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Basic Science Aspects

Methane Attenuates Hepatic Ischemia/Reperfusion Injury in Rats Through Antiapoptotic, Anti-Inflammatory, and Antioxidative Actions

Ye, Zhouheng*; Chen, Ouyang; Zhang, Rongjia*; Nakao, Atsunori; Fan, Danfeng*; Zhang, Ting*; Gu, Zhengyong*; Tao, Hengyi§; Sun, Xuejun*

Author Information
doi: 10.1097/SHK.0000000000000385

Abstract

INTRODUCTION

Ischemia/reperfusion (I/R) injury consists of a course of several events, including an initial interruption of blood and oxygen supply to an organ and subsequent restoration of perfusion and oxygenation (1). Hepatic I/R injury occurs in several situations such as liver surgery (2), liver transplantation (3), and circulation shock (4). Liver I/R can introduce severe tissue damage and diverse types of cell death, which results in increased mortality (3).

Different molecular mechanisms contribute to the pathophysiology of hepatic I/R injury. The predominant mechanism in hepatic I/R injury is the production of reactive oxygen species (ROS) and accumulation of proinflammatory cytokines during the reperfusion period (5). Increased ROS, especially hydroxyl radicals, play an important role in the early part of cell death and tissue damage, causing chaotic microvascular liver dysfunction. Furthermore, the inflammatory response derived from Kupffer cells, the resident macrophages of the liver, and infiltrating leukocytes aggravate injury. Reactive oxygen species and the inflammatory response constitute severe I/R damage to the liver (6). Although multiple candidate injury and inflammatory pathways have been identified in the investigation of I/R theories, no clinically applicable therapeutic strategy has been established to prevent or treat this disorder.

Methane, the simplest alkane, is the most abundant organic gas in the atmosphere (7) and the most abundant greenhouse gas after carbon dioxide (8). It can help control the amount of hydroxyl in the troposphere (9) and also plays a role in global warming. Characterized as biologically inactive, methane has had little known function in the field of disease treatment. However, methane has recently been suggested to have positive effects on small bowel transit time (10) and Boros and colleagues (11) suggest that methane inhalation has potent protective effects on animal intestinal I/R. Nonetheless, the effect of methane on liver I/R injury is still unknown. Meanwhile, the effectiveness of methane-rich saline, which is more clinically applicable than inhaled methane, needs to be clarified.

Accordingly, we hypothesized that methane-rich saline has a protective effect on warm liver I/R injury. Using a rat hepatic I/R model, we investigated liver function and cell death to exhibit the protective effect of methane, examining its effect on apoptosis, oxidation level, and the inflammatory response.

MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley rats weighing 200 to 250 g (Experimental Animal Center of the Second Military Medical University, Shanghai, China) were housed in a temperature-controlled room with free access to food and water under a natural day/night cycle. All experimental procedures were approved by the Second Military Medical University Institutional Animal Care and Use Committee.

Experimental protocol

Under chloral hydrate anesthesia, rats received hepatic I/R operations. In the experiments of effective dosage, 40 rats were randomly divided into seven groups: the sham group received laparotomies without blood supply interference; the I/R group underwent hepatic I/R surgery; the I/R + Saline group suffered I/R injury with 5-mL/kg saline administration; groups receiving I/R surgery with 1, 5, 20, or 40 mL/kg of methane saline (MS). Methane saline was intraperitoneally administered with a syringe in one bolus injection. The volumes for rats in each treatment group are as follows. Relatively, the rats in 1-, 5-, 20-, and 40-mL/kg MS groups received 0.2 to 0.25 mL, 1 to 1.25 mL, 4 to 5 mL, and 8 to 10 mL, separately. To ensure that each rat accepts one shot, we used syringes with different capacities (1, 5, or 10 mL). At the end of reperfusion, all rats were sacrificed for blood sample. In the subsequent test for protective effect of methane, 30 rats were randomly separated to three groups: sham group, I/R group, and I/R with 10-mL/kg dose of methane-rich saline group. Saline or methane-rich saline was injected at the beginning of reperfusion. At the end of reperfusion, 15 rats were sacrificed for tissue imaging, blood sample, and liver tissue collection. The other 15 rats were sacrificed for liver and fixed in phosphate-buffered formalin for histological analysis (Fig. 1).

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Fig. 1:
Diagram of protocol of experiments.

