{"id":27386,"date":"2024-01-03T21:46:11","date_gmt":"2024-01-03T19:46:11","guid":{"rendered":"https:\/\/hho-bulgaria.com\/h2-gas-reduces-intestinal-injury-in-sepsis\/"},"modified":"2024-02-04T21:13:20","modified_gmt":"2024-02-04T19:13:20","slug":"h2-gas-reduces-intestinal-injury-in-sepsis","status":"publish","type":"post","link":"https:\/\/hho-bulgaria.com\/en\/h2-gas-reduces-intestinal-injury-in-sepsis\/","title":{"rendered":"H2 Gas Reduces Intestinal Injury in Sepsis"},"content":{"rendered":"
\n

INTRODUCTION<\/h2>\n

Sepsis is the result of a systemic inflammatory response syndrome caused by infection. Severe sepsis is characterized by development of multiple-organ dysfunction syndrome (MODS) and\/or tissue hypoperfusion (1<\/a><\/sup>). Despite decreasing mortality of sepsis in recent years, the risk of death remains high (2<\/a><\/sup>). The intestine is one of the organs that are most vulnerable to the effects of sepsis (3<\/a><\/sup>). When intestinal dysfunction occurs, it may accelerate the dysfunction of other organs. The intestinal mucosal barrier is composed of connections between intestinal epithelial cells and adjacent cells and represents the first line of defense, preventing intestinal bacteria and endotoxin from entering the body (4<\/a><\/sup>). In conditions of oxidative stress, however, the structure and function of the intestinal mucosa are damaged, and the permeability of the intestinal wall increases, leading to intestinal bacterial translocation. Furthermore, increased release of proinflammatory mediators, including interleukin-6 (IL-6), tumor necrosis factor-\u03b1 (TNF-\u03b1), and high-mobility group box 1 protein (HMGB1) is one of the most important causes of sepsis-related intestinal injury. High-mobility group box 1 protein, an important \u201clate\u201d proinflammatory mediator involved in the pathogenesis of sepsis, plays a role as a signal of tissue damage and in particular in activating defense programs. It is also an important biomarker in the process of sepsis (5<\/a><\/sup>).<\/p>\n

There are many potential methods to limit the intestinal injury caused by sepsis. Heme oxygenase-1 (HO-1), also called heat shock protein 32 (hsp32), plays a critical role in the oxidative degradation of heme to free iron, biliverdin, and carbon monoxide (6<\/a><\/sup>). It is up-regulated by severe inflammatory reactions and acts as a cytoprotective enzyme during inflammatory processes, oxidant injury, and ischemia-reperfusion injury (7, 8<\/a><\/sup>). Another important molecule is nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), a basic leucine zipper protein, which plays an important role in regulating the expression of antioxidant proteins that protect against the oxidative damage triggered by injury and inflammation and mitochondrial biogenesis. Under oxidative stress, Nrf2 travels to the nucleus, where it binds to a DNA promoter and initiates transcription of antioxidant genes and their proteins, such as HO-1 (9<\/a><\/sup>).<\/p>\n

Hydrogen gas (H2<\/sub>), which has antioxidative, anti-inflammatory, and antiapoptotic effects, may be effective in the treatment of sepsis (10<\/a><\/sup>). In an earlier study by our group, we showed that inhalation of H2<\/sub> improved survival in a variety of septic models and reduced damage and dysfunction of various organs, including the lung, the liver, and the kidney (11\u201313<\/a><\/sup>). Hydrogen gas treatment has also been shown to significantly reduce intestinal edema and hemorrhage in mice with sepsis (14<\/a><\/sup>). We also demonstrated that HO-1 plays an important role in the anti-inflammatory effect of H2<\/sub> in lipopolysaccharide-stimulated RAW264.7 macrophages (15<\/a><\/sup>). Using the cecal ligation and puncture (CLP) model, a classical septic model, the present study was designed to further investigate whether the protective effect of H2<\/sub> on intestinal injury in septic mice is mediated via HO-1.<\/p>\n

MATERIALS AND METHODS<\/h2>\n

Animals<\/h3>\n

Adult male Institute for Cancer Research mice weighing 18 to 25 g were used in this experiment, purchased from the Laboratory Animal Center of the Military Medical Science Academy of the Chinese People\u2019s Liberation Army (PLA). All the animals were housed in cages in a 12-h light\/dark cycled room at 22\u00b0C to 25\u00b0C and fed with a standard laboratory diet and free access to water. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University and performed in accordance with the guidelines of the National Institutes of Health for the care and use of experimental animals.<\/p>\n

