Donor lung inflation with hydrogen gas during cold storage alleviates energy metabolism disorder in lung grafts by protecting mitochondrial function in rats

doi:10.21203/rs.3.rs-1627962/v1

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Donor lung inflation with hydrogen gas during cold storage alleviates energy metabolism disorder in lung grafts by protecting mitochondrial function in rats

https://doi.org/10.21203/rs.3.rs-1627962/v1

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Background

Lung inflation with hydrogen improves the quality of donor lungs by mitigating ischemia-reperfusion injury. However, the underlying mechanism of hydrogen remains uncertain. We investigated whether the protective effects of lung inflation with 3% hydrogen during the cold ischemia phase is related to affecting energy metabolism in lung grafts of rats.

Method

Adult male Wistar rats were randomized into four groups: sham group (underwent thoracotomy without lung transplantation), control group (donor lungs were deflated at 4 for 2 hours), oxygen group (donor lungs were inflated with 40% oxygen mixed gas for 2 hours during cold storage), and hydrogen group (inflating the donor lungs with 3% hydrogen + 40% oxygen containing mixed gas). Orthotopic left lung transplantation was performed in control group, oxygen group, and hydrogen group, followed by 2 hours of reperfusion. Lung grafts were assayed for static compliance, histological, lactic acid content, pyruvic acid content, ATP content, mitochondrial respiratory complex I to IV activity, and reactive oxygen species content.

Results

Compared with the control group and oxygen group, hydrogen group reduced pyruvate and lactic acid contents and increased adenosine triphosphate contents in lung grafts. Furthermore, hydrogen improved mitochondrial morphology, increased mitochondrial complex enzyme I, II, and III activity, and reduced reactive oxygen species contents in lung grafts when comparing with the control group and the oxygen group. The severity of pulmonary injury, static compliance, and the PaO₂/FiO₂ of lung grafts in the hydrogen group and the oxygen group were comparable.

Conclusion

Donor Lung inflation with 3% hydrogen during cold storage is associated with reduced anaerobic metabolism level and improvements of mitochondrial morphology and function in lung grafts.

lung transplantation

hydrogen

ischemia-reperfusion injury

energy metabolism

mitochondria

lung inflation

Improving donor quality remains one of the urgent needs to expand the donor pool. The quality of donor preservation determines the severity of ischemia-reperfusion injury (IRI) and affects allograft outcomes [1, 2]. Many strategies have been used to attenuate donor lung injury prior to lung transplantation (LTx), including hypothermic preservation, lung preservation solution, and protective gas [3]. Among them, lung inflation with gas can attenuate graft injury and improve pulmonary function after LTx, although the protective mechanisms of most kinds of gas require investigation [4, 5].

As an antioxidant gas, molecular hydrogen can selectively scavenge cytotoxic oxygen radicals [6]. Due to its antioxidative, anti-inflammatory and antiapoptotic properties, hydrogen exhibits efficacies in IRI models of multiple organs, including myocardial renal, hepatic, and lung IRI [7], [8]. Hydrogen is currently under investigation for clinical applications, but the primary molecular target of hydrogen remains unknown [9].

Mitochondria are one of the main sources of free reactive oxygen species (ROS). Oxidative stress resulting from excessive free radical production may affect mitochondrial integrity. Hydrogen can pass through the membranous structures of cells and organelles and act on subcellular structures, such as mitochondria and the endoplasmic reticulum [10, 11]. Hydrogen-rich saline was shown to increase the mitochondrial membrane potential (MMP) and significantly attenuate mitochondrial permeability transition pore (mPTP) opening in cerebral I/R rats [12]. Mitochondria are the major energy sources of the cell, producing ATP through oxidative phosphorylation. Energy metabolism disorders, especially high-energy phosphate and glycogen deficiency during cold storage, are one of the main causes of IRI [13]. However, the impact of hydrogen inflation during the cold preservation phase on energy metabolism and mitochondria in lung grafts remains unclear.

