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EFFECT OF INTRAPERITONEAL RESUSCITATION WITH DIFFERENT CONCENTRATIONS OF SODIUM PYRUVATE ON INTESTINAL ISCHEMIA REPERFUSION INJURY IN HEMORRHAGIC SHOCK RAT

Jiang, Lin-Lin; Zhang, Jing-Jing; Zhang, Zong-Ze; He, Xiang-Hu; Chen, Dong-Ling; Wang, Yan-Lin

doi: 10.1097/SHK.0000000000000515
Basic Science Aspects
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Objective: To determine the effects of intraperitoneal resuscitation (PR) with different concentrations of sodium pyruvate (PY) on intestinal ischemia reperfusion injury in rats hemorrhagic shock (HS).

Methods: Sixty rats were randomly assigned to six groups. These included: group SHAM, intravenous resuscitation only (VR) group, and four PR groups based on resuscitation fluid: glucose-lactate-based peritoneal dialysis solution (LA), and PY-1.1%, PY-1.6%, and PY-2.2% (concentrations in grams/dL). Mean arterial pressure (MAP) was monitored continuously. Blood pH, base excess (BE), lactate, intestinal myeloperoxidase (MPO), malondialdehyde (MDA), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), activated caspase-3, and zonula occludens-1 (ZO-1) were measured; intestinal mucosal damage index (IMDI) and subcellular changes were observed; apoptotic index (AI) was calculated.

Results: Three hours after resuscitation, in PY groups, MPO, MDA, IMDI, AI, TNF-alpha, and IL-6 were significantly lower than VR and LA groups, while pH and BE were higher. PY groups showed less expression of activated caspase-3 but elevated ZO-1. Among PY groups, group PY-1.1% had the lowest MPO, MDA and TNF-alpha, and had less pathological damage and subcellular changes than other experimental groups.

Conclusions: PR using PY solution combined with VR provided protection against intestinal ischemia-reperfusion injury following HS and resuscitation. Under the same hypertonic condition, 1.1% PY solution showed significant advantages compared with 2.2% and 1.6% solutions. The underlying mechanisms may include the maintenance of hemodynamic stability, regulation of homeostasis, inhibition of oxidative stress and inflammation, and protection of intestinal epithelial tight junction barrier function.

Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China

Address reprint requests to Yan-Lin Wang, MD, PhD, Department of Anesthesiology, Zhongnan Hospital of Wuhan University, No. 169 Donghu Road, Wuhan 430071, Hubei, China. E-mail: wyl0342@sina.com.

Received 30 August, 2015

Revised 15 September, 2015

Accepted 14 October, 2015

The authors report no conflicts of interest.

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INTRODUCTION

Hemorrhagic shock (HS) is the leading cause of early death in trauma patients (1). If patients with HS fail to receive prompt fluid resuscitation, near half of them will die of multiple organ failure (MOF) (2). During early intravenous resuscitation (VR), the splanchnic vasculature remains contracted, and, as ischemia persists, reperfusion injury may occur. In this setting, intestinal trypsin, increased intestinal permeability, and intestinal barrier dysfunction together facilitate the translocation of intestinal flora and toxins that can damage distant organs, leading to systemic inflammatory response syndrome (SIRS) or Multiple Organ Dysfunction Syndrome (MODS) (3, 4). Reducing intestinal ischemia-reperfusion injury and preventing intestinal barrier dysfunction could improve prognosis after trauma.

Intraperitoneal resuscitation (PR) can ameliorate visceral vasoconstriction following intravenous resuscitation (VR) (5, 6), regulate microcirculation, protect endothelial cells, and reduce third spacing of fluid. Indeed PR combined with VR can effectively improve the survival rate and outcomes after resuscitation (7). However, the protective effect of PR following resuscitation depends on the characteristic of the fluid used, such as osmotic pressure, glucose concentration, and other factors (7, 8).

As a metabolic intermediate, pyruvate can regulate glucose metabolic processes, inhibit the release of inflammatory cytokines, reduce oxygen-free radical formation, and exhibit anti-inflammatory and antioxidant effects. Pyruvate can protect cells and organs (9–11). In animal experiments, PR using fluid containing pyruvate combined with VR has proven better than normal saline or Lactated Ringer's solution fluid resuscitation and can prevent gut barrier dysfunction and improve survival and prognosis (12, 13).