CLP model<\/h3>\n

Cecal ligation and puncture was performed as previously described (16<\/a><\/sup>). Anesthesia was induced using intraperitoneal injection of 2% sodium pentobarbital (50 mg\/kg) in saline. To induce severe sepsis, the cecum was exposed through a 1-cm midline abdominal incision, and then ligated below the ileocecal valve in the distal three quarters of the cecum. It was then punctured with a 20-gauge needle, and the fecal contents were squeezed gently through the puncture point. The cecum was returned to the abdomen and the incision closed with a sterile 3\u20130 silk suture. The animals in the control groups were subjected to laparotomy without CLP. All the operations were performed under sterile surgical conditions. All the animals were given a subcutaneous injection of 1-mL saline solution for resuscitation.<\/p>\n

H2<\/sub> treatment<\/h3>\n

As in our previous studies (11<\/a><\/sup>), the animals in the H2<\/sub> treatment groups were placed in a plastic box with an inlet and outlet. The H2<\/sub> was administered through a TF-1 gas flowmeter (YUTAKA Engineering Corp, Tokyo, Japan) and mixed with air in the box. The concentration of H2<\/sub> in the box was monitored by a detector (HY-ALERTA Handheld Detector Model 500; H2<\/sub> Scan, Valencia, Calif) and kept at 2% throughout the treatment process. The animals in the H2<\/sub> groups inhaled 2% H2<\/sub> for 60 min at 1 and 6 h after CLP or sham operation. The animals in the control groups without H2<\/sub> breathed room air.<\/p>\n

Reagents<\/h3>\n

Zinc protoporphyrin IX (ZnPPIX), a HO-1 inhibitor, was obtained from Merck Millipore (Darmstadt, Germany). Tin protoporphyrin IX (TinPPIX), also an HO-1 inhibitor, was obtained from Frontier Scientific (Logan, Utah). High-mobility group box 1 protein enzyme-linked immunosorbent assay (ELISA) kits were obtained from IBL (Hamburg, Germany). The activities of malondialdehyde (MDA) and 8-iso-prostaglandin F2\u03b1 (8-iso-PGF2\u03b1), as indicators of the degree of oxidative stress, were assayed using commercial kits purchased from Cayman Chemical Company (Ann Arbor, Mich). The DeadEnd fluorometric terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) system was obtained from Promega (Madison, Wis). RNAprep pure tissue kit was obtained from TIANGEN (Beijing, China). RevertAid first strand cDNA synthesis kit was obtained from ThermoFisher Scientific Inc (Austin, Tex). RealMasterMix (SYBR Green) was obtained from TIANGEN. Mouse anti-Nrf2 and HO-1 antibodies were obtained from Santa Cruz Biotechnology, Inc (Dallas, Tex) and Abcam (Cambridge, UK), respectively. Rabbit anti-HMGB1 antibody was obtained from Abcam. \u03b2-actin antibody was purchased from Sigma-Aldrich (St. Louis, Mo). Horseradish peroxidase\u2013conjugated goat antimouse IgG antibodies and horseradish peroxidase\u2013conjugated goat antirabbit IgG antibodies were obtained from Jackson ImmunoResearch (Newmarket, Suffolk, UK). Diaminobenzidine was purchased from Sigma-Aldrich.<\/p>\n

Experimental Protocols<\/h3>\n

Experiment 1: Effects of H2<\/sub> treatment and HO-1 inhibitor on the survival rate of mice in different groups<\/h4>\n

One hundred and twenty animals were randomly assigned to 6 groups (n = 20 per group): sham, sham + H2<\/sub>, severe sepsis, severe sepsis + H2<\/sub>, severe sepsis + ZnPPIX, and severe sepsis + H2<\/sub> + ZnPPIX. Zinc protoporphyrin IX is an inhibitor of HO-1. Animals in the ZnPPIX groups received an intraperitoneal injection of ZnPPIX (40 mg\/kg) 1 h before CLP. Zinc protoporphyrin IX was dissolved in N,N-dimethylformamide (DMF). The survival rate was observed on days 1, 2, 3, 5, and 7 after CLP or sham operation. To further clarify the role of HO-1 exactly, we used a more specific HO-1 inhibitor, TinPPIX. Another 80 mice were divided into 4 groups (n = 20 per group): severe sepsis, severe sepsis + H2<\/sub>, severe sepsis + TinPPIX, and severe sepsis + H2<\/sub> + TinPPIX. Tin protoporphyrin IX was administered to mice (50 mg\/kg) at 6 h before CLP through subcutaneous injection (17<\/a><\/sup>). We also monitored the survival rate on days 1, 2, 3, 5, and 7 after CLP or sham operation.<\/p>\n

