[Investigation on the signaling pathways in the mechanism of hyperoxia-induced acute lung injury based on transcriptomics sequencing].

OBJECTIVE: To observe and verify the changes of transcriptome in hyperoxia-induced acute lung injury (HALI), and to further clarify the changes of pathways in HALI. METHODS: Twelve healthy male C57BL/6J mice were randomly divided into normoxia group and HALI group according to the random number table, with 6 mice in each group. The mice in the normoxia group were fed normally in the room, and the mice in the HALI group was exposed to 95% oxygen to reproduce the HALI animal model. After 72 hours of hyperoxia exposure, the lung tissues were taken for transcriptome sequencing, and then Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analysis was performed. The pathological changes of lung tissue were observed under light microscope after hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to verify the key molecules in the signal pathways closely related to HALI identified by transcriptomics analysis. RESULTS: Transcriptomic analysis showed that hyperoxia induced 537 differentially expressed genes in lung tissue of mice as compared with the normoxia group including 239 up-regulated genes and 298 down-regulated genes. Further KEGG pathway enrichment analysis identified 20 most significantly enriched pathway entries, and the top three pathways were ferroptosis signaling pathway, p53 signaling pathway and glutathione (GSH) metabolism signaling pathway. The related genes in the ferroptosis signaling pathway included the up-regulated gene heme oxygenase-1 (HO-1) and the down-regulated gene solute carrier family 7 member 11 (SLC7A11). The related genes in the p53 signaling pathway included the up-regulated gene tumor suppressor gene p53 and the down-regulated gene murine double minute 2 (MDM2). The related gene in the GSH metabolic signaling pathway was up-regulated gene glutaredoxin 1 (Grx1). The light microscope showed that the pulmonary alveolar structure of the normoxia group was normal. In the HALI group, the pulmonary alveolar septum widened and thickened, and the alveolar cavity shrank or disappeared. RT-RCR and Western blotting confirmed that compared with the normoxia group, the mRNA and protein expressions of HO-1 and p53 in lung tissue of the HALI group were significantly increased [HO-1 mRNA (2-ΔΔCt): 2.16±0.17 vs. 1.00±0.00, HO-1 protein (HO-1/ß-actin): 1.05±0.01 vs. 0.79±0.01, p53 mRNA (2-ΔΔCt): 2.52±0.13 vs. 1.00±0.00, p53 protein (p53/ß-actin): 1.12±0.02 vs. 0.58±0.03, all P < 0.05], and the mRNA and protein expressions of Grx1, MDM2, SLC7A11 were significantly decreased [Grx1 mRNA (2-ΔΔCt): 0.53±0.05 vs. 1.00±0.00, Grx1 protein (Grx1/ß-actin): 0.54±0.03 vs. 0.93±0.01, MDM2 mRNA (2-ΔΔCt): 0.48±0.03 vs. 1.00±0.00, MDM2 protein (MDM2/ß-actin): 0.57±0.02 vs. 1.05±0.01, SLC7A11 mRNA (2-ΔΔCt): 0.50±0.06 vs. 1.00±0.00, SLC7A11 protein (SLC7A11/ß-actin): 0.72±0.03 vs. 0.98±0.01, all P < 0.05]. CONCLUSIONS: HALI is closely related to ferroptosis, p53 and GSH metabolism signaling pathways. Targeting the key targets in ferroptosis, p53 and GSH metabolism signaling pathways may be an important strategy for the prevention and treatment of HALI.

Annexin-A1 short peptide alleviates septic myocardial injury by upregulating SIRT3 and inhibiting myocardial cell apoptosis.