Methane-rich saline production and measurements

Methane stored in a gas canister was dissolved in physiological saline for 3 h under high pressure (0.4 MPa) to a supersaturated level. The saturated methane-rich saline was stored under atmospheric pressure at 4°C for 24 h. Methane-rich saline was freshly prepared 1 day before animal experiments to ensure a steady concentration of injection. Gas chromatography (Gas Chromatography-9860; Qiyang Co., Shanghai, China) was implemented to disclose the content of methane in saline solute 1 day after the preparation. Admittedly, de-gas of methane in the process of measurement, particularly in the process of becoming normothermic and in the operation of using a syringe for injection MS, could happen that would bring impact on the result of methane concentration in the process of detection (see Figure, Supplemental Digital Content 1, at https://links.lww.com/SHK/A302). To minimize the impact of de-gassing, methane concentration was measured at the time immediately before administration. Briefly, 24 h after the preparation of MS, it was arranged to reach the normothermic situation for 2 h. Then, methane-rich saline was injected with a syringe into the 20-mL test tube. The test tube, using particularly in the headspace gas chromatography, was loaded with nitrogen gas to reduce disturbance in advance. After heating in the headspace machine for 20 min, the test tube was assumed to totally contain methane, which was stored in the MS. Upper gas of the tube was mechanically introduced using negative pressure into the gas chromatography for analysis. According to the calculation, the concentration of the methane-rich saline, using both method of production and measurement previously mentioned, was 0.99 mmol/L using methane-containing standard gas (Shanghai Jiliang Standard Gas Ltd., Shanghai, China) as comparison. In addition, the concentration of methane in blood was detected according to the same protocol previously mentioned.

Hepatic I/R procedure

Rats were fasted for 12 h before the experiments but were provided free access to tap water. Anesthesia was induced by intraperitoneally administering chloral hydrate (0.3 mL/100 g). Hepatic I/R procedures were performed as previously described (12). Briefly, an incision parallel to the central axis was made 3 cm away from the median line of the abdomen under chloral hydrate anesthesia. The blood supply to the left lateral and median lobes of the liver was occluded using an atraumatic vascular clamp for 60 min. A heat blanket with rectal temperature detection was applied to maintain 37°C body temperature. Reperfusion was initiated by removal of the vascular clamp. In the treatment group, methane-rich saline was injected into the abdominal cavity at the beginning of reperfusion with the indicated dose after the closure of the incision. The administration time of methane-rich saline was determined by the result of methane concentration in blood (see Figure, Supplemental Digital Content 2, at https://links.lww.com/SHK/A303). Reperfusion lasted for 6 h before rats were sacrificed without supplemental anesthesia during the process of reperfusion.

Serum analysis

Blood samples were placed in a 4°C environment for 12 h. Then, serum was separated by centrifugation (10 min at 4,000 rpm at 4°C) in preparation for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) analysis with an autoanalyzer (Hitachi 7600–20, Japan). The concentrations of ALT, AST, and LDH are expressed as units per liter. Serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Rat ELISA kit; Fantibody, NY) according to the manufacturer’s instruction.

MDA and SOD

Whereas malondialdehyde (MDA) levels were measured as a marker of lipid oxidation, superoxide dismutase (SOD) levels were used to indicate antioxidant level. Briefly, liver tissue was collected and immediately frozen in liquid nitrogen and kept at −80°C. One gram tissue was homogenized with 10 mL physiological saline using a glass homogenizer followed by 2,000 rpm centrifugation. Supernatant was collected. Malondialdehyde concentration was calculated using the thiobarbituric acid method (Jiancheng Institute of Biotechnology, Nanjing, China). The results are expressed as nanomoles per gram. Superoxide dismutase activity (units per mg protein) in hepatic tissue homogenate was estimated by evaluating the rate of inhibition of nucleotide oxidation according to the assay kit (Jiancheng Institute of Biotechnology).

Western blot

Tissue was homogenated in tissue lysate (Beyotime Chemical Co., China) containing 50 mmol/L Tris (pH 8.0), 150 mmol NaCl, 1% Triton X-100, and 100 μg/mL phenylmethyl sulfonylfluoride. Two minutes of centrifugation combined with 1 min of cooling was repeated until the tissue was well split. Protein concentration was measured by bicinchoninic acid assay (Beyotime Chemical Co., China). Protein samples (20 μg) were denatured for 4 min at 95°C in sample buffer. Electrophoresis was performed in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by protein transfer to nitrocellulose membrane (Beyotime Chemical Co., China). The membrane was blocked in 5% nonfat dry milk in TBST (10 mmol Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20) overnight at 4°C followed by incubation in primary rabbit–antirat caspase-3 polyclonal antibody (dilution 1:1,000; Abcam, USA). Using antirabbit horseradish peroxidase secondary antibody (dilution 1:15,000; Abmart, Shanghai, China) for 1 h at room temperature, the probed protein was detected with the ECL chemiluminescence system (Bestbio, Shanghai, China). Blots were quantified using Eppendorf equipment.

