Metformin mitigates gas explosion-induced blast lung injuries through AMPK-mediated energy metabolism and NOX2-related oxidation pathway in rats

Abstract

Gas explosions are a recurrent event in coal mining that cause severe pulmonary damage due to shock waves, and there is currently no effective targeted treatment. To illustrate the mechanism of gas explosion-induced lung injury and to explore strategies for blast lung injury (BLI) treatment, the present study used a BLI rat model and supplementation with metformin (MET), an AMP-activated protein kinase (AMPK) activator, at a dose of 10 mg/kg body weight by intraperitoneal injection. Protein expression levels were detected by western blotting. Significantly decreased expression of phosphorylated (p)-AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and metabolic activity were observed in the BLI group compared with those in the control group. However, the mitochondrial stability, metabolic activity and expression of p-AMPK and PGC1α were elevated following MET treatment. These results suggested that MET could attenuate gas explosion-induced BLI by improving mitochondrial homeostasis. Meanwhile, high expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX2) and low expression of catalase (CAT) were observed in the BLI group. The expression levels of NOX2 and CAT were restored in the BLI + MET group relative to changes in the BLI group, and the accumulation of oxidative stress was successfully reversed following MET treatment. Overall, these findings revealed that MET could alleviate BLI by activating the AMPK/PGC1α pathway and inhibiting oxidative stress caused by NOX2 activation.

Keywords: AMP-activated protein kinase; blast lung injury; gas explosion injury; metformin; mitochondria; nicotinamide adenine dinucleotide phosphate oxidase.

Figure 1

Gas explosions cause lung tissue damage and loss of pulmonary function. (A and B) Changes in lung appearance and pulmonary function measured using (C) MV and (D) PIFR. Yellow arrow, bleeding; blue arrow, alveolar collapse. n=8. ^*P<0.05 compared with the control group. MV, ventilation per minute rate; PIFR, peak inspiratory flow rate; BLI, blast lung injury.

Figure 2

MET reduces pathological and endothelial factor damage to lungs caused by gas explosions. (A) Hematoxylin and eosin staining of lung tissue in each group at 1, 3 and 7 days after gas explosion (magnification, x200; scale bar, 100 µm). (B) Acute lung injury score (from 0 to 4). n=6. (C) Expression of RAGE in lung tissue of rats, measured using RT-qPCR. The relative quantitative expression of RAGE mRNA was normalized to that of GAPDH. n=6. ^*P<0.05 compared with control group; ^#P<0.05 compared with the BLI group. RT-qPCR, reverse-transcription-quantitative PCR; RAGE, renal tumor antigen 1; BLI, blast lung injury; MET, metformin.

Figure 3

MET regulates the energy metabolic disturbances caused by gas explosions. (A) Changes in ATP content in lung tissue. Changes in (B) α-KGDH, (C) ICDHm and (D) PFK activity in lung tissue. n=8. ^*P<0.05 compared with the control group; ^#P<0.05 compared with the BLI group. ICDHm, micro isocitrate dehydrogenase mitochondrial; α-KGDH, micro α-ketoglutarate dehydrogenase; PFK, phosphofructokinase; BLI, blast lung injury; MET, metformin.

Figure 4

MET activates the AMPK/PGC1α mitochondrial protection pathway. (A) Expression of p-AMPK, t-AMPK and PGC-1α. Protein levels of (B) p-AMPK, (C) PGC-1α and (D) p-AMPK/t-AMPK were quantified using Image J Software. n=8. RT-qPCR analysis of the relative quantitative expression levels of (E) PGC-1α and (F) AMPK and (G) TFAM in lung tissues of rats, and the relative quantitative expression of each gene was normalized to that of GAPDH. n=6. ^*P<0.05 compared with the control group; ^#P<0.05 compared with the BLI group. RT-qPCR, reverse-transcription-quantitative; BLI, blast lung injury; MET, metformin; p-, phosphorylated; t-, total; AMPK, AMP-activated protein kinase; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; TFAM, transcription factor A of mitochondrial.

Figure 5

MET maintains mitochondrial morphological stability. Representative images of lung tissue as observed using transmission electron microscopy on 1, 3 and 7 days after the gas explosion. Images were obtained with TEM (magnification, x8,000; scale bar, 1.0 µm). MC, mitochondria; LB, lamellate bodies; BLI, blast lung injury; MET, metformin.

Figure 6

MET downregulates NOX2-induced oxidative stress caused by gas explosions. (A) Protein expression levels of NOX2 and CAT. Protein expression levels of (B) NOX2 and (C) CAT were quantified. n=8. RT-qPCR analysis for the relative quantitative expression of (D) GP91 and (E) CAT in lung tissue of rats, and the relative quantitative expression was normalized to that of GAPDH. n=6. (F) Changes in the total antioxidant capacity (T-AOC) in the serum of rats with gas explosion-induced injury. n=6. ^*P<0.05 compared with the control group; ^#P<0.05 compared with the BLI group. RT-qPCR, reverse-transcription-quantitative; MET, metformin; NOX2, nicotinamide adenine dinucleotide phosphate oxidase; CAT, catalase; GP91, gp91 phox; T-AOC, total antioxidant capacity; BLI, blast lung injury.