Heliox preconditioning exerts pulmonary protection effects on neonatal acute respiratory distress syndrome by inhibiting oxidative stress and apoptosis

Abstract

Objective: This study aimed to investigate whether heliox preconditioning (HePC) alleviates neonatal acute respiratory distress syndrome (ARDS) by inhibiting oxidative stress and apoptosis, and to explore its potential mechanism.

Methods: Blood samples and bronchoalveolar lavage fluid (BALF) were collected from rat pups were randomly divided into control group, sham group, ARDS group, ARDS + CaMKII^- group, ARDS + CaMKII⁺ group, and ARDS + HePC group. We also investigated the role of CaMKII by manipulating its expression in vitro. Inflammatory markers, oxidative stress, apoptosis and activation of signaling pathways were assessed using histological staining, ELISA, Western blotting, qRT-PCR, Ca²⁺, immunofluorescence staining, and flow cytometry.

Results: In vivo, HePC significantly reduced the expression of CaMKII, inhibited the activation of CaMKII/RyR2, ameliorated the LPS-induced lung histopathological changes in rat pups, reduced lung wet/dry ratios, ROS and MDA levels, and pro-inflammatory cytokine levels, and significantly increased the expression of antioxidant proteins (Nrf2, HO-1 and SOD) and reduced LPS-induced apoptosis. In vitro, overexpression of CaMKII increases oxidative stress and activates RyR2, leading to cytoplasmic Ca²⁺ overload and increased apoptosis. HePC can reverse the above reactions by inhibiting the expression of CaMKII.

Conclusion: HePC may attenuate oxidative stress through CaMKII and alleviate cytoplasmic Ca²⁺ overload by regulating CaMKII/RyR2, which inhibits apoptosis, exerting lung protection against ARDS.

Keywords: CaMK-II; anti-apoptosis; anti-oxidative stress; heliox preconditioning; neonatal acute respiratory distress syndrome.

FIGURE 1

HePC protects against LPS-induced lung injury and inflammation. (A) TNF-α and IL-1β levels in rat BALF. (B) TNF-α and IL-1β levels in the cell supernatant. (C) Lung wet/dry weight ratios were evaluated at 48 h post LPS treatment. (D) H&E staining of lung sections (×100). **P < 0.01, ***P < 0.001.

FIGURE 2

HePC ameliorates pulmonary inflammation via promoting antioxidant defenses. (A) TNF-α and IL-1β levels in rat BALF. (B) Quantitative ROS fluorescence intensity of each group in rats and cell models. (C) MDA levels in both rat BALF and cell supernatants. (D) Lung wet/dry weight ratios were evaluated at 48 h post LPS treatment. (E) HO-1, Nrf2 and SOD levels in rat lung. (F) H&E staining of lung sections (×100). *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 3

HePC Regulates the Expression of CaMKII. (A) Representative protein blot images of CaMKII and p-CaMKII in rat lung. Data are presented as mean ± SD (n = 5 per group). (B) Representative protein blot images of CaMKII and p-CaMKII in cells. Data are presented as mean ± SD (n = 5 per group). (C) Expression of CaMKII mRNA in rat lung and cell were detected by qRT-PCR. Values are means ± SD (n = 5 per group). (D) Immunofluorescence for p-CaMKII (green) in rat lung sections. Nuclei stained with DAPI (blue) (scale bar = 50 μm). *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 4

HePC Regulates Apoptosis through the CaMKII/RyR2 Signaling Pathway. (A) Expression of RyR mRNA in rat lung and cell were detected by qRT-PCR. Values are means ± SD (n = 5 per group). (B) Representative protein blot images in rat lung and cells. Data are presented as mean ± SD (n = 5 per group). (C) Calcium ionophore response in A549 lung carcinoma cells loaded with Fluo-4. Representative figure from nine experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 5

The effects of HePC on LPS-Induced Apoptosis. (A) Representative images of TUNEL assay staining to detect apoptotic cells in the lung sections of rat at 48 h. Scale bar = 50 μm. (B) The apoptosis of A549 cells was detected by flow cytometry in vitro. *P < 0.05, **P < 0.01.