Several studies have investigated the effect of pyruvate, but only a few of them explored the role of different concentrations. Hu Sen and his colleagues provided rats with PR fluids of the same hypertonicity, and they found that PR using simple 2.2% sodium pyruvate (PY) solution had better outcomes than pyruvate dialysate (14). To follow up on this, we wonder whether 2.2% PY is the best concentration. In Petrat's study, the effect of three different intravenous doses (50 mg/kg, 250 mg/kg, and 1000 mg/kg) of pyruvate was investigated. The results showed that only at a dose of 250 mg /kg or above, systemic effects of PY might become apparent. But if the concentration of pyruvate was too high, it would have great influence on internal environment (15). Because the plasma concentration increases more slowly when PY is administered through the intraperitoneal compared with intravenous route, and in order to conveniently compare with 2.2%, we chose to use 1.1% as the lowest concentration in this present study. 1.6% is the intermediate concentration so that we can explore how the concentration affects the protective advantages of PY.

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MATERIALS AND METHODS

Reagents

Sodium pyruvate, glucose, and sodium pentobarbital were purchased from Sigma (St Louis, MO); myeloperoxidase (MPO), malondialdehyde (MDA), lactic acid assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); TUNEL assay kit for apoptosis was purchased from Roche Biotech (Basel, Switzerland); 2.5% Glu-LA-peritoneal dialysis solution (Glu-LA-PDS) was purchased from Baxter Healthcare Ltd., Guangzhou, China.

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Preparation of solutions

Sodium pyruvate solutions were freshly prepared before experiment. Sodium pyruvate powder was dissolved in distilled water to prepare 1.1% (100 mM), 1.6% (145 mM), and 2.2% (200 mM) solutions. An FM-8P automatic freezing point osmometer (Shanghai, China) was used to measure osmotic pressure, glucose was added to adjust the osmotic pressure of sodium pyruvate solution to 400 mosm/L, and HCl and NaOH were used to adjust pH to 5.4. All solutions were sterilized by filtration and warmed to room temperature before use.

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Animals

Male Sprague-Dawley rats (60, SPF grade, weighing 250–300 g) were provided by the Animal Center of Wuhan University, Wuhan, China. The animals were housed in the Experimental Animal Center, Zhongnan Hospital of Wuhan University, Wuhan for 1 week before the experiment, under standardized conditions of temperature (22°C ± 1°C), humidity (55% ± 5%), and 12 h/12 h light–dark cycles, with water and food ad libitum. Experimental procedures were approved by the animal experiments committee of Wuhan University. All procedures complied with the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health).

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Experimental procedure

All animals were fasted 12 h but allowed free access to water until 4 h before surgery. The rats were anesthetized using 1% sodium pentobarbital intraperitoneal injection at a dose of 40 mg/kg, then continuously given 0.7% isoflurane during the entire process to maintain anesthesia and spontaneous breathing. The right carotid artery was cannulated and a biological monitor was attached in-line to record the mean arterial blood pressure (MAP). The right femoral vein was cannulated for blood and fluid infusion and left femoral artery was cannulated for hemorrhage. All punctures were carried out under sterile conditions. Rats were then injected with 300 U/kg heparin sodium to achieve systemic heparinization and all indwelling tubes were prefilled with 100 U/mL of heparin saline. The animals were allowed to stabilize for a period of 15 to 20 min. A heating apparatus was used to maintain the animal's rectal temperature at 37°C.

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Experimental groups and procedure

Animals were randomly assigned to six groups, n = 10 in each group, as presented in Table 1.

Table 1

Table 1

In all groups except the group SHAM, blood was withdrawn through the left femoral artery to drop MAP to 40 mmHg within 10 min, then blood was either withdrawn or administered to maintain MAP at 40 ± 5 mmHg for 60 min. After the shock phase, animals were provided intravenous resuscitation as delineated above, and intraperitoneal injection was given simultaneously, using a syringe pump. Both were completed in 30 min. From the start of bleeding until 3 h after resuscitation, MAP was monitored and recorded continuously. Three hours following resuscitation, blood was sampled and ileum tissue was dissected, 5 cm of the tissue was soaked in 4% neutral-buffered formalin, 0.5 cm was placed in 2.5% glutaraldehyde for electron microscopy fixation, and 10 cm was placed in cryovials and snap frozen in liquid nitrogen. The procedure is presented in Figure 1.