Septic myocardial injury is a common complication of severe sepsis, which occurs in about 50% of cases. Patients with this disease may experience varying degrees of myocardial damage. Annexin-A1 short peptide (ANXA1sp), with a molecular structure of Ac-Gln-Ala-Tyr, has been reported to exert an organ protective effect in the perioperative period by modulating sirtuin-3 (SIRT3). Whether it possesses protective activity against sepsis-induced cardiomyopathy is worthy of study. This study aimed to investigate whether ANXA1sp exerts its anti-apoptotic effect in septic myocardial injury in vitro and in vivo via regulating SIRT3. In this study, we established in vivo and in vivo models of septic myocardial injury based on C57BL/6 mice and primary cardiomyocytes by lipopolysaccharide (LPS) induction. Results showed that ANXA1sp pretreatment enhanced the seven-day survival rate, improved left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), and cardiac output (CO), and reduced the levels of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and lactate dehydrogenase (LDH). Western blotting results revealed that ANXA1sp significantly increased the expression of SIRT3, Bcl-2, and downregulated Bax expression. TUNEL staining and flow cytometry results showed that ANXA1sp could attenuate the apoptosis rate of cardiomyocytes, whereas this anti-apoptotic effect was significantly attenuated after SIRT3 knockout. To sum up, ANXA1sp can alleviate LPS-induced myocardial injury by reducing myocardial apoptosis via SIRT3 upregulation.

Gpnmb silencing protects against hyperoxia-induced acute lung injury by inhibition of mitochondrial-mediated apoptosis.

Background: Hyperoxia-induced acute lung injury (HALI) is a complication to ventilation in patients with respiratory failure, which can lead to acute inflammatory lung injury and chronic lung disease. The aim of this study was to integrate bioinformatics analysis to identify key genes associated with HALI and validate their role in H2O2-induced cell injury model.Methods: Integrated bioinformatics analysis was performed to screen vital genes involved in hyperoxia-induced lung injury (HLI). CCK-8 and flow cytometry assays were performed to assess cell viability and apoptosis. Western blotting was performed to assess protein expression.Results: In this study, glycoprotein non-metastatic melanoma protein B (Gpnmb) was identified as a key gene in HLI by integrated bioinformatics analysis of 4 Gene Expression Omnibus (GEO) datasets (GSE97804, GSE51039, GSE76301 and GSE87350). Knockdown of Gpnmb increased cell viability and decreased apoptosis in H2O2-treated MLE-12 cells, suggesting that Gpnmb was a proapoptotic gene during HALI. Western blotting results showed that knockdown of Gpnmb reduced the expression of Bcl-2 associated X (BAX) and cleaved-caspase 3, and increased the expression of Bcl-2 in H2O2 treated MLE-12 cells. Furthermore, Gpnmb knockdown could significantly reduce reactive oxygen species (ROS) generation and improve the mitochondrial membrane potential.Conclusion: The present study showed that knockdown of Gpnmb may protect against HLI by repressing mitochondrial-mediated apoptosis.

ANXA1sp attenuates sepsis-induced myocardial injury by promoting mitochondrial biosynthesis and inhibiting oxidative stress and autophagy via SIRT3 upregulation.

Sepsis-induced myocardial injury is one of the most difficult complications of sepsis in intensive care units. Annexin A1 (ANXA1) short peptide (ANXA1sp) protects organs during the perioperative period. However, the protective effect of ANXA1sp against sepsis-induced myocardial injury remains unclear. We aimed to explore the protective effects and mechanisms of ANXA1sp against sepsis-induced myocardial injury both in vitro and in vivo. Cellular and animal models of myocardial injury in sepsis were established with lipopolysaccharide. The cardiac function of mice was assessed by high-frequency echocardiography. Elisa assay detected changes in inflammatory mediators and markers of myocardial injury. Western blotting detected autophagy and mitochondrial biosynthesis-related proteins. Autophagic flux changes were observed by confocal microscopy, and autophagosomes were evaluated by TEM. ATP, SOD, ROS, and MDA levels were also detected.ANXA1sp pretreatment enhanced the 7-day survival rate, improved cardiac function, and reduced TNF-α, IL-6, IL-1ß, CK-MB, cTnI, and LDH levels. ANXA1sp significantly increased the expression of sirtuin-3 (SIRT3), mitochondrial biosynthesis-related proteins peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM). ANXA1sp increased mitochondrial membrane potential (△Ψm), ATP, and SOD, and decreased ROS, autophagy flux, the production of autophagosomes per unit area, and MDA levels. The protective effect of ANXA1sp decreased significantly after SIRT3 silencing in vitro and in vivo, indicating that the key factor in ANXA1sp's protective role is the upregulation of SIRT3. In summary, ANXA1sp attenuated sepsis-induced myocardial injury by upregulating SIRT3 to promote mitochondrial biosynthesis and inhibit oxidative stress and autophagy.

