| ObjectiveLung ischemia-reperfusion injury often occurs in patients with sudden cardiac arrest（CA）-cardiopulmonary resuscitation（CPR）-return of spontaneous circulation（ROSC）.In recent years,it has been found that acute lung injury（ALI）in patients with ROSC is an independent risk factor for the prognosis of out-of-hospital cardiac arrest（OHCA）.However,there is no study reporting the relationship between ALI after ROSC and death during hospitalization in patients with in-hospital cardiac arrest（IHCA）.Inflammatory infiltration and diffuse pulmonary edema are the main pathophysiological features of acute lung injury.Pulmonary microvascular endothelial cells（PMVECs）are the important barrier to prevent water molecules in the vascular,inflammatory mediators and other harmful substances from entering the pulmonary septum and alveolar cavity.When PMVECs are influenced by ischemia,hypoxia and stimulation of pro-inflammatory mediators,the expression of adhesion molecules including intercellular adhesion molecule-1（ICAM-1）and vascular adhesion molecule-1（VCAM-1）,and the expression of tight junction（TJ）protein including claudin-5（CLDN5）and occludin（OCLN）in PMVECs decrease.These factors increase the permeability of the gas-blood barrier,enhance the inflammatory response and then aggravate acute lung injury.At present,the molecular mechanism of lung ischemia-reperfusion injury after ROSC is not clear,and some studies have confirmed that protein kinase A（PKA）plays an anti-injury role in ischemia-reperfusion injury of endothelial cells.In addition,a large number of studies have confirmed signal transduction pathways including PKA-Nuclear factor kappa-B（NF-κB）and PKA-Nuclear factor erythroid 2-related factor 2（Nrf2）play an important role in the injury of vascular endothelial cells in different research models.Therefore,we argue that PKA-Nrf2/NF-κB could be involved in lung ischemia-reperfusion injury after ROSC.Adiponectin is an active protein secreted mainly by adipocytes,which plays an important role in biological changes by alleviating various cellular inflammatory responses and inhibiting inflammatory factors.Previous studies have confirmed that adiponectin could alleviate the inflammatory injury of vascular endothelial cells by activating PKA,and inhibiting the activation of NF-κB and its downstream signal pathway.In addition,we have proved that adiponectin could regulate the inflammatory injury of vascular endothelial cells through PKA-Nrf2 signal pathway.However,the mechanism of whether it can alleviate the injury of pulmonary microvascular endothelial cells after ROSC by regulating PKA-Nrf2/NF-κB signal pathway remains to be further confirmed.Therefore,we conducted a retrospective clinical study to explore whether acute lung injury was an independent risk factor for death during hospitalization in patients with ROSC.Further animal experiments were carried out to determine the possible mechanism of acute lung injury after ROSC and whether adiponectin could alleviate acute lung injury mediated by pulmonary microvascular endothelial cells by regulating PKA-Nrf2/NF-κB signal pathway.Materials and MethodsChapter 1 Study on the relationship between acute lung injury and the prognosis of adult patients with return of spontaneous circulation after in-hospital cardiac arrestA single-center retrospective observational study was conducted,and we collected the data of consecutive adult patients with ROSC after IHCA from July 5,2012 to July 4,2019 in Zigong fourth people’s Hospital according to the inclusion and exclusion criteria.The clinical data and lung imaging data of the patients were collected.The patients were divided into death group and survival group,andΔlung injury score（ΔLIS=LIS within 24 hours after resuscitation-LIS within 24 hours before resuscitation）was calculated.Logistic regression analysis was used to explore the independent risk factors related to the prognosis of patients with ROSC.