Fig. 1

Fig. 1

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Measurement of MAP and blood pH, BE, and lactate levels

The right carotid artery catheter was connected to a pressure transducer, and data were displayed and continuously monitored. Data were recorded at baseline, the beginning and end of hemorrhagic shock, and 5, 10, 30, 60, 90, 120, 150, and 180 min post-resuscitation (H0, H60, R5, R10, R30, R60, R90, R120, R150, R180). Blood was collected from the right carotid artery catheter 3 h after resuscitation, blood pH and BE were analyzed using an automatic biochemical analyzer, and lactate was measured using a kit according to manufacturer's instruction.

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Observation of morphological changes by light microscopy

Fixed intestinal tissues were removed from 4% paraformaldehyde, embedded in paraffin and sectioned. After hematoxylin eosin staining, slides were observed under a microscope. Damage of intestinal tissue was assessed by intestinal mucosal damage index (IMDI) according to Chiu's scoring system (16). Briefly, the scores were determined as follows. 0: normal mucosa; 1: development of subepithelial space at villus tips; 2: expansion of subepithelial space with moderate lifting; 3: denuded tips with an exposed lamina propria and villous blunting; 4: epithelial shedding from both the apex and the mid-region of the villi associated with a shortened and widened villous structure; 5: disintegration of the lamina propria of the intestinal mucosa with ulceration.

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Observation of subcellular structures by electron microscopy

Intestinal tissues fixed in 2.5% glutaraldehyde solution were removed, rinsed in washing buffer 15 min three times, and left overnight. 0.2 mL of sample buffer and 0.2 mL of osmium tetroxide were then added to the sample. Two hours later, the samples were rinsed with washing buffer again, dehydrated in acetone and infiltrated in acetone resin for 2 h, then embedded in resin. Semithin sections were made and positioned in the light microscope, and then double stained with uranyl acetate and lead citrate. Subcellular structures were then examined with a transmission electron microscopy.

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Measurement of MPO and MDA in intestinal tissue

Frozen intestinal tissues were homogenized and centrifuged at 4°C. MPO and MDA were measured according to manufacturer's instructions.

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Detection of TNF-alpha and IL-6 in intestinal tissues

Frozen intestinal tissues were homogenized and centrifuged at 4°C. TNF-alpha and IL-6 were measured using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. All kits were bought from Elabscience Biotechnology Co., Ltd., Wuhan, China.

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Detection of active caspase-3 and ZO-1 in the tissues

The expression of active caspase-3 and ZO-1 was detected by Western blot analysis. About 50 mg of intestinal tissue was placed into 500 μL of lysis buffer and mixed thoroughly, and then centrifuged at 4°C. Supernatant was collected and protein concentration was determined using the BCA method. Lysates were then loaded to SDS-PAGE gel, electroporated at 80 V in stacking gel and 120 V in separating gel. Protein was then transferred to a PVDF membrane that was pre-activated using methanol. The membrane was then blocked in 5% non-fat milk at room temperature for 60 min. Primary antibodies were diluted: anti- Glyceraldehyde-3-phosphate dehydrogenase (1:10000, Abcam Inc., Cambridge, UK), anti-zonula occludens-1 (1:500, Santa Cruz Biotechnology, Dallas, Tex), anti-active-caspase3 (1:1,000, Abcam Inc.). Blocked membrane was incubated with diluted primary antibody at 4°C overnight. Secondary antibody HRP-labeled goat anti-rabbit IgG (KPL Biotechnology, Shanghan,China) was diluted to 1:10,000 and incubated with membrane at 30°C for 30 min. Proteins were then visualized using chemiluminescence, and the optical density value of the target band was analyzed using the alpha software (Alpha Innotech Company, Fremon, Calif) processing system.

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Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay

Intestinal tissues fixed in 4% paraformaldehyde were embedded in paraffin and sectioned. Sections were deparaffinized, processed with proteinase K and 3% H2O2, stained with DAB and hematoxylin, then rinsed and dehydrated. TUNEL positive cells appear brown under a light microscopy. Under 400 times magnification, three random vision fields were chosen. AI was calculated as the percentage of positive cell numbers to total cell number.

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Statistical analysis

All measurement data are presented as mean ± standard deviation. Data analysis was performed using SPSS for Windows (Version 19.0, SPSS Inc, Chicago, Ill). One-way ANOVA analysis was used for comparison of means of multiple samples and Student–Newman–Keuls test was used for multiple comparisons. P ≤ 0.05 was considered statistically significant.

