Purpose: Gut permeability and microvascular injur y following ischaemia/reperfusion (IR) have been implicated in the systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Taurine (TAU), a sulfur-containing amino acid, is a powerful antioxidant and regulator of intracellular calcium and several studies have established that treatment with TAU protects cerebral, cardiac and testicular tissue from (IR) injury . This study investigates the protective effect of taurine in an experimental model of I/R-induced gut injury in rats . Methods: Sprague-Dawley rats were randomized into three groups: Control, I/R, TAU + I/R. TAU was given by gavage or intravenous injection before I/R. Ischaemia was induced by cross-clamping superior mesenteric and coeliac vascular pedicle for 20 - 30 min, followed by 60 - 180 min reperfusion. Gut permeability, blood flux, tissue oedema, leucocytes infiltration and eNOS expression were measured at 3 hrs following reperfusion using FD4. Leukocyte-endothelial interactions were determined by intra-vital microscopy during I/R. <i> In vitro </i> studies assessed the protective effect of TAU on endothelial cell function and survival. Results: Treatment with TAU significantly attenuated IR-induced gut hyper permeability, tissue oedema, leukocyte adhesion and infiltration. TAU also prevented the reduction in gut blood flow, leukocyte rolling velocity and eNOS expression induced by IR. TAU protects against I/R -induced endothelial cell injury by reduced anti-oxidant activity and modulation of eNOS expression and intracellular calcium fluxes. Conclusions: TAU protects the gut from intestinal barrier dysfunction induced by surgical I/R.
Interruption or reduction of blood and oxygen supply to tissue results in cellular dysfunction and death leading ultimately to organ failure. Restoration of blood supply and thereby oxygen to ischaemic tissues; reperfusion, is essential to the survival and recovery of damaged tissue. However, reperfusion may paradoxically induce further cell death [
I/R injury is characterised by the development of increased microvascular permeability, oedema and tissue necrosis. The injury cascade is mediated mostly by neutrophil-endothelial adherence and subsequent neutrophil-mediated organ damage [
The endothelium, a cellular monolayer that forms a vital interface between circulating blood and tissue, not only plays an important role in the regulation of vascular tone and the maintenance of vascular integrity, but is also a major contributor to vessel repair. Vascular endothelial cells (EC) are the initial and prime targets of I/R injury [
Therapeutic interventions initiated at the onset of reperfusion are as effective in attenuating I/R injury as treatments introduced during the pre-ischaemic period [
This work tests the hypothesis that taurine can augment cellular responses to I/R injury in the perioperative period reducing inflammation and end organ tissue damage. Therefore the primary aim of this work is to evaluate taurine as a potential adjunct therapy to surgery in a rodent model of I/R injury.
Medium 199, Phosphate buffered saline (PBS), P/S/F (penicillin, streptomycin, fungimycin) glutamine, 0.05% trypsin-0.02% EDTA solution and FCS (fetal calf serum) (Gibco Laboratories Paisley, Scotland, UK). Collagenase (type II) (Worthington Freehold, New Jersey). 2% gelatin, endothelial cell growth supplement, glycerol, taurine, heparin, NaCl, Dextran Hepes, KCl, MgCl2, Glucose, CaCl2, sodium deoxycholate, Triton-100, acrylamide, SDS, Tris, EDTA, ammonium persulphate, TEMED, glycine, bromophenol blue, BSA, Tetrabutylammonium hydroxide (PIC-A) and 2-mercaptoethanol (Sigma ,St. Louis, MO, USA). Fluo-3 AM and DCFH-DA (fluorescent probe dichlorfluorescent diacetate bis (acetoxymethyl) (Molecular Probes, Eugene, Oregon USA). TACS® Annexin V-FITC Apoptosis Detection Kits (R & D systems, U.K) Ficoll-PaqueÒ (Pharmacia, Uppsala, Sweden). E-Lyse (Cardinal Associates, USA). FACScan (Beckton Dec- kinson, Mountain View). 12-well, flat-bottom plates (NUNC, Denmark). Culture flasks (Falcon, Lincoln Park, NJ). Peristaltic pump controller (Masterflex, Cole Parmer Instrument, Co). Bichinoic acid (BCA) protein assay kit (Rockford, Illinois, USA). Mouse antihuman IgG1 eNOS antibody (Transduction Laboratories, USA). Goat antimouse IgG1 conjugated with horseradish peroxidase (DAKO, UK), SuperSignal West Pico Chemiluminescent substrate (Pierce, Rockford, USA). CPU 8720, UV/VIS Scanning Spectrophotometer, (Philips, Eindhoven, Netherlands). Laser-doppler monitor (Moor instrument Ltd, England). Intravital microscope (Nikon Diaphot, Tokyo, Japan), 40× objective lens (Flour, Nikon). Video camera (Mitsubishi, CCD-100E, Japan). Monitor (Sony, KVM2150L, Tokyo, Japan) Videocassette recorder (Phillips, VR347, Netherlands).