ΔLIS was used to predict the mortality of patients with ROSC during hospitalization by ROC curve.Finally,Spearman correlation analysis was used to explore the correlation between acute lung injury and white blood cell（WBC）,neutrophil（NEU）,neutrophil percentage（NEU%）and their changes before and after resuscitation.Chapter Ⅱ Adiponectin attenuating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with return of spontaneous circulation2.1 Characteristics of acute lung injury in mice with ROSCThe mice were randomly divided into Sham group and CPR group.Mice of each group were killed at 0.5h,3h,6h and 24h after ROSC in CPR group,and mice in sham group were killed at the corresponding time point.Before the animals were killed,the lungs were scanned by computed tomography（CT）and scored according to the criterion of CT.After the animals were killed,bronchoalveolar lavage was performed,and bronchoalveolar lavage fluid and lung tissue were taken for follow-up research.The wet/dry weight ratio of lung tissue was calculated and hematoxylin-eosin（HE）staining was performed.The concentrations of total protein and tumor necrosis factor-α（TNF-α）in bronchoalveolar lavage fluid（BALF）were measured by bicinchoninic acid（BCA）and enzyme linked immunosorbent assay（ELISA）respectively.The peak time of acute lung injury after CPR was observed for subsequent research.2.2 The role of pulmonary microvascular endothelial cell injury in acute lung injury in mice with ROSCThe mice were randomly divided into Sham group and CPR group.The mice were killed and samples were taken at the time point determined in this chapter 2.1.The number of neutrophils in the alveolar space immunofluorescently stained with Ly6G+was compared.Expression of adhesion molecules including ICAM-1 and VCAM-1 in pulmonary microvascular endothelial cells was evaluated by double immunofluorescence（IF）staining with an anti-CD31 antibody.Expression of tight junction proteins including CLDN5 and OCLN in pulmonary microvascular endothelial cells was evaluated by double immunofluorescence staining with an anti-CD31 antibody.2.3 Adiponectin attenuating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with ROSCThe mice were randomly divided into Sham group,CPR group and APN group.The mice were killed at the time point determined in this chapter 2.1.The general indexes of lung injury（CT score,wet/dry weight ratio and HE staining）,the expression of protein（total protein and TNF-α）in BALF,the number of neutrophils in the alveolar space immunofluorescently stained with Ly6G+,and the levels of adhesion molecules（ICAM-1 and VCAM-1）and tight junction proteins（CLDN5 and OCLN）in PMVECs were detected and compared among the three groups.Chapter Ⅲ Adiponectin alleviating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with return of spontaneous circulation by regulating the PKA-Nrf2/NF-κB signaling pathway3.1 The effects of different doses of PKA agonists and inhibitors on acute lung injury mediated by pulmonary microvascular endothelial cells in mice with ROSCThe mice were randomly divided into 8 groups:Sham group,CPR group,CPR+forskolin（FSK）group（FL group,FM group and FH group）and CPR+H-89 group（HL group,HM group and HH group）.The mice were killed at the time point determined in this chapter 2.1.The general indexes of lung injury（CT score,wet/dry weight ratio and HE staining）and the expression of protein（total protein and TNF-α）in BALF were detected and compared among the eight groups.According to the above results,the best intervention doses of PKA agonists and inhibitors were selected for subsequent research.3.2 APN attenuating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with ROSC by regulating the PKA-Nrf2/NF-κB signaling pathwayThe mice were divided into 5 groups,including Sham group,CPR group,APN group,APN+FSK group and APN+H-89 group.The mice were killed at the time point determined in this chapter 2.1.The general indexes of lung