Kai-Ying Xu a, Chuang-Yan Wu a, b, Song Tong a, Peng Xiong c, **, Si-Hua Wang a, *

ABSTRACT
Lung ischemia-reperfusion (IR) occurs in many circumstances and leads to impaired lung function. The NACHT, LRR and PYD domains-containing protein 3 (Nlrp3) inflammasome is reportedly activated during lung IR. Mcc950 is a recently developed Nlrp3 inhibitor. The aim of our study was to test the eficacy of Mcc950 on lung IR injury and to investigate the role of reactive oxygen species (ROS) in Nlrp3 inflam- masome activation using a murine lung IR model.The results of the current study conirmed that Nlrp3 was upregulated and activated during lung IR, and inhibiting oxidative stress by the ROS scavenger edaravone attenuated Nlrp3 inflammasome acti- vation. Mcc950 pretreatment signiicantly alleviated IR-induced lung injury by reducing production of the proinflammatory cytokines Il-1β and Il-18 and inhibiting neutrophil iniltration and cell apoptosis. Protein coimmunoprecipitation revealed that Mcc950 partially blocked the interaction between Nlrp3 and Nek7 (NimA-related protein kinase 7). Therefore, we conclude that ROS-dependent activation of the Nlrp3 inflammasome contributed to lung IR injury. Mcc950 signiicantly reduced lung IR injury by blocking Nlrp3 inflammasome activation, and the mechanism was partially attributed to inhibition of the interaction between Nlrp3 and Nek7. Thus, Mcc950 is a promising treatment for the prevention of lung IR injury.

Keywords:Nlrp3 inflammasome;Mcc950;Ischemia reperfusion;Lung injury;Inflammation

1.Introduction
Lung IR injury frequently occurs in many circumstances and leads to impaired lung function [1]. Many mechanisms of IR injury have been proposed, such as oxidative stress, sterile immunity, endothelial dysfunction, cell death, complement activation, and over-activation of coagulation pathways [1,2]. However, the exact molecular mechanisms are not completely elucidated, and effective prevention and treatment measures are still lacking.The NACHT, LRR and PYD domains-containing protein 3 (Nlrp3) inflammasome is an intracellular polyprotein complex that is well documented for its capacity to control the proteolytic activation of caspase-1; subsequently, activated caspase-1 induces the produc- tion of mature interleukin-1β (Il-1β) and Il-18 and the inflammatory cell death process known as pyroptosis [3]. The Nlrp3 inflammasome complex mainly consists of Nlrp3,apoptosis- associated speck-like adapter protein (Asc), pro-caspase-1, and the recently reported protein NIMA-related kinase 7 (Nek7). Nek7 is a member of the mammalian NEK family, and it can bind to Nlrp3 to regulate the oligomerization and activation of the Nlrp3 inflammasome [4e6]. The Nlrp3 inflammasome can be activated by pathogen-associated concomitant pathology molecular patterns(PAMPs),damage- associated molecular patterns (DAMPs), and certain crystalline material (for example uric acid crystals and asbestos). Although the exact molecular mechanisms triggering Nlrp3 inflammasome activation by these structurally different substances remain unde- ined, the accumulation of reactive oxygen species (ROS) metastatic infection foci is considered to be a crucial mediator for Nlrp3 inflammasome acti- vation [7]. Mcc950 is a recently developed small molecule com- pound that can selectively inhibit Nlrp3 inflammasome activation both in vitro and in vivo [8]. Previous studies have reported that Mcc950 exerts protective effects in animal models of steroid- resistant asthma [9], nonalcoholic fatty liver disease [10], myocar- dial infarction [11], and intracerebral hemorrhage [12]. Recently, researchers have hypothesized that Mcc950 might act by disrupt- ing Nek7-Nlrp3 interactions [13]. However, this notion has not been tested in vivo.Pulmonary IR injury is accompanied by a sterile inflammatory response. Recently, one study reported that the Nlrp3 inflamma- some participated in the inflammation response during lung IR [14]. However, the mechanism by which the Nlrp3 inflammasome was activated and the eficacy of Mcc950 during lung IR has not been discussed. This study aims to investigate the eficacy and mechanism of Mcc950 using a mouse lung IR injury model. In addition, ROS promote Nlrp3 inflammasome activation, but whether ROS is theirst signal of Nlrp3 inflammasome activation or the second signal remains to be determined [7]. Therefore, we examined the role of ROS in Nlrp3 inflammasome activation by using the ROS scavenger edaravone.

