Fusion Expression and Fibrinolytic Activity of rPA/SP-B

2021 ◽  
Vol 28 ◽  
Author(s):  
Yi-Shan Tang ◽  
Xiao-Jun Zhang ◽  
Wan-Neng Wang ◽  
Ting Wang ◽  
Wu-Long Cao ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Acute Lung Injury ◽  
Cell Membrane ◽  
Lung Injury ◽  
Fusion Protein ◽  
Surface Activity ◽  
Fibrinolytic Activity ◽  
Cell Model ◽  
Fusion Expression ◽  
Recombinant Plasmids

Background: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

scholarly journals Pulmonary Capillary Endothelium-Bound Angiotensin-Converting Enzyme Activity in Acute Lung Injury

Circulation ◽  
2000 ◽  
Vol 102 (16) ◽  
pp. 2011-2018 ◽  
Author(s):  
Stylianos E. Orfanos ◽  
Apostolos Armaganidis ◽  
Constantinos Glynos ◽  
Ekaterini Psevdi ◽  
Panagiotis Kaltsas ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Angiotensin Converting Enzyme ◽  
Capillary Endothelium ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme Activity ◽  
Pulmonary Capillary

scholarly journals Lipopolysaccharide Inhibits Alpha Epithelial Sodium Channel Expression via MiR-124-5p in Alveolar Type 2  Epithelial Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-9 ◽  
Author(s):  
Yan Ding ◽  
Yong Cui ◽  
Zhiyu Zhou ◽  
Yapeng Hou ◽  
Xining Pang ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Epithelial Cells ◽  
Lung Injury ◽  
Sodium Channel ◽  
Target Genes ◽  
Cell Model ◽  
Epithelial Sodium Channel ◽  
Alveolar Epithelial Cells ◽  
Luciferase Assay ◽  
Alveolar Type

Mesenchymal stem cells (MSCs) have been a potential strategy in the pretreatment of pulmonary diseases, while the mechanisms of MSCs-conditioned medium (MSCs-CM) involved with microRNAs on the regulation of lung ion transport are seldom reported. We investigated the role of miR-124-5p in lipopolysaccharide-involved epithelial sodium channel (ENaC) dysfunction and explored the potential target of miR-124-5p. We observed the lower expression of miR-124-5p after the administration of MSCs-CM, and the overexpression or inhibition of miR-124-5p regulated epithelial sodium channel α-subunit (α-ENaC) expression at protein levels in mouse alveolar type 2 epithelial (AT2) cells. We confirmed that α-ENaC is one of the target genes of miR-124-5p through dual luciferase assay and Ussing chamber assay revealed that miR-124-5p inhibited amiloride-sensitive currents associated with ENaC activity in intact H441 monolayers. Our results demonstrate that miR-124-5p can decrease the expression and function of α-ENaC in alveolar epithelial cells by targeting the 3′-UTR. The involvement of MSCs-CM in lipopolysaccharide-induced acute lung injury cell model could be related to the downregulation of miR-124-5p on α-ENaC, which may provide a new target for the treatment of acute lung injury.

Dexamethasone ameliorates H2S-induced acute lung injury by increasing claudin-5 expression via the PI3K pathway

2017 ◽  
Vol 37 (6) ◽  
pp. 626-635 ◽  
Author(s):  
P Geng ◽  
T Ma ◽  
J Xing ◽  
L Jiang ◽  
H Sun ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Protective Effect ◽  
Cell Model ◽  
Umbilical Vein ◽  
Pi3k Pathway ◽  
Protective Effects ◽  
Sprague Dawley ◽  
Protective Mechanisms ◽  
Hazardous Gases

Acute lung injury (ALI) is a major outcome of exposure to high levels of hydrogen sulfide (H2S). Dexamethasone (DXM) has been used to treat ALI. However, the mechanisms involved in H2S-induced ALI and the protective mechanisms of DXM in treating ALI are still nebulous. To explore the mechanisms involved, we evaluated the role of claudin-5 in the protective effect of DXM against H2S-induced ALI. Sprague-Dawley rats were exposed to H2S to establish the ALI model. In parallel with the animal model, a cell model was also established by incubating human umbilical vein endothelial cells (HUVECs) with NaHS. Lung hematoxylin–eosin staining, electron microscope assay, and wet/dry ratio were used to identify whether the ALI was successfully induced by H2S, and changes in claudin-5 expression were detected in both rats and HUVECs. Our results revealed that claudin-5 was markedly decreased after H2S exposure and that DXM significantly attenuated the H2S-induced downregulation of claudin-5 in both rats and HUVECs. In the animal experiment, p-Akt and p-FoxO1 presented a similar tendency as claudin-5, but their levels decreased 6 h prior to the levels of claudin-5. In a further investigation, the DXM-induced protective effect on ALI and rescue effect on downregulation of claudin-5 were both blocked by LY294002. The current study demonstrated that claudin-5 was involved in the development of H2S-induced ALI and that DXM exerted protective effects through increasing claudin-5 expression by activating the phosphatidylinositol 3-kinase pathway. Therefore, claudin-5 might represent a novel pharmacological target for treating ALI induced by H2S and other hazardous gases.