Experiment 2: Effects of H2<\/sub> treatment and HO-1 inhibitor on CLP-induced intestinal injury in mice<\/h4>\n

Additional 36 animals were randomly divided into 6 groups (n = 6 per group): sham, sham + H2<\/sub>, severe sepsis, severe sepsis + H2<\/sub>, severe sepsis + ZnPPIX, and severe sepsis + H2<\/sub> + ZnPPIX. The detailed experimental protocols were the same as described before. Mice were killed 24 h after CLP or sham operation. Peritoneal lavage fluid (PLF) from each mouse was collected for measuring the colony-forming unit (CFU) numbers to reflect bacterial loads. The oxidative products, including MDA and 8-iso-PGF2\u03b1 and proinflammatory mediator, HMGB1, in serum and intestine were measured. Moreover, the intestine samples were resected for measuring the number of apoptotic cells in them and observing the histopathological changes in the different groups. In addition, to reflect the organ damage apart from the intestine, we also measured some biochemical markers, such as the following: alanine aminotransferase (ALT), aspartate transaminase (AST), creatinine (Cr), and serum urea nitrogen (SUN). To further clarify the role of HO-1 exactly, we used a more specific HO-1 inhibitor: TinPPIX. Another 24 mice were divided into 4 groups (n = 6 per group): severe sepsis, severe sepsis + H2<\/sub>, severe sepsis + TinPPIX, and severe sepsis + H2<\/sub> + TinPPIX. We also monitored the intestinal histopathological scores of mice in these groups after CLP or sham operation.<\/p>\n

Experiment 3: Effect of H2<\/sub> treatment and ZnPPIX on the expressions of HO-1, Nrf2, and the downstream molecule, HMGB1<\/h4>\n

Additional 108 animals were adopted in this part of the experiment and were randomly divided into 6 groups (n = 18 per group): sham, sham + H2<\/sub>, severe sepsis, severe sepsis + H2<\/sub>, severe sepsis + ZnPPIX, and severe sepsis + H2<\/sub> + ZnPPIX. The late inflammatory mediator HMGB1 in intestine, as well as antioxidant molecules Nrf2 and HO-1 in the intestine were detected at 6, 12, and 24 h by Western blot and quantitative polymerase chain reaction (PCR). In addition, the changes of HMGB1 in different groups were also observed by Immunohistochemical staining.<\/p>\n

Determination of CFU<\/h3>\n

Mice were killed at 24 h after CLP or sham operation. As in the previous study (18<\/a><\/sup>), 12 \u03bcL of PLF from each mouse were collected in sterile tubes and serially diluted with sterile saline. Twelve microliters of each dilution were plated on agar plates and incubated overnight at 37\u00b0C. Bacterial loads in PLF were counted using the number of CFU.<\/p>\n

Measurement of ALT, AST, Cr, and SUN<\/h3>\n

Blood samples were obtained from the left ventricle and immediately used to measure the levels of ALT, AST, Cr, and SUN using an automatic biochemical analyzer (Hitachi Autoanalyzer 7150, Tokyo, Japan).<\/p>\n

Histopathology analysis<\/h3>\n

The intestinal tissues of the groups were fixed in 10% paraformaldehyde for 1 week and then embedded in paraffin. The specimens were sliced at 5 \u03bcm and stained with hematoxylin-eosin (H-E) for light microscopy (Olympus, Tokyo, Japan). A scoring system was used to evaluate the degree of intestinal injury (19<\/a><\/sup>). Intestinal histopathological scores were assessed by 2 pathologists who were blinded to the treatment groups.<\/p>\n

Measurement of MDA and 8-iso-PGF2\u03b1<\/h3>\n

Blood samples were obtained from the left ventricle and centrifuged at 3000 rpm for 10 min. The supernatants were separated from the blood and transferred to freezing tubes and stored at \u221280\u00b0C. Intestinal tissue homogenates were also centrifuged at 3000 rpm for 10 min, and supernatants were stored at \u221280\u00b0C. Levels of MDA and 8-iso-PGF2\u03b1 were measured using a microplate reader (CA 94089; Molecular Devices, Sunnyvale, Calif).<\/p>\n

