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.
Gas explosions accidents in coal mining threaten public safety and social economy. The mortality associated with gas accidents reached 54.5% of the total coal industry-associated mortalities from 2010 to 2019 in China (
As a cavity and gas-bearing organ, the lung is affected by in gas explosion-induced blast injury. Thus it can be severely torn and fragmented by gas explosion-induced shockwaves (
AMP-activated protein kinase (AMPK), a major energy sensor at both cellular and whole-body levels (
Metformin, an activator of AMPK, is a key regulator of energy metabolism and balance, and it improves mitochondrial respiration, restores the mitochondrial life cycle by activating the protein kinase AMPK and inhibits oxidative stress through the upregulation of PGC1α and superoxide dismutase 1 in acute lung injury (
The present study aimed to explore the role of the AMPK/NOX2 pathway in gas blast-induced lung injury by designing experiments that simulated the effects of a gas explosion on a real roadway with a rat model, which provided evidence to demonstrate the pulmonary-protective effects of metformin and its specific molecular mechanism with respect to targeting and alleviating gas explosion-induced blast lung injury.
Metformin (MET) was purchased from Bristol-Myers Squibb Pharmaceuticals Ltd. The total antioxidant capacity (T-AOC) detection kit (cat. no. #A015-3-1) was purchased from the Nanjing Jiancheng Bioengineering Institute. The micro isocitrate dehydrogenase mitochondrial (ICDHm) assay kit (cat. no. #BC2165), micro α-ketoglutarate dehydrogenase (α-KGDH) assay kit (cat. no. #BC0715), phosphofructokinase (PFK) activity assay kit (#BC0535), ATP content assay kit (cat. no. #BC0305) were purchased from Beijing Solarbio Science & Technology Co., Ltd. TRIzol® was purchased from Ambion (Thermo Fisher Scientific, Inc.). The Pierce™ BCA protein assay kit and SuperScript III reverse transcriptase were purchased from Thermo Fisher Scientific, Inc. SDS-PAGE sample loading buffer was purchased from Beyotime Institute of Biotechnology. β-actin (cat. no. #AF7018), AMPK (cat. no. #AF6423) and p-AMPK antibodies (cat. no. #AF3423) were purchased from Affinity Biosciences, Ltd.; NOX2 antibody (cat. no. #A19701) and PGC-1α (cat. no. #A19674) antibody were purchased from ABclonal Biotech Co., Ltd.; and catalase (CAT; cat. no. #12980) antibody was purchased from Cell Signaling Technology, Inc. SYBR® GreenER qPCR SuperMix Universal was purchased from Invitrogen (Thermo Fisher Scientific, Inc.).
In total, 90 six-month-old male specific pathogen-free (SPF) Sprague-Dawley rats with an average weight of 200±20 g were purchased from Speifu (Beijing) Biotechnology Co., Ltd. and the animal license number was SCXK 2019-0010. The animals were housed in a SPF animal room and maintained under a controlled environment (24±1˚C temperature, relative humidity of 40-50% and 12 h light/dark cycle) for 1 week with adaptive feeding to ensure rats adaptation to the new environment and free diet before commencement of the experiments. The rats were randomly divided into three groups (n=30/group) as follows: Control group, gas explosion injury (BLI) group and gas explosion injury + metformin (BLI + MET) group. A total of 10 rats, randomly selected from each group, were euthanized on days 1, 3 and 7 after treatment. Rats were dissected and the lungs of rats were isolated and sectioned. The animal experiments were performed in accordance with Guide for the Care and Use of Laboratory Animals (
Preliminary experiments confirmed that the explosion flame range was 160 m (data not shown); thus, rats were placed 240 m away from the center of the explosion to ensure that they were only affected by shock waves. The explosion chamber, with a total volume of 100 m3 contained air with 9.3% methane gas, and the ignition energy was 20 J. Rats in the BLI and MET groups were deeply anesthetized with 1% sodium pentobarbital (50 mg/kg) and placed in a custom iron cage. After the explosion, rats of the BLI + MET group were administered metformin daily by intraperitoneal injection (10 mg/kg). A total of 10 rats from each group were randomly selected and euthanized on the 1st, 3rd and 7th day. Before collecting the blood, rats were deeply anesthetized as aforementioned. The rats were cervically dislocated after the blood was collected and the organs were retained after death was confirmed. Lung tissues were isolated and weighed. The right lower lung tissue was fixed in 4% paraformaldehyde at 25˚C for 24 h, and the remaining lung tissue was frozen in liquid nitrogen at -80˚C for further tests.
Rats were placed in the test room for at least 30 min for habituation. Indicators of lung function, peak inspiratory flow rate (PIFR) and minute ventilation (MV), were measured using a whole-body plethysmography system (Buxco Electronics, Inc.).
