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Itraq-Based Quantitative Proteomic Analysis of Lungs in Murine Polymicrobial Sepsis with Hydrogen Gas Treatment

Bian, Yingxue; Qin, Chao; Xin, Yuchang; Yu, Yang; Chen, Hongguang; Wang, Guolin; Xie, Keliang; Yu, Yonghao

Author Information
doi: 10.1097/SHK.0000000000000927



Sepsis is considered to be a systemic inflammatory response syndrome triggered by microbial invasion, and severe sepsis was defined as poor organ function secondary to documented or suspected infection (1, 2). When sepsis occurs, the most severely injured organ is lung, which develops to acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) (3). Moreover, sepsis-associated ALI—characterized by inflammatory changes, surfactant dysfunction, tissue damage, and abnormal coagulation—can result in high morbidity, mortality, and inpatient expenditure (4). Therefore, novel therapeutic strategies that aimed at reducing lung damage in septic patients have been implemented. However, there is still no successful treatment for sepsis (2, 4, 5).

It is widely accepted that hydrogen gas (H2) exerts an effective therapeutic role in many diseases, including sepsis, ischemia–reperfusion injury, organ transplantation, multiple organ dysfunction syndrome (MODS), and neurodegenerative diseases (6, 7). Our previous studies have indicated that 2% of H2 can effectively ameliorate multiple organ damage and increase survival rate in septic mice (8, 9, 12). The mechanism of the protective effects of H2 may closely relate to the anti-inflammatory, antioxidant, and antiapoptotic effects, which can be regulated by some signaling pathways like NF-κB or Nrf2/HO-1 (7, 8). However, the underlying mechanisms of H2 treatment remain still unclear.

With the development of molecular biology techniques, proteomic analysis, a powerful screening technology for the global evaluation of protein expression in complex samples, has been widely applied in various studies. Previous studies have investigated the proteome alterations of sepsis (10). The aim of this study was to identify the differential proteins of lung in septic mice with H2 treatment using the isobaric tags for relative and absolute quantitation (iTRAQ) technology coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS), which might clarify the molecular mechanism of H2 in the treatment of sepsis.


Animals and experimental design

Adult male ICR (institute of cancer research) mice, weighing 20 to 25 g and aged 6 weeks, were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). Mice were fed with sufficient water and food in a controlled environment (temperature 21–23°C, humidity 50%–60%, 12:12 h light–dark cycle). A total of 176 mice were used in this study and were randomly divided into four groups (n = 44 per group): sham, sham+H2, sepsis, and sepsis+H2. Septic mice were induced by cecal ligation and puncture (CLP). Two percent of H2 was inhaled for 1 h beginning at 1 and 6 h after sham or CLP operation. 80 mice were used for the assessment of survival rate (n = 20 per group), 32 mice were used for the bacterial load quantification (n = 8 per group), 40 mice were used for lung proteomics analysis (n = 10 per group), and 24 mice were used for western blot analysis (n = 6 per group). After sham or CLP operation, the 7-day survival rate was evaluated. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University General Hospital (Tianjin, China).

CLP model

CLP was performed as described in a previous study (11). After fasting for 8 h with water supply, animals were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg) in saline and were placed on a sterile operation table. A midline abdominal incision was cut to expose cecum, which was subsequently ligated at the proximal one quarter of cecum. The distal cecum was punctured with a 20-gauge needle and excrement was squeezed out through the puncture point, and then cecum was replaced gently and incision was sutured with a sterile 3-0 silk suture. Animals in sham and sham+H2 groups underwent laparotomy without CLP. All animals were given a subcutaneous injection of 1 mL normal saline after operation.

H2 treatment

As what we described in our previous study (12), animals were put into a leaktight plexiglass box with an inlet and an outlet. H2 was delivered by a TF-1 gas flowmeter (YUTAKA Engineering Corp) which mixed with air into the box at a rate of 4 L/min. The concentration of H2 was monitored by a detector (HY-ALERTA Handheld Detector Model 500; H2 Scan). Carbon dioxide exhaled by animals was cleared away by Baralyme. Animals were given a 2% H2 treatment for 1 h at 1 and 6 h after CLP or sham operation, while animals in sham or sepsis groups were placed in the same box without H2 treatment.

