Dexmedetomidine and argon in combination against ferroptosis through tackling txnip-mediated oxidative stress in dcd porcine livers

ABSTRACT Graft availability from donation after circulatory death (DCD) is significantly limited by ischaemia reperfusion (IR) injury. Effective strategies to mitigate IR injury in DCD

grafts are essential to improve graft quality and expand the donor pool. In this study, liver grafts from DCD pigs were preserved in the University of Wisconsin (UW) solution saturated with

0.1 nM dexmedetomidine (Dex) and various concentrations of noble gases Argon (Ar) and/or Xenon (Xe) at 4 °C for 24 or 72 h. The combined 50% Ar and Dex provided maximum protection to liver

grafts by reducing morphological damage, apoptosis, necroptosis, ferroptosis, hepatocyte glycogen depletion, reticulin framework collapse, iron deposition, and oxidative stress. In vitro,

human liver Hep G2 cells were preserved in the UW solution saturated with 0.1 nM Dex and 50% Ar in combination at 4 °C for 24 h, followed by recovery in medium at 37 °C for up to 48 h to

mimic clinical IR injury. This treatment significantly increased the expression of anti-oxidative stress proteins by promoting the translocation of thioredoxin-interacting protein (TXNIP) to

mitochondria, thereby inhibiting ferroptosis, increasing plasma membrane integrity, and maintaining cell viability.In summary, The combination of 0.1 nM Dex and 50% Ar may be a promising

strategy to reduce ferroptosis and other form cell death, and preserve liver grafts. SIMILAR CONTENT BEING VIEWED BY OTHERS HYPOTHERMIC OXYGENATED PERFUSION INHIBITS HECTD3-MEDIATED TRAF3

POLYUBIQUITINATION TO ALLEVIATE DCD LIVER ISCHEMIA-REPERFUSION INJURY Article Open access 24 February 2021 PRE-ARREST DOXYCYCLINE PROTECTS DONATION AFTER CIRCULATORY DEATH KIDNEYS Article

Open access 17 December 2020 HYPOTHERMIC OXYGENATED PERFUSION INHIBITS CLIP1-MEDIATED TIRAP UBIQUITINATION VIA TFPI2 TO REDUCE ISCHEMIA‒REPERFUSION INJURY OF THE FATTY LIVER Article Open

access 02 December 2024 INTRODUCTION Due to advancements in surgical techniques, immunosuppressant developments, and post-operative care, the success rate of liver transplantation has

significantly increased [1]. However, the demand for liver grafts greatly exceeds the available supply, resulting in lengthy waiting lists for patients requiring a liver transplant. In 2019,

approximately 118,000 patients were waiting for an organ transplant, with the majority of them awaiting a kidney or liver [2]. Efforts are underway to expand the availability of liver donor

organs, with a strategy being the increase in donation after circulatory death (DCD) donors [3]. There is a growing understanding of how mitochondrial metabolism contributes to ischaemia

reperfusion (IR) injury and poor outcomes in liver transplantation. Due to the imbalance between the production of reactive oxygen species (ROS) and antioxidant defence mechanisms,

mitochondrial oxidative stress poses a significant challenge in liver transplantation [4]. Oxidative stress negatively affects various cellular components, including membranes, lipids,

lipoproteins, and deoxyribonucleic acid (DNA) [5, 6], initiating signalling cascades that activate regulated cell death pathways, ultimately damaging liver grafts [7,8,9]. The

supplementation of noble gases such as xenon (Xe) or argon (Ar) during cold preservation has been proposed as a strategy to reduce IR injury [10, 11]. Dexmedetomidine (Dex), a selective