The lower lobe of right lung tissue was fixed with 4% formaldehyde at 25˚C for 24 h, dehydrated, embedded in paraffin, cut into 4-µm sections and kept at 60˚C for 1 h. Tissue was deparaffinized with xylene for twice 20 min and then rehydrated, stained with hematoxylin at 25˚C for 5 min and eosin at 25˚C for 3 min before histopathological observation. Overall, five visual fields were randomly selected for each section under a light microscope and scored based on the following four aspects: Alveolar wall thickness, congestion, hemorrhage and inflammatory cell infiltration. A five-point scale from 0 to 4 was used to generate a systematic scoring system for acute lung injury as follows: 0=No damage; l=mild damage; 2=moderate damage; 3=severe damage; and 4=very severe damage (
Lung tissues frozen at -80˚C were used for biochemical analyses using the aforementioned ICDHm, α-KGDH, PFK activity and ATP content assay kit according to the manufacturer's instructions. Approximately 30 mg of lung tissue was added to 300 µl of homologous extraction solution for ice-bath grinding and then centrifuged at 8,000-11,000 x g at 4˚C for 10-15 min. The supernatant was collected and placed on ice for further testing. Working reagents and the sample supernatant were added to each well of a 96-well plate and incubated for several minutes at 37˚C according to the manufacturer's instructions. Product formations were detected using a standard microplate reader (PerkinElmer, Inc.; EnSpire Multimode Plate Reader) at a specific absorbance wavelength (340 and 505 nm).
Fresh rat lung tissue was obtained and cut into cubes of ~1 mm3 to minimize mechanical damage, such as pulling, contusion and extrusion. The small lung tissue cubes were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer overnight at 4˚C and then washed in ice-cold PBS three times. The fixed samples were post-fixed in 1% osmium tetroxide in 0.1 M PBS overnight at 25±3˚C and rinsed in PBS as previously described. Next the samples were dehydrated, infiltrated, embedded in resin and polymerized overnight in an oven at 60˚C. The embedded tissue was cut into ultrathin sections of 60-80 nm using an ultrathin slicer (Leica UC7; Leica Microsystems GmbH). After staining with 2% uranium acetate saturated alcohol solution and 2% lead citrate solution (25˚C for 15 min each), the ultrastructures of cells were observed under a transmission electron microscope (HT7700; Hitachi, Ltd.) and the captured images were analyzed by Image J 1 (National Institutes of Health).
Blood was obtained from the abdominal aorta before the animals were euthanized. The collected blood samples were clotted at 25±3˚C for 30 min and then centrifuged at 4,000 x g at 4˚C for 15 min. The serum was removed and stored in the tube at -80˚C. Freshly prepared FeSO4-7H2O standard solutions were used; the remaining steps in the experiment were performed according to the instructions of the T-AOC detection kit.
Total RNA from lung tissues was extracted using TRIzol®. The extracted RNA was reverse-transcribed into cDNA using SuperScript III reverse transcriptase according to the manufacturer's instructions. All mRNA levels were detected using SYBR® GreenER™ qPCR SuperMix Universal. The cycling conditions were as follows: 95˚C Pre-denaturation for 1 min, 40 cycles of 95˚C for 15 sec, 60˚C for 15 sec and 72˚C for 30 sec. Finally, 65˚C for 1 min, 95˚C for 20 sec and 37˚C for 1 min. Fold-changes in mRNA expression levels were calculated using 2-ΔΔCq values (
Rat lung tissues were rapidly homogenized using an ultrasonic tissue disruptor (SCIENTZ-48; Ningbo Xinzhi Biotechnology Co., Ltd.) with cold RIPA extraction buffer (Beyotime Institute of Biotechnology) to ensure complete homogenization. Lung tissue remained on ice for 30 min, and then it was centrifuged at 15,000 x g at 4˚C for 15 min to obtain the supernatant. Protein concentration was measured using the Pierce™ BCA protein assay kit and denatured with SDS-PAGE sample loading buffer at 100˚C for 5 min. 30 ng denatured protein samples were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (30 min at 90 V, 90 min at 120 V), and electro-transferred to a 0.45 µm polyvinylidene difluoride membrane for 70 min at 300 mA. The membranes were incubated in blocking solution (5% skim milk) at 25±3˚C for 90 min, and subsequently incubated with the antibodies targeting the following proteins at 4˚C overnight: β-actin (1:3,000), AMPK (1:2,000), p-AMPK (1:2,500), NOX2 (1:2,000), PGC-1α (1:2,000) and CAT (1:2,000). The next day, the membrane was washed three times with 5% tris-buffered saline with tween for 30 min (10 min each) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3,000, cat. no. #S0001, Affinity Biosciences, Ltd.) for 2 h at 25±3˚C. Finally, the membranes were washed three times for 10 min each, and chemiluminescence detection was performed using the Amersham Image Quant800 (Cytiva) system. The images were analyzed by Image J 1 (National Institutes of Health).
All data were analyzed using SPSS 20.0 (IBM Corp.). Continuous variables of normally distributed were expressed as the mean ± SD, and significant differences between groups were determined using one-way ANOVA followed by Tukey's post hoc test. Ordinal variables were expressed as the median with interquartile range and were analyzed using Kruskal-Wallis test and Dunn's test. P<0.05 was considered to indicate a statistically significant difference.