Survival rate

The survival rate of different groups was evaluated within 7 days after sham or CLP operation. All the experiments were performed twice.

Sample collection

Animals in different groups were deeply anesthetized at 24 h after CLP or sham operation. Then, 2 mL of sterile phosphate buffer saline (PBS) was injected into each animal's peritoneal cavity. Peritoneal cavity was then opened and peritoneal lavage was aseptically collected. Moreover, thoracic cavity was opened, and blood was obtained through cardiac puncture using a sterile 18-gauge needle and collected in a heparinized syringe. After auricula dextra was punctured, isotonic saline solution was perfused from left ventricle to remove blood. Lung tissues were then harvested. Left lung tissue was homogenized in 1 mL of 0.9% sterile saline for quantification of bacterial load, and right lung tissue was quickly frozen by liquid nitrogen and stored at −80 °C for proteomics analysis. Five samples were mixed together in each group as an analysis sample to eliminate the individual differences. A total of 8 samples of 4 groups which included 10 biological replicates and 2 technical replicates were disposed by iTRAQ-based quantitative proteomic analysis.

Bacterial load quantification

Blood, peritoneal lavage, and lung homogenates were serially diluted (1:10) in sterile PBS. For bacterial culture, 100 μL of each dilution was plated on 5% goat blood agar plates (Becton, Dickinson and Company, Germany). Plates were incubated at 37 °C in aerobic conditions for 24 h, and then the number of colony-forming units (CFU) was counted.

Protein extraction and digestion

The frozen lung tissue was powered with liquid nitrogen and lysed in 8 M urea, 1% NP40, 1% sodium deoxycholate supplemented with 5 mM DTT, 2 mM EDTA and protease inhibitor cocktail (Calbiochem, San Diego, Calif). Unbroken cells and debris were removed by centrifugation at 4 °C for 10 min at 20,000 g. Protein content in supernatant was determined with a two-dimensional Quant kit (GE Healthcare, Piscataway, NJ). An equal amount of protein was reduced with 5 mM DTT, alkylated with 25 mM IAM, and then precipitated with ice-cold acetone. The precipitate was washed twice with acetone, suspended in 0.1 M TEAB, and digested with 1/25 trypsin (Promega, Madison, Wis) for 12 h at 37 °C. The digestion was terminated with 1% TFA, and the resulting peptide was cleaned with Strata X C18 SPE column (Phenomenex, Torrance, Calif) and vaccum-dried in scanvac maxi-beta (Labogene, lynge, Denmark).

iTRAQ labeling

Peptides were reconstituted in 20 μL of 0.5 M TEAB and processed according to the manufacturer's protocol for iTRAQ 8-plex kit (AB Sciex, Foster City, Mass). Briefly, iTRAQ reagent was thawed and reconstituted in 70 μL isopropanol. Peptides were labeled with iTRAQ reagent by incubation at room temperature for 2 h and then dried by vacuum centrifugation.

Fractionation by basic reverse-phase chromatography

The labeled peptides were pooled and reconstituted in buffer A (100% H2O, pH 10.0) and loaded onto a 4.6 × 150 mm XBridge Shield C18 RP column containing 3.5 μm particles (Waters, Milford, Mass) with LC20AD HPLC (Shimadzu, Kyoto, Japan). The peptides were eluted at a flow rate of 1 mL/min with a gradient of 2% to 10% buffer B (80% ACN, pH 10.0) for 5 min, 10% to 35% buffer B for 50 min and 35% to 90% buffer B for 10 min. The system was then maintained in 90% buffer B for 5 min before equilibrating with 2% buffer B. Elution was monitored by measuring absorbance at 214 nm, and fractions were collected every 1 min. The eluted peptide was pooled as 21 fractions and vacuum-dried.

