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 . Fukuse T et al. found that pulmonary storage at 50% or 100% FiO2 could induce mitochondrial dysfunction and promote lipid peroxidation . 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 . 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 . 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 . 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 .
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 . 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 . 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 .
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 . 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 . 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 . 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 , promoting mPTP closure , protecting mitochondrial DNA , and targeting mitochondrial ROS neutralization . Noda K et al. found that hydrogen improved the energy metabolism of donors by upregulating mitochondrial complex I, II and IV activities . In a model of sepsis-induced lung injury, hydrogen therapy increased mitochondrial complex I activity . 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 . 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% . Besides, Gong Z et al. found that H2 inhalation can reduce air pollution-induced airway inflammation and oxidative stress . 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 . 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.