Intranasal administration of Lactobacillus johnsonii attenuates hyperoxia-induced lung injury by modulating gut microbiota in neonatal mice

Abstract

Background: Supplemental oxygen impairs lung development in newborn infants with respiratory distress. Lactobacillus johnsonii supplementation attenuates respiratory viral infection in mice and exhibits anti-inflammatory effects. This study investigated the protective effects of intranasal administration of L. johnsonii on lung development in hyperoxia-exposed neonatal mice.

Methods: Neonatal C57BL/6N mice were reared in either room air (RA) or hyperoxia condition (85% O₂). From postnatal days 0 to 6, they were administered intranasal 10 μL L. johnsonii at a dose of 1 × 10⁵ colony-forming units. Control mice received an equal volume of normal saline (NS). We evaluated the following four study groups: RA + NS, RA + probiotic, O₂ + NS, and O₂ + probiotic. On postnatal day 7, lung and intestinal microbiota were sampled from the left lung and lower gastrointestinal tract, respectively. The right lung of each mouse was harvested for Western blot, cytokine, and histology analyses.

Results: The O₂ + NS group exhibited significantly lower body weight and vascular density and significantly higher mean linear intercept (MLI) and lung cytokine levels compared with the RA + NS and RA + probiotic groups. At the genus level of the gut microbiota, the O₂ + NS group exhibited significantly higher Staphylococcus and Enterobacter abundance and significantly lower Lactobacillus abundance compared with the RA + NS and RA + probiotic groups. Intranasal L. johnsonii treatment increased the vascular density, decreased the MLI and cytokine levels, and restored the gut microbiota in hyperoxia-exposed neonatal mice.

Conclusions: Intranasal administration of L. johnsonii protects against hyperoxia-induced lung injury and modulates the gut microbiota.

Keywords: Hyperoxia; Mean linear intercept; Microbiota; Probiotics; Vascular endothelial growth factor.

Fig. 1

Intranasal L. johnsonii administration increased the body weights and intestinal junctional protein expression in hyperoxia-exposed neonatal mice on postnasal day 7. Intranasal L. johnsonii or NS was administered to neonatal mice reared in RA or O₂ from postnatal days 0–6. A Birth body weights and body weights on postnatal day 7, and B representative Western blots and semiquantitative analysis of intestinal ZO-1 and occludin expression on postnatal day 7. n = 7–11. *p < 0.05, ***p < 0.001, two-way ANOVA followed by Bonferroni post hoc test

Fig. 2

Intranasal L. johnsonii administration improved alveolarization and increased VEGF expression and lung angiogenesis on postnatal day 7. A Representative lung histology and MLI on postnatal day 7. B Representative immunohistochemical staining for vWF (black arrow) and representative Western blots and semiquantitative analysis of VEGF and vascular density. Treatment with L. johnsonii significantly diminished the hyperoxia-induced increase in the MLI and significantly augmented the hyperoxia-induced decrease in VEGF protein levels and vascular density. n = 7–11. *p < 0.05, ***p < 0.001, two-way ANOVA followed by Bonferroni post hoc test

Fig. 3

Intranasal L. johnsonii administration decreased the hyperoxia-induced increase in lung cytokines. The mice reared in hyperoxia and treated with NS exhibited significantly higher lung IL-1, IL-6, and TNF-α levels compared with those reared in RA and treated with NS or L. johnsonii. Treatment with L. johnsonii significantly inhibited the hyperoxia-induced increase in lung IL-1 and IL-6 levels on postnatal day 7. n = 7–11. **p < 0.01, ***p < 0.001, two-way ANOVA followed by Bonferroni post hoc test

Fig. 4

Intranasal L. johnsonii administration–modulated gut microbiota in hyperoxia-exposed neonatal mice. A Bacterial composition at the phylum level. B Bacterial composition at the genus level. C α-diversity. D β-diversity. E Linear discriminant analysis. Intranasal L. johnsonii administration altered the bacterial composition and diversity of the gut microbiota in the O₂ + NS (n = 10), RA + NS (n = 7), RA + probiotic (n = 10), and O₂ + probiotic (n = 9) groups. Two-way ANOVA followed by Bonferroni post hoc test. PNR: RA + NS, PLR: RA + probiotic, PNH: O₂ + NS, PLH: O₂ + probiotic

Fig. 5

Effects of intranasal L. johnsonii administration on the lung microbiota. A Bacterial composition at the phylum level. B Bacterial composition at the genus level. C α-diversity. D β-diversity. E Linear discriminant analysis. Intranasal L. johnsonii administration altered the bacterial composition and diversity of the lung microbiota in the O₂ + NS (n = 10), RA + NS (n = 7), RA + probiotic (n = 10), and O₂ + probiotic (n = 9) groups. Two-way ANOVA followed by Bonferroni post hoc test. PNR: RA + NS, PLR: RA + probiotic, PNH: O₂ + NS, PLH: O₂ + probiotic

Fig. 6

Correlation heatmap of the dominant gut microbial genera, lung development, and lung injury parameters. The blue color indicates a positive correlation. The red color indicates a negative correlation

Fig. 7

Vitamin K2-related biosynthetic pathways of differentially abundant predicted metabolic pathways in the comparison between hyperoxia or L. johnsonii treatment (A) in intestine and (B) in lung. Abundance analysis was performed using ALDEx2 (*p < 0.05; **p < 0.01; ***p < 0.001)