LC–ESI–MS/MS analysis

The lyophilized peptides were resuspended in buffer A (2% ACN, 0.1% FA), loaded onto an Acclaim PepMap 100 C18 trap column (Dionex, 75 μm × 2 cm) by Ultimate 3000 nanoUPLC (Dionex, Germering, Germany) and eluted onto an Acclaim PepMap RSLC C18 analytical column (Dionex, 75 μm × 25 cm). A 45 min linear gradient was run at 300 nL/min, starting from 11% to 20% buffer B (80% ACN, 0.1% FA), followed by gradient to 80% buffer B at 2 min, and maintenance at 80% buffer B for 3 min.

The peptides were subjected to NSI source in mass spectrometry Q Exactive plus (Thermo Scientific, Bremen, Germany) coupled online to UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000 and the m/z scan range was from 350 to 1,500. A data-dependent procedure that alternated between 1 MS scan followed by 15 MS/MS scans was applied for the top 15 precursor ions above a threshold of 1E5 with 15 s dynamic exclusion. Peptides were selected and fragmented for MS/MS using 28% NCE. Ion fragments were detected in the Orbitrap at a resolution of 17,500. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 3E6 ions were accumulated for generation of MS spectra and 1E5 ions for MS/MS spectra. The max injection time was 250 ms for MS scan and 100 ms for MS/MS scan.

Protein identification and quantitation

The resulting raw data were converted to mascot generic file with Proteome Discoverer (Thermo Scientific, v1.4.1.14) and processed by Mascot search engine (Matrix Science, London, UK, v.2.3.02). Tandem mass spectra were searched against swissprot mouse database (16,724 sequences). Mass error was set to 20 ppm for precursor ions and 0.02 Da for fragment ions. Trypsin was selected for enzyme specificity and two missed cleavages were allowed. Carbamidomethylation on Cys, iTRAQ 8-plex tag on Lys, and peptide N-terminal was specified as fixed modification; oxidation on Met and iTRAQ 8-plex tag on Tyr was specified as a variable modification. Decoy (reverse) database were searched again to estimate and ensure the false discovery rate (FDR) was less than 1%. The calculating results were revalued by algorithm percolator, and peptide-spectrum matches (PSMs) with P < 0.05 and e-value < 0.05 were accepted. For quantitation, every kind of protein must have at least two unique peptides above identity. The protein ratio type was set to be weighted, and the normalization method was median.

Western blot

Lung tissues were obtained at 24 h after CLP or sham operation, and then lysed in RIPA buffer and protease inhibitor to extract total protein to detect the expression of semaphorin 7A (Sema 7A), Transferrin, ubiquitin thioesterase (OTULIN), and mitogen-activated protein kinase kinase kinase 1 (MAP3K1). The total protein was quantified by BCA protein assay kit (Well-bio, Shanghai, China). The same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Mass), blocked using 5% nonfat milk in Tris-Tween buffer saline for 2 h, and incubated overnight at 4 °C with the following primary antibodies against Sema 7A (1:400; Abcam, Cambridge, UK), Transferrin (1:2,000; Abcam, Cambridge, UK), OTULIN (1:1,000; Cell Signaling Technology, Mass), MAP3K1 (1:1,000; Abcam, Cambridge, UK), and β-actin (1:6,000; Proteintech, Chicago, Ill). After five washes with Tris-buffered saline+Tween, membranes were incubated for 1 h with goat antirabbit or antimouse secondary antibodies (1:5,000; KPL, Mass) at room temperature. Finally, protein bands were treated with a chemiluminescence plus reagent and visualized with an enhanced chemiluminescence (ECL) reagent. The intensity of each band was calculated by Gene Tools Match software (Syngene, Cambridge, UK). The relative levels of Sema 7A, Transferrin, OTULIN, and MAP3K1 were normalized to β-actin. The experiment was repeated 6 times.

Statistical analysis

The SPSS 16.0 software was used in this study. The survival rate was expressed as percentages and analyzed by Fisher exact probability method. Measurement data were expressed as mean ± standard deviation (SD). One-way ANOVA was used to analyze the comparison of multiple groups and Tukey's multiple comparison test was used to analyze intergroup comparison. P < 0.05 was considered significant.