Real-time reverse transcription polymerase chain reaction

Gene sequences were obtained from NCBI, and RNA sequences of TNF-α, IL-1β, and IL-6 were designed by Oligo and primer 5 and were synthesized by Invitrogen Co. RNA was extracted from tissue homogenization by Trizol (Invitrogen, Carlsbad, Calif). Complementary DNA was synthesized using Reverse Transcriptase (Promega, USA). Real-time polymerase chain reaction (PCR) mixes were prepared with TaKaRa kit (TaKaRa, Tokyo, Japan) and run on the Real-time PCR system (ABI Steponeplus, Applied Biosystems, USA). The procedure was implemented at 95°C for 30 s, 95°C for 5 s, and 60°C for 30 s and repeated 40 times. The 22DDCt method was used to analyze results. Detections were repeated at least twice independently. Primers of β-actin gene expression were ordered (Invitrogen, Shanghai) and measured as an endogenous control.

Histological examination

Isolated liver tissues were fixed in 10% formalin and embedded in paraffin. The tissues were cut into 5-μm sections and stained with hematoxylin and eosin (H&E) for histological examination. The terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assays (In Situ Cell Death Detection Kit; Fluorescein, Roche, Switzerland) were performed on paraffin-embedded sections for cell apoptosis detection. Immunohistochemistry was executed for 8-hydroxyguanosine (8-OHG) and CD68 measurement.

Data analysis

Values are presented as mean ± SD. Statistical analysis was performed using the SPSS software package (SPSS Inc., Chicago, USA) with one-way analysis of variance followed by Student-Newman-Keuls or least significant difference tests. A value of P < 0.05 was considered statistically significant.

RESULTS

Methane-rich saline protects the liver from I/R injury

Experiments were applied according to the protocol shown in Figure 1. To have a solid proof for methane protective function, ALT, AST, and LDH levels of liver I/R injury are applied, which are presented in Figure 2A, B, and C, respectively. Subjects receiving I/R procedures suffered a sharp escalation in levels from 160.4 ± 25.94 to 794.71 ± 150.94 (P < 0.001). As doses of methane-rich saline escalated, ALT levels continuously retreated according to serum detection. Compared with the I/R + Saline group, ALT levels of the 5-mL/kg group was significantly reduced (602.47 ± 107.92 vs. 833.175 ± 176.49). The doses of 20 and 40 mL/kg decreased ALT to 547.94 ± 64.67 and 421.74 ± 117.82, respectively, which both were significantly lower compared with the I/R + Saline group (Fig. 2A; P < 0.01). Meanwhile, AST levels decreased significantly after administration of MS at different dosages, displaying a similar tendency according to ALT level (Fig. 2A, B). Lactate dehydrogenase did not change dramatically, even at the 40-mL/kg dose (Fig. 2C). In addition, H&E staining agreed with both liver function results and liver picture that rodents injected with MS showed less cell death and less tissue damage than rats in the I/R group (Fig. 2E). The proportion of necrosis in the I/R + MS group was much lower than that of the I/R group (15.32 ± 10.15 vs. 59.70 ± 16.0; P < 0.05; Fig. 2D).

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Fig. 2:
Changes in ALT level (A), AST level (B), and LDH level (C) represent liver function in sham, I/R, I/R + 1 mL/kg, IR + 5 mL/kg, I/R + 20 mL/kg, and I/R + 40 mL/kg groups. The reductive effect of ALT and AST showed a dose-dependent tendency. Data represent means ± SD of n = 4 – 9 animals per group; *P < 0.05, **P < 0.01. D, The percent of necrosis was evaluated by comparing the areas of necrosis with area of whole microscopic fields. The percentage of one slice was calculated from two results of microscopic field counts. E, H&E staining of tissue showed cell necrosis in sham, I/R, and I/R + 10 mL/kg groups. Areas in the white polygon are damaged tissue. Data represent means ± SD of n = 3 – 6 per group; **P < 0.01.