scholarly journals Protective effects of a bacterially expressed NIF-KGF fusion protein against bleomycin-induced acute lung injury in mice

2010 ◽  
Vol 42 (8) ◽  
pp. 548-557
Author(s):  
X. Li ◽  
S. Li ◽  
M. Zhang ◽  
X. Li ◽  
X. Zhang ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Fusion Protein ◽  
Protective Effects

scholarly journals Treatment of acute lung injury by targeting MG53-mediated cell membrane repair

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Yanlin Jia ◽  
Ken Chen ◽  
Peihui Lin ◽  
Gissela Lieber ◽  
Miyuki Nishi ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Cell Membrane ◽  
Lung Injury ◽  
Membrane Repair

Effect of exogenous surfactant instillation on experimental acute lung injury

1989 ◽  
Vol 66 (4) ◽  
pp. 1846-1851 ◽  
Author(s):  
J. D. Harris ◽  
F. Jackson ◽  
M. A. Moxley ◽  
W. J. Longmore
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Surface Activity ◽  
Pulmonary Surfactant ◽  
Exogenous Surfactant ◽  
Protein Ratio ◽  
Surfactant Replacement ◽  
Lung Injury Model ◽  
Surfactant Phospholipids ◽  
The Individual

Pulmonary surfactant replacement has previously been shown to be effective in the human neonatal respiratory distress syndrome. The value of surfactant replacement in models of acute lung injury other than quantitative surfactant deficiency states is, however, uncertain. In this study an acute lung injury model using rats with chronic indwelling arterial catheters, injured with N-nitroso-N-methylurethane (NNNMU), has been developed. The NNNMU injury was found to produce hypoxia, increased mortality, an alveolitis, and alterations in the pulmonary surfactant system. Alterations of surfactant obtained by bronchoalveolar lavage included a reduction in the phospholipid-to-protein ratio, reduced surface activity, and alterations in the relative percentages of the individual phospholipids compared with controls. Treatment of the NNNMU-injured rats with instilled exogenous surfactant (Survanta) improved oxygenation; reduced mortality to control values; and returned the surfactant phospholipid-to-protein ratio, surface activity, and, with the exception of phosphatidylglycerol, the relative percentages of individual surfactant phospholipids to control values.

scholarly journals Long non-coding RNA OIP5-AS1 aggravates acute lung injury through promoting inflammation and cell apoptosis via regulating miR-26a-5p/TLR4 axis

2021 ◽  
Author(s):  
Qingsong Sun ◽  
Man Luo ◽  
Zhiwei Gao ◽  
Xiang Han ◽  
Weiqin Wu ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Cell Apoptosis ◽  
Cell Model ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Wide Range ◽  
Pulmonary Disorder ◽  
Proinflammatory Factors

Abstract Background: Acute lung injury (ALI) is a pulmonary disorder that leads to acute failure of respiration and thereby results in a high mortality worldwide. Increasing studies have verified that TLR4 is a promoter in ALI, however, the underlying upstream mechanisms of TLR4 was still rarely investigated. Methods: Lipopolysaccharide (LPS) was used to induce cell model and animal model. A wide range of experiments including RT-qPCR, Western blot, ELISA, flow cytometry, H&E staining, RIP, luciferase activity and caspase-3 activity were carried out to figure out the expression status, specific role and potential upstream mechanism of TLR4.Result: RT-qPCR identified that TLR4 expression was upregulated in ALI mice and LPS-induced WI-38 cells. Moreover, miR-26a-5p was confirmed to target TLR4 according to luciferase reporter assay. Besides, miR-26a-5p overexpression decreased the contents of proinflammatory factors (TNF-α and IL-1β) and restrained cell apoptosis, while upregulation of TLR4 reversed these effects of miR-26a-5p mimics, implying that miR-26a-5p alleviated ALI through regulating TLR4. Afterwards, OIP5-AS1 was identified to bind with miR-26a-5p by RNA immunoprecipitation (RIP) and luciferase reporter assay. Functionally, OIP5-AS1 upregulation accelerated the inflammation injuries and miR-26a-5p overexpression counteracted the influence of OIP5-AS1 upregulation on proinflammatory factors and cell apoptosis.Conclusion: OIP5-AS1 accelerated ALI through regulating miR-26a-5p/TLR4 axis in ALI mice and LPS-induced cells, which indicates a promising insight into diagnostics and therapeutics in ALI.