Survival rate

After CLP operation, mice were observed to have several significant physiopathological changes such as messy hair, loose stool, reduced physical activity, and increased respiratory rate. During 7-day experimental observation, no mice died in sham and sham+H2 groups. However, the 7-day survival rates in sepsis and sepsis+H2 groups were markedly decreased, respectively (P < 0.05). Two percent H2 inhalation markedly increased the 7-day survival rate of septic mice (20% vs. 50%, P < 0.05) (Fig. 1).

Fig. 1
Fig. 1:
H2 inhalation meliorated the 7-day survival rate in septic mice.

Bacterial load quantification in blood, peritoneal lavage, and lungs

At 24 h after CLP or sham operation, blood, peritoneal lavage, and lung samples were harvested to determine the CFU. The increased bacterial burden was found in blood, peritoneal lavage, and lung samples of sepsis-challenged animals (P < 0.05), which were significantly ameliorated by H2 treatment (P < 0.05) (Fig. 2).

Fig. 2
Fig. 2:
H2 enhanced the clearance of bacterial burden in the blood, peritoneal lavage, and lung tissue of septic mice.

Data characterization

A high-throughput iTRAQ-based quantitative proteomics workflow was employed to identify and quantify the proteome of lung tissues in different groups that we mentioned above. Finally, 5,688 proteins were identified, of which 4,472 proteins were quantifiable. The change of protein expression was considered as significant when the upregulated quantitative ratio was more than 1.3-fold or the downregulated ratio was less than 0.77-fold, and coefficient of variance (CV) <30% among technical replicates.

A total of 168 changed proteins, including cystatin-C, superoxide dismutase (SOD) 1, SOD 3, atrial natriuretic peptide (ANP), and heme oxygenase-1 (HO-1), were identified by iTRAQ-based quantitative analysis in sepsis/sham group (Supplemental Digital Content 1,, which demonstrates the ratio of 168 sepsis-associated proteins.), in which 112 proteins were at higher levels and 56 proteins were at lower levels.

Based on pairwise comparisons in four groups, differential proteins of H2 treatment alleviating ALI in septic mice should at least meet one of these three conditions below. Proteins should be significantly different both in sepsis/sham and sepsis+H2/sepsis groups, and the alteration trends of these proteins in sepsis/sham group should be contrary to the trends in sepsis+H2/sepsis group (class 1, Supplemental Digital Content 2,, which demonstrates the ratio of 192 H2-related proteins). Proteins should be significantly different in sepsis/sham group, while these proteins have no significant differences in sepsis+H2/sham+H2 group (class 2, Supplemental Digital Content 2,, which demonstrates the ratio of 192 H2-related proteins). The first and second conditions suggest that the therapeutic targets of H2 treatment are the same as the pathogenic targets of sepsis and that H2 treament can inhibit the change of protein expression induced by sepsis. Proteins should have no significant differences in sepsis/sham group, while these have significant differences in sepsis+H2/sham+H2 group (class 3, Supplemental Digital Content 2,, which demonstrates the ratio of 192 H2-related proteins), which indicates that the target of H2 treatment is different from the target of sepsis and the functional antagonism of these proteins may reduce the damage of sepsis. Based on the above three conditions, a total of 192 proteins were identified and considered to be closely related to the mechanism of H2 treatment alleviating ALI in septic mice. Several significantly differential proteins, according with the first, second and third conditions, were, respectively, shown in Tables 1–3.

Table 1
Table 1:
List of the several H2-related significantly differential proteins according with condition one
Table 2
Table 2:
List of the several H2-related significantly differential proteins according with condition two
Table 3
Table 3:
List of the several H2-related significantly differential proteins according with condition three

Functional enrichment analysis of H2-related proteins

To further understand the function and role of H2-related proteins, we performed Gene Ontology (GO) and pathway enrichment analysis using PANTHER database ( (binomial test was used to test the statistical significance). Among the 192 H2-related proteins, 184 proteins had annotation information in database. The whole GO and pathway analysis of H2-related proteins were shown in Supplemental Digital Content 3,

For a biological process GO term, the percentages of top three terms were metabolic process (23.38%), cellular process (16.92%), and biological regulation (10.20%). The top 10 significantly enriched biological process terms were shown in Figure 3A. There were four terms closely related to muscle contraction (muscle system process, muscle contraction, striated muscle contraction, and heart contraction).