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RESULTS

Mean arterial pressure

MAP was measured until 3 h after resuscitation, and the data are shown in Figure 2. Before resuscitation (H60), MAP showed no significant differences among groups (P > 0.05). During the process of fluid resuscitation, MAP increased significantly in each group from H60 to R30. MAP in the PY groups were higher than group LA and group VR at R5, R10, R60, R90, R120, R150, and R180 (P < 0.05). But at R5 and R10, MAP showed no significant difference among PY groups. At R30, R60, R90, R120, R150, and R180, group PY-1.1% had higher MAP than group PY-1.6% and group PY-2.2% (P < 0.05). The MAP in group PY-1.6% was not significantly different from that of group PY-2.2% at any point.

P

P

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Change of arterial blood pH, BE, and lactate

To understand the change of the internal environment, arterial blood pH, BE, and lactate were analyzed (Table 2). Compared with group SHAM, lactate values in the other groups were increased (P < 0.05). With the increase of the concentration of the PY solutions, the blood lactate value increased as well. The lactate value was higher in group PY-2.2% than in group PY-1.6% and group PY-1.1% (P < 0.05) and lower in group PY-1.1% than in group LA and group VR (P < 0.05). BE value was higher in all PY groups than in group LA and group VR (P < 0.05); it was significantly higher in group PY-2.2% and group PY-1.6% than in group PY-1.1% (P < 0.05). The trend of arterial blood pH was similar to that of BE.

Table 2

Table 2

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Histopathological changes under light microscopy

IMDI was ascertained. As shown in Table 3, all the PY groups had lower IMDI than group VR or group LA (P < 0.05), and group PY-1.1% had the lowest IMDI (P < 0.05).

Table 3

Table 3

As shown in Figure 3, in group VR, villi appeared to be swelling severely, most of which revealed evidence of necrosis and fracturing; inflammatory cells infiltrated the lamina propria (Fig. 3B). In group LA, some villi were broken, and the continuity of striated border was severely damaged, with loss of epithelial cells and destruction of villi (Fig. 3C). In group PY-1.1%, the structure of villi remained intact, damage of striated border continuity was mild, only a little of the lamina propria showed edema, and a few inflammatory cells were observed (Fig. 3D). In group PY-1.6%, villi structure remained, most appeared edematous, only a small portion of villi showed fractures, and infiltration of inflammatory cells was observed (Fig. 3E). In group PY-2.2%, most villi were complete and the lamina propria was mildly edematous; some epithelial cells lost polarity and breakage of some villi was observed (Fig. 3F).

Fig. 3

Fig. 3

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Subcellular structure change under electron microscopy

As shown in Figure 4, in group VR (Fig. 4B), there were severe separations of gap junctions between cells, and mitochondria appear swollen severely or even disappear. In group LA (Fig. 4C), separations of gap junctions between cells appear, and some mitochondria were severely swollen. In group PY-1.1% (Fig. 4D), microvilli are arranged tightly, organelles structures appear normal, slight separation is evident in a small amount of cells gap junctions. In group PY-1.6% (Fig. 4E), there is aggregation of chromosomes to the edge of the nucleus. In group PY-2.2% (Fig. 4F), microvilli are sparse, mitochondria appear normal.

Fig. 4

Fig. 4

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MDA and MPO level in intestinal tissues

Malondialdehyde (MDA) is an indicator of lipid peroxidation. The intestinal MDA concentrations in group LA and PY groups were lower than that in group VR (P < 0.05). Among the PY groups, group PY-1.1% had the lowest MDA level which was not significantly different from group SHAM (P > 0.05). MPO is a protease primarily produced by neutrophil, and can be used to measure level of neutrophil activation. The relative expression of MPO among the groups was generally similar to that of MDA, except that MPO in group PY-1.6% was lower than that in group PY-2.2% (P < 0.05), as shown in Table 3.

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Pro-inflammatory cytokines TNF-α and IL-6 in intestinal tissues

TNF-α and IL-6 are pro-inflammatory cytokines. TNF-α and IL-6 expression levels in intestinal tissues 3 h after resuscitation are shown in Figures 5 and 6. Levels of TNF-α and IL-6 in all experimental groups were higher than that in group SHAM (P < 0.05), and the PY groups have significantly lower levels than group LA and group VR (P < 0.05). Among PY groups, there was no significant difference in IL-6 expression (P > 0.05), while the group PY-1.1% revealed the lowest TNF-α level.