2.Methods and materials
2.1.Animals
Speciic pathogen-free male C57BL/6 mice (8e10 weeks, 20e24 g) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and were kept in a speciic pathogen-free facility (Tongji Medical College, Wuhan, China). Animal operations were performed strictly according to the guidelines of the National In- stitutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

2.2. Lung ischemia reperfusion model
Mice were anesthetized with 100 mg/kg 1% sodium pentobar- bital (Sigma-Aldrich, USA) intraperitoneally (i.p.) and 4.0 mg/kg atropine (Hefeng, Shanghai, China) i.p. The mice received anti- coagulation treatment with 500 U/kg heparin sodium (Qianhong, Changzhou, China) i.p 15 min before operation.Endotracheal intubation was performed with a 20G intravenous catheter con- nected to a small animal ventilator (ALC-V8, Shanghai, China); the tidal volume was adjusted to 3.0 ml room air, the respiratory rate to 120 per minute, and the inspiratory/expiratory ratio to 1:2. After opening the left thoracic cavity between the 3rd and 4th rib, a microvascular clamp was used to clamp the lung hilum. The clamp was removed 1 h later, and the chest cavity was closed. After reperfusion for 1 h, 2 h, 6 h or 12 h, mice were sacriiced for spec- imen analysis. A sham group received thoracotomy only.

2.3.Treatment protocols
Grouping was as follows: sham operation group (sham), IR group (IR), IR þ Mcc950 pretreatment group (IR þ Mcc950), and IR þ edaravone pretreatment group (IR þ edaravone). According to different administration time points, the IR þ Mcc950 group was further divided into the following subgroups: 15 min, 1 h, and 3 h before ischemia. Mcc950 (50 mg/kg, i.p.) (Selleckchem, Houston, USA) was diluted to 5 mg/ml with normal saline before use [8]. Edaravone (10 mg/kg, i.p.) (Simcere, Nanjing, China) was diluted to 1 mg/ml with normal saline before use [15,16]. Blood was collected through the postcava and centrifuged at 3000 rpm for 10 min at 4 o C; the plasma was stored in a —80 o C refrigerator. Bron- choalveolar lavage fluid (BALF) was obtained by lavaging the left lung with 2 ml of normal saline through a tracheal catheter (re- covery rate > 80%) three times and centrifuged at 3000 rpm for 10 min at 4 o C; the supernatant was stored at —80 o C. The pulmo- nary artery was lavaged with 2 ml normal saline at a pressure of 15 cmH2O to remove the residual blood in the pulmonary blood vessel. The top half of the left lung was used to measure the wet/dry (W/D) weight ratio, and the remaining lung tissue was stored in liquid nitrogen or ixed in 10% paraformaldehyde for further use.

2.4. Histological assessment of lung injury
Parafin-embedded lung sections (5 mm) were stained with he- matoxylin and eosin. Lung injury scoring was independently per- formed by two pathologists according to published criteria [17].

2.5. Lung tissue wet/dry weight (W/D) ratio
Fresh top halves of the left lung were weighed and were then placed into an electric oven at 65 o C for 48 h. The dry weight was then obtained, followed by calculation of the W/D ratio.

2.6. Detection of total protein content in BALF
The protein concentration in the BALF supernatant was detected using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China).

2.7. Measurement of MDA and GSH in lung tissue
The levels of malondialdehyde (MDA) and glutathione (GSH) in lung tissue were detected by two commercial kits. A Lipid Peroxi- dation MDA Assay Kit (Beyotime Biotechnology, Shanghai, China) was used for the measurement of MDA, which was calculated as nmol/mg protein. A GSH and GSSG Assay Kit (Beyotime Biotech- nology, Shanghai, China) was used for the assay of GSSG and total GSH; the GSH levels were calculated using the following method: GSH ¼ total GSH —2 X GSSH, and the results were indicated as the percentage of GSH in total GSH.