Antiapoptotic effect of methane-rich saline in hepatic I/R injury

We also used Western blots of caspase-3 content in the tissue of liver to show how methane affects the apoptotic process (Fig. 3A). The gray-scale ratio in the I/R + MS group was reduced significantly compared with that in the I/R group (0.898 ± 0.148 vs. 1.397 ± 0.153; P < 0.05; Fig. 3B). Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining showed that apoptotic cell numbers were higher in the I/R group than those in the I/R + MS group (Fig. 3D). Quantification of apoptotic cells, shown in Figure 3C, demonstrates less cell death in the I/R + MS group compared with that in the I/R group, which matched the caspase-3 results.

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Fig. 3:
A, Western blot of caspase-3 in sham, I/R, and I/R + 10 mL/kg groups. B, Relative density of caspase-3 in sham, I/R, and I/R + 10 mL/kg groups. Data represent means ± SD of n = 4 – 6 per group; *P < 0.05, # P < 0.05. C, TUNEL-positive cell counts of hepatocytes. The percentage of TUNEL cells were measured by comparing positive cells with cell number in total in one 400× microscopic field. Data represent means ± SD of n = 2 – 6 per group; *P < 0.05, # P < 0.05. D, Photograph of fluorescence TUNEL cells in 400× microscopic field. The first row of the picture was nucleus presenting with blue color. The second row of the photo was damaged DNA presenting with green. The third row of the picture was merging by first and second rows of the image. Name of group from left to right: Sham, I/R, and I/R + MS. n = 2 – 6 per group.

Antioxidative effect of methane-rich saline in hepatic I/R injury

To explore the potential mechanism of methane-rich saline protection on hepatic I/R, we measured MDA, SOD, and 8-OHG levels. The three of them are regular representatives for oxidant change in injury. The highest levels of MDA (2 ± 0.23 nmol/mg) were detected in the I/R group; MDA levels of 1.39 ± 0.26 nmol/mg were detected in the I/R + MS group (Fig. 4A). Regarding SOD content, the I/R group consumed lots of SOD, which was much lower than that in the Sham group (13.88 ± 2.41 vs. 17.73 ± 1.52; P < 0.05). Methane saline significantly increased SOD level to 22.60 ± 4.15 compared with that in the I/R group (Fig. 4B; P < 0.01). 8-Hydroxyguanosine staining also showed a decreased number of positive cells in the I/R + MS group compared with that in the I/R group (Fig. 4C). These results highly pointed that methane interferes with the injury process by the antioxidant pathway.

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Fig. 4:
A, Malondialdehyde level of liver tissue in sham, I/R, and I/R + 10 mL/kg groups. B, Superoxide dismutase level of liver tissue in sham, I/R, and I/R + 10 mL/kg groups. C, Immunohistochemistry of 8-OHG in sham, I/R, and I/R + 10 mL/kg groups. The second row of the result is the amplification (400×) of the first row (200×). Data represent means ± SD of n = 5 – 6 per group; *P < 0.05, # P < 0.05.

Anti-inflammatory effect of methane-rich saline in I/R injury

To explore the possibility that methane also reduced inflammatory levels for a protective effect on I/R injury, we measured major indicators in inflammation to show the alteration. Tumor necrosis factor-α levels of the I/R group were significantly higher in both in situ mRNA levels and serum protein levels after I/R injury. Methane saline administration significantly reduced TNF-α lower than that of the I/R group in both tissue and serum detection (Fig. 5A, D). The level of IL-6 in PCR detection and content of serum exhibited a similar trend (Fig. 5B, E). Although the result of IL-1β displayed an enormous reduction after MS administration compared with that in the I/R group (Fig. 5C), serum content detection failed to show any differences between the three groups (Fig. 5F). Also, Figure 6 suggested that methane reduced infiltration of inflammatory cells in injury tissue. These results indicate, besides an antioxidant way, that methane prevents liver I/R injury by an anti-inflammatory mechanism.

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Fig. 5:
Relative expression of TNF- α (A), IL-6 (B), and IL-1β (C) in sham, I/R, and I/R + 10 mL/kg groups. Data represent means ± SD of n = 4 – 6 per group; *P < 0.05, # P < 0.05. The content of TNF-α (D), IL-6 (E), and IL-1β (F) in sham, I/R, and I/R + 10 mL/kg groups. NS indicates not significant. Data represent means ± SD of n = 6 per group; *P < 0.05, # P < 0.05.
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Fig. 6:
A, B, C, CD68-positive cells in microscopic field with 400× augmentation of sham, I/R, and I/R + MS groups, respectively. Cells with dark brown color indicate CD68 expressed positively. D, Calculative result of positive CD68 cells in sham, I/R, and I/R + MS groups. Data represent as means ± SD of n = 4 – 6 per group; *P < 0.05.