α2-adrenergic receptor agonist with a sedative effect, has been reported to mitigate IR injury by reducing oxidative stress and inflammatory responses [12, 13]. Accordingly, in this study,

we investigated whether Ar, Xe, or Dex, alone or in combination, provided increased protection against IR injury, thereby enhancing liver graft quality. The underlying molecular mechanisms

were also explored. RESULTS DEX AND NOBLE GASES IMPROVED THE MORPHOLOGY OF COLD-PRESERVED DCD LIVERS When compared to non-cold preserved negative controls (NC), 24- or 72-h cold-preserved

livers (CI24h or CI72h) exhibited significant morphological changes, including cellular swelling, plasma membrane rupture, nuclei breakdown and loss of staining (Fig. 1A, C). After 24 h cold

preservation, the liver injury score increased to 3.21 ± 0.84 (_p_ < 0.01). However, Dex combined with 30% or 50% Ar significantly reduced hepatocyte necrosis and decreased liver injury

score to 1.96 ± 0.43 (_p_ < 0.05) or 1.93 ± 0.43 (_p_ < 0.05), respectively (Fig. 1B). After 72 h of cold preservation, the liver injury score rose to 3.23 ± 0.52 (_p_ < 0.001); Dex

combined with 50% Ar significantly preserved hepatocytes and reduced the liver injury score to 2.32 ± 0.53 (_p_ < 0.05, Fig. 1D). DEX AND NOBLE GASES REDUCED APOPTOSIS IN COLD-PRESERVED

DCD LIVERS The average number of TUNEL+ apoptotic cells significantly increased from 33.04 ± 7.43 (NC) to 229.41 ± 65.28 cells per field after 24 h of cold preservation (_p_ < 0.0001,

Fig. 2A, C), and from 51.22 ± 10.58 (NC) to 515.84 ± 159.71 cells per field after 72 h of cold preservation (_p_ < 0.0001, Fig. 2B, D). Significant decreases in TUNEL+ cells at 24 h were

observed when cold preservation solutions were supplemented with Dex combined with 30% Ar (_p_ < 0.05), 15% Xe and 15% Ar, (_p_ < 0.05), 50% Xe (_p_ < 0.0001), 50% Ar (_p_ < 

0.0001), or 25% Xe and 25% Ar (_p_ < 0.0001) (Fig. 2C). After 72 h, significant reductions in TUNEL+ cells were noted with Dex combined with 50% Xe (_p_ < 0.01), 50% Ar (_p_ < 

0.001), or 25% Xe and 25% Ar (_p_ < 0.01) (Fig. 2D). DEX AND NOBLE GASES REDUCED NECROPTOSIS IN COLD-PRESERVED DCD LIVERS After 24 h of cold preservation, the expression of necroptosis

biomarker, pMLKL, significantly increased by 51.6% (_p_ < 0.0001), while the solutions supplemented with Dex combined with 30% Ar (_p_ < 0.05), 50% Ar (_p_ < 0.01), or 25% Xe and

25% Ar (_p_ < 0.05) decreased pMLKL expression (Fig. 3A, C). After 72 h of cold preservation, pMLKL expression increased 2.1-fold (_p_ < 0.0001), while supplementation with 50% Xe or

50% Ar significantly reduced pMLKL expression (_p_ < 0.05, separately; Fig. 3B, D). DEX AND NOBLE GASES REDUCED FERROPTOSIS IN COLD-PRESERVED DCD LIVERS Cold preservation significantly

increased GPX4 expression at 24 h (_p_ < 0.001, Fig. 4A, C). While 72 h of cold preservation significantly decreased GPX4 expression (_p_ < 0.05, Fig. 4B, D), demonstrating the

occurrence of ferroptosis. In addition, Dex combined with 50% Ar (_p_ < 0.05) or 25% Xe and 25% Ar (_p_ < 0.05) significantly reduced ferroptosis by increasing GPX4 expression (Fig.

4D). DEX AND NOBLE GASES REDUCED GLYCOGEN DEPLETION, RETICULIN FRAMEWORK COLLAPSES, AND IRON DEPOSITION IN COLD-PRESERVED DCD LIVERS PAS staining (Fig. 5A) revealed moderate glycogen

depletion after 24 h of cold preservation, indicated by hepatocytes with lighter purple cytoplasm. The glycogen content improved when solutions were supplemented with Dex combined with 50%

Xe or 50% Ar. After 72 h, severe glycogen depletion was observed, while Dex combined with 50% Xe or 50% Ar attenuated this depletion. Reticulum staining (Fig. 5B) showed reticular fibre

collapse and massive hepatocyte necrosis after 24 and 72 h of cold preservation, which was alleviated by solutions supplemented with Dex combined with 50% Xe or 50% Ar. Iron staining (Fig.