Measurement of HMGB1<\/h3>\n

The serum and intestinal tissue homogenates were also used to detect the level of HMGB1 using ELISA kits with the microplate reader (CA 94089).<\/p>\n

Western blot<\/h3>\n

Intestinal protein was extracted at 6, 12, and 24 h after CLP or sham operation using precooled sodium dodecyl sulfate sample buffer and protease inhibitors. After centrifugation at 12,000 rpm for 10 min, the supernatant was obtained as the total protein. The protein was fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. These membranes were blocked with 5% nonfat milk and enclosed in the sealed bag with primary antibodies: \u03b2-actin (1:2000 dilution), Nrf2 (1:2000 dilution), HO-1 (1:1000 dilution) and HMGB1 (1:1000 dilution). After incubation at 4\u00b0C overnight, the membranes were washed 3 times in Tris-buffered saline with Tween and incubated in the specific second antibodies for 1 h. Finally, the membranes were treated with chemiluminescence plus reagent and observed using a chemiluminescence imaging system (Syngene, Cambridge, UK). The density of each band was calculated by Gene Tools Match software (Syngene). The results are shown as a percentage of the value in the sham group.<\/p>\n

Real-time quantitative PCR<\/h3>\n

Samples were obtained 6, 12, and 24 h after the CLP or sham operation. Three kinds of messenger RNA (mRNA) were detected in this experiment: Nrf2 mRNA, HO-1 mRNA, and HMGB1 mRNA. Total mRNA was extracted using an RNAprep pure tissue kit. A RevertAid first strand cDNA synthesis kit was used for the reverse transcriptase reaction. The PCR amplifications were performed with RealMasterMix (SYBR Green) by iQ\u21225 under the following conditions: 95\u00b0C for 2 min, 40 cycles at 95\u00b0C for 15 s, 60\u00b0C for 30 s, and 68\u00b0C for 30 s. The levels of the 3 kinds of mRNA were quantitatively normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. Results were calculated using the delta-delta-CT method. The results are shown as the percentage of the sham group value. Gene primer sequences were (Nrf2 Forward 5\u2032-C G A C A G A A A C C T C C A T C T A C T G A A-3\u2032, Reverse 5\u2032-C C T C A T C A C G T A A C A T G C T G A A G-3\u2032; HO-1 Forward 5\u2032-A C A G A T G G C G T C A C T T C G-3\u2032, Reverse 5\u2032-T G A G G A C C C A C T G G A G G A-3\u2032; HMGB1 Forward 5\u2032-A G C C C T G T C C T G G T G G T A T T T T C A A-3\u2032, Reverse 5\u2032-G C T G T G C A C C A A C A A G A A C C T G C-3\u2032; GAPDH Forward 5\u2032-C A A G G T C A T C C A T G A C A A C T T T G-3\u2032, Reverse 5\u2032-G T C C A C C A C C C T G T T G C T G T A G-3\u2032) (20\u201322<\/a><\/sup>).<\/p>\n

TUNEL assay<\/h3>\n

TUNEL staining locates apoptotic cells by DNA fragmentation. In the process of apoptosis, the DNA strand breaks, and large amounts of viscous 3\u2019-OH ends are created and labeled by fluorescein-12-2\u2019-deoxyuridine 5\u2019-triphosphate, which is connected by recombinant TerminalTransferase. TUNEL staining can accurately locate specific apoptotic cells observed under a fluorescence microscope. By contrast, normal nuclei are not stained by this method. In this study, the positive cells were strained green. Images were obtained and analyzed using a Leica DM 400B (Leica, Germany).<\/p>\n

Immunohistochemical staining<\/h3>\n

The mice were anesthetized and infused with isotonic sodium chloride solution followed by 4% paraformaldehyde. The intestinal tissues were removed and fixed in 4% paraformaldehyde for 6 h. These intestinal tissues were then embedded in paraffin and cut into 5-\u03bcm slices for immunohistochemical staining. After dewaxing, hydration, and antigen retrieval, the sections were blocked in 5% goat serum and then with anti-HMGB1 rabbit polyclonal antibody (1:500) overnight at 4\u00b0C. After washing with PBS 3 times, the sections were incubated with secondary antibody (1:200) at 37\u00b0C for 1 h and stained with diaminobenzidine. Hematoxylin was used to stain the nuclei for 5 min. The intracellular localization of HMGB1 was then observed using an Olympus eclipse 80i microscope (Olympus), and the images were analyzed with Image-Pro Plus 6.1 System. All slides were analyzed in a blinded fashion.<\/p>\n