Fig. 5

Fig. 5

Fig. 6

Fig. 6

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Expressions of ZO-1 and caspase-3 proteins in intestinal tissues and TUNEL apoptotic index

ZO-1 is one of the most important and critical components in the structural and functional organization of tight junctions. As shown in Figure 7, compared with group SHAM, all experimental groups presented significantly reduced ZO-1 levels (P < 0.05). The PY groups expressed significantly more ZO-1 than group VR and group LA (P < 0.05), and group PY-1.1% had the highest level. In regard to active caspase-3 expression, the PY groups did not significantly differ from group SHAM (P > 0.05), while group PY-2.2% had the highest level among all PY groups, which was significantly higher than that of group PY-1.1% (P < 0.05), but did not significantly differ from that of group PY-1.6% (P = 0.46, P > 0.05). As shown in Table 2, apoptotic index (AI) in PY groups is significantly lower than that in group VR and LA group (P < 0.05). All the PY groups had higher AI compared with group SHAM (P < 0.05). However, the AI of group PY-1.1% was the closest to that of group SHAM.

P

P

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DISCUSSION

Treatment of HS is aimed at supplying effective circulating blood volume, restoring and maintaining the blood flow of organs, and correcting tissue ischemia and hypoxia. However, inappropriate fluid resuscitation may fail in these tasks and contribute to tissue edema (8).

PR activates cell potassium ion channels and adenosine receptor A1 (17), protects vascular endothelial cell, promotes the release of nitric oxide, dilates visceral blood vessels, and decreases neutrophil adhesion (6). The effect of PR on microcirculation can improve tissue edema caused by shock resuscitation, prevent lung and intestinal fluid transfer, and reduce third spacing of fluid (8).

The protective effect of pyruvate has been shown in multiple vital organs. For example, increasing the content of pyruvate in coronary artery can effectively improve the cardiac index and stroke volume without increasing heart rate in patients with acute heart failure caused by acute myocardial infarction (9). In a rat stroke model, pyruvate can regulate the expression of HIF-1alpha and EPO in brain neuron and glial cells, activate AKT, reduce intracellular DNA cleavage, and relieve ischemia-reperfusion injury of brain. By enhancing glucose metabolism and oxidative phosphorylation, pyruvate can increase ATP generation and lower the degree of cognitive deficits after trauma (10, 18). During the process of fluid resuscitation after HS, pyruvate can correct lactic acidosis (19), and improve the prognosis and survival rate (20). Pyruvate has protective effects on intestinal mucosal ischemia-reperfusion injury (15, 21). When administering fluid resuscitation in HS, the addition of PR using pyruvate solution to VR can effectively inhibit the inflammatory response, scavenge oxygen-free radicals, protect the intestinal mucosal barrier, reduce intestinal ischemia-reperfusion injury following resuscitation, and prevent the occurrence of MODS (12–14). Zakaria et al. attributed the beneficial effects of PR to the hypertonicity and the contained glucose of the peritoneal dialysis fluid used (5, 6). Hu et al. compared two kinds of dialysate containing pyruvate with commonly used 2.5% Lac-PDS in an experimental design that excluded the impact of the osmotic pressure (14). Their results showed PY had significant advantages for visceral protection, especially for the small intestine. And the 2.2% PY aqueous solution was superior to isotonic pyruvate dialysate. Our present study was designed to further investigate PY concentration in PR.

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Effect of different concentrations on hemodynamics and blood pH and BE

Pyruvate prevents ATP depletion, maintains cell ion channels functions, and reduces the loss of Ca2+ in vascular smooth muscle. These factors promote the maintenance of vascular tone (9, 22). Pyruvate also suppresses myocardial nitrative stress following HS, increases cardiac output, and improves cardiac function. In our study, the PY groups exhibited better hemodynamic stability than group VR and group LA. At R5 and R10, there were no significant differences in MAP among PY groups. However, at R30, R60, R90, R120, R150, and R180, group PY-1.1% demonstrated a significantly higher MAP than group PY-1.6% and group PY-2.2%. During the shock phase, anaerobic glycolysis in tissue cells enhances, lactic acid concentration elevates, and acidic substances accumulate, causing metabolic acidosis. Administration of exogenous pyruvate might enhance the activity of lactate dehydrogenase (LDH). During this process, hydrogen ions are consumed. In this way, pyruvate casuses systemic alkalization (19). Blood lactate levels increased in group PY-1.6% and group PY-2.2%, which were even higher than group LA. However, in group PY-1.1% the lactate level was relatively low, and both pH and BE were within normal range. Frank Petrat reported that high concentrations of pyruvate solution increased blood lactate concentration, causing blood pH to become alkaline (15). This was consistent with our blood gas test results.