5C) indicated extensive iron deposition in hepatocytes after 24 or 72 h of cold preservation, which was reduced by solutions supplemented with Dex combined with 50% Xe or 50% Ar. DEX AND

NOBLE GASES REDUCED OXIDATIVE STRESS AND TXNIP EXPRESSION IN COLD-PRESERVED DCD LIVERS Both oxidative stress and TXNIP expression increased after 24 and 72 h of cold preservation and were

ameliorated by treatment with Dex combined with 50% Xe or 50% Ar (Fig. 6A–D). Notably, the distribution of TXNIP expression overlapped significantly with the location of oxidative stress,

indicating a strong relationship between oxidative stress and TXNIP expression. DEX AND AR INHIBITED FERROPTOSIS THROUGH ANTI-OXIDATIVE STRESS IN HUMAN HEPG2 CELLS Hypothermic hypoxia

reoxygenation (HR) significantly increased GPX4 expression in a time-dependent manner up to 48 h of reoxygenation (_p_ < 0.05, Fig. 7A, B). ACSL4 expression also increased at 24 (_p_ <

 0.05) and 48 h (_p_ < 0.0001) (Fig. 7A, C) after reoxygenation, indicating ferroptosis. Grp78 expression, indicating endoplasmic reticulum (ER) stress, increased time-dependently after

reoxygenation for 6, 24, and 48 h (_p_ < 0.05, respectively; Fig. 7A, D). Intracellular HMGB1 (Fig. 7A, D) also increased after reoxygenation at 24 (_p_ < 0.01) and 48 h (_p_ < 

0.001) (Fig. 7A, E). After 48 h of reoxygenation, the expression of GPX4 (_P_ < 0.001) and ACSL4 (_p_ < 0.0001) was increased significantly, while Dex and 50% Ar in combination

decreased ACSL4 expression (_p_ < 0.05, Fig. 7F–H). In addition, crystal violet staining (Fig. 7I) demonstrated that Dex combined with 50% Ar increased cell number and viability after 24 

h of hypothermic hypoxia and 24 h of reoxygenation. Antioxidant proteins, catalase, thioredoxin, and SOD-1 were detected to assess the effects of Dex and 50% Ar on early-oxidative stress at

4 h and late-oxidative stress at 24 h of reoxygenation (Fig. 7J). At the late-oxidative stress stage, Dex combined with 50% Ar significantly upregulated the expression of catalase and

thioredoxin (_p_ < 0.05, respectively; Fig. 7K, L) rather than SOD-1 (Fig. 7M). DEX AND AR MODULATED TXNIP TRANSLOCATION AND MAINTAINED PLASMA MEMBRANE INTEGRITY IN HEPG2 CELLS In HepG2

cells, TXNIP was primarily located in the nuclei and cytoplasm under normal conditions (NC) and after 24 h of hypothermic hypoxia (H24). After 4 h of reoxygenation (HR4), TXNIP translocated

to the mitochondria. In contrast, when the UW solution was saturated with 0.1 nM Dex (HR4 + D) or 50% Ar (HR4 + A), TXNIP remained primarily in the nucleus (Fig. 8A). In addition, the

expression of TXNIP significantly increased after 4 h of reoxygenation (_p_ < 0.0001), but 50% Ar reduced this increase significantly (_p_ < 0.05, Fig. 8B, C). Membrane staining (Fig.

8D) showed that, in normal HepG2 cells, the cell membrane was stained bright yellow. Hypothermic hypoxia and reoxygenation increased the permeability of the cell membrane, allowing more DiLC

dye to enter the cells and stain the intracellular organelle membranes bright yellow. Membrane vesicles labelled with DilC were found when the UW solution was saturated with Dex or 50% Ar.

