MicroRNA21-5p alleviates hyperoxia-induced acute lung injury in rats through activating phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway by regulating type Ⅱ alveolar epithelial cell apoptosis / 中华危重病急救医学

Objective:To investigate whether microRNA-21-5p (miR-21-5p) alleviates hyperoxia-induced acute lung injury (HALI) through activating the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway by regulating apoptosis of type Ⅱ alveolar epithelial cell (AECⅡ).Methods:Seventy-two male Sprague-Dawley (SD) rats were divided into normozone-controlled group, HALI group, PI3K/Akt signaling pathway inhibitor LY294002+HALI group (LY+HALI group), miR-21-5p overexpression+LY294002+HALI group (miR-21-5p+LY+HALI group), miR-21-5p overexpression+HALI group (miR-21-5p+HALI group), and dimethyl sulfoxide (DMSO)+HALI group by random number table method with 12 rats in each group. Animal models of HALI were prepared using 95% concentrations of oxygen. The animals in the normozone-controlled group were fed normally under normoxia. Transfection of lung tissue by miR-21-5p adeno-associated viral vector AAV6-miR-21-5p was performed by instillation of 200 μL titer (1×10 12 TU/mL) through a tracheal catheter 3 weeks prior to modeling. DMSO and LY294002 were administered via the tail vein at 0.3 mg/kg 1 hour before modeling. After 48 hours of modeling, carotid artery blood was collected to detect oxygenation index (OI) and respiratory index (RI), and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect miR-21-5p expression. Lung tissue was collected, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA), and the ratio of pulmonary wet/dry weight (W/D) was determined, and the pathological changes of lung histopathology were observed under the light microscopy after hematoxylin-eosin (HE) staining. Each group was purified AECⅡ cells from 6 rats, the apoptosis rate was detected by flow cytometry, and the expression levels of phosphatase and tensin homologous gene (PTEN), and proteins from the PI3K/Akt signaling pathway were detected by Western blotting. Results:Compared with the normozone-controlled group, alveolar septal thickening and massive inflammatory cell infiltration were found after hyperoxia exposure, RI, inflammatory factors, lung W/D ratio, pathological score, AECⅡ cells early apoptosis rate, PTEN protein expression and phosphorylation level of Akt were increased, while OI and miR-21-5p expression were decreased, indicating the successful preparation of the model. After pretreatment, LY294002 could aggravate the pathological injury of lung tissue in HALI rats, RI, inflammatory factors and lung W/D ratio were further increased, and OI was further reduced compared with HALI group. At the same time, it could promote the AECⅡ cell apoptosis, further up-regulate the expression of PTEN, and reduce the phosphorylation of Akt. However, miR-21-5p pretreatment could negatively regulate PTEN, activate PI3K/Akt signal pathway, inhibit AECⅡ cell apoptosis, and reduce HALI, which was shown by the decreased level of inflammatory factors in miR-21-5p+LY+HALI group compared with LY+HALI group [TNF-α (μg/L): 100.33±3.48 vs. 116.55±2.53, IL-6 (ng/L): 141.06±3.70 vs. 161.31±3.59, IL-1β (μg/L): 90.82±3.69 vs. 112.23±2.87, all P < 0.05], RI, lung injury pathology score, lung W/D ratio, and AECⅡ cell early apoptosis rate were significantly decreased [RI: 0.81±0.02 vs. 1.05±0.07, pathology score: 0.304±0.008 vs. 0.359±0.007, lung W/D ratio: 5.29±0.03 vs. 5.52±0.08, apoptosis rate: (27.20±2.34)% vs. (34.17±1.49)%, all P < 0.05], OI and expressions of miR-21-5p were significantly increased [OI (mmHg, 1 mmHg≈0.133 kPa): 266.71±2.75 vs. 230.12±4.04, miR-21-5p (2 -ΔΔCt): 2.21±0.13 vs. 0.33±0.03, both P < 0.05], and PTEN protein expression in AECⅡ cell was significantly reduced (PTEN/GAPDH: 0.50±0.06 vs. 0.93±0.06, P < 0.05), and phosphorylation level of Akt was significantly increased [phosphorylated Akt (p-Akt) protein (p-Akt/GAPDH): 0.86±0.05 vs. 0.56±0.06, P < 0.05]. Conclusion:miR-21-5p attenuates HALI by inhibiting AECⅡ cell apoptosis, possibly through negative regulation of PTEN to activate PI3K/Akt signaling pathway.