This study was conducted with the approval of the Beaumont Hospital Ethics Committee. The animal procedures were carried out under license from Department of Health, Republic of Ireland.
Human saphenous vein endothelial cells (HSVECs) were isolated from human saphenous vein by enzymatic dissociation as described previously [
HSVECs were incubated in the airtight chamber with a gas inlet and outlet (Billups-Rothenburg). A hypoxia microenvironment was achieved by purging the chamber with 95% N2, 5% CO2 for 15 min prior to the experiment. The resulting dissolved oxygen concentration in the chamber was 2%. Replacing the HSVEC in humidified 5% CO2 condition at 37˚C after hypoxia simulated reoxygenation. HSVECs were cultured to 90% confluence and plated at 2 × 105 cells per well on 2% gelatin-coated 12-well, flat-bottom plates. The final concentration of taurine was 0.5 mg/ml [
HSVECs apoptosis and necrosis were assayed by flow cytometry using the TACS® kit according to the manufacturer’s instructions. Briefly, after centrifugation, the pellets of HSVECs (2 × 105 cells) was resuspended in 100 µl fluorochrome solution (Propidium Iodide 10 µl, Annexin V-FITC 1 µl) and incubated for 15 min at room temperature in the dark and analyzed by FACScan. The minimum number of 10,000 events was collected and Cell quest® software was used for analysis.
ROS in HSVEC was detected by using the fluorescent probe dichloroflurescein diacetate bis (DCFH-DA) as described previously [
Intracellular Ca2+ was determined using Fluo-3 AM staining by a FACScan HSVECs were treated with hypoxia for 48 hours followed by 24 hours reoxygenation either in the presence or absence of taurine. Control cells were incubated in normoxia condition without taurine. 1 ul of (2 × 105) cells were washed once with buffer (10 mM Hepes, 5 mM KCl, 145 mM NaCl, 1 mM MgCl2, 10 mM Glucose, 1 mM CaCl2, pH 7.4) and resuspended in 100 µl buffer. After adding 0.42 µl of Fluo-3 AM (1.0 mM in DMSO with 1% pluronic F-127), cells were incubated at room temperature for 45 min. LMCF intensity of stained cells was detected with FL1. The minimum number of 10,000 events was collected and Cell quest® software was used for analysis.
HSVECs were treated with 300 μl lysis buffer (50 mM Tris-HCL, 150 mM NaCl, 5 mM EDTA, 0.5% (w/v) sodium deoxychlolate, 0.5% (v/v) triton X-100, pH 7.5) for 45 min on ice. Segments of ileum were snap frozen in liquid nitrogen and stored at −80˚C. Tissues were thawed, homogenized in PBS. Lysed samples were centrifuged at 12,000 rpm for 10 minutes at 4˚C. Protein concentration was quantified using a BCA assay kit. The final protein concentration of samples was adjusted to 200 µg/100µl and the samples were then suspended in SDS-glycerol loading buffer (pH 6.8, 62.5 mmol/L Tris, 2% SDS, 10% glycerol, 5% mercaptoethanol, 0.01% bromophenol blue). Protein was denatured at 100˚C for 10 minutes and separated by 12% SDS-PAGE with 20 µg of total protein loaded per lane. Proteins were then transferred to a nitrocellulose membrane and labeled with a primary monoclonal antibody, mouse antihuman IgG1, specific for the eNOS. After the secondary monoclonal antibody was added, goat antimouse IgG1 conjugated with horseradish peroxidase and the immunoblots were developed using Super Signal West Pico Chemiluminescent substrate and visualized by exposure of X-ray film.