DISCUSSION The present study demonstrated that DCD porcine livers suffered severe injury after prolonged cold preservation. Supplementation of Dex and noble gases (Xe or Ar) in the UW

solution significantly mitigated the activation of regulated cell death signalling pathways, attenuated glycogen depletion, reticulin framework collapse, iron deposition, and reduced

oxidative stress. Meanwhile, the UW solution containing 0.1 nM Dex combined with 50% Ar provided a significant protection to maintain liver graft quality during cold preservation. In vitro,

HepG2 cells subjected to “hypothermic hypoxia-reoxygenation” experienced ferroptosis; however, treatment with 0.1 nM Dex and 50% Ar significantly increased the expression of antioxidative

stress proteins, catalase and thioredoxin, and inhibited TXNIP translocation, thereby preventing ferroptosis and maintaining cell viability. Moreover, Dex and Ar, in combination, repaired

the damaged cell membrane by forming membrane vesicles. Collectively, our data suggested that the combination of 0.1 nM Dex and 50% Ar against ferroptosis may present a potential therapeutic

strategy to improve the quality of DCD livers for clinical use, although further studies are warranted. Iron is essential for liver function, contributing to various physiological

processes, such as iron storage, heme synthesis, and detoxification [14]. The liver stores about 10% of the body’s iron, while excess free iron catalyses the formation of free radicals _via_

the Fenton reaction, leading to oxidative stress and cellular damage [15]. The liver is particularly susceptible to oxidative stress due to its role in metabolism and detoxification [15].

Therefore, ferroptosis, a form of cell death characterised by lipid peroxidation and iron-dependent processes, may significantly impact liver transplantation injury. This study focused on

investigating the role of ferroptosis in liver IR injury, providing insights into disease mechanisms and identifying potential therapeutic targets to improve patient outcomes. During cold

preservation, the liver is deprived of oxygen and nutrients, leading to decreased ATP production and compromising cell viability and function [16, 17]. However, cold preservation also causes

mitochondrial enlargement and membrane damage, resulting in the accumulation of ROS that causes oxidative damage to cellular components [17]. Several studies showed that oxidative stress

was elevated in patients undergoing liver transplantation, associated with an increased risk of complications such as graft failure and infection [18, 19]. Reperfusion injury, characterised

by the sudden influx of oxygen molecules and nutrients into previously ischaemic tissue, contributes to graft dysfunction and failure by causing additional cellular injury and inflammation

[20, 21]. Dex has been shown to protect the liver against IR injury by reducing cellular oxidative stress. This is achieved by reducing the activity of ROS-producing enzymes such as NADPH

oxidase and xanthine oxidase, while increasing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase, which act to neutralise ROS [22,23,24]. However, the

molecular mechanisms of noble gases, such as Xe and Ar, in protecting against liver IR injury remain largely undefined. It has been proposed that noble gases may play a role in opening

mitochondrial ATP-sensitive K (mitoKATP) channels, thus enhancing mitochondrial function and reducing ROS production [25]. Our study showed that the combination of Dex with noble gases,

particularly Xe or Ar, had a stronger protective effect in reducing oxidative stress and regulated cell death, including apoptosis, necroptosis, and ferroptosis, in long-term cold preserved

DCD porcine livers. Our data indicated that hypothermic hypoxia reoxygenation significantly increased TXNIP expression; however, Dex and Ar significantly reduced TXNIP expression and

translocation to mitochondria, while increasing thioredoxin expression. TXNIP and thioredoxin are closely related proteins that regulate cellular redox homoeostasis [26]. Thioredoxin is a

ubiquitous oxidoreductase critical for maintaining cellular redox balance, catalysing the reduction of disulfide bonds in proteins, and reducing ROS levels in cells [27]. In contrast, TXNIP

is shuttled to the mitochondria under oxidative stress; high levels of TXNIP inhibit thioredoxin redox activity, leading to increased hydrogen peroxide levels within the mitochondria.