Fig. 3
Fig. 3:
Significant enrichment analysis of differentially expressed proteins.

For cellular component, cell part (34.68%), organelle (26.61%), and extracellular region (13.71%) were ranked as the three largest proportions. Among the top 10 significantly enriched cellular component terms (Fig. 3B), there were 4 extracellular terms (extracellular space, extracellular matrix, extracellular region part, and extracellular region) and 4 terms about muscle fiber (contractile fiber, myofibril, contractile fiber part, and sarcomere).

For the molecular function GO term percentages, we found that binding (29.57%), catalytic activity (27.83%), and structural molecule activity (16.52%) were the top three terms. Among the top 10 significantly enriched molecular function terms (Fig. 3C), half of them were about oxygen transport (oxygen transporter activity, heme binding, tetrapyrrole binding, oxygen binding, and transporter activity). Based on the final optimal set, four significantly enriched pathways were blood coagulation, plasminogen-activating cascade, nicotinic acetylcholine receptor signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathway (Fig. 3D).

We also performed functional interaction network analysis through STRING database ( There were 4 enriched interaction clusters in STRING functional association networks (Fig. 4): 6 ribosomal proteins, 10 myosin- and troponin-related proteins, 7 collagen- and adhesion-related proteins, and 10 coagulation-related proteins.

Fig. 4
Fig. 4:
Top four clusters of highly interconnected protein networks.

Validation of Sema 7A, Transferrin, OTULIN, and MAP3K1

Western blot analysis was done to validate the results of quantitative proteomics. On the basis of estimation of protein function and the availability of commercial antibodies, proteins involving Sema 7A, Transferrin, OTULIN, and MAP3K1 were selected. Figure 5 shows the expression of four proteins measured by western blot. Compared with sham group, the expression levels of Sema 7A, OTULIN, and MAP3K1 were increased in sepsis group, while the expression level of Transferrin was decreased (P < 0.05). Compared with sepsis group, the expression levels of Sema 7A, OTULIN, and MAP3K1 were decreased in sepsis+H2 group, while the expression level of Transferrin was increased (P < 0.05). The expression ratios of four validated proteins were shown in Supplemental Digital Content 4,

Fig. 5
Fig. 5:
Effects of H2 on the protein expression of Sema 7A, Transferrin, OTULIN, and MAP3K1 in lung tissue of septic mice.


CLP, a gold standard model for sepsis, was used in the present study. Particularly, several biomarkers of sepsis, including Cystatin-C, SOD 1, SOD 3, ANP, and HO-1, were identified in 168 sepsis-related proteins. Cystatin-C, SOD 1, and SOD 3 were served as biomarkers in the early diagnosis of sepsis (13, 14). ANP had prognostic and diagnostic significance in sepsis (15). Moreover, our previous studies have found that H2 alleviated inflammatory response in septic mice by activating Nrf2/ARE signaling pathway and upregulating the expression of HO-1 (16, 17), which was also the candidate protein in our proteomics data. The expression trends of these five proteins in our MS analysis were identical with the results reported in literature, implying that the septic model performed by CLP was successful.

Currently, it is generally accepted that H2 and hydrogen-rich saline both exert an effective therapeutic role in sepsis. Our previous studies have indicated that H2 treatment provides a beneficial effect on sepsis and sepsis-induced organ damages, including lung, liver, kidney, and brain (12). As H2 inhalation has direct effects on lung, in the present study, mice in the H2 group received 2% H2 inhalation for 1 h beginning at 1 and 6 h after CLP or sham operation. Moreover, in this study, inhalation of H2 was found to effectively increase the 7-day survival rate in septic mice. The mechanisms shown in our previous studies were associated with the regulation of oxidative stress, inflammatory response and apoptosis, which might be mediated through NK-κB and Nrf2/HO-1 signaling pathway (7). In this study, we also found that H2 enhanced the clearance of bacterial burden from blood, peritoneal lavage, and lung tissue of septic mice.