Statistical analysis<\/h3>\n

All data are presented as mean \u00b1 SD except for survival rates. The survival rates are expressed as percentage. The survival rates were analyzed using the Fisher exact probability method, and the intergroup differences of the remaining data were tested using one-way analysis of variance followed by the least significant difference t<\/em> test for multiple comparisons. The data were analyzed using SPSS 18.0 software (SPSS Inc, Chicago, Ill). P<\/em> < 0.05 was considered statistically significant.<\/p>\n

RESULTS<\/h2>\n

Survival rate<\/h3>\n

No mice died in the sham or sham + H2<\/sub> groups during the 7-day study period. Survival rate was reduced in the severe sepsis groups (P<\/em> < 0.05). Inhalation of H2<\/sub> was associated with improved survival rate in mice with sepsis (P<\/em> < 0.05), but septic mice that received ZnPPIX had a significantly lower survival rate than those that did not (P<\/em> < 0.05) (Fig. 1). In accordance with the results of using ZnPPIX, when we used TinPPIX, a more specific inhibitor of HO-1, we found that compared with the severe sepsis + H2<\/sub> group, the survival rate of mice in the severe sepsis + H2<\/sub> + TinPPIX group decreased (P<\/em> < 0.05; Fig. 2).<\/p>\n

\n
"F1-14"<\/a>
Fig. 1: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on survival rate<\/strong>. The survival rate of mice was monitored for 7 days. *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group and &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group.<\/div>\n<\/figcaption><\/figure>\n
"F2-14"<\/a>
Fig. 2: <\/a><\/p>\n
Effects of H2<\/sub> and TinPPIX on survival rate<\/strong>. The survival rate of mice was monitored for 7 days. #<\/sup> P<\/em> < 0.05 versus the severe sepsis group and &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

CFU numbers in the peritoneal fluid<\/h3>\n

As shown in Figure 3, the numbers of bacterial CFU in the peritoneal cavity of the mice were significantly increased in the severe sepsis group at 24 h after CLP operation (P<\/em> < 0.05). Therapy with H2<\/sub> increased bacterial clearance and decreased CFU numbers in the severe sepsis group (P<\/em> < 0.05). However, septic mice that received ZnPPIX had a significantly higher level of CFU numbers than those that did not (P<\/em> < 0.05). In accordance with the results of using ZnPPIX, when we used TinPPIX, we found that compared with the severe sepsis + H2<\/sub> group, the CFU numbers in the severe sepsis + H2<\/sub> + TinPPIX group increased (P<\/em> < 0.05).<\/p>\n

\n
"F3-14"<\/a>
Fig. 3: <\/a><\/p>\n
Effects of H2<\/sub>, ZnPPIX, and TinPPIX on the CFU numbers in the PLF<\/strong>. The mice were killed at 24 h after CLP or sham operation. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group, and \u20ac<\/sup> P<\/em> < 0.05 versus the severe sepsis + TinPPIX group.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

Organ damage<\/h3>\n

To assess the severity of intestinal injury, we observed the intestines by H-E staining and assessed the severity of intestinal injury in different groups using histopathological scores. In the sham and sham + H2<\/sub> groups, the intestinal mucosa showed no abnormal morphological changes in the epithelial cells, and no evidence of congestion, edema, or inflammatory cell infiltration; in the severe sepsis group, the intestinal mucosal villi were shortened and atrophic with massive epithelial lifting from the lamina propria and exposure of dilated capillaries to the intestinal lumen. There was hemorrhage and ulceration on the surface (P<\/em> < 0.05). In the severe sepsis + H2<\/sub> group, intestinal injury was less marked than in the severe sepsis group, with only slight epithelial lifting from the lamina propria and moderate villus edema (P<\/em> < 0.05). However, these protective effects of H2<\/sub> were reduced by ZnPPIX, with animals in the severe sepsis + H2<\/sub> + ZnPPIX group showing more severe intestinal injury (P<\/em> < 0.05). Furthermore, the intestinal histopathological score of mice in the severe sepsis + H2<\/sub> + TinPPIX group was much higher than the severe sepsis + H2<\/sub> group (P<\/em> < 0.05; Fig. 4).<\/p>\n

\n
"F4-14"<\/a>
Fig. 4: <\/a><\/p>\n
Effects of H2<\/sub>, ZnPPIX, and TinPPIX on pathological changes in the intestine<\/strong>. The intestinal tissues were obtained from mice 24 h after CLP or sham operation. A, Hematoxylin-and-eosin staining (original magnification \u00d7200). B, Intestine histopathological scores. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group, and \u20ac<\/sup> P<\/em> < 0.05 versus the severe sepsis + TinPPIX group.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