Hydrogen peroxide is a potent oxidising agent that damages proteins, lipids, and DNA within the mitochondria, triggering cell death [28]. Therefore, the inhibition of TXNIP expression and

translocation decreases hydrogen peroxide production, oxidative damage, and mitochondrial dysfunction. The combination of Dex and Ar showed potent protection for liver grafts associated with

IR injury by reducing TXNIP-mediated oxidative stress and maintaining mitochondrial function. Our study also revealed that the combination of Dex and Xe or Ar significantly reduced glycogen

depletion and hepatocyte reticulin framework collapse. The lack of oxygen during cold preservation activates glycolysis, which is essential for cell survival [29]; regulated cell death may

occur if glycolysis is inhibited due to glycogen deficiency [30]. Since most donors are cadaveric, optimising their nutritional status before transplantation through pretreatment is

practically impossible. Therefore, reducing glycogen depletion is important to maintain hepatocyte survival during cold preservation [29]. Our data showed that supplementation with Dex and

noble gases, in particular Ar, significantly reduced hepatocyte glycogen depletion during cold preservation, potentially providing energy to hepatocytes and reducing graft injury. We also

found significant damage to the liver reticulin framework during cold preservation. The reticulin fibres, composed of collagen and glycoprotein, provide structural support to liver cells and

help maintain liver architecture [31]. Our work suggested that cold preservation might cause collagen fibres to contract, disrupting the reticulin framework, which led to loss of liver

function and increased risk of post-transplantation complications. The combination of Dex with Xe or Ar may help maintain the integrity of the reticulin framework by increasing collagen and

glycoprotein content and reducing cell death. Our study has several limitations. Firstly, DCD livers were only cold preserved without reperfusion during engraftment that mainly causes ROS

generation and cell injury. However, the hypothermic hypoxia reoxygenation model to simulate IR injury in an in vitro model was implemented in the current study to investigate the

progression of ferroptosis after reperfusion. This is at least in part to compensate this shortfall. Secondly, our study used the cancer cell line HepG2 which are not normal liver cells

although it is not nontumorigenic with high proliferation rate and epithelial-like morphology with many differentiated hepatic functions [32]. It has been used in previous studies on liver

injury using different models [33, 34]. Even so, in future studies, we will actively consider using a normal liver cell line for further analysis. Lastly, unlike the animal study that

evaluated several types of regulated cell death, we only assessed cell viability and ferroptosis in the in vitro model; more cell injurious types should be evaluated in future to demonstrate

the impact of Dex and Ar under hypothermic hypoxia reoxygenation condition. Nevertheless, our study holds significant clinical implications. It is well-recognised that organs procured from

donation after brain death (DBD) donors are of higher quality due to the continued circulation at the time of brain death declaration. However, with the growing reliance on marginal or DCD

donors to meet the increasing demand for transplantation, it is crucial to enhance graft preservation strategies. DCD organs are particularly vulnerable to the impacts of IR, and effective

strategies to mitigate IR injury of DCD organs are urgent for enlarging the donor pool. While this preservation strategy may increase the availability of liver grafts, it is important to

note that this study is a proof of concept one. Further validation is required in more clinically relevant settings, such as in large animal (pig) liver transplant models. In conclusion, our

work provided evidence supporting the clinical potential use of Dex and noble gas saturated cold preservation for transplant graft preservation. The combination of Dex with a high

concentration of noble gas, especially 50% Ar, showed the most significant benefits in maintaining liver graft morphology and reducing regulated cell death during cold preservation. Clinical

translation of Dex and Ar in combination may expand the donor pool by including more liver grafts, shorting waiting times, and improving the quality of life for patients. However, further

validation is needed to assess outcomes after engraftment with this preservation strategy. METHODS EX VIVO SAMPLE ANALYSIS ANIMALS AND LIVER GRAFT COLD PRESERVATION In total 5 male 15–17 kg

experimental mini pigs were obtained from the Laboratory Animal Centre of Army Medical University, Chongqing, China. The animal study was approved by the Army Medical University Biomedical

Ethics Committee (Approval No. AMUWEC20210209) and all procedures followed the ARRIVE guidelines. The animals fasted for 8 h and were subcutaneously injected with 500 µg/kg midazolam