injury（CT score,wet/dry weight ratio and HE staining）,the expression of protein（total protein and TNF-α）in BALF,the number of neutrophils in the alveolar space stained with Ly6G+,the levels of adhesion molecules（ICAM-1 and VCAM-1）and tight junction proteins（CLDN5 and OCLN）in PMVECs,and the levels of channel proteins（p-PKA,Nrf2 and NF-κB）were detected and compared among the groups.Chapter Ⅳ Mechanism research of acute lung injury mediated by pulmonary microvascular endothelial cells in Adipoq-/-and Nrf2-/-mice with return of spontaneous circulation4.1 Acute lung injury mediated by pulmonary microvascular endothelial cells in Adipoq-/-mice with ROSCWild type（WT）and adiponectin gene knockout(Adipoq-/-)C57BL/6 mice were divided into four groups:WT Sham group,Adipoq-/-Sham group,WT CPR group and Adipoq-/-CPR group.The mice were killed at the time point determined in this chapter 2.1.The general indexes of lung injury（CT score,wet/dry weight ratio and HE staining）,the expression of protein（total protein and TNF-α）in BALF,the number of neutrophils in the alveolar space stained with Ly6G+,the levels of adhesion molecules（ICAM-1 and VCAM-1）and tight junction proteins（CLDN5 and OCLN）in PMVECs,and the levels of channel proteins（p-PKA,Nrf2 and NF-κB）were detected and compared among the groups.4.2 Effects of adiponectin on acute lung injury mediated by pulmonary microvascular endothelial cells in Nrf2-/-mice with ROSCWT and Nrf2 gene knockout(Nrf2-/-)C57BL/6 mice were divided into six groups:WT Sham group,Nrf2-/-Sham group,WT CPR group,Nrf2-/-CPR group,WT CPR+APN group and Nrf2-/-CPR+APN group.The mice were killed at the time point determined in this chapter 2.1.The general indexes of lung injury（CT score,wet/dry weight ratio and HE staining）,the expression of protein（total protein and TNF-α）in BALF,the number of neutrophils in the alveolar space stained with Ly6G+,the levels of adhesion molecules（ICAM-1and VCAM-1）and tight junction proteins（CLDN5 and OCLN）in PMVECs,and the levels of channel proteins（p-PKA and NF-κB）were detected and compared among the groups.ResultsChapter 1 Study on the relationship between acute lung injury and the prognosis of adult patients with return of spontaneous circulation after in-hospital cardiac arrestA total of 173 patients after ROSC were included in this study.Multivariate logistic regression analysis showed thatΔLIS（OR=1.331,95%CI:1.061-1.671,P<0.05）was an independent risk factor for predicting mortality during hospitalization in patients with ROSC.The results of correlation analysis showed thatΔNEU（r=0.308,P<0.001）and the timing of LIS scoring after ROSC（r=0.356,P<0.001）were moderately correlated withΔLIS.Therefore,the changes of the number of neutrophils in the circulatory system before and after resuscitation and the timing of LIS scoring after resuscitation were moderately correlated acute lung injury after ROSC.Chapter Ⅱ Adiponectin attenuating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with return of spontaneous circulation2.1 Characteristics of acute lung injury in mice with ROSCCompared with Sham group,lung CT score,wet/dry weight ratio,pathological injury of lung tissue,and the concentration total protein and TNF-αin BALF in CPR group increased at each time point,especially at 3h and 6h after ROSC.Considering the availability of the experiment,3 hours after ROSC was selected as the time point of taking samples in subsequent research.2.2 The role of pulmonary microvascular endothelial cell injury in acute lung injury in mice with ROSCCompared with the Sham group,the number of neutrophils in the alveolar space in the CPR group increased significantly（P<0.05）,the levels of ICAM-1and VCAM-1 in the CPR group increased significantly（P<0.05）,and the levels of CLDN5 and OCLN in the CPR group decreased significantly（P<0.05）.2.3 Adiponectin attenuating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with ROSCCompared with Sham group,the general indexes