Compared with the sham groups, levels of ALT, AST, Cr, and SUN were significantly higher in the severe sepsis groups (P<\/em> < 0.05). The levels of these markers were significantly lower in septic animals that inhaled H2<\/sub> than in those that did not (P<\/em> < 0.05). Compared with the severe sepsis + H2<\/sub> group, levels of ALT, AST, Cr, and SUN were higher in the severe sepsis + H2<\/sub> + ZnPPIX group (P<\/em> < 0.05; Fig. 5).<\/p>\n

\n
"F5-14"<\/a>
Fig. 5: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on serum biochemical parameters<\/strong>. The intestinal tissues were obtained from mice 24 h after CLP or sham operation. A, Alanine aminotransferase (ALT). B, Aspartate transaminase (AST). C, Creatinine (Cr). D, Serum urea nitrogen (BUN). Results are shown as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, and $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

Inflammatory mediator and oxidative products<\/h3>\n

As shown in Figures 6 and 7, the levels of oxidative products (MDA and 8-iso-PGF2\u03b1) and inflammatory mediator (HMGB1) were higher in the severe sepsis groups than in the sham groups (P<\/em> < 0.05). In the severe sepsis + H2<\/sub> group, these levels were lower than in the animals with severe sepsis that did not receive H2<\/sub> (P<\/em> < 0.05). In addition, use of ZnPPIX abrogated this effect of H2<\/sub> in the severe sepsis + H2<\/sub> + ZnPPIX group (P<\/em> < 0.05; Figs. 6 and 7).<\/p>\n

\n
"F6-14"<\/a>
Fig. 6: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on the serum levels of oxidative products and HMGB1 protein<\/strong>. The intestinal tissues were obtained from mice 24 h after CLP or sham operation. A, MDA. B, 8-iso-PGF2. C, HMGB1. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, and $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group.<\/div>\n<\/figcaption><\/figure>\n
"F7-14"<\/a>
Fig. 7: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on the intestinal levels of oxidative products and HMGB1 protein<\/strong>. The intestinal tissues were obtained from mice 24 h after CLP or sham operation. A, MDA. B, 8-iso-PGF2. C, HMGB1. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, and $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

Intestinal apoptotic cells<\/h3>\n

TUNEL staining showed that there were significantly more apoptotic cells in the severe sepsis groups than in the sham groups (P<\/em> < 0.05). Compared with the severe sepsis group, the number of apoptotic cells was lower in the severe sepsis + H2<\/sub> group (P<\/em> < 0.05), but this effect of H2<\/sub> was abrogated by ZnPPIX (P<\/em> < 0.05; Fig. 8).<\/p>\n

\n
"F8-14"<\/a>
Fig. 8: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on intestinal cell apoptosis<\/strong>. The intestinal tissues were obtained from mice 24 h after CLP or sham operation. A, TUNEL staining (original magnification \u00d7400). B, Number of TUNEL-positive cells. Apoptotic areas are indicated by white arrows. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, and $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

Expression of HO-1 and HMGB1<\/h3>\n

The levels of mRNA and protein expression of HO-1 in the sham groups were much lower than those in the severe sepsis groups at all time points (P<\/em> < 0.05). Levels were higher in the severe sepsis + H2<\/sub> group than in the severe sepsis group (P<\/em> < 0.05). The expression of HO-1 was lower in the severe sepsis + H2<\/sub> + ZnPPIX group than in the severe sepsis + H2<\/sub> group (P<\/em> < 0.05). The levels of HMGB1 in the severe sepsis group were higher than those in the sham group at 12 and 24 h (P<\/em> < 0.05), but this increase was not seen in the severe sepsis + H2<\/sub> group (P<\/em> < 0.05). The levels of mRNA and protein expression of HMGB1 in the severe sepsis + H2<\/sub> + ZnPPIX group were much higher than those in the severe sepsis + H2<\/sub> group (P<\/em> < 0.05). However, there were no significant changes among these groups at 6 h (P<\/em> > 0.05; Figs. 9 and 10). High-mobility group box 1 protein immunohistochemistry showed that the level of HMGB1 was higher in the severe sepsis groups than in the sham groups (P<\/em> < 0.05). The level of HMGB1 was lower in the severe sepsis + H2<\/sub> group than in the severe sepsis group (P<\/em> < 0.05). Furthermore, the level of HMGB1 was higher in the severe sepsis + H2<\/sub> + ZnPPIX group than in the severe sepsis + H2<\/sub> without ZnPPIX (P<\/em> < 0.05; Fig. 11).<\/p>\n