Adult male Sprague-Dawley rats weighing 280 - 350 g were randomized into one of three groups: Control (sham I/R), I/R or taurine + I/R. They were not allowed solid food, but had free access to water 12 h prior to the experiment. For the intestinal permeability assay taurine @ 200 mg/kg/day was given by gavage for 5 days prior to the experiment. For the examination of microvascular injury taurine was given through jugular vein as a bolus. The rat was anaesthetized with sodium pentobarbitone (50 mg/kg, ip.) and following anaesthesia, a tracheotomy was performed to facilitate breathing during the experiment. The right jugular vein was cannulated for taurine (200 mg/kg body weight) or saline administration. After midline laparotomy, the coeliac and superior mesenteric vascular pedicle was isolated near their aortic origins.
After a midline abdominal incision, a segment of ileum (10 cm long), supplied by 3 - 4 blood vessel arcades was isolated from the remaining part of the intestine by incising the mesentery and the gut lumen was closed by tying it proximally and distally. Intestinal I/R was induced by separating and clamping the superior mesenteric and coeliac vascular pedicle with an traumatic clamp for 30 min, followed by reperfusion for 180 min. Sham operation included separation of the pedicle without clamping, followed by 180 min sham reperfusion. After I/R, the aorta was cannulated at abdomen and perfusion with saline to remove the blood in the tissue. The tissue was further perfused at 40 ml/min using a peristaltic pump controller with physiologic saline containing 20 µg/ml FD4 (4000 Dalton fluorescent dextran) for 5 min. At the end of the experiment, the segment was filled up with 1ml saline, and then the samples were taken from the segment of ileum and perfusion solution and centrifuged at 10 ×g for 10 min at 4˚C. After the plasma (perfusion solution) and luminal solution had been diluted (1:200), the concentration of FD4 was determined with the Perkin Elmer luminescence spectrophotometer (excitation wavelength: 485 nm; emission wavelength: 535 nm). In order to calculate intestinal permeability, the following equation was used:
Percentage ratio of plasma to lumen expression of FD4 = luminal FD4 concentration (µg/ml)/perfusion solution FD4 concentration (µg/ml) × 100% [
Intestinal mucosal permeability was determined by using an everted gut sac method, as previously described [
where M is the mass (ng) of FD4 in the gut sac at the 30 min incubation period, [FD4] ser is the FD4 concentration in the serosal fluid aspiration from the sac at the end of the 30 min incubation period, F is the flux of FD4 (in ng/min) across the mucosa, [FD4] muc is the FD4 concentration measured in the beaker at the beginning of the 30 min incubation period, A is the calculated area (in cm2) of the mucosal surface, and C is the clearance of FD4 (nl/min/cm2) across the mucosa.
Myeloperoxidase activity, an index of PMN accumulation, was determined as previously described [
(Highest absorbance/10)/weight of tissue used/0.0113 = MPO/gram of tissue
0.0113 = constant.
Intestinal wet: dry weight ratio was used as a measure of tissue oedema. A segment of intestine was harvested from each rat. The wet: dry weight ratio was calculated by weighing the freshly harvested intestinal tissue (wet weight), heating at 90˚C in gravity convection oven for 72 hours and weighing the residuum that became constant (dry weight).
Gut blood flow was determined via a laser-doppler monitor and expressed as flux by placing a probe on the surface of gut and recording at 0 min (baseline), before reperfusion (30 min of ischeamia) and at 180 min after reperfusion, respectively.