We hypothesized that donor lung inflation using hydrogen during the cold storage period exerts protective effects on lung grafts by improving energy metabolism disorders through preservation of mitochondrial structure and function.

Animals

Adult male Wistar rats (240–300 g) were obtained from the Animal Center of Harbin Medical University, Harbin, China, under production license SCXK (black) 2011006. All research protocols in this study were approved by the Institutional Animal Care and Use Committee of Harbin Medical University.

Donor preparation and groups

Donor rats were anesthetized and ventilated via tracheostomy (respiratory rate, 50–60/min; positive end-expiratory pressure, 2 cm H₂O; and tidal volume, 10 mL/kg) with 40% oxygen (O₂) and 60% nitrogen (N₂) for 10 min (Harvard Apparatus, South Natick, MA). Five minutes after administration of heparin (1000 U/kg) through the tail vein, the donor rats were sacrificed by exsanguination and subjected to thoracotomy. Then, the donor lungs were perfused with 20 mL of 4°C low-potassium dextran solution (prepared by Harbin Medical University according to previous studies) at a pressure of 20 cm H₂O.

During the cold ischemic period, donor lungs were randomly divided into 4 groups (n = 8): the sham group, the control group, oxygen inflation (O₂) group, and hydrogen inflation (H₂) group. The rats in the sham group underwent left thoracotomies without LTx, and the left lung was extracted after 2 hours of ventilation. Donor lungs in the control group were deflated without gas. In the O₂ and H₂ groups, donor lungs were statically inflated with 40% O₂ + 60% N₂ and 3% H₂ + 40% O₂ + 57% N₂, respectively (Li Ming Gas Corporation, Harbin, China) at 10 mL/kg. The trachea was sealed, and the inflating gas was refreshed every 20 minutes. After stored in 4° C for 2 hours, the right lung lobule was frozen and stored for testing. The left lungs were extracted for LTx.

LTx

Recipients were ventilation followed the same protocol as donors before sacrifice. Left thoracotomy through the fourth intercostal space was performed, followed by orthotopic left LTx using a cuff technique [14]. Two hours after reperfusion, heparinized recipients were sacrificed by exsanguination to extract the left lung.

Blood gas analysis

Arterial blood samples were measured 10 min before transplantation (T0) and 10 min (T1), 30 min (T2), 60 min (T3), 90 min (T4), and 120 min (T5) after reperfusion using a conventional analyzer (Rapid Lab 348, Bayer, Medfield, USA). In addition, 2 hours after reperfusion, left pulmonary venous blood was collected for pulmonary venous oxygen tension (PvO₂)/FiO₂ measurement.

Static compliance of the lung grafts

Median thoracotomy was performed immediately after sacrifice. Then, the lung grafts were connected to a homemade apparatus to obtain pressure-volume (P-V) curves. The lungs were incrementally inflated to 30 cm H₂O and then deflated to 0 cm H₂O in a stepwise manner with 1 min of equilibration at 5-cm H₂O intervals each time. The volume was corrected for gas compression in the apparatus [15].

Histopathologic analysis

Formalin-fixed, paraffin-embedded sections of the lung grafts were stained with hematoxylin and eosin. The assessment was based on 5 histological features: neutrophil infiltration, airway epithelial cell damage, interstitial edema, hyaline membrane formation, and hemorrhage [16]. Each item was graded on a 5-point scale according to lung injury severity (normal = 0, minimal change = 1, slight change = 2, moderate change = 3, severe change = 4). Each standard lung injury score (LIS) was recorded. Pathologists who were blinded to the study evaluated the histological changes.

Transmission electron microscopy (TEM)

A 2-mm portion of lung tissue was immediately fixed in 3% glutaraldehyde, and mitochondria in left lung type II alveolar epithelial cells in resin-embedded sections were imaged using an electron microscope (H-7650, Hitachi, Tokyo, Japan). The level of mitochondrial damage was assessed using the Flameng scoring standard [17]. The evaluation criteria were as follows: 0, normal structure, complete particles; 1, normal structure, loss of particles; 2, mitochondrial swelling but a clear matrix; and 3, sputum rupture, matrix concentration, extensive destruction, and membrane rupture.