2.8. Immunohistochemical staining of Ly6G þ neutrophils
Parafin-embedded lung sections (5 mm) were prepared. Then, sections were incubated with rat anti-mouse lymphocyte antigen 6 complex locus g6D (Ly6G) antibody (1:100 dilution, BD Biosciences, catalog number: 551459) at 4 o C overnight. Subsequently, the sec- tions were incubated with horseradish peroxidase (HRP)-conju- gated secondary antibody for 30 min at room temperature. Fresh 3,30 -diaminobenzidine (DAB) solution was added to the sections, and the sections were counterstained with hematoxylin. Images were captured under microscopy, and the total number of Ly6G þ cells were counted with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Silver Spring, MD).

2.9. TUNEL assay
Parafin-embedded lung sections (5 mm)were prepared. Apoptotic cells were detected using a commercial kit (Roche Applied Science, catalog number: 11684817910). Nuclei were stained with 40 , 6-diamidino-2-phenylindole (DAPI). Fluorescent pictures were captured using a fluorescence microscope (Olympus, Tokyo, Japan) and DP2-BSW software. TUNEL-positive cells were counted manually with Image-Pro Plus 6.0 software.

2.10. ELISA
The levels of Il-18 in plasma were measured using a commercial kit (Abclonal, China). Absorbance at 450 nm was measured using a microplate reader (Thermo Scientiic),and the 450-nm absorbance values were corrected by subtracting the reading at 570 nm.

2.11. Western blot analysis
K.-Y. Xu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7

2.12.Coimmunoprecipitation assay
Total proteins were extracted from the lung tissue homogenates, and the concentrations were measured using a BCA protein assay kit (Beyotime Biotechnology, China). Proteins were separated by electrophoresis on 10% SDS-PAGE gels and transferred to a PVDF membrane. The membranes were blocked using 5% skim milk formulated with TBST for 1 hat room temperature. Next, the membranes were incubated at 4 。C overnight with the following primary antibodies: Nlrp3 (1:1000, Cell Signaling Technology, #15101), caspase-1 (1:500, Santa Cruz Biotechnology, sc-398715), IL-β (1:1000, Cell Signaling Technology, #12242), Nek7 (Abcam, ab133514), and glyceraldehyde 3-phosphate (GAPDH; 1:3000, Proteintech, catalog number: 10494-1-AP). After washing, the membranes were incubated for 1 hat room temperature with the following secondary antibodies: horseradish peroxidase- conjugated goat anti-rabbit immunoglobulin G (IgG; 1:3000, Pro- teintech, catalog number: SA00001-2) or horseradish peroxidase- conjugated goat anti-mouse IgG (1:3000, Proteintech, catalog number: SA00001-1). Blots were detected by a commercial kit (ECL, GE Healthcare Biosciences, Pittsburgh, PA, USA). The optical density of the bands was analyzed with ImageJ software. GAPDH was used as the internal control for each group, and protein expression levels were semi-quantiied relative to the levels of the sham group, which were set to 1.Lung tissue was homogenized on ice with RIPA Lysis Buffer (Beyotime Biotechnology, China) and centrifuged at 12000 g for 10 min at 4 。C. Supernatants containing equal amounts of protein were incubated at 4 。C overnight with the following antibodies: control immunoglobulin G (1:100, Beyotime Biotechnology, China, A7016), Nek7 (1:100, Abcam, ab133514), and Nlrp3 (1:200, Cell Signaling Technology, #15101). Then, the samples were incubated with protein A/G beads (Santa Cruz, sc-2003) for 3 hat 4 。C with gentle rotation. Beads were obtained by centrifugation and washed with lysis buffer. After proteins were eluted by boiling the samples, the proteins were detected by western blotting.

2.13. Real-time quantitative PCR
Total RNA was extracted using TRIzol® reagent (Invitrogen), and 2 μg total RNA was reverse-transcribed into cDNA using a cDNA Synthesis Kit (Takara, Code NO. RR036A). Real-time quantitative PCR was implemented on an ABI StepOne Plus System (Applied Biosystems, Foster City, CA) using SYBR Premix Ex Taq (Takara, Code NO. RR82LR). The primers used are as follows: Nlrp3, 50 -AACTGT- CATAGGGTCAAAACGC-30 (forward) and 50 -CACGGCAGAAGCTA- GAAGTGAG-30 (reverse); caspase-1, 50 -CCCAGGCAAGCCAAATCT-30 (forward) and 50 -TTGAGGGTCCCAGTCAGTCC-30 (reverse); Il-1β, 50 -