In the present study, quantitative proteomic techniques including iTRAQ labeling in vitro coupled with LC–MS/MS were applied. A total of 192 proteins were identified and considered to be closely related to the mechanism of H2 alleviating ALI in septic mice. GO, PANTHER database, and STRING network were utilized to annotate the function and pathway of these 192 proteins.

The GO analysis indicated that the H2-related differentially expressed proteins were mainly involved in muscle contraction (muscle system process, muscle contraction, striated muscle contraction, and heart contraction) and oxygen transportation (oxygen transporter activity, heme binding, tetrapyrrole binding, oxygen binding, and transporter activity). Severe sepsis elicited mitochondrial dysfunction and depressed biogenesis in skeletal muscles. Butler's study indicated that oxygen delivery index in dogs with sepsis was significantly lower (18). The transportation and utilization of oxygen are poor by an impaired mitochondrial electron transport chain in septic patients. In our previous study, H2 could increase the expression of HO-1 in septic mice, but reduce the expression of inflammatory cytokines such as HMGB1 (17). In the present study, there were 4 terms about muscle contraction in the top 10 significantly enriched biological process and 5 terms of those about oxygen transport in the top 10 significantly enriched molecular function, demonstrating that the protective role of H2 on sepsis-induced ALI may be mediated by alleviating the mitochondrial injury and the abnormal metabolism of skeletal muscle, accordingly strengthening the contraction of skeletal muscle of diaphragm and limbs to improve the oxygen transport capacity of septic mice, thereby improving the respiration and circulation.

In our pathway analysis, H2-related differential proteins were significantly enriched in nicotinic acetylcholine receptor signaling pathway. Some studies have found that the activation of alpha7 nicotinic acetylcholine receptor provided an anti-inflammatory role through the modulation of HO-1 expression, TNF release and transalveolar neutrophil migration, and then improved sepsis-induced ALI (19), suggesting that H2 alleviates ALI in septic mice perhaps through the anti-inflammatory effect of nicotinic acetylcholine receptor and this signaling pathway.

There were four clusters in STRING protein–protein interaction networks. The first cluster was six ribosomal proteins. In previous researches, sepsis induced the reduction of protein synthesis, ribosomal number, and translation factor abundance in muscle (20, 21). The finding of ribosomal proteins cluster suggested that the protective mechanism of H2 against sepsis might through improving protein synthesis by upregulating the expression of ribosomal proteins.

Second, a cluster of proteins related to myosin and troponin were found in STRING. It is well known that myosin and troponin are cytoskeletal proteins. Myosin regulatory proteins may explain vascular hyporeactivity or vasoplegia, which is closely associated with calcium desensitization during muscle system process in sepsis (22). Interestingly, the top 10 significantly enriched biological process terms included 4 terms related to muscle contraction process (muscle system process, muscle contraction, striated muscle contraction, and heart contraction) and 1 term response to metal ion. Moreover, the top 10 significantly enriched cellular component terms included four terms related with the contraction of muscle fibers (contractile fiber, myofibril, contractile fiber part, and sarcomere). These results suggest that the therapeutic mechanism of H2 inhalation against sepsis may involve electrically stimulated muscle contraction modulated by calcium ion and the stability of cytoskeleton.

Third, the STRING network analysis also identified seven proteins of collagen and adhesion related to barrier membranes. From the distribution of cellular component terms, 4 of the top 10 terms were associated with extracellular structures (extracellular space, extracellular matrix, extracellular region part, and extracellular region), which are relevant to cell adhesion and locomotion (23, 24). Moreover, collagen and adhesion proteins are also associated with the significantly enriched pathway—inflammation mediated by chemokine and cytokine signaling pathway (25).

The last cluster was 10 coagulation-related proteins. Previous study has revealed a strong association between coagulation function and sepsis (26). In our pathway analysis, the top two significantly enriched pathways were blood coagulation and plasminogen activator cascade. In view of these clusters of proteins which mentioned above, the protective role of H2 treatment against sepsis was related to protein synthesis, muscle contraction, collagen barrier membranes, cell adhesion, and coagulation function.