Hyperoxia can Induce Lung Injury by Upregulating AECII Autophagy and Apoptosis Via the mTOR Pathway.

Oxygen therapy is a crucial medical intervention, but it is undeniable that it can lead to lung damage. The mTOR pathway plays a pivotal role in governing cell survival, including autophagy and apoptosis, two phenomena deeply entwined with the evolution of diseases. However, it is unclarified whether the mTOR pathway is involved in hyperoxic acute lung injury (HALI). The current study aims to clarify the molecular mechanism underlying the pathogenesis of HALI by constructing in vitro and in vivo models using H2O2 and hyperoxia exposure, respectively. To investigate the role of mTOR, the experiment was divided into five groups, including normal group, injury group, mTOR inhibitor group, mTOR activator group, and DMSO control group. Western blotting, Autophagy double labeling, TUNEL staining, and HE staining were applied to evaluate protein expression, autophagy activity, cell apoptosis, and pathological changes in lung tissues. Our data revealed that hyperoxia can induce autophagy and apoptosis in Type II alveolar epithelial cell (AECII) isolated from the treated rats, as well as injuries in the rat lung tissues; also, H2O2 stimulation increased autophagy and apoptosis in MLE-12 cells. Noticeably, the experiments performed in both in vitro and in vivo models proved that the mTOR inhibitor Rapamycin (Rapa) functioned synergistically with hyperoxia or H2O2 to promote AECII autophagy, which led to increased apoptosis and exacerbated lung injury. On the contrary, activation of mTOR with MHY1485 suppressed autophagy activity, consequently resulting in reduced apoptosis and lung injury in H2O2-challenged MLE-12 cells and hyperoxia-exposed rats. In conclusion, hyperoxia caused lung injury via mTOR-mediated AECII autophagy.

ROS-mediated MAPK activation aggravates hyperoxia-induced acute lung injury by promoting apoptosis of type II alveolar epithelial cells via the STAT3/miR-21-5p axis.

Inhibition of type II alveolar epithelial (AE-II) cell apoptosis is a critical way to cure hyperoxia-induced acute lung injury (HALI). It has been reported that miR-21-5p could reduce H2O2-induced apoptosis in AE-II cells. However, the upstream molecular mechanism remains unclear. Herein, we established a cellular model of HALI by exposing AE-II cells to H2O2 treatment. It was shown that miR-21-5p alleviated H2O2-induced apoptosis in AE-II cells. ROS inhibition decreased apoptosis of H2O2-evoked AE-II cells via increasing miR-21-5p expression. In addition, ROS induced MAPK and STAT3 phosphorylation in H2O2-treated AE-II cells. MAPK inactivation reduces H2O2-triggered AE-II cell apoptosis. MAPK activation inhibits miR-21-5p expression by promoting STAT3 phosphorylation in H2O2-challenged AE-II cells. Furthermore, STAT3 activation eliminated MAPK deactivation-mediated inhibition on the apoptosis of AE-II cells under H2O2 condition. In conclusion, ROS-mediated MAPK activation promoted H2O2-triggered AE-II cell apoptosis by inhibiting miR-21-5p expression via STAT3 phosphorylation, providing novel targets for HALI treatment.

ANXA1sp Protects against Sepsis-Induced Myocardial Injury by Inhibiting Ferroptosis-Induced Cardiomyocyte Death via SIRT3-Mediated p53 Deacetylation.