Fig. 1. The expression of Nlrp3 was consistent with the severity of lung IR injury and Mcc950 pretreatment alleviated IR-induced lung injury. (a) Representative lung tissue sections stained by HE (original magniication x200, scale bar ¼ 100 μm) and (b) lung injury score (n ¼ 6 per group). (c) The expression of Nlrp3 mRNA in lung tissue (n ¼ 3 per group). (d~e) The protein level of Nlrp3 in lung homogenate (n ¼ 3 per group). (f) Representative lung tissue sections stained by HE and (g) lung injury score with or without Mcc950 treatment. (h) Lung wet/dry ratio and (i) Total protein concentration in bronchoalveolar lavage fluid (BALF) with or without Mcc950 treatment. The results are expressed as the means ± SEM, *P < 0.05 compared with the sham group, #P < 0.05 compared with the I/R 2 h group (The IR group in Fig. 1f~i is same to the I/R 2 h group).ATGGGCTGGACTGTTTCTAATG-30 (forward) and 50 -CTTGTGACCCT- GAGCGACC-30(reverse);Il-18,50 -GCCTCAAACCTTCCAAATCA-30 (forward) and 50 - TACAGTGAAGTCGGCCAAAG-30 (reverse); GAPDH, 50 -GTCATCCCAGAGCTGAACG-G-30(forward) and 50 -TACTTGGG- CAGGTTTCTCCAGG-30 (reverse). The mRNA levels of target genes were normalized to GAPDH using the 2 —ΔΔCT method.

2.14.Statistical analysis
All data are indicated as the means ± SEM (standard errors of the mean). Comparisons between two groups were performed using paired or unpaired t tests, while one-way or two-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls (SNK) post hoc test was used for comparisons between multiple groups. Statistical analyses were completed using SPSS software (version 22.0), and P < 0.05 indicated signiicance.

3.Results
3.1.The expression of Nlrp3 reflected the severity of lung injury during lung IR
To study the role of the Nlrp3 inflammasome during lung IR injury, we irst studied the expression of Nlrp3 during lung IR. Mice were grouped as follows: the sham operation group (sham), the ischemia 1 h group (Ischemia), the ischemia 1 h plus reperfusion 1 h
group (I/R 1 h), the ischemia 1 h plus reperfusion 2 h group (I/R 2 h), the ischemia 1 h plus reperfusion 6 h group (I/R 6 h), and the ischemia 1 h plus reperfusion 12 h group (I/R 12 h). We found that the lung IR injury was transient, and the severity of lung injury after reperfusion 2 h was greater than that after reperfusion 1 h, 6 h, and 12 h (p < 0.05) (Fig.1a and b). Similarly, the mRNA and protein levels of Nlrp3 showed a transient increase during lung IR, and both levels peaked in the reperfusion 2 h group (Fig. 1c~e). Therefore, we chose ischemia 1 h and reperfusion 2 h for the remaining experiments.

3.2. Mcc950 pretreatment alleviated ischemia-reperfusion induced lung injury
To conirm the role of the Nlrp3 inflammasome in lung IR injury, we pretreated mice with Mcc950 before IR. In HE-stained sections, the sham group displayed normal lung histological structure. However, the IR group had signiicantly damaged alveolar structure with widespread atelectasis, interstitial edema, evident inflam- matory cell iniltration, and alveolar hemorrhage (Fig. 1f). Mcc950 pretreatment at 1 h and 3 h before IR markedly alleviated these pathological changes, and the IR þ Mcc950 3 h group exhibited the strongest improvement; however, the IR þ Mcc950 15-min group showed no signiicant improvement (Fig.1f). The lung injury scores were inline with the pathohistological changes (Fig. 1g).