Detection of inflammatory cytokines

Venous blood samples from graft recipients were centrifuged at 6,000 rpm for 5 min for plasma collection. Interleukin (IL)-8, IL-10, and tumor necrosis factor-α (TNF-α) in serum were detected by enzyme-linked immunosorbent assay (ELISA) kits (Boster, wuhan, china).

Measurements of the energy metabolism level

A lactic acid content assay kit, pyruvic acid content assay kit, and ATP content assay kit (Solarbio, Beijing, China) were used to measure the lactic acid level, pyruvic acid content, and ATP level in lung grafts, respectively, following the manufacturer’s instructions.

Analysis of the ROS content in lung grafts

Cryosections from lung grafts were stained with dihydroethidium (DHE) (Beyotime, Shanghai, China) at 37°C for 30 min in a light-protected incubator. After washing with PBS three times (5 min each time), slides were cover slipped and fixed with an anti-fluorescent quencher. Images were taken with a fluorescence microscope at an excitation wavelength of 490 nm and an emission wavelength of 590 nm. The exposure time was 30 milliseconds. DHE fluorescence intensity was quantified by ImageJ (v1.33, NIH, Bethesda, MD, United States).

Lung mitochondrial complex activity determination

The right lung of the donor and the left lung graft were homogenized to extract mitochondrial protein according to the instructions of a mitochondrial isolation kit (Beyotime, Shanghai, China). The mitochondrial protein concentration was detected, and mitochondrial respiratory complex I to IV activity was measured according to mitochondrial respiratory chain complex activity assay kits (Solarbio, Beijing, China). Fluorescence was detected using a fluorescence microplate reader (Infinite M200Pro, Tecan, Switzerland).

Statistical analysis

All data are expressed as the mean value ± standard error of measurement (SEM). Group comparisons were analyzed using one-way analysis of variance (ANOVA) followed by the Student-Newman–Keuls test or the Kruskal–Wallis test. Repeated measurement data were analyzed by repeated-measures ANOVA. All statistical analyses were performed using SPSS version 22.0. P < 0.05 was considered statistically significant.

Gas Inflation Ameliorated Lung Graft Dysfunction

Improvement in gas change function with increased oxygenation index (arterial oxygen partial pressure [PaO₂]/inspired oxygen fraction [FiO₂]) after LTx was observed in lung inflation treated mice, relative to control group. The PaO₂/FiO₂ ratios in the O₂ group and H₂ group were significantly higher than that in the control group after reperfusion. No significant difference in the PaO₂/FiO₂ ratio was found between the O₂ group and H₂ group (Table 1).

Table 1

PaO₂/FiO₂ values in each time point (mean ± SD, n = 8).
	Group	T₀	T₁	T₂	T₃	T₄	T₅
PaO₂/FiO₂ (mm Hg)	Sham	443 ± 16	442 ± 8	441 ± 12	437 ± 12	440 ± 12	443 ± 17
	Control	431 ± 23	375 ± 15*	376 ± 20*	371 ± 17*	356 ± 18*	343 ± 19*
	O₂	437 ± 23	395 ± 18*^#	389 ± 36*^#	385 ± 20*^#	379 ± 27*^#	381 ± 18*^#
	H₂	432 ± 31	399 ± 18*^#	386 ± 27*^#	386 ± 28*^#	384 ± 27*^#	383 ± 18*^#
PaO₂/FiO₂ values were decreased gradually after reperfusion in the O₂ group and the H₂ group were attenuated in the H₂ group further. PaO₂/FiO₂, arterial oxygen partial pressure [PaO₂]/inspired oxygen fraction (FiO₂). T₀-T₅ represented the following time points: baseline before transplantation, and 10 min, 30 min, 60 min, 90 min, 120 min after reperfusion. ^*P < 0.05 vs. sham group; ^#P < 0.05 vs. control group.