We selected four differentially expressed proteins (Sema 7A, Transferrin, OTULIN, and MAP3K1) to be the candidate proteins. The expression tendency of these proteins confirmed by western blot was in good agreement with the proteomics data, suggesting the accuracy of high throughput quantitative proteomics analysis based on iTRAQ and LC–MS/MS. But the difference of protein expression in proteomics data was sometimes smaller than the difference measured by western blot. This phenomenon is consistent with the character that iTRAQ technology tends to underestimate proteins’ fold change because of contaminating near-isobaric ions co-isolated and fragmented (27). So it is necessary to verify expression level with low-throughput molecular biology techniques especially for those proteins of biological significance.

The membrane-associated GPI (glycosylphosphatidylinositol)-linked semaphorin Sema 7A is a negative regulator of T-cell response (28, 29). Sema 7A stimulates cytokine production in monocytes and macrophages through α1β1 integrin, which is critical for the effector phase of inflammatory response and T cell-mediated immune response (30). Sema 7A is distributed in lung epithelial cells and vascular endothelial cells, mediating the migration of neutrophils and the production of pulmonary proinflammatory cytokines. Interestingly, integrin αm and integrin α2b were identified in the 192 H2-related differential proteins. The mass spectrometric data showed that the expression ratio of Sema 7A in sepsis/sham group was 1.708, while in sepsis+H2/sepsis group was 0.764. Sema 7A possibly stimulates inflammatory response, cytokine production in monocytes, and macrophages through integrin in CLP-induced septic mice. However, H2 treatment can significantly downregulate the expression of Sema 7A, which can suppress cytokine production and inflammatory response.

Transferrin is the main iron protein in plasma that involves in iron transport, absorption and the degradation, store, and recycle of heme (31). Serum Transferrin and its sialylation could reflect the intensity of inflammatory response, and this new feature is a potential marker of sepsis severity (32). In 2011, Aman et al. (33) found that, in critically ill patients, the decreased level of plasma Transferrin is associated with an increased pulmonary vascular permeability. Through the pathway analysis, we found that Transferrin participated in mineral absorption and HIF-1 signaling pathways. The mass spectrometric data showed that the ratio of Transferrin expression in sepsis/sham group was 0.401, while in sepsis+H2/sepsis group was 1.755, hinting that Transferrin may play an important role in the mechanism of H2 treatment against sepsis by decreasing pulmonary vascular permeability and involving in mineral absorption and HIF-1 signaling pathway.

OTULIN is a deubiquitinase that acts not only as a regulator of angiogenesis, but also as an innate immune response (34). MAP3K1 composes a protein kinase signal transduction cascade and activates the ERK and JNK kinase pathways (35). According to our proteomic data, the expression ratio of OTULIN and MAP3K1 in sepsis/sham group was 1.399 and 1.515, while in sepsis+H2/sepsis group was 0.719 and 0.747, respectively. OTULIN and MAP3K1 may play certain functions during H2 alleviating lung injury in septic mice.

In conclusion, we performed quantitative proteomics approaches of iTRAQ labeling coupled with LC–MS/MS to find out 192 differentially expressed proteins which associated to the protective effects of H2 against sepsis. Through functional enrichment analysis, the identified proteins could be classified through their different functions, such as muscle contraction, oxygen transport, protein synthesis, collagen barrier membrane, cell adhesion, and coagulation function. Furthermore, H2-related differential proteins were significantly enriched in four signaling pathways, and two of them are associated with coagulation. In addition, H2 alleviates ALI in septic mice through downregulating the expression of Sema 7A, OTULIN, MAP3K1, and upregulating the expression of Transferrin. Future studies about these differentially abundant proteins will be performed to understand the mechanism and clinical application of H2 in sepsis.


The authors thank Xin Li (ProteinT (Tianjin) Biotechnology Co. Ltd., Tianjin, China) for his technical assistance in mass spectrometry and bioinformatics analysis, and Dr. Jie Li for finally discussing and editing the manuscript.


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Acute lung injury; hydrogen gas; isobaric tags for relative and absolute quantitation (iTRAQ); proteomics; sepsis

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