of lung injury（CT score,wet/dry weight ratio and HE score）and the levels of protein（total protein and TNF-α）in BALF in the CPR group increased significantly（P<0.05）;the index of injury in PMVECs（the number of neutrophils in the alveolar space,and the levels of ICAM-1 and VCAM-1）in the CPR group aggravated significantly（P<0.05）,and the levels of CLDN5 and OCLN in the CPR group were significantly lower in the CPR group（P<0.05）.Compared with CPR group,the levels of CLDN5 and OCLN in APN group were significantly higher（P<0.05）,and the other indexes were significantly lower（P<0.05）,but did not return to the levels of Sham group.Chapter Ⅲ Adiponectin alleviating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with return of spontaneous circulation by regulating the PKA-Nrf2/NF-κB signaling pathway3.1 The effects of different doses of PKA agonists and inhibitors on acute lung injury mediated by pulmonary microvascular endothelial cells in mice with ROSCCompared with Sham group,the general indexes of lung injury（CT score,wet/dry weight ratio and HE score）in CPR group increased significantly（P<0.05）,the levels of protein（total protein and TNF-α）in BALF in the CPR group increased significantly（P<0.05）.Compared with the CPR group,the above-mentioned indexes of mice in different doses of FSK group were significantly lower（P<0.05）,but there were no statistical differences among three dose groups,so the low-dose group was selected to complete subsequent research.The above-mentioned indexes increased more significantly in different doses of H-89 group,especially in HH group.Therefore,the follow-up study chose high dose to complete subsequent research.3.2 APN attenuating acute lung injury mediated by pulmonary microvascular endothelial cells in mice with ROSC by regulating the PKA-Nrf2/NF-κB signaling pathway3.2.1 Comparison of acute lung injury in each groupCompared with Sham group,the general indexes of lung injury（CT score,wet/dry weight ratio and HE score）,the levels of protein（total protein and TNF-α）in BALF,the number of neutrophils in the alveolar space,and the levels of ICAM-1 and VCAM-1 in CPR group increased significantly（P<0.05）,and the levels of CLDN5 and OCLN in the CPR group were significantly lower in the CPR group（P<0.05）.Compared with CPR group,the levels of CLDN5 and OCLN in APN group were significantly higher（P<0.05）,and the other indexes were significantly lower（P<0.05）,but did not return to the levels of Sham group.Compared with APN group,the levels of CLDN5 and OCLN in APN+FSK group were significantly higher（P<0.05）,and the other indexes were significantly lower（P<0.05）.Compared with APN group,the levels of CLDN5and OCLN in APN+H-89 group were significantly lower（P<0.05）,and the other indexes were significantly higher（P<0.05）.3.2.2 Comparison of protein levels of signal transduction pathway in PMVECs in each groupCompared with Sham group,the levels of NF-κB p-p65,Nrf2 and p-PKA in CPR group increased significantly（P<0.05）;compared with CPR group,the levels of NF-κB p-p65 in APN group decreased significantly（P<0.05）,and the levels of Nrf2 and p-PKA in APN group increased significantly（P<0.05）.Compared with APN group,the levels of NF-κB p-p65 in APN+FSK group decreased further significantly（P<0.05）,and the levels of Nrf2 and p-PKA in APN+FSK group increased further significantly（P<0.05）.Compared with APN group,the levels of NF-κB p-p65 in APN+H-89 group increased significantly（P<0.05）,and the levels of Nrf2 and p-PKA in APN+H-89 group decreased significantly（P<0.05）.Chapter Ⅳ Mechanism research of acute lung injury mediated by pulmonary microvascular endothelial cells in Adipoq-/-and Nrf2-/-mice with return of spontaneous circulation4.1 Acute lung injury mediated by pulmonary microvascular endothelial cells in Adipoq-/-mice with ROSC4.1.1 Comparison of acute lung injury mediated by pulmonary microvascular endothelial cells in each groupCompared with WT Sham group,there