Chitosan oligosaccharide ameliorates acute lung injury induced by blast injury through the DDAH1/ADMA pathway

Abstract

Objective: To investigate the protective effect of chitosan oligosaccharide (COS) on acute lung injury (ALI) caused by blast injury, and explore possible molecular mechanisms.

Methods: A mouse model of blast injury-induced ALI was established using a self-made explosive device. Thirty mice were randomly assigned to control, ALI and ALI + COS groups. An eight-channel physiological monitor was used to determine the mouse physiological index. Enzyme linked immunosorbent assay was used to measure serum inflammatory factors. Hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluorescence staining, real time-polymerase chain reaction and western blot assay were used to detect inflammatory reactions, oxidative stress and apoptosis.

Results: Mice were sacrificed 24 hours after successful model induction. Compared with the ALI group, the heart rate, respiration and PCO2 were significantly lower, but the PO2, TCO2 and HCO3- were significantly higher in the ALI + COS group. Compared to ALI alone, COS treatment of ALI caused a significant decrease in the wet/dry lung weight ratio, indicating a reduction in lung edema, inflammatory cell infiltration, levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-4, IL-6 and nuclear factor kappa B mRNA and protein expression were reduced and IL-10 mRNA and protein expression was increased (P < 0.05). COS significantly inhibited reactive oxygen species, MDA5 and IREα mRNA and protein expressions, cell apoptosis and Bax and Caspase-3 mRNA and protein expressions, and significantly increased superoxide dismutase-1 mRNA expression, and Bcl-2 and Caspase-8 mRNA and protein expression (all P<0.05). COS significantly increased dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein expression, and reduced ADMA and p38 protein expression (P< 0.05).

Conclusion: Blast injury causes inflammation, oxidative stress and apoptosis in the lung tissues of mice. COS has protective effects on blast injury-induced ALI, possibly by promoting DDAH1 expression and inhibiting ADMA and mitogen-activated protein kinase pathways.

Fig 1. COS effects on mouse weight, heart rate, respiratory rate and blood gas analysis.

The MPA2000 eight-channel physiological monitor was used to record pulmonary function. Lung function parameters were calculated by esophageal pressure, airway pressure and gas flow. After left femoral artery catheterization, connecting heart function analyzer and blood pressure sensor, mouse heart rate was continuously monitored. *P < 0.05, vs. control group; ^#P < 0.05, vs. the ALI group. COS: chitosan oligosaccharide; ALI: acute lung injury.

Fig 2. COS effects on the wet/dry weight ratio and pathological changes in the lung of mice.

*P < 0.05, vs. control group; ^#P < 0.05, vs. ALI group. COS: chitosan oligosaccharide; ALI: acute lung injury.

Fig 3. COS effects on inflammatory factor expression in the lung.

The expression of TNF-α, IL-1β, IL-4, IL-6 and IL-10 in serum were determined using ELISA kit. The expression of inflammatory factors was detected using real time-PCR and western blot. *P < 0.05, vs. control group; ^#P < 0.05, vs. ALI group. COS: chitosan oligosaccharide; ALI: acute lung injury; TNF: tumor necrosis factor; IL: interleukin.

Fig 4. COS effects on oxidative stress in the lung.

Oxidative stress in the lung was determined using immunofluorescence staining, real time-PCR and western blot. *P < 0.05, vs. control group; ^#P < 0.05, vs. ALI group. COS: chitosan oligosaccharide; ALI: acute lung injury.

Fig 5. COS effects on apoptotic protein expression in the lung.

Apoptotic protein expression in the lung was detected using TUNEL, real time-PCR and western blot. *P < 0.05, vs. control group; ^#P < 0.05, vs. ALI group. COS: chitosan oligosaccharide; ALI: acute lung injury; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Fig 6. COS effects on pathway protein expression in the lung.

Pathway protein expression in the lung was measured using immunofluorescence staining, real time-PCR and western blot. *P < 0.05, vs. control group; ^#P < 0.05, vs. ALI group. COS: chitosan oligosaccharide; ALI: acute lung injury.