Wedelolactone alleviates hyperoxia-induced acute lung injury by regulating ferroptosis / 中华危重病急救医学

OBJECTIVE@#To study whether wedelolactone can reduce hyperoxia-induced acute lung injury (HALI) by regulating ferroptosis, and provide a basic theoretical basis for the drug treatment of HALI.@*METHODS@#A total of 24 C57BL/6J mice were randomly divided into normal oxygen control group, HALI model group and wedelolactone pretreatment group, with 8 mice in each group. Mice in wedelolactone pretreatment group were treated with wedelolactone 50 mg/kg intraperitoneally for 6 hours, while the other two groups were not given with wedelolactone. After that, the HALI model was established by maintaining the content of carbon dioxide < 0.5% and oxygen > 90% in the molding chamber for 48 hours, and the normal oxygen control group was placed in indoor air. After modeling, the mice were sacrificed and lung tissues were collected. The lung histopathological changes were observed under light microscope and pathological scores were performed to calculate the ratio of lung wet/dry mass (W/D). The levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in lung tissues of mice in each group were determined. The protein expression of glutathione peroxidase 4 (GPX4) in lung tissue was detected by Western blotting.@*RESULTS@#Under light microscope, the alveolar structure of HALI model group was destroyed, and a large number of neutrophils infiltrated the alveolar and interstitial lung, and the interstitial lung was thickened. The pathological score of lung injury (score: 0.75±0.02 vs. 0.11±0.01) and the ratio of lung W/D (6.23±0.34 vs. 3.68±0.23) were significantly higher than those in the normal oxygen control group (both P < 0.05). Wedelolactone pretreated mice had clear alveolar cavity and lower neutrophil infiltration and interstitial thickness than HALI group. Pathological scores (score: 0.43±0.02 vs. 0.75±0.02) and W/D ratio (4.56±0.12 vs. 6.23±0.34) were significantly lower than HALI group (both P < 0.05). Compared with the normal oxygen control group, the levels of SOD (kU/g: 26.41±4.25 vs. 78.64±3.95) and GSH (mol/g: 4.51±0.33 vs. 12.53±1.25) in HALI group were significantly decreased, while the levels of MDA (mmol/g: 54.23±4.58 vs. 9.65±1.96), TNF-α (μg/L: 96.32±3.67 vs. 11.65±2.03), IL-6 (ng/L: 163.35±5.89 vs. 20.56±3.63) and IL-1β (μg/L: 72.34±4.64 vs. 15.64±2.47) were significantly increased, and the protein expression of GPX4 (GPX4/β-actin: 0.44±0.02 vs. 1.00±0.09) was significantly decreased (all P < 0.05). Compared with the HALI group, the levels of SOD (kU/g: 53.28±3.69 vs. 26.41±4.25) and GSH (mol/g: 6.73±0.97 vs. 12.53±1.25) were significantly higher in the wedelolactone pretreatment group, and the levels of MDA (mmol/g: 25.36±1.98 vs. 54.23±4.58), TNF-α (μg/L: 40.25±4.13 vs. 96.32±3.67), IL-6 (ng/L: 78.32±4.65 vs. 163.35±5.89), and IL-1β (μg/L: 30.65±3.65 vs. 72.34±4.64) were significantly lower (all P < 0.05), and protein expression of GPX4 was significantly higher (GPX4/β-actin: 0.68±0.04 vs. 0.44±0.02, P < 0.05).@*CONCLUSIONS@#Wedelolactone attenuates HALI injury by regulating ferroptosis.

Role and mechanism of signal transducer and activator of transcription protein STAT1/3/5 in hyperoxia-induced acute lung injury / 中华危重病急救医学