was no significant difference in the general indexes of lung injury（CT score,wet/dry weight ratio and HE score）,the levels of protein（total protein and TNF-α）in BALF,the number of neutrophils in the alveolar space stained with Ly6G+,the levels of adhesion molecules（ICAM-1 and VCAM-1）and tight junction proteins（CLDN5 and OCLN）in PMVECs in Adipoq-/-Sham group（P>0.05）.Compared with Adipoq-/-Sham group,the levels of CLDN5 and OCLN in Adipoq-/-CPR group decreased significantly（P<0.05）,and the other indexes in Adipoq-/-CPR group increased significantly（P<0.05）.Compared with WT CPR group,the levels of CLDN5 and OCLN in Adipoq-/-CPR group decreased significantly（P<0.05）,and the other indexes in Adipoq-/-CPR group increased significantly（P<0.05）.4.1.2 Comparison of protein levels of signal transduction pathway in PMVECs in each group of miceCompared with WT Sham group,there was no significant difference in the levels of NF-κB p-p65,Nrf2 and p-PKA in PMVECs in Adipoq-/-Sham group（P>0.05）.Compared with Adipoq-/-Sham group,the levels of NF-κB p-p65,Nrf2 and p-PKA in Adipoq-/-CPR group increased significantly（P<0.05）.Compared with WT CPR group,the levels of NF-κB p-p65 in Adipoq-/-CPR group increased significantly（P<0.05）,and the levels of Nrf2 and p-PKA in Adipoq-/-CPR group decreased significantly（P<0.05）.4.2 Effects of adiponectin on acute lung injury mediated by pulmonary microvascular endothelial cells in Nrf2-/-mice with ROSC4.2.1 Comparison of acute lung injury mediated by pulmonary microvascular endothelial cells in each groupCompared with WT Sham group,there was no significant difference in the general indexes of lung injury（CT score,wet/dry weight ratio and HE score）,the levels of protein（total protein and TNF-α）in BALF,the number of neutrophils in the alveolar space stained with Ly6G+,the levels of adhesion molecules（ICAM-1 and VCAM-1）and tight junction proteins（CLDN5 and OCLN）in PMVECs in Nrf2-/-Sham group（P>0.05）.Compared with Nrf2-/-Sham group,the levels of CLDN5 and OCLN in Nrf2-/-CPR group decreased significantly（P<0.05）,and the other indexes in Nrf2-/-CPR group increased significantly（P<0.05）.Compared with WT CPR group,the levels of CLDN5and OCLN in Nrf2-/-CPR group decreased significantly（P<0.05）,and the other indexes in Nrf2-/-CPR group increased significantly（P<0.05）.Compared with Nrf2-/-CPR group,the levels of CLDN5 and OCLN in Nrf2-/-CPR+APN group increased significantly（P<0.05）,and the other indexes in Nrf2-/-CPR group decreased significantly（P<0.05）.4.2.2 Comparison of protein levels of signal transduction pathway in PMVECs in each groupCompared with WT Sham group,there was no significant difference in the levels of NF-κB p-p65 and p-PKA in PMVECs in Nrf2-/-Sham group（P>0.05）.Compared with Nrf2-/-Sham group,the levels of NF-κB p-p65 and p-PKA in Nrf2-/-CPR group increased significantly（P<0.05）.Compared with WT CPR group,the levels of NF-κB p-p65 in Nrf2-/-CPR group increased significantly（P<0.05）,and the levels of p-PKA in Nrf2-/-CPR group were similar（P>0.05）.Compared with Nrf2-/-CPR group,the levels of NF-κB p-p65 in Nrf2-/-CPR+APN group decreased insignificantly（P>0.05）,and the levels of p-PKA in Nrf2-/-CPR group increased significantly（P<0.05）.Conclusion1.Acute lung injury after return of spontaneous circulation in patients with in-hospital cardiac arrest is an independent risk factor for predicting the prognosis.The more severe the lung injury,the worse the prognosis.2.Pulmonary microvascular endothelial cell injury is an important factor of lung injury after return of spontaneous circulation in mice with cardiac arrest,and adiponectin can alleviate acute lung injury of mice by attenuating the acute injury of pulmonary microvascular endothelial cells.3.Adiponectin can regulate the PKA-Nrf2/NF-κB signaling pathway to attenuate acute lung injury mediated by pulmonary microvascular endothelial cells in mice with return of spontaneous circulation. |
PDF Full Text Request