(Pfizer, New York, USA) for sedation [35]. They were then admitted to the animal operating room, receiving non-invasive blood pressure and electrocardiogram monitoring. A peripheral

intravenous line was established through the left auricular vein, and 1–2 L/min oxygen was given _via_ an animal face mask. After intravenous injection of 20 µg/kg atropine (Mingsheng

Medicine, Zhejiang, China), 2 mg/kg propofol (AstraZeneca, Cambridge, UK), and 100 µg fentanyl (Renfu Medicine, Hubei, China) [36], heparin (Dongcheng Pharma, Shandong, China) at 300 IU/kg

was administered intravenously 5 min before circulatory death induction [35]. Circulatory death was induced by intravenous injection of 600 mg propofol (AstraZeneca) and then 10 mg

atracurium (Hengrui Medicine, Jiangsu, China) [37]. A clamshell thoracotomy was performed to expose the lungs and heart. The left atrial appendage and the precava and postcava were incised

for venting blood. 12-F catheters (Byram Healthcare, New York, USA) connecting the infusion apparatus were inserted into the main pulmonary artery and the aorta to flush pulmonary vascular

and hepatic vascular [37]. After flushing, the pigs were left at room temperature for in situ warm ischaemia. The liver was extracted 30 min after circulatory death, then placed in the

University of Wisconsin solution (UW solution, Bridge to Life Ltd, Columbia, USA) for 24 or 72 h at 4 °C. Non-cold preservation control livers were fixed using 4% paraformaldehyde

(Sigma-Aldrich, Poole, UK) immediately after extraction. COLD PRESERVATION SOLUTIONS Gas mixtures (Heruijia BioMed, Chongqing, China) were prepared in cylinders with 45% O2, 5% CO2 and

balanced with N2 in treatment groups as follows: (1) 45% O2, 50% N2, and 5% CO2; (2) 0.1 nM Dex, 45% O2, 50% N2, and 5% CO2; (3) 0.1 nM Dex, 30% Xe, 45% O2, 25% N2, and 5% CO2; (4) 0.1 nM

Dex, 30% Ar, 45% O2, 25% N2, and 5% CO2; (5) 0.1 nM Dex, 15% Xe, 15% Ar, 45% O2, 20% N2, and 5% CO2; (6) 0.1 nM Dex, 50% Xe, 45% O2, and 5% CO2; (7) 0.1 nM Dex, 50% Ar, 45% O2, and 5% CO2;

(8) 0.1 nM Dex, 25% Xe, 25% Ar, 45% O2, and 5% CO2. Gas mixtures were bubbled into 250 mL sterile UW solutions supplemented with 0.1 nM Dex [38] (Sigma-Aldrich, Poole, UK) at 0.5 L/min for

20 min in a gas-washing bottle (ZAUBEE, Chengdu, China). H&E STAINING The liver sections were deparaffinised. Nuclei were stained with haematoxylin (Sigma-Aldrich), and the extracellular

matrix and cytoplasm were stained with eosin (Sigma-Aldrich). The liver injury criteria were evaluated with cell disruption, nuclei loss, and tissue structure damage. Scores 0: normal, 1:

<25%, 2: 25–50%, 3: 50–75%, 4: >75% percentage area. PAS STAINING Glycogen was measured in liver sections using a PAS staining kit (Abcam, ab150680, Cambridge, UK) according to the

instructions of the manufacturer. RETICULUM STAINING Liver reticulin fibres were measured in liver sections using a reticulum staining kit (Abcam, ab236473, Cambridge, UK) according to the

instructions of the manufacturer. IRON STAINING Iron deposition was measured in liver sections using an iron staining kit (Abcam, ab150674, Cambridge, UK) according to the instructions of

the manufacturer. OXYIHC OXIDATIVE STRESS ASSAY Oxidative stress of livers was measured using an OxyIHC™ Oxidative Stress Detection kit (EMD Millipore, S7450, Poole, UK) according to the

instructions of the manufacturer. TUNEL ASSAY The sections were detected with in situ TUNEL assay (Abcam, ab66108, Cambridge, UK. TUNEL+ nuclei were visualised by green FITC fluorescence.