scholarly journals The Key Target and Molecular Mechanism of the Volatile Component of Scutellaria baicalensis Georgi in Acute Lung Injury Based on Network Pharmacology

2021 ◽  
Vol 12 ◽  
Author(s):  
Guosong Zhu ◽  
Jiaqiang Zhang ◽  
Yali Yang ◽  
Haoran Zhang ◽  
Wenwen Jin ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Response ◽  
Signaling Pathways ◽  
Molecular Mechanism ◽  
Cell Model ◽  
Volatile Component ◽  
Scutellaria Baicalensis ◽  
Inflammatory Factors ◽  
Regulation Mechanism

Ethnopharmacological relevance:Scutellaria baicalensis georgi is one of the most widely studied TCMs; its effects in ALI have been studied in a large number of experiments, and the efficacy of volatile oil from TCM remains to be studied.Aim: The volatile component of Scutellaria baicalensis georgi was selected to act on the key target of acute lung injury and was preliminarily studied for its specific molecular mechanism.Methods: The volatile active substances of Scutellaria baicalensis georgi were extracted by GC–MS, and the active ingredients related with the occurrence and development of acute lung injury were searched and matched by the TCMSP database. The pharmacologic data and analysis platform of TCM were used to retrieve and screen for the volatile active components and the possible therapeutic targets of Scutellaria baicalensis georgi. In addition, acute lung injury was searched in the disease target database to identify the corresponding disease target proteins, thereby establishing a protein–protein interaction network. Finally, the effects of wogonin on the apoptotic and inflammatory factors in the acute lung injury cell model were analyzed experimentally.Results: We identified 100 candidate targets and successfully constructed a complex target network. The targets identified by the above gene enrichment analysis played important roles in the autoimmune disease cell cycle apoptosis and related signaling pathways. The KEGG pathway analysis showed that most of the target genes were involved in the inflammatory response regulation of the TRP, PI3K-Akt, and IL-17 signaling pathways. The participation of wogonin in the specific regulatory pathways of PI3K-Akt signaling and IL-17 signaling was verified through experiments. In the lung-injured cell model, the results showed that wogonin inhibited the apoptosis of injured lung cells by inhibiting the expression of BAD gene and the activation of cleaved caspase-3 gene while increasing Bcl-2 expression. In addition, wogonin inhibited the expression of the abovementioned inflammatory factors and further inhibited the inflammatory response in the lung injury cells.Conclusion: The results of pharmacological network analysis can predict and explain the regulation mechanism of multi-target and multi-pathway of TCM components. This study identified the potential target and important pathway of wogonin in regulating acute lung injury. At the same time, the accuracy of network pharmacological prediction is also preliminarily verified by molecular biology experiment.

scholarly journals Apelin-13 Administration Protects Against LPS-Induced Acute Lung Injury by Inhibiting NF-κB Pathway and NLRP3 Inflammasome Activation

2018 ◽  
Vol 49 (5) ◽  
pp. 1918-1932 ◽  
Author(s):  
Hailin Zhang ◽  
Sha Chen ◽  
Meichun Zeng ◽  
Daopeng Lin ◽  
Yu Wang ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Nlrp3 Inflammasome ◽  
Structural Damage ◽  
Cell Model ◽  
Weight Ratio ◽  
Inflammatory Factors ◽  
Mouse Lung ◽  
Inflammasome Activation ◽  
Ros Formation

Background/Aims: Acute lung injury (ALI) is induced by a variety of external and internal factors and leads to acute progressive respiratory failure. Previous studies have shown that apelin-13 can decrease the acute lung injury induced by LPS, but the specific mechanism is unclear. Therefore, a mouse lung injury model and a cell model were designed to explore the mechanism of how apelin-13 alleviates the acute lung injury caused by LPS. Methods: The effect of apelin-13 on LPS-induced structural damage was determined by H&E staining and by the wet/dry weight ratio. The related inflammatory factors in BALF were examined by ELISA. The apoptotic pathway and the NF-κB and NLRP3 inflammasome pathways were evaluated by using Western blotting and immunofluorescence staining. Results: LPS induced the structural damage and the production of inflammatory cytokines in the lung tissues of mice. These deleterious effects were attenuated by apelin-13 administration. The protective effects of apelin-13 were associated with decreased reactive oxygen species (ROS) formation and the inhibition of the activation of the NF-κB and NLRP3 inflammasome pathways in mice and in Raw264.7 cells. Conclusion: Taken together, these data suggest that apelin-13 administration ameliorates LPS-induced acute lung injury by suppressing ROS formation, as well as by inhibiting the NF-κB pathway and the activation of the NLRP3 inflammasome in the lungs.

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