The PvO₂/FiO₂ ratios in the control group (294 ± 32 mm Hg), O₂ group (358 ± 37 mm Hg) and H₂ group (397 ± 29 mm Hg) were lower than that in the sham group (443 ± 17 mm Hg), and the PvO₂/FiO₂ ratio in the H₂ group was significantly higher than that in the O₂ group (P < 0.05).

In addition, lung inflation improved the recipient lung static compliance (Fig. 1). At a pressure of 30 cm H₂O, the volumes in the sham group, control group, O₂ group, and H₂ group were 23.12 ± 4.64 ml/kg, 12.46 ± 2.82 ml/kg, 13.2 ± 2.95 ml/kg, and 16.81 ± 3.08 ml/kg, respectively. The static compliance values from the static P-V curve were obviously higher in the H₂ group and O₂ group than in the control group (P < 0.05). Static compliance in the H₂ group was higher than that in the O₂ group, although the difference was not statistically significant.

Gas inflation improved the histopathology scores of lung grafts

LTx induces progressive histologic damage, including moderate to severe edema in the alveolar septum and interstitium, clear membrane formation and extensive alveolar hemorrhage. The lung grafts in the O₂ group and H₂ group displayed a relatively intact alveolar structure with mild edema and less leukocyte infiltration. Accordingly, LISs were significantly lower in O₂ and H₂ group than control group. LISs were comparable between the O₂ group and the H₂ group (Fig. 2).

Hydrogen inflation alleviated the inflammatory response in recipients

Compared with control treatment, oxygen or hydrogen inflation caused a notable reduction in the serum levels of IL-8 and TNF-α. The serum levels of IL-8 and TNF-α in the H₂ group were significantly lower than those in the O₂ group. The serum levels of IL-10 were higher in the O₂ and H₂ groups than in the control group. The H₂ group showed notably higher IL-10 levels than those in the O₂ group (Table 2).

Table 2

The indices of inflammatory response in each group (mean ± SD, n = 8).
Group	IL-8 (pg/ml)	TNF-α (pg/ml)	IL-10 (pg/ml)
Sham	256 ± 32.8	217 ± 28.3	242 ± 29.9
Control	499 ± 29.7*	544 ± 59.2*	30 ± 8.4*
O₂	407 ± 18.4*^#	423 ± 49.9*^#	123 ± 28.7*^#
H₂	333 ± 28.5^#&	329 ± 51.1^#&	187 ± 37.8^#&
IL, Interleukin; TNF-α, tumor necrosis factor-α. *P < 0.05 vs. sham group. ^#P < 0.05 vs. control group. ^&P < 0.05 vs. O₂ group.

The energy metabolism level in lung grafts

Elevated lactic acid and pyruvic acid production resulting from LTx were prevented in H₂ group and O₂ group. Furthermore, a notable reduction in lactic acid and pyruvic acid levels were observed in H₂ group compared with the O₂ group. Besides, in the H₂ group and the O₂ group, gas inflation treatment prevented the I/R-induced reduction in the ATP content compared with the control group. Additionally, ATP levels were significantly higher in the H₂ group than in the O₂ group (Fig. 3).

Analysis of the ROS content in lung tissue

ROS levels were detected at the end of cold storage and 2 h after reperfusion. At the end of cold ischemia phase (CIP), the ROS content in the non-transplanted right donor lung was significantly increased in O₂ and H₂ groups compared with the control group. The ROS content in the H₂ group was slightly lower than that in the O₂ group but with no statistical significance (Fig. 4). However, 2 hours after reperfusion, the situation changed significantly. In O₂ group and H₂ groups, gas inflation markedly inhibited the increase in ROS contents. The application of hydrogen in the H₂ group substantially decreased the ROS content compare with the O₂ group (Fig. 5).

Effects of hydrogen gas inflation on the structure and function of mitochondria

As shown in Fig. 6, mitochondria in the control group were visibly swollen and vacuolated, the lamellar corpuscle structure fused or disappeared, part of the crest broke or disappeared, the matrix was lighter, and the crest structure disappeared. Mitochondrial damage in lung grafts was alleviated in both the O₂ group and H₂ group. The improvement in mitochondrial morphology was more obvious in the H₂ group than in the O₂ group.