IMMUNOHISTOCHEMISTRY For ex vivo fluorescence staining, tissue sections were fixed and permeabilised. They were then blocked with 4% donkey serum (Invitrogen) and incubated with mouse

monoclonal anti-GPX4 (1:100, Abcam) or rabbit monoclonal anti-phosphorylated MLKL (1:100, Abcam) overnight at 4 °C. Samples were then washed and incubated with fluorochrome-conjugated

secondary antibodies (1:300, Abcam). For in vitro fluorescence staining, the cells were fixed and incubated in 1% normal donkey serum, and subsequently incubated overnight with the 1:100

concentration mouse anti-TXNIP primary antibody (Abcam). Then the second fluorochrome-conjugated secondary antibodies (1:300, Abcam) were incubated. Mitochondria were stained with

MitoTracker probes (Sigma-Aldrich, M22426, Poole, UK). Nuclei were stained with DAPI and mounted with VECTASHIELD Mounting Medium (Invitrogen). IN VITRO EXPERIMENT CELL CULTURE AND

HYPOTHERMIC HYPOXIA REOXYGENATION INJURY Human Hepatoblastoma cells (HepG2 cell line; European Cell Culture Collection, Salisbury, UK) were maintained in DMEM medium (Gibco, Billings, USA)

supplemented with 10% fetal bovine Serum (Gibco) and 100 U/mL penicillin-streptomycin (Gibco) at 37 °C in a humidified incubator until confluent. For hypothermic hypoxia reoxygenation

injury, the cell culture medium was replaced with the UW cold preservation solution, and the cells were placed at 4 °C for 24 h. Then, the UW solution was replaced with warm fresh medium,

and the cells were incubated at 37 °C for another 6, 24 or 48 h. IN VITRO DEX AND AR TREATMENT Gas mixture component with 50% Ar, 45% O2, and 5% CO2 was bubbled into the UW solution

supplemented with 0.1 nM Dex for 5 min in an aseptic container. Hep G2 cells were incubated with Dex-50% Ar-saturated UW solution and placed in an air-tight chamber with 50% Ar, 45% O2 and

5% CO2 at 4 °C for 24 h. After that, the Dex-50%Ar-saturated UW solution was replaced by warm fresh DMEM cell culture medium (Invitrogen), and the cells were incubated for up to 48 h at 37 

°C in an incubator. WESTERN BLOT Cell cultures were homogenised to remove cell debris. After being heated, denatured, and loaded onto a NuPAGE 4 to 12% Bis-Tris gel for electrophoresis, the

protein extracts (40 g/sample) were transferred to a polyvinylidene difluoride membrane and blocked with 5% BSA in TBST for 1 h at room temperature. Membranes were probed with 1:1000 primary

antibodies GPX4 (Abcam), ACSL4 (Abcam), Grp78 (Abcam), HMGB1 (Abcam), catalase (Abcam), thioredoxin (Abcam), SOD1 (Abcam), TXNIP (Abcam) in TBST overnight at 4 °C, followed by

HRP-conjugated secondary antibody for 1 h (Cell Signalling, Technology). The loading control was the constitutively expressed protein, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell

Signalling, Technology) or Smooth muscle actin (SMA, Cell Signalling, Technology). The blots were visualised using improved chemiluminescence (Santa Cruz, Dallas, USA). MEMBRANE LIPID

LABELLING The cells were incubated with DilC-AM dye (DilC-AM stain kit, D3911, Paisley, UK) at 5 μl/1 mL in medium for 20 min at 37 °C in the dark. Then, the cells were washed and fixed.

CRYSTAL VIOLET STAINING The cells were fixed with ice-cold 100% methanol (Sigma). A 0.5% crystal violet solution in 25% methanol (Sigma) was added, and the cells were incubated for 10 min.

