AMPK/MTOR/TP53 Signaling Pathway Regulation by Calcitonin Gene-Related Peptide Reduces Oxygen-Induced Lung Damage in Neonatal Rats through Autophagy Promotion.

Our previous studies indicated that calcitonin gene-related peptide (CGRP) alleviates hyperoxia-induced lung injury and suggested the possible involvement of autophagy in this process. Herein, we aimed to further explore the potential involvement of tumor protein p53 (TP53) and autophagy in the mode of action of CGRP against hyperoxia-induced lung injury in vitro and in vivo. The study conducted tests on type II alveolar epithelial cells (AECII) and rats that were subjected to hyperoxia treatment or combined treatment of hyperoxia with CGRP, CGRP inhibitor, rapamycin (an autophagy agonist), 3-methyladenine (3-MA, an autophagy inhibitor), TP53 silencing/inhibitor (pifithrin-α), or expression vector/activator (PRIMA-1 (2,2-bis(hydroxymethyl)-3-quinuclidinone)) and their corresponding controls. We found that oxidative stress, apoptosis, and autophagy were all increased by hyperoxia treatment in vitro. However, treating AECII cells with CGRP reversed hyperoxia-induced oxidative stress and apoptosis but further promoted autophagy. In addition, the combined treatment with rapamycin or TP53 silencing with CGRP promoted the effect of CGRP, while contrary results were obtained with combined therapy with 3-MA or TP53 overexpression. In vivo, the number of hyperoxia-induced autophagosomes was promoted in the lung tissue of neonatal rats. Furthermore, hyperoxia increased the expression levels of AMP-activated protein kinase (AMPK) alpha 1 (also known as protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1)) but inhibited TP53 and mechanistic target of rapamycin (MTOR); these expression trends were regulated by CGRP treatment. In conclusion, we showed that CGRP can attenuate hyperoxia-induced lung injury in neonatal rats by enhancing autophagy and regulating the TP53/AMPK/MTOR crosstalk axis.

Calcitonin Gene-Related Peptide Attenuates Hyperoxia-Induced Oxidative Damage in Alveolar Epithelial Type II Cells Through Regulating Viability and Transdifferentiation.

As a stem cell of alveolar epithelium, the physiological status of alveolar epithelium type II cells (AECII) after hyperoxia exposure is closely related to the occurrence of hyperoxia-induced lung injury and the restoration of normal morphological function of damaged alveolar epithelium. However, the relevant mechanisms involved are not very clear. Therefore, this study aimed to explore the effect of calcitonin gene-related peptide (CGRP) on AECII exposed to hyperoxia and its potential mechanisms. The AECII viability was detected using MTT assay. The malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected by spectrophotometry. The transdifferentiation capacity of AECII was evaluated by flow cytometry. The expression levels of Notch1, Hes, HERP, and AECII markers were detected using immunohistochemistry and/or RT-qPCR or immunofluorescence. ELISA was used for the determination of inflammatory markers. The results showed that CGRP significantly promoted cell viability, and markedly suppressed hyperoxia-induced transdifferentiation of AECII; these biological alterations were coincided with decreased MDA level, increased SOD activity, and activated Notch signaling pathway (upregulated expression levels of Notch1, Hes, and HERP). Notably, the in vitro effects of CGRP on Notch signaling pathway were further investigated in animal model, and the HE staining results showed that CGRP reduced in vivo oxidative injury and inflammation in hyperoxia-treated AECII through the promotion of structural and functional regeneration, accompanied by elevated Notch1 expression and activated Notch signaling cascade as shown by immunohistochemistry and QPCR, respectively. Immunohistochemistry of APQ-5 and SPC indicated that CGRP reversed the transdifferentiation of AECIIs in vivo. Our current results were consistent across both in vitro and in vivo settings, and provide a new direction for the prevention and treatment of bronchopulmonary dysplasia (BPD).

Efficacy of human immunoglobulin injection and effects on serum inflammatory cytokines in neonates with acute lung injury.

The present study aimed to explore the efficacy of intravenous immunoglobulin (IVIG) injection in neonates with acute lung injury (ALI) and assess its effects on serum inflammatory cytokine levels. The research subjects were 140 neonates with ALI who were evenly distributed into a control group (COG) and a study group (STG). The COG patients were treated routinely, whereas patients in the STG were administered IVIG in addition to the standard treatment received by the COG. The arterial partial pressure of oxygen (PaO2), PaO2/fraction of inspired oxygen (FIO2), mechanical ventilation time and hospitalization time were compared between the two groups. ELISA was used to determine the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the patients before treatment and at 12, 24 and 36 h after treatment. The Kaplan-Meier method was used to analyze the survival of the patients, including their survival for 30 days after treatment. The patients were divided into high and low cytokine expression groups based on their mean expression levels of serum IL-6 and TNF-α before treatment. After treatment, PaO2 and PaO2/FiO2 were significantly higher and mechanical ventilation and hospitalization time were reduced in the STG in comparison with the COG (all P<0.001). At 12, 24 and 36 h after treatment, serum IL-6 and TNF-α levels in the STG were lower than those in the COG (both P<0.05). The 30-day survival rate after treatment was not significantly different between the two groups (P>0.05). The 30-day survival rate in the high IL-6 and high TNF-α expression COG was lower than that in the low IL-6 and low TNF-α expression COG (both P<0.05). The results of the present study indicate that IVIG may improve pulmonary gas exchange, shorten the course of disease and reduce the inflammatory response in neonates with ALI.

Knockdown of Long Noncoding RNAs of Maternally Expressed 3 Alleviates Hyperoxia-Induced Lung Injury via Inhibiting Thioredoxin-Interacting Protein-Mediated Pyroptosis by Binding to miR-18a.

Long-term hyperoxia exposure may cause lung damage with characteristic inflammation. Long noncoding RNA of maternally expressed 3 (MEG3) is up-regulated in lung tissues exposed to hyperoxia; however, the underlying mechanism is unclear. Hyperoxia-induced cells and mouse models were used to study these mechanisms. Molecular assays were used to detect cell viability, cytotoxicity, and expression of miR-18a, MEG3, and inflammatory cytokines. The interaction among MEG3, miR-18a, and thioredoxin-interacting protein (TXNIP) was verified; and pyroptosis-related proteins were analyzed. The in vivo model was established by exposing MEG3 knockdown mice to hyperoxia. Hematoxylin and eosin staining was used to assess pathologic alterations of lung tissues. Hyperoxia suppressed cell viability, induced cell damage, and exacerbated the secretion of IL-1ß and IL-18. Hyperoxia inhibited miR-18a, with increased expression of MEG3, TXNIP, and nonobese diabetic-like receptor family pyrin domain containing 3 (NLRP3). MEG3 aggravated TXNIP expression by binding to miR-18a. Knockdown of MEG3 rescued hyperoxia-induced pyroptosis by up-regulating miR-18a. Furthermore, knockdown of MEG3 inhibited NLRP3 inflammasome activity and caspase-1 signaling by miR-18a. In vivo knockdown of MEG3 and overexpression of miR-18a relieved hyperoxia-induced lung injury via restraining NLRP3 inflammasome-mediated pyroptosis, whereas miR-18a inhibition reversed these effects. In conclusion, knockdown of MEG3 inhibits pyroptosis to alleviate hyperoxia lung injury by suppressing NLRP3 inflammasome and caspase-1 signaling via regulating miR-18a-TXNIP axis.