A segment of mesentery was exteriorised and was observed with an inverted intravital microscope with an 40× objective lens. The mesentery was carefully positioned to minimize the influence of respiratory movements and was moistened with BBS (NaCl: 131.9 mM, KCl: 4.7 mM, CaCl2: 2.0 mM, MgSO4: 1.2 mM and NaHCO3: 20 mM, pH 7.4, 36.5˚C - 37˚C). A video camera mounted on the microscope projected the images which were recorded on videocassette. Single unbranched venules with diameter of 20 - 35 µm and length >100 µm were chosen for study. Rolling leucocytes was determined by measuring the time required for individual leukocyte to transverse a 100-µm length of the venule expressed as µm/second. A leukocyte was considered to be adherent to the endothelial cell if it remained stationary for a period of 30 seconds or longer. Leukocyte adherence was expressed as number of leucocytes adhered to vessel wall per 100 µm of venule. Leukocyte transmigration was expressed as the difference between the number of extravasated leucocytes in the field of view the beginning and at the end of the reperfusion period.
Data is expressed as mean ± standard error of the mean (SEM). Statistical difference between groups was determined using Data Desk® (version 4) software, univariate ANOVA with Scheffe’s post hoc correction. Significant difference was established at P < 0.05.
48 hours of hypoxia followed by 24 hours of re-oxygenation significantly reduced HSVECs survival, as measured by flow cytometry (49.4% ± 2.76%, control vs 73.5% ± 1.13% I/R. P < 0.01). Supplementation with taurine (0.5 mg/ml) before or after hypoxia significantly attenuated I/R-induced HSVECs death (Tau + I/R: 71.6% ± 2.91% and I + Tau/R: 64.7% ± 4.01%, P < 0.01 vs I/R group) (
either before or after hypoxia significantly attenuated I/R-induced HSVECs necrotic death (Tau + I/R: 9.7% ± 1.47%, and I + Tau/R: 11.2% ± 1.31%) (
Intracellular ROS was significantly increased (358.62 ± 22.61 MCF vs 245.24 ± 18.25 control) following I/R. Administration of taurine (0.5 mg/ml) before or after 48 hours of hypoxia significantly reduced I/R-induced generation of ROS in HSVECs (Tau + I/R: 235.3 ± 14.3 and I + Tau/R: 279.3 ± 8.12, P < 0.01 and P < 0.05 vs I/R group, respectively) (
I/R injury significantly elevated intracellular calcium [Ca2+] compared to control cells (114.3 ± 18.96 MCF vs 42.7 ± 9.54 control). Supplementation with taurine (0.5 mg/ml) before or after the 48 hours of hypoxia significantly reduced I/R- induced Ca2+ influx in HSVECs (Tau + I/R: 54.7 ± 7.0; I + Tau/R: 70.3 ± 13.0) (
48 hours hypoxia followed by 24 hours of re-oxygenation decreased the EC expression of eNOS. The addition of taurine either before hypoxia or after hypoxia prevented this decrease. (
Intestinal endothelial barrier integrity was evaluated by measuring percent ratio of the FD4 transfer from blood to the intestinal lumen (Control n = 6, I/R and Tau + I/R n = 7). Permeability of FD4 from blood to the intestinal lumen significantly increased in after 30 min ischeamia followed by 180 min reperfusion, as evidenced by a marked increase in the flux of the fluorescent dye FD4 in I/R groups (37.6% ± 7.68%) p < 0.01 vs. Control group (6.0% ± 1.23%). Treatment with taurine reduced the increase in the permeability after I/R (11.4% ± 2.29%). P < 0.01 vs. I/R group (
Sham operated rats (n = 6) had mesentery-associated MPO concentrations at 8.98 ± 1.59 U/g. Gut ischeamia/reperfusion increased these concentrations to 15.79 ± 1.61 U/g9 (n = 6). (P < 0.05 vs Control group). The administration of taurine (n = 7) significantly decreased the MPO concentration to 9.35 ± 1.45 U/g. (p < 0.05 vs. I/R group) (
Wet and dry weight ratio of gut tissue in the animals with I/R (n = 4) was significantly higher than in sham operated animals (n = 4) (5.26 ± 0.54 p < 0.01 vs. 3.69 ± 0.15 for Control). This increase in wet and dry weight of gut was not observed in those animals receiving taurine (n = 6) (3.65 ± 0.29), p < 0.01 vs. I/R) (
The gut blood flux as assessed by Laser-Doppler (
Taurine prevented this I/R altered gut blood flux (n = 4) (69.20 ± 4.16, p < 0.05 vs. I/R group).