\n
"F9-14"<\/a>
Fig. 9: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on the mRNA expression of HO-1, HMGB1 protein, and Nrf2 in the intestine<\/strong>. The intestinal tissues were obtained from mice 6, 12, and 24 h after CLP or sham operation. A, mRNA levels of HO-1, HMGB1, and Nrf2 in the intestine at 6 h. B, mRNA levels of HO-1, HMGB1, and Nrf2 in the intestine at 12 h. C, mRNA levels of HO-1, HMGB1, and Nrf2 in the intestine at 24 h. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, and $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group.<\/div>\n<\/figcaption><\/figure>\n
"F10-14"<\/a>
Fig. 10: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on the protein expression of HO-1, HMGB1 protein, and Nrf2 in the intestine<\/strong>. The intestinal tissues were obtained from mice 6, 12, and 24 h after CLP or sham operation. A, Levels of HO-1, HMGB1, and Nrf2 proteins in the intestine at 6 h. B, Levels of HO-1, HMGB1, and Nrf2 proteins in the intestine at 12 h. C, Levels of HO-1, HMGB1, and Nrf2 proteins in the intestine at 24 h. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, and $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group.<\/div>\n<\/figcaption><\/figure>\n
"F11-14"<\/a>
Fig. 11: <\/a><\/p>\n
Effects of H2<\/sub> and ZnPPIX on the HMGB1 protein in the intestine assessed using immunohistochemistry<\/strong>. The intestinal tissues were obtained from mice 24 h after CLP or sham operation. A, Immunohistochemistry of HMGB1 in different groups (original magnification \u00d7200). B, Comparison of the average integrated optical density in HMGB1-positive staining intestinal tissues among the groups. Results are expressed as mean \u00b1 SD (n = 6). *P<\/em> < 0.05 versus the sham group, #<\/sup> P<\/em> < 0.05 versus the severe sepsis group, &<\/sup> P<\/em> < 0.05 versus the severe sepsis + H2<\/sub> group, and $<\/sup> P<\/em> < 0.05 versus the severe sepsis + ZnPPIX group. IOD\/area, Integrated optical density per stained area.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

Nrf2 expression<\/h3>\n

As shown in Figures 9 and 10, the expression of Nrf2 was higher in the severe sepsis group than in the sham group (P<\/em> < 0.05). Moreover, septic animals that received H2<\/sub> had an even greater expression of Nrf2 (P<\/em> < 0.05; Figs. 9 and 10).<\/p>\n

DISCUSSION<\/h2>\n

Mice with severe sepsis induced by CLP had greater intestinal injury than nonseptic animals. As shown by H-E staining, mice with severe sepsis had shortened and atrophic intestinal mucosal villi with massive epithelial lifting from the lamina propria, and more marked edema and hemorrhage than animals without sepsis. Treatment with H2<\/sub> reduced the degree of intestinal injury, but ZnPPIX and TinPPIX reversed these protective effects of H2<\/sub>. Similarly, therapy with H2<\/sub> decreased the number of apoptotic cells and the levels of biochemical parameters, oxidative products, and HMGB1 in the mice with severe sepsis, effects that were again reversed by ZnPPIX. Furthermore, the protein and mRNA expressions of HO-1 and Nrf2 in the intestine were higher in the mice with severe sepsis compared to those without, and higher still in septic mice treated with H2<\/sub>. Zinc protoporphyrin IX inhibited the expression of HO-1 protein.<\/p>\n

Hydrogen gas was inhaled by the mice for 1 h at 1 and 6 h after CLP or sham operation. To observe the function of H2<\/sub>, we also examined the levels of oxidative products\u2014MDA and 8-iso-PGF2\u03b1\u2014in the different groups; and to clarify the protective effect of H2<\/sub>, we assessed the expression of HMGB1. The results showed that the levels of HMGB1 protein and mRNA expression were in accordance with the severity of intestinal injury. These data suggest that HMGB1 may be a key proinflammatory mediator in the intestinal injury caused by severe sepsis and that the protective effects of inhaled H2<\/sub> on the septic intestinal injury of mice may be mediated by inhibition of HMGB1.<\/p>\n