Objective:To investigate whether signal transducer and activator of transcription (STAT1/3/5) have a protective effect on hyperoxia-induced acute lung injury (HALI) and its mechanism.Methods:Seventy C57BL/6J mice were randomly divided into five groups: normoxia control group, HALI group, and STAT1/3/5 inhibitor groups, with 14 mice in each group. The HALI model was established by exposure to more than 90% hyperoxia for 48 hours; three STAT inhibitor groups were pretreated by intraperitoneal injection of STAT1 inhibitor 40 mg/kg and STAT3 inhibitor 5 mg/kg, and STAT5 inhibitor 10 mg/kg for 1 week. Six blood samples were randomly collected from each group, and microRNA-21 (miR-21) expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Lung tissue of the sacrificed mice was obtained, and enzyme linked immunosorbent assay (ELISA) was used to detect the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β), superoxide dismutase (SOD), malonic dialdehyde (MDA), and matrix metalloproteinase 9 (MMP9). The water content of lung tissue was calculated. The pathological changes in lung tissue were observed under the light microscope, and the pathological score of lung injury was performed. Western blotting was used to detect the expression of phosphorylated STAT (p-STAT1, p-STAT3, p-STAT5) in lung tissue. The 7-day cumulative survival rates of the remaining 8 mice in each group were analyzed using Kaplan-Meier survival curves.Results:Under the light microscope, the alveolar structures in the HALI group and the STAT1 inhibitor group were destroyed, a large number of neutrophils (NEU) infiltrated in the alveoli and lung interstitium, which were thickened. The pathological score of lung injury and the water content of the lung tissue was significantly increased. In STAT3 inhibitor and STAT5 inhibitor groups, the alveolar cavity was clear, the degree of NEU infiltration and the thickness of lung interstitium were lower than those in HALI group, the pathological score of lung injury and the water content of lung tissue were significantly decreased, especially in STAT3 inhibitor group. Compared with the normoxia control group, the contents of TNF-α, IL-6, IL-1β, MDA, and MMP9, and the expression levels of p-STAT3 and p-STAT5 in the HALI group were significantly increased. In contrast, the content of SOD and the expression of miR-21 were significantly decreased. Compared with the HALI group, the contents of TNF-α, IL-6, IL-1β, MDA, and MMP9 in the STAT3 inhibitor group and STAT5 inhibitor group were significantly decreased. At the same time, the content of SOD and the expression of miR-21 were significantly increased, especially in STAT3 inhibitor group [TNF-α (μg/L): 42.53±3.25 vs. 86.36±5.48, IL-6 (ng/L): 68.46±4.28 vs. 145.00±6.89, IL-1β (μg/L): 28.74±3.53 vs. 68.00±5.64, MDA (μmol/g): 20.33±2.74 vs. 42.58±3.45, and MMP9 (ng/L): 128.55±6.35 vs. 325.13±6.65, SOD (kU/g): 50.53±4.19 vs. 22.53±3.27, miR-21 (2 -ΔΔCt): 0.550±0.018 vs. 0.316±0.037, all P < 0.05]. Kaplan-Meier survival curve analysis showed that the 7-day cumulative survival rates of the STAT3 inhibitor group and STAT5 inhibitor group were significantly higher than those of the HALI group [62.5% (5/8), 37.5% (3/8) vs. 12.5% (1/8), both P < 0.05]. Conclusion:Inhibition of STAT3 hyperactivation may suppress the inflammatory response, regulate oxidative stress, improve lung permeability through regulating the expression of miR-21, which exert lung protection in HALI.

Research progress of extracellular vesicle microRNA in acute lung injury / 中华危重病急救医学

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.

Advance in research of microRNA-21-5p regulate autophagy by targeting gene / 中华危重病急救医学

Autophagy is a dynamic process that degrades intracellular proteins and damaged organelles, and maintains environmental stability within the cell and provides good conditions for cell survival. Hyperoxic acute lung injury (HALI) is one of the serious complications of clinical oxygen therapy. The pathogenesis of HALI is still unclear. There are studies having shown that autophagy is involved in the pathogenesis of HALI. There are many pathway mechanisms that regulate autophagy, including phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway, mitogen-activated protein kinase/extracellular signaling-regulated protein kinase (MAPK/ERK) signaling pathway, adenosine 5'-monophosphate-activated protein kinase/unc-51 like autophagy activating kinase 1 (AMPK/ULK1) signaling pathway, transforming growth factor β (TGF-β) and forkhead box O1 (FoxO1) and Ras guanosine triphosphatease superfamily member Rab11a, each of which is referred to as microRNA-21-5p (miR-21-5p) target gene having a role in regulating autophagy activity in many diseases. In this paper, the above-mentioned signaling pathways of miRNA-21-5p target genes regulating autophagy were reviewed in order to find clues about the mechanism of miRNA-21-5p regulating autophagy in HALI and provide a theoretical basis for subsequent basic research.

Research progress on autophagic mechanism in acute lung injury/acute respiratory distress syndrome / 中国中西医结合急救杂志

Autophagy is a fundamental cellular process that plays a role in the turnover of sub-cellular organelles to protein. The activation of autophagy may prevent the pathophysilogical progression of oxidative stress-induced protein damage or organelle damage. The selective form of autophagy can maintain organelle renewal by degrading damaged proteins. In this article, the research progress on the mechanism of autophagy in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) was summarized in order to understand more deeply on this topic and arouse more researchers to pay attention to the role of autophagy in lung protection of ALI/ARDS.