Fig. 2.Mcc950 inhibited Nlrp3 inflammasome activation but had no influence on the priming process. (a) Representative western blots and semiquantitative analysis of cleaved caspase-1 (b), Nlrp3 (c) and mature Il-1β (d) (n ¼ 4 per group). (e) The concentration of mature Il-18 in plasma (n ¼ 6 per group). (~fi) Relative mRNA expression level of Nlrp3, caspase-1, Il-1β and Il-18 (n ¼ 4 per group). The results are indicated as the means ± SEM, *P < 0.05 compared with the sham group, #P < 0.05 compared with the IR group.Furthermore, the W/D ratios of the lung tissue and total protein content in the BALF were measured to reflect the permeability of the alveolar capillary membrane. The W/D ratios were markedly elevated after lung IR compared with those after sham operation (P < 0.01); Mcc950 pretreatment lowered the W/D ratios after IR, but only the IR + Mcc950 3 h group reached statistical signiicance (P < 0.05) (Fig. 1h). Similarly, the total protein content in the BALF were also markedly increased after IR compared with the sham operation (P < 0.01); administration of Mcc950 1 hand 3 hbefore IR signiicantly reduced the total protein content in BALF (P < 0.05) (Fig. 1i).

3.3.Mcc950 inhibited Nlrp3 inflammasome activation but had no influence on the priming of Nlrp3 inflammasome components during lung IR
To determine the effect mechanism of Mcc950, we measured the expression of Nlrp3 inflammasome components. The protein levels of Nlrp3, cleaved caspase-1, mature Il-1β, and mature Il-18 were signiicantly increased after IR compared with sham opera- tion (P < 0.05); the IR + Mcc950 1 h and IR + Mcc950 3 h groups showed signiicant decreases in cleaved caspase-1, mature Il-1β and mature Il-18 (P < 0.05); however, Mcc950 had no influence on the increased level of Nlrp3 protein during lung IR (Fig. 2ae). The mRNA expression levels of Nlrp3, caspase-1, and Il-1β were mark- edly increased after IR compared with the sham operation (P < 0.05), but the mRNA level of Il-18 did not markedly change; additionally, Mcc950 pretreatment had no substantial influence on the transcription of Nlrp3 inflammasome components (Fig. 2 ).

3.4. Mcc950 decreased neutrophil infiltration and cell apoptosis during lung IR
To further study the mechanism of Mcc950 in lung IR injury, we examined neutrophil iniltration and cell apoptosis in lung tissue. Consistent with its effect on Nlrp3 inflammasome activation, Mcc950 signiicantly inhibited neutrophil iniltration (Fig. 3a and b) and cell apoptosis (Fig. 3c and d) in the IR + Mcc950 3 h group compared with those in the IR group (P < 0.05).

3.5. Nlrp3 inflammasome activation was ROS-dependent during lung IR
To determine the role of ROS on Nlrp3 inflammasome activation during lung IR, we used the powerful ROS scavenger edaravone. Edaravone signiicantly decreased the production of MDA and oxidation of GSH (P < 0.05), but Mcc950 did not exert these effects (Fig. 4a and b). Edaravone also suppressed Nlrp3 inflammasome activation (Fig. 4c and d), but, edaravone had no remarkable in- fluence on the mRNA expression of Nlrp3 inflammasome compo- nents (Fig. 4e~h).

3.6. Mcc950 partially inhibited the interaction of Nlrp3 and Nek7
To determine the effect of Mcc950 on the interaction between Nlrp3 and Nek7 in vivo, we performed protein coimmunoprecipi- tation experiments on lung tissue lysates, followed by western blotting. The results showed that the interaction of Nlrp3 and Nek7 was markedly enhanced after lung IR and partially inhibited by Mcc950 but not edaravone pretreatment (Fig. 4i and j). Thus,

Fig. 3.Mcc950 decreased neutrophil iniltration and cell apoptosis during lung IR. (a) Immunohistochemical staining of LY6G + neutrophils in lung tissue sections, (b) Quanti- ication of LY6G + neutrophils per high-power ield (HPF; original magniication 根400, scale bar = 50 μm). (c) Detection of apoptosis via a TUNEL assay in lung tissue sections, (d) Quantiication of TUNEL + cells per HPF (original magniication 根400, scale bar = 50 μm). The results are indicated as the means ± SEM, *P < 0.05 compared with the sham group, #P < 0.05 compared with the IR group. inhibition of Nlrp3 inflammasome activation by Mcc950 was partially attributed to inhibition of Nlrp3 and Nek7 interactions.