Compared with those in the sham group, the mitochondrial complex enzyme I, II, III, and IV activities in the control group, O₂ group, and H₂ group were significantly decreased. In the H₂ group and the O₂ group gas inflation treatment restored the decreased mitochondrial complex enzyme I, II, and III activities after LTx. Moreover, a notable increase in mitochondrial complex enzyme I activity was observed in H₂ group compared to the O₂ group. However, the promotional effects of hydrogen on mitochondrial complex enzyme II, III, and IV activities were not statistically significant compared with those of oxygen treatment (Fig. 7).

In this study, we investigated whether hydrogen using in the way of donor lung inflation in the CIP can protect mitochondria and mitigate energy metabolism dysfunction in lung grafts. The results showed that 40% oxygen lung inflation mitigated the mitochondrial damage resulting from I/R and restored mitochondrial complex enzyme I activity. Additionally, lung inflation with oxygen decreased lactic acid and pyruvic acid contents and increased ATP production in lung grafts. The addition of 3% hydrogen further improved all the parameters mentioned above and notably reduced ROS production.

In contrast to solid organs, the lung contains gas in alveoli. The conditions of alveoli during cold preservation, including gas volume, gas composition, and pressure, are important factors affecting the quality of donor lungs. Conflicting effects of hyperinflation or hypoinflation conditions during storage on pulmonary functions have been reported [18, 19]. However, the deflation state contributes to alveolar cell and alveolocapillary membrane damage and subsequently results in mechanical injury owing to shear stresses during re-expansion [20]. Fukuse T et al. found that pulmonary storage at 50% or 100% FiO₂ could induce mitochondrial dysfunction and promote lipid peroxidation [21]. As shown by Haniuda M et al., 100% oxygen inflation during 24-hour preservation may increase pulmonary capillary permeability in rabbits, and optimal preservation was achieved by room air inflation [22]. Although there is controversy about the optimum O₂ concentration for lung inflation during CIP, high O₂ concentrations might induce the production of oxygen free radicals, increasing the risk of PGD during reperfusion [23]. Thus, most groups inflate donor lungs with a FiO₂ of 50%, and to 50% of total lung capacity during pulmonary preservation [24, 25].

In the present study, compared with atelectatic lungs, lungs inflated with 40% oxygen during the CIP showed mitigated allograft dysfunction, decreased systemic inflammatory responses, and attenuated lung IRI after LTx. The addition of 3% hydrogen further enhanced the effect of 40% oxygen lung inflation, which is consistent with our previous findings [26]. Hydrogen treatment could also restrain the inflammatory response in lung tissues. The administration of 3% hydrogen during cold storage was observed to reduce the expression of the proinflammatory cytokines TNF-α, IL-6, and ICAM-1 in rat pulmonary microvascular endothelial cells in an LTx model [27].

Inconsistent with our previous study, the addition of hydrogen did not significantly improve the static compliance of lung grafts and the PaO₂/FiO₂ ratio compared with oxygen inflation alone, although the trend showed an improvement [26]. The reason may lie in the insufficient sample size. These results may suggest that the protective effect of hydrogen is not sufficiently powerful to protect against LTx-induced pulmonary dysfunction. Interestingly, the PvO₂/FiO₂ ratio, a more directly index to the pulmonary oxygenation function, was improved by hydrogen addition. Hydrogen seems to exert beneficial effects in a concentration-dependent manner in chronic obstructive pulmonary disease and traumatic brain injury [28, 29]. The influence of different hydrogen concentrations for lung inflation on lung grafts remains to be explored. Considering the application safety, here we used the most widely used concentration of 3% hydrogen.