High-mobility group box 1 protein is an intracellular protein that translocates to the nucleus and combines with DNA then regulates gene expression. Various studies have shown that when HMGB1 is activated and secreted into the extracellular milieu, it mediates downstream inflammatory responses in endotoxemia and sepsis (23<\/a><\/sup>). High-mobility group box 1 protein mediates proinflammatory responses in many kinds of inflammatory cells during the inflammatory response in vivo and is necessary for the full process of inflammation in animal models of endotoxemia and sepsis (24, 25<\/a><\/sup>). Previous studies have shown that HMGB1 is a diagnostic biomarker of bacteremia or severe sepsis (26, 27<\/a><\/sup>). Heme oxygenase-1 is one of the heme oxygenase isoforms and has a protective effect on many organs during severe sepsis (28, 29<\/a><\/sup>). It contributes to the protection of the intestinal mucosa. Takamiya et al. (30<\/a><\/sup>) showed that HMGB1 contributed to the lethality of endotoxemia in HO-1 deficient mice. Moreover, in lipopolysaccharide-treated HO-1\u2212\/\u2212<\/sup> mice, Tracz et al. (31<\/a><\/sup>) showed that genetic deficiency of HO-1 led to kidney disease and early death. To confirm the role of HO-1 in septic intestinal injury, we compared the protein and mRNA expressions of HO-1 in animals before and after administration of ZnPPIX, a HO-1 inhibitor. Therapy with H2<\/sub> for 1 h at 1 and 6 h after CLP increased the expression of HO-1 in the intestine, which led to the down-regulation of HMGB1. Zinc protoporphyrin IX reversed the protective effect of inhaled H2<\/sub> not only in the inflammatory response but also in the apoptosis of intestinal epithelial cells. All the results indicate that inhibition of HO-1 may aggravate the inflammatory response and damage the intestine. We can, therefore, conclude that inhaled H2<\/sub> reduces the intestinal injury induced by CLP by increasing HO-1.<\/p>\n

Nuclear factor-erythroid 2 p45-related factor is one of the upstream factors of HO-1 (32<\/a><\/sup>), as shown previously (33<\/a><\/sup>). Here, we also detected the levels of Nrf2 protein and mRNA expression and showed that the changes in Nrf2 in different groups were in accordance with those of HO-1. Hence, we can confirm that Nrf2 is one of the factors that may regulate the activity of HO-1. Nuclear factor-erythroid 2 p45-related factor is a redox-sensitive transcription factor that has the structure of a leucine zipper. It is a pleiotropic protein that controls the expression of cytoprotective genes by connecting to the antioxidant response element (34<\/a><\/sup>). Expression of Nrf2 is low under normal conditions; however, when the body is stimulated by oxidative stress, transcription of Nrf2 increases, resulting in increased levels of Nrf2 and its downstream factors. These factors are usually protective proteins that may reduce the damage caused by oxidative stress (35<\/a><\/sup>). Nuclear factor-erythroid 2 p45-related factor, therefore, plays an important role in protecting against oxidative stress by stimulating the expression of HO-1. Thus, we infer that H2<\/sub> may exert its effects by activation of Nrf2 and its downstream factor, HO-1. However, further study is needed to confirm this hypothesis.<\/p>\n

In conclusion, we have shown that inhaled H2<\/sub> alleviates the intestinal injury induced by severe sepsis and decreases the expression of the late proinflammatory factor, HMGB1. The mechanism of this effect may be partly related to the Nrf2\/HO-1 pathway, which stimulates the expression of HO-1 and its upstream molecule, Nrf2. We believe that further studies are needed to evaluate the potential role of inhaled H2<\/sub> in the therapy of severe sepsis.<\/p>\n<\/section>\n","protected":false},"excerpt":{"rendered":"

Hydrogen Gas Alleviates the Intestinal Injury Caused by Severe Sepsis in Mice by Increasing the Expression of Heme Oxygenase-1<\/p>\n","protected":false},"author":1,"featured_media":17899,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[130],"tags":[],"disease":[843],"body-organ":[1025],"applications":[679],"test_subjects":[1518],"report-topic":[1329],"class_list":["post-27386","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrogen-health","disease-sepsis-2","body-organ-intestine-2","applications-inhalation-2","test_subjects-mouse-2","report-topic-multiple-organ-dysfunction-syndrome-mods-2"],"acf":[],"yoast_head":"\nH2 Gas Reduces Intestinal Injury in Sepsis<\/title>\n<meta name=\"description\" content=\"Hydrogen Gas Alleviates the Intestinal Injury Caused by Severe Sepsis in Mice by Increasing the Expression of Heme Oxygenase-1\" 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