In the present study, lung injuries were clearly observed in rats exposed to gas explosions. Compared with that in the control group, gas explosion exerted marked effects on the lungs of rats in the BLI group, characterized by evident lung bleeding points (yellow arrows) and lung alveolar collapse (the blue arrows) (
MET attenuates pathological and endothelial factor damage to lungs caused by gas explosion. The lungs of the rats in the control group had intact alveolar structures, thin, clear alveolar walls and no edema (
Pulmonary ATP levels were significantly lower in the BLI group compared with in the control group, and MET treatment significantly increased pulmonary ATP levels in gas explosion-induced rats lung injury (
Similarly, the enzymatic activities of rate-limiting enzymes α-KGDH and ICDHm in the TCA cycle (
The aforementioned results showed that MET alleviated pathological damage and regulated energy metabolism in the blast-injured lung of rats. Therefore, the present study detected the expression of AMPK and PGC-1α using western blotting and RT-qPCR to confirm the effect of MET on the mitochondrial protective pathway (
Ultrastructural changes in the lung tissue were observed using TEM (
Western blotting revealed that NOX2 levels in lung tissues were significantly increased in the BLI group compared with those in the control group, and MET treatment decreased NOX2 levels, the main source of ROS in acute lung injury (
The shock wave pressure caused by a gas explosion increases following multiple reflections, resulting in extensive damage and serious harm due to the complex structure of the mine. The high-temperature flame from a gas explosion can cause serious damage to the upper and lower respiratory tracts and alveolae (
The present study used an actual roadway to imitate a real gas explosion situation in a tunnel. There were bleeding points in the lungs and a large number of free red blood cells were observed under the microscope; this indicated that shock waves caused by a gas explosion could induce pulmonary hemorrhage and decreased MV and PIFR, indicating that a gas explosion in a real roadway environment could lead to a decrease in respiratory function and lung tissue damage in a rat model. The peak of inflammatory cell influx was related to the impairment of lung function. Pulmonary edema induced by inflammation often shows an enhanced respiration pause (
To investigate whether MET could mitigate gas explosion-induced blast injury, 10 mg/kg of MET was administered to rats in the present study. The results revealed that MET alleviated the pathological injury induced by exposure to a gas explosion. To demonstrate that MET could positively regulate the energy metabolism associated with injured alveolar cells in gas explosion, the changes in enzyme activity were measured. Under normal circumstances, >80% of ATP required by the human body is provided by oxidative phosphorylation (
In the present study, MET promoted the phosphorylation of AMPKα and increased the expression of PGC1α. In addition,
The results of the present study are consistent with the results aforementioned. Ultrastructural changes in lung tissue demonstrated the improvement in the structural stability of the mitochondria after MET treatment. In previous studies, considerable damage to the right lower lobe, mitochondrial abnormalities and the loss of a lamellar body structure have been observed after acute primary blast injury by an electron microscope. Moreover, the lesions noted in the lung may be progressive in the first 24 h after injury (
Finally, the present study demonstrated that MET downregulated oxidative stress caused by NOX2, which was activated in gas explosion-induced BLI. Oxidative stress is known to induce mitochondrial functional protein destruction or mtDNA damage (
On a temporal level, the damage to the lung function due to gas explosions is continuously aggravated. The results from the present experiment indicated that the situation was the most severe on day 3 after the injury. This is consistent with the pre-experimental results of our previous studies. However, between days 3 and 7 after the gas explosion, there was a weak tendency of abnormal molecular indicators to return to their normal levels, but this self-recovery was particularly limited. The present results provided evidence that if MET was administered as early as possible after a gas explosion-induced injury, it could reduce the persistent aggravation of intrapulmonary injury caused by a gas explosion. In summary, MET mitigated gas explosion-induced blast lung injuries through AMPK-mediated energy metabolism and NOX2-related oxidase pathway in rats.
In the present study, only one dose intervention group was used. Future experiments should use multiple dose groups of metformin to determine the best dose of treatment.
In conclusion, gas explosions can cause the energy metabolism abnormalities associated with acute lung injury and lead to the onset of oxidative stress in the lungs. MET activates AMPK and downregulates NOX2 expression. This maintains the mitochondrial structure, enhances intrapulmonary antioxidant capacity, promotes intrapulmonary ATP production and finally alleviates the extent of BLI induced by a gas explosion.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
MZ, WJR and SQY conceived the study and designed experiments. CJD and SH performed animal experiments. MZ, YZS and XWD performed the main experiments. SQY and WJR confirm the authenticity of all the raw data. NL, YG and LZ were responsible for data acquisition and analysis. WY and JC analyzed and interpreted data. MZ and SQY wrote and revised the manuscript. WY, JC, WJR and SQY were accountable for all aspects of the work in ensuring that questions are appropriately investigated and resolved. All authors read and approved the final manuscript.
The animal study protocol was approved by the Medical Ethics Committee of Xinxiang Medical University (approval no. XYLL-2019001; Jan 13, 2019).
Not applicable.
The authors declare that they have no competing interests.
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.
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
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.
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)
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.
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.