During cold storage, anaerobic metabolism alone could not meet pulmonary energy consumption requirements, which might be associated with a more severe IRI. Donor lungs require oxygen for aerobic metabolism to maintain energy levels [30]. In this study, due to the limited oxygen supply in the deflated donor lung, ATP was mostly provided by anaerobic glycolysis, as evidenced by increased lactate acid and pyruvic acid levels. The oxygen in alveoli compensated for lung tissue hypoxia and thus improved aerobic metabolism in lung grafts, as evidenced by increased ATP contents, reduced lactic acid and pyruvic acid accumulation. The effect of inhibiting energy depletion was more pronounced in the presence of 3% hydrogen. These results are consistent with reports by Liu Q et al., who showed that intraperitoneal administration of hydrogen-rich saline inhibited depletion of ATP levels in hepatocytes of mice with obstructive jaundice [31].

The underlying mechanism of the favorable effect of hydrogen on graft energy metabolism requires further investigation. We speculated that by maintaining the function of mitochondria, hydrogen can promote glucose use by mitochondria for aerobic oxidation and reduce anaerobic glycolysis in lung tissue.

Large amount of ROS resulting from I/R is an important mediator that injure the lung grafts in LTx [32]. In this study, inflation with 40% oxygen before reperfusion elevated ROS production in donor lungs, suggesting that oxidative stress was induced by oxygen directly absorbed by lung tissues from alveoli. Oxygen-containing gas inflation compensated for hypoxia and mitigated metabolic disorders, but oxygen promotes ROS production, which may help to explain why the high intra-alveolar oxygen concentrations (50% or 100% oxygen) in cold-preserved lungs aggravated lung graft injury [21]. Whether oxygen supply by lung inflation can produce reperfusion injury-like effects during the CIP requires further study.

The addition of hydrogen reduced ROS production in the CIP to a certain extent, although the difference was not statistically significant. Interestingly, hydrogen exposure resulted in a notable decrease in ROS levels after 2 h of reperfusion. These findings may indicate that hydrogen exerted a more obvious antioxidant effect during the reperfusion phase than during the CIP [33]. Hydrogen suppresses oxidative stress injury due to its hydroxyl radical (the most cytotoxic of all ROS) scavenging capacity and by inducing numerous antioxidative proteins in the Nrf2 signaling pathway, such as heme oxygenase and SOD [34–36]. In addition, hydrogen was observed to decrease lipid peroxidation [37, 38].

In previous experiments, the effects of hydrogen on mitochondria included preventing the loss of MMP [12], promoting mPTP closure [39], protecting mitochondrial DNA [40], and targeting mitochondrial ROS neutralization [41]. Noda K et al. found that hydrogen improved the energy metabolism of donors by upregulating mitochondrial complex I, II and IV activities [42]. In a model of sepsis-induced lung injury, hydrogen therapy increased mitochondrial complex I activity [43]. Ishihara G et al. showed that hydrogen suppressed superoxide generation in complex I in A549 cells (a human lung cell line) and neutralized semiquinone radicals to reduce superoxide production in complex III [44]. In this experiment, lung inflation with 3% hydrogen in the CIP exerted a better effect on maintaining a normal mitochondrial morphology. Moreover, hydrogen markedly increased mitochondrial complex I activity and ATP contents in lung grafts, indicating improved mitochondrial energy metabolism. Investigating the impact of hydrogen on mitochondrial structure and function may help to reveal the protective mechanism of hydrogen.

The mitochondrial complexes are important enzyme complex in the aerobic respiratory chains of mitochondria. Measurement of the mitochondrial complex activity can reflect the state of respiratory electron transport chain and the generation of ROS. Our results suggested that LTx induced impairment of mitochondrial function, leading to the overproduction of mitochondrial ROS, oxidative stress, and reduced intracellular ATP levels. Oxygen inflation attenuated the mitochondrial malfunction in lung grafts. The addition of 3% hydrogen has shown more significant effect than oxygen alone.