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Morphological changes under light microscopy and subcellular change under electron microscopy

Pyruvate can prevent ATP depletion and inhibit the activation of inflammatory cells and the release of inflammatory cytokines. Sileri's study showed that pyruvate could maintain intestinal motility and absorption following ischemia-reperfusion injury (23). By light microscopy, gut tissue damage was significantly milder in PY groups than group VR and group LA. The morphological structures of group PY-1.1% were essentially normal. By electron microscopy, microvillus, mitochondria, and cell junctions were well protected in PY groups, especially in group PY-1.1%, which further confirmed its protective effect on intestinal barrier systems.

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Effect of different concentrations of pyruvate on oxidative stress and inflammation

Reactive oxygen species (ROS) and free radicals are produced after gut ischemia-reperfusion injury. ROS can damage plasma membranes and subcellular structure; lipid peroxidation produces MDA, which has cytotoxic effects. All these factors can lead to intestinal barrier system dysfunction (24). IL-6 and TNF-alpha are the most common pro-inflammatory cytokines. When IL-6 expression is increased, ileal tight junctions are damaged, which increases the permeability of intestinal wall, leading to bacterial translocation (25). TNF-alpha plays an important role in uncontrolled inflammation during ischemia-reperfusion, and affects the expression of tight junction protein ZO-1 in in vitro experiments (26, 27). At a certain level, the release of IL-6 is dependent on TNF (28). Use of the anti-TNF-alpha antibody Infliximab can alleviate the increases of gut permeability caused by inflammation (29). The synergistic effect of gut inflammation and oxidative stress can injure the integrity of intestinal barrier system and promote the translocation of bacterial and toxic soluble substances to parenteral tissues, leading to SIRS and MODS eventually.

Pyruvate is an alpha-keto acid. It can be decarboxylated and react directly with hydrogen peroxide to form water and carbon dioxide, and thus may serve as a scavenger of free radicals. It also reduces neutrophil infiltration and MPO activity, elevates the intracellular fluid oxidation–reduction potential, reduces the formation of ROS and superoxide, indirectly increases GSH content (22), prevents oxidation of the biomembrane system, and reduces the generation of MDA. Pyruvate can inhibit the release of pro-inflammatory cytokines IL-6 and TNF-alpha (30), and even the activation of the NF-κB pathway (31). Through free radical scavenging, pyruvate can reduce the formation of ROS, increase the content of antioxidants, and reduce the intestinal tissue damage caused by oxidative stress. Through inhibition of inflammatory cell infiltration and pro-inflammatory cytokines release, pyruvate can also regulate the inflammatory response and play an antioxidation and anti-inflammatory role. In this present study, although the three different concentrations of PY solutions showed no significant difference on their inhibitory effect on inflammatory mediators IL-6 and TNF-alpha, the results indicated that group PY-1.1% had the most obvious effect on TNF-alpha inhibition. Since TNF-alpha is related to tight junction protein expression (29), the inhibitory effect of group PY-1.1% on TNF-alpha not only has anti-inflammatory prospects, but also has potentially a role in enhancing tight junction and preventing epithelial cell sloughing off.

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Effect of different concentrations of pyruvate on intestinal epithelia tight junction barrier

Intestinal epithelial tight junction barrier consists of intestinal epithelial cells and tight junction proteins. Under normal circumstances, intestinal epithelial cell layers keep self-renewing and apoptosis will occur in some of the epithelial cells, mainly in the crypt or villi tips and will not cause barrier function disruption (32). Proteasome-dependent apoptosis has two signaling pathways with the same final effector caspase-3. Under oxidative stress, intestinal epithelial cells often exhibit excessive activation of caspase-3, leading to intestinal barrier dysfunction (24). Thus, the activation level of caspase-3 can be used to describe cell apoptosis level and reveal information relevant to the integrity of intestinal mucosal. ZO-1 is the first confirmed tight junction protein, it can form a complex with other membrane-associated guanylate kinase homologs (MAGUKs), and attach to actin cytoskeleton (33), and hence, its expression level is closely related to the function of tight junctions. As shown in Figure 5, the PR groups had less active caspase-3 and more ZO-1 than other experimental groups. Group PY-1.1% had levels of caspase-3 and ZO-1 similar to those of group SHAM. TUNEL assay results also showed that the intestinal tissue AI was lower in group PY-1.1% than in the other groups. All these data indicate that pyruvate can effectively inhibit excessive apoptosis, increase ZO-1 expression, and maintain intestinal mucosal barrier function.