At baseline, there was no significant difference in leukocyte rolling velocity
between control (51.57 ± 2.78 µm/s) vs. I/R (48.48 ± 2.64 µm/s) vs. Tau + I/R (50.94 ± 0.96 µm/s). After ischemia for 20 min, the rolling velocity (y-axis) per reperfusion interval (x-axis) for three experimental groups is represented in
10 min, 30 min and 60 min (26.11 ± 2.64, 32.25 ± 2.86, and 29.09 ± 3.78 µm/s, respectively, p < 0.01 vs. Control group). Tau + I/R group (n = 5) maintains velocity at the corresponding time interval. (44.58 ± 3.92, 53.37 ± 4.62, and 55.38 ± 6.37 µm/s, respectively, p < 0.01 vs. I/R group).
The quantity of adhesive leucocytes (y-axis) per reperfusion interval (x-axis) for the same experimental groups is represented in
The intestinal mucosa is extremely sensitive to I/R injury which can tissue damage and remote organ failure. Hypoxia-mediated endothelial damage results in vascular dysfunction leading to leukocyte trafficking, tissue oedema and impaired vasomotor responses. Several studies have shown that EC are resistant to hypoxia, requiring a prolonged period of reduced oxygen to induce cell death [
cells eventually leads to death by either apoptosis or necrosis depending on the intensity and duration of the I/R insult [
Nitric oxide (NO) at low concentrations is cytoprotective for endothelial cells modulating vascular tone attenuating increases in albumin leakage into venules, preventing platelet aggregation and neutrophil infiltration following I/R [
I/R induced adverse alterations in intestinal permeability resulting in gut oedema in vivo, which was accompanied by leukocyte accumulation in the intestinal tissue. Intestinal endothelial permeability significantly increased after 30 min ischaemia followed by 180 min reperfusion, facilitating leucocyte transmigration from the blood to the intestinal lumen as evidenced by increased MPO activation. Intestinal epithelial barrier permeability significantly increased following I/R which could allow bacterial translocation. Treatment with taurine preserved both endothelial and epithelial barrier function permeability. I/R injury resulted in significant gut tissue oedema which was not observed in those animals receiving taurine. Taurine prevented I/R induced reduction of gut blood flux and prevented increased MPO activity. These results correlate well with recent findings from Sukhotnik et al. who demonstrated that taurine preserved mucosal parameters and intestinal histology following IR injury [
Leukocyte rolling is an essential prerequisite for adherence leading to tethering between the leucocytes and endothelial cells, allowing leucocytes to become activated [
In conclusion, the main findings of this work are that taurine in clinically relevant doses effectively inhibits intestinal hyperpermeability and tissue edema and maintains normal microvascular function, following I/R of the coeliac and superior mesenteric vascular pedicle in rodents. Taurine can protect the gut barrier function during surgery and warrants further investigation as a peri-operative intervention, which can help to maintain gut barrier integrity and improve outcomes for patients undergoing surgery.
This work was funded by The Department of Surgery, Royal College of Surgeons in Ireland.
Chen, H., Chen, G. and Condron, C. (2017) Taurine Protects Gut Barrier Function and Prevents Endothelial Cell Injury Induced by Ischaemia- Reperfusion. Food and Nutrition Sciences, 8, 678-698. https://doi.org/10.4236/fns.2017.86048