERS,protects HPMVECs and alleviated cell apoptosis and necroptosis induced by TG.3.Knockdown the RELA gene expression,down-regulated TG-induced ATF6 protein expression in HPMVECs,and ERS,cell apoptosis and necroptosis were aggravated.4.Knockdown the ATF6 gene expression,down-regulated TG-induced GRP78,GRP94 protein and up-regulated RELA pathway protein expression in HPMVECs,and ERS,cell apoptosis and necroptosis were aggravated.5.NF-κB signal attenuated ERS-mediated apoptosis and necroptosis by regulating ATF6. |
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