DISCUSSION

Although methane is a common gas with unknown activity on human disease, recent research has shown its unexpected protective effects on organ I/R injury (11). Chemically, methane holds four stable carbon hydrogen bonds that underlie inactive reactions with other compounds. In nature, such as in the troposphere, methane neutralizes ozone and radical hydroxyl to help balance the gas distribution of the atmosphere (9). In the late 1960s, curiosity gave rise to an experiment analyzing radioactive methane on the metabolism of sheep. Radioactive methane was administered to the blood circulation of sheep, and radioactive carbon dioxide was detected in the breath of animals. This scene shows us that methane is used in biological metabolism, but the physiological metabolism of this gas still needs to be clarified (13).

The relationship between methane and intestinal disease has been addressed in recent research. Diagnosis, detection, and effectiveness are always hot controversies for scientists to investigate. Pimentel et al. (14) supposed this detection could draw a bright prospect for clinical application, and Attaluri et al. (15) supported this by assuring diagnosis ratio on real patients with intestinal disease. Mechanism research has shown that methane plays a role in small intestinal and ileum constriction as well as velocity of the tract (16). The resulting hypothesis is that methane influences the physiological motion of the intestinal tract, thus contributing to all kinds of clinical intestinal conditions such as diarrhea (17). In this setting, methane correlated with the progress of disease. Furthermore, results show us that methane may protect against intestinal I/R injury in dogs (11). Indicators of methane’s protective effect against I/R injury show that methane inhalation is of potential therapeutic interest in disease. The inflammation involved in I/R injury has been modulated to a less harmful level.

In our study, we hypothesized that methane-rich saline may play a protective role in hepatic I/R injury. Liver function tests verified that ALT and AST levels may be reduced by methane-rich saline administration. This effect could be enhanced by high doses of methane-rich saline. Also, H&E staining showed that necrosis is dramatically affected by methane. Meanwhile, the number of apoptotic hepatocytes was reduced by methane in TUNEL detection. Western blot of caspase-3 substantiated this hypothesis. These results confirmed our assumption that methane-rich saline protects the liver from I/R injury through anti-inflammatory and antioxidant ways. Although the possibility of de-gassing may exist in the administration process (see Figures, Supplemental Digital Content 1 at https://links.lww.com/SHK/A302, which demonstrates methane concentration in blood), the methane absorbed by objects is still effective according to the results above. The de-gassing problem of methane should be taken into consideration in future experiments.

Methane protects the liver from I/R injury through its anti-inflammatory and antioxidant properties. Methane reduced MDA levels and enhanced SOD to a high level compared with the I/R group. The residual ROS attack implied that methane-rich saline attenuated oxidation injury. This was verified by a reduction of MDA levels and 8-OHG content. In addition, the anti-inflammatory effects of methane-rich saline were demonstrated by a reduction of TNF-α and IL-6 levels both in real-time PCR and ELISA detection. Generally, we reached the conclusion that methane protection functioned in an anti-inflammatory and antioxidant manner.

Inflammation is regarded as a major component of I/R injury (18). Details of inflammation involvement have been illustrated in many studies (19). Therapy against inflammation protects organs from I/R injury (20). Our results confirmed that representative factors of inflammation were dramatically changed in I/R injury. Methane-rich saline downregulated this tendency by immediate injection at the beginning of the reperfusion period. Thus, methane-rich saline may have a potential protective effect against I/R injury.

Oxidants were considered to be a principal factor in hepatic I/R injury both in the early stage and later period (21). Reversing oxidant levels strongly improved the condition of organs suffering from I/R injury (5). Results of our study support the view that oxidant levels increase dramatically in I/R injury. Methane-rich saline decreased oxidant levels tremendously after administration. These results are consistent with the finding that antioxidants bring promising hepatic I/R injury treatment (5). Thus, methane-rich saline may impact the amount of oxidants for I/R injury protection.

In summary, our study suggests that methane might be an encouraging approach to protecting the liver against hepatic I/R injury; however, this warrants further study and verification. Methane provided in saline may more easily translate into clinical application.

CONCLUSIONS

Therefore, we hypothesized that methane-rich saline treatment protects against liver I/R injury. The mechanism of this promising effect could be the antiapoptotic, antioxidative, and anti-inflammatory actions of methane.

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Keywords:

Methane; hepatic I/R injury; apoptosis; oxidation; inflammation

Supplemental Digital Content

© 2015 by the Shock Society