Sepsis-induced myocardial injury (SIMI), a common complication of sepsis, may cause significant mortality. Ferroptosis, a cell death associated with oxidative stress and inflammation, has been identified to be involved in SIMI. This study sought to investigate the role of ANXA1 small peptide (ANXA1sp) in SIMI pathogenesis. In this study, the mouse cardiomyocytes (H9C2 cells) were stimulated with lipopolysaccharide (LPS) to imitate SIMI in vitro. It was shown that ANXA1sp treatment substantially abated LPS-triggered H9C2 cell death and excessive secretion of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6). ANXA1sp pretreatment also reversed the increase of ROS and MDA generation as well as the decrease of SOD and GSH activity in H9C2 cells caused by LPS treatment. In addition, ANXA1sp considerably eliminated LPS-caused H9C2 cell ferroptosis, as revealed by the suppression of iron accumulation and the increase in GPX4 and FTH1 expression. Furthermore, the ameliorative effects of ANXA1sp on LPS-induced H9C2 cell damage could be partially abolished by erastin, a ferroptosis agonist. ANXA1sp enhanced SIRT3 expression in LPS-challenged H9C2 cells, thereby promoting p53 deacetylation. SIRT3 knockdown diminished ANXA1sp-mediated alleviation of cell death, inflammation, oxidative stress, and ferroptosis of LPS-treated H9C2 cells. Our study demonstrated that ANXA1sp is protected against LPS-induced cardiomyocyte damage by inhibiting ferroptosis-induced cell death via SIRT3-dependent p53 deacetylation, suggesting that ANXA1sp may be a potent therapeutic agent for SIMI.

[Research progress of phosphoglycerate mutase 5-mediated mitophagy and necroptosis].

Mitophagy is the selective degradation of damaged mitochondria, and it is of great significance to maintain the normal quantity and quality of mitochondria to ensure cell homeostasis and survival. Necroptosis is a type of programmed cell necrosis that can be induced by excessive mitophagy. Reactive oxygen species (ROS) are produced mainly by mitochondria and can damage mitochondria. Hyperoxic acute lung injury (HALI) is a serious complication of clinical oxygen therapy, and its pathogenesis is not clear. Existing studies have shown that mitophagy and necroptosis are involved in the occurrence of HALI. There are many mechanisms regulating mitophagy and necroptosis, including tumor necrosis factor-α (TNF-α), E3 ubiquitin protein ligase (PINK1/Parkin) protein pathway encoded by PTEN-induced kinase 1/PARK2 gene, phosphoglycerate mutase 5 (PGAM5), etc. PGAM5 has been proved to be a key factor linking mitophagy and necroptosis. Previous studies of our team found that the mechanism of microRNA-21-5p (miR-21-5p) alleviating HALI was related to its pGAM5-mediated inhibition of mitophagy, but the mechanism of PGAM5-mediated mitophagy and necroptosis remains unclear. Therefore, this paper reviews the targets of PGAM5-mediated mitophagy and necroptosis, in order to find clues of lung protection of pGAM5-mediated mitophagy and necroptosis in HALI, and provide theoretical basis for subsequent basic research.

[Research progress of extracellular vesicle microRNA in acute lung injury].

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by the destruction of the barrier function of alveolar epithelial cells and capillary endothelial cells and the recruitment of inflammatory cells, which leads to alveolar and interstitial edema, hyaline membrane formation and inflammatory infiltration of the lungs, etc. The mechanism is not completely defined. The current treatment plan focuses on comprehensive treatments such as ventilator support treatment, fluid management, and nutritional support, but the prognosis is still poor. Studies have shown that extracellular vesicle microRNA (miRNA) from different sources participate in regulating the function of epithelial cells, endothelial cells and phagocytes in different ways, thus aggravating or improving ALI, and have diagnostic, differential diagnosis and the therapeutic value. In this article, the mechanism, diagnostic and differerntial value of extracellular vesicle miRNA from different sources in ALI and the therapy of extracellular vesicle miRNA from stem cell in ALI are reviewed.