STATISTICAL ANALYSIS All numerical data were expressed as mean ± standard deviation (SD) or median with interquartile range if not normally distributed as appropriate and presented with a

scatter plot. Data were analysed using the two-tailed Student’s t-test or analysis of variance (ANOVA) followed by the Turkey test for comparisons where appropriate (GraphPad Prism 5.0,

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yields enhanced protection in lung grafts. Theranostics. 2023;13:4376–90. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references FUNDING The National Natural Science

Foundation of China, Beijing, China, (No. 81801900). The Chongqing Talent Foundation, Chongqing, China, (No.CQYC20220303588). The Chelsea-Westminster Hospital Joint Research Committee Grant,

London, UK. 2021 Key Specialist Project of Clinical Medicine, Shunde Hospital of Southern Medical University (No. SRSP2021039). AUTHOR INFORMATION Author notes * These authors contributed

equally: Qian Chen, Jiashi Sun, Xiangfeng Liu. AUTHORS AND AFFILIATIONS * Department of Anesthesiology, Perioperative and Systems Medicine, Children’s Hospital, Zhejiang University School of

Medicine, National Clinical Research Centre for Child Health, Hangzhou, Zhejiang, China Qian Chen & Daqing Ma * Department of Anesthesiology, Southwest Hospital, Army Medical

University, Chongqing, China Qian Chen, Xiangfeng Liu, Zhigang Qin, Jieyu Li, Jianbo Ma, Zhengwei Xue, Yirong Li, Ziheng Yang & Jianteng Gu * Division of Anaesthetics, Pain Medicine and

Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea & Westminster Hospital, London, UK Qian Chen, Jiashi Sun, Qizhe Sun, Lingzhi Wu, 

Enqiang Chang, Hailin Zhao, Yiwen Zhang & Daqing Ma * Department of Anesthesiology, Shunde Hospital, Southern Medical University, Shunde, Guangdong, China Yiwen Zhang Authors * Qian Chen

View author publications You can also search for this author inPubMed Google Scholar * Jiashi Sun View author publications You can also search for this author inPubMed Google Scholar *

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inPubMed Google Scholar * Ziheng Yang View author publications You can also search for this author inPubMed Google Scholar * Qizhe Sun View author publications You can also search for this

author inPubMed Google Scholar * Lingzhi Wu View author publications You can also search for this author inPubMed Google Scholar * Enqiang Chang View author publications You can also search

for this author inPubMed Google Scholar * Hailin Zhao View author publications You can also search for this author inPubMed Google Scholar * Yiwen Zhang View author publications You can also

search for this author inPubMed Google Scholar * Jianteng Gu View author publications You can also search for this author inPubMed Google Scholar * Daqing Ma View author publications You

can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceptualisation: Daqing Ma, Qian Chen, Hailin Zhao. Methodology: Xiangfeng Liu, Qian Chen, Jiashi Sun. Investigation:

Qian Chen, Xiangfeng Liu, Jiashi Sun, Jianbo Ma, Zhigang Qin, Ziheng Yang, Yirong Li, Jieyu Li, Zhengwei Xue, Jianteng Gu, Yiwen Zhang. Visualisation: Qian Chen, Xiangfeng Liu, Jiashi Sun,

Qizhe Sun, Lingzhi Wu, Enqiang Chang, Hailin Zhao, Jianteng Gu, and Yiwen Zhang. Supervision: Daqing Ma, Jianteng Gu. Writing—original draft: Qian Chen, Jiashi Sun, Xiangfeng Liu, Daqing Ma.

Writing—review & editing: Qian Chen and Daqing Ma. CORRESPONDING AUTHORS Correspondence to Jianteng Gu or Daqing Ma. ETHICS DECLARATIONS COMPETING INTERESTS The data reported here are

subjected for further development of clinical application by some of the authors. ETHICS APPROVAL The animal study was approved by the Third Military Medical University Biomedical Ethics

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licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chen, Q., Sun, J., Liu, X. _et al._ Dexmedetomidine and argon in

combination against ferroptosis through tackling TXNIP-mediated oxidative stress in DCD porcine livers. _Cell Death Discov._ 10, 319 (2024). https://doi.org/10.1038/s41420-024-02071-7

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