In this present study, when osmotic pressure was constant, 1.1% aqueous solution of PY has shown obvious advantages not only in its anti-inflammatory and antioxidative effects but also in its protective effect on barrier function. The 1.6% and 2.2% therapeutic effects were not as beneficial as the 1.1%. One possible explanation is pyruvate has a strong alkalizing effect, and when high concentrations of PY solution are administered, the blood pH may be raised too high, resulting in a slightly alkaline internal environment that can alter enzyme function relevant to numerous metabolic and inflammatory pathways. D. Mathioudakis's research showed low concentration of PY solution had a nutritional role for neutrophil and enhanced neutrophil inflammatory response (34). Frank Petrat's study also demonstrated that when the dose of pyruvate administered was 250 mg/kg or more, the local and systemic effects of pyruvate could fully manifest (15). Therefore, only when the concentration of PY solution is appropriate, can it ensure an effective blood concentration of pyruvate without causing metabolism disorder due to excessive substrate provision.

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Clinical significance

In this present study, PR using PY solution effectively prevented intestinal ischemia-reperfusion injury after HS in a rat model. Even though we did not observe splanchnic blood flow and survival rate in this study, other studies have already confirmed that the administration of PR using solutions containing pyruvate could significantly improve those indexes (13, 14). Compared with lactate-based peritoneal dialysis solution, PR fluid of pyruvate showed significant protective advantages on its protective effect in cells and organs. PR using pyruvate solution combined with VR can effectively dilate visceral blood vessels in liver, kidney, and gut relieve visceral ischemia and hypoxia and improve organ function. By investigating three different concentrations of pyruvate solutions, our study provides information relevant for selecting an ideal concentration of the PY used in the PR fluid in HS. The electrolytes analysis in our preliminary study showed that none of these three concentrations of sodium pyruvate peritoneal resuscitation solutions caused sodium overload, indicating that the use of high concentrations of sodium pyruvate peritoneal resuscitation will not cause electrolyte imbalance. Nonetheless, clinical evidence needs to be collected.

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Limitations

This study focused on the therapeutic effect of different concentrations of PY peritoneal resuscitation fluid, but the concentration of pyruvate in blood was not measured, so it was not clear how the change of concentrations of PY peritoneal resuscitation fluid would affect the active pyruvate in blood. Regarding anti-apoptosis effects, we only detected active caspase-3, but did not measure caspase-10 and 9. Analyzing the expression of all three proteases will further clarify the signaling pathway involved in the anti-apoptotic effect of pyruvate.

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CONCLUSIONS

In the current study, PR using PY solutions (1.1%, 1.6%, and 2.2%) combined with VR provided protection against intestinal ischemia-reperfusion injury following HS and resuscitation. Under the same hypertonic condition, 1.1% PY solution was found to be superior to 1.6% and 2.2%. The possible mechanisms may include the maintenance of hemodynamic stability, regulation of homeostasis, inhibition of oxidative stress and inflammation, and protection of the intestinal epithelial tight junction barrier function.

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Acknowledgment

The authors thank Fang-Qiang Zhou, MD, from Fresenius Dialysis Centers at Chicago, for his assistance with pharmacology in this work.

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Keywords:

Apoptosis; barrier function; gut injury; inflammation; oxidative stress; BE; base excess; HS; hemorrhagic shock; IL-6; interleukin-6; IMDI; intestinal mucosal damage index; MAGUKs; membrane-associated guanylate kinase homologs; MAP; mean arterial pressure; MDA; malondialdehyde; MODS; multiple organ dysfunction syndrome; MOF; multiple organ failure; MPO; myeloperoxidase; PR; intraperitoneal resuscitation; PY; sodium pyruvate; SIRS; Systemic Inflammatory Response Syndrome; TNF-α; tumor necrosis factor; VR; intravenous resuscitation; ZO-1; zonula occludens-1

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