Many clinical trials have established the protective effects of hydrogen on many organs and systems. For respiratory diseases, the application of hydrogen gas is recommended in the7th and 8th edition of Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment issued by China National Health Commission. A multicenter, randomized, parallel controlled clinical trial showed that hydrogen-oxygen mixture inhalation treatment for 2 days alleviated the symptoms in COVID-19 patients, such as dyspnea, chest tightness, chest pain and cough. Oxygen saturation at rest was increased by 50% [45]. Besides, Gong Z et al. found that H₂ inhalation can reduce air pollution-induced airway inflammation and oxidative stress [46]. For patients with asthma and COPD, inhaling 2.4% H₂ for 45 min reduced airway inflammation, characterized by a decrease in monocyte chemoattractant protein-1 levels and a decrease in IL-4 and IL-8 levels in exhaled breath condensate.

Compare with other antioxidants, hydrogen is low-cost and easy to access. The usage of H₂ is so convenient that H₂ can be easily administered through various ways, including inhalation, drinking H₂-rich water (HW), injection of H₂-rich saline (HRS), bathing in HW.

This study has several limitations. First, we only evaluated the point of 2 hours after perfusion, which is not the usual time point of IR injury. Thus, the continuous effect of hydrogen on lung IRI cannot be clarified. Second, only 3% hydrogen was detected in this work. The dose-specific effects of hydrogen and optimal hydrogen concentration for lung inflation during cold storage cannot be clarified. Furthermore, we did not compare the effect of hydrogen with other antioxidants, such as ROS inhibitors. Further limiting this study, we assessed the mitochondrial morphology only in the alveolar type II cells. The influence of hydrogen on other types of pulmonary cells was not assessed. Since 99% of the internal surface area of the lung is covered with alveolar type I (ATI) and AT-II epithelial cell, the inflating gas first directly contact with alveolar epithelial cells and act on them [47]. In addition, the number of AT-II epithelial cells is more than that of AT-I, and the cytoplasm of AT-II cells contain plenty of mitochondria and other organelles. Thus, we selected more representative AT-II epithelial cells for observation.

In conclusion, our findings suggested that lung inflation with 3% hydrogen gas during cold storage attenuated lung IRI in a rat LTx model. These effects of hydrogen may be associated with improvements of mitochondrial morphology and function and reduced anaerobic metabolism level. Hydrogen inflation might be an effective option for improving the quality of donor lungs quality during cold preservation and transportation.

Term	Meaning
ANOVA	one-way analysis of variance
CIP	cold ischemia phase
DHE	dihydroethidium
IRI	ischemia-reperfusion injury
LTx	lung transplantation
LIS	lung injury score
MMP	mitochondrial membrane potential
mPTP	mitochondrial permeability transition pore
ROS	reactive oxygen species

Ethics approval and consent to participate

All animal procedures were performed in accordance with the guidelines of “Animals (Scientific Procedures) Act 1986”. Research protocols in this study were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. This study is reported in accordance with ARRIVE guidelines.

Consent for publication

Not applicable

Availability of data and materials

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: National Nature Science Foundation of China (Grant 81570088).

Authors' contributions

LQ, EX, HZ, PZ contributed to performing experiments, data analysis, drafting or revising the article, gave final approval of the version to be published. CN, JK analyzed the data and edited the manuscript. All authors have read and approved the manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

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Donor lung inflation with hydrogen gas during cold storage alleviates energy metabolism disorder in lung grafts by protecting mitochondrial function in rats

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Abstract

Background

Method

Results

Conclusion

Figures

Background

Methods

Animals

Donor preparation and groups

LTx

Blood gas analysis

Static compliance of the lung grafts

Histopathologic analysis

Transmission electron microscopy (TEM)

Detection of inflammatory cytokines

Measurements of the energy metabolism level

Analysis of the ROS content in lung grafts

Lung mitochondrial complex activity determination

Statistical analysis

Results

Gas Inflation Ameliorated Lung Graft Dysfunction

Gas inflation improved the histopathology scores of lung grafts

Hydrogen inflation alleviated the inflammatory response in recipients

The energy metabolism level in lung grafts

Analysis of the ROS content in lung tissue

Effects of hydrogen gas inflation on the structure and function of mitochondria

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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