4.Discussion
The Nlrp3 inflammasome is widely involved in respiratory dis- eases [18]. Here, we conirmed increased activation of the Nlrp3 inflammasome after lung IR.In addition, pretreating mice with Mcc950 3 h before ischemia signiicantly improved lung histological damage,lung edema, and alveolar capillary membrane permeability. Differences in the eficacy of Mcc950 caused by different treatment time points may be attributed to the speciic pharmacokinetics of Mcc950, route of administration and the ischemia method used in this study. In mice, Mcc950 has an oral bioavailability of 68% and a half-life of 3.27 h; the blood concen- tration peaks at 1 h after administration and can maintain peak concentration for 4 h [8,19].Consistent with its protective effect on lung IR injury, Mcc950 signiicantly reduced the production of proinflammatory Il-1β and Il-18 and the iniltration of neutrophils. These inflammatory cyto- kines and neutrophils are crucial mediators of acute lung injury[20,21]. Mcc950 also dramatically reduced cell death during lung IR. As mentioned above, inflammasome activation can induce cell pyroptosis [22]. In our experiment, a subset of the cell death induced by lung IR may be pyroptosis. Thus, we believe that the protective effect of Mcc950 during lung IR may be partially attrib- uted to inhibition of cell pyroptosis. Although Mcc950 signiicantly inhibited Nlrp3 inflammasome activation, it had no signiicantef- fect on the elevated mRNA expression of Nlrp3 inflammasome buy Tween 80 components induced by lung IR. Hence, we suggest that Mcc950 blocked Nlrp3 inflammasome activation rather than the priming process [8,23]. Notably, the transcription level of Il-18 did not markedly increase after lung IR, likely because the Il-18 gene is constitutively expressed in cells and has little demand for priming signals [24,25].The crucial role of oxidative stress during IR has been well documented. ROS is the main mediator of oxidative stress and has been reported to participate in Nlrp3 inflammasome activation [7]. In our present study, inhibition of oxidative stress by the ROS scavenger edaravone, which has been widely applied in clinical treatment [26,27], markedly alleviated Nlrp3 inflammasome acti- vation.

Fig. 4.Nlrp3 inflammasome activation was ROS-dependent during lung IR; Mcc950 blocked Nlrp3 inflammasome activation partially by inhibiting the interaction of Nlrp3 and NEK7. (a) MDA content (nmol/mg protein) and (b) the percentage of GSH to total GSH (%) (n = 6 per group). (c) Representative western blots of cleaved caspase-1, Nlrp3 and mature Il-1β(n = 4 per group). (d) The concentration of mature Il-18 in plasma (n = 6 per group). (e~h) Relative mRNA expression level of Nlrp3, caspase-1, Il-1β and Il-18 (n = 4 per group). (i) Representative coimmunoprecipitation and western blots of Nlrp3 and Nek7. Lung tissue homogenate from the IR group containing the same amount of protein was used as an IgG control. (j) The optical densities of Nlrp3 bands were standardized by its input protein level. The results are expressed as the means ± SEM, *P < 0.05 compared with the sham group, #P < 0.05 compared with the IR group priming process of Nlrp3 inflammasome components. These results suggest that ROS act as a second signal for Nlrp3 inflammasome activation during lung IR [28,29].Finally, we analyzed the effect of Mcc950 on the interactions of Nek7 and Nlrp3. Lung IR promoted binding between Nek7 and Nlrp3; Mcc950 but notedaravone signiicantly reduced this inter- action. However, when we used the Nlrp3 antibody to coprecipitate Nek7, we did not detect Nek7 bands in the subsequent western blot analyses. We speculate that this may be due to the low expression of Nlrp3 protein, resulting in less coimmunoprecipitation of Nek7. Despite this latter result, our results support the hypothesis that Mcc950 might act by blocking interactions between Nlrp3 and Nek7.

In summary, we conirmed that ROS-dependent Nlrp3 inflam- masome activation exacerbated lung IR injury and that Mcc950 had a protective effect against lung injury. This protective role was mediated by its ability to inhibit inflammation and cell death, and was partially attributed to the disruption of interactions between Nlrp3 and Nek7. Thus, Mcc950 is a promising future therapeutic candidate for preventing lung IR injury. However, the exact mechanism of action, optimal route of administration, and appro- priate dose of Mcc950 need to be further clariied.