Key words : Hypoxia-inducible factor 1-α, Microcirculation, Reactive oxygen species

Introduction

Hepatic ischemia-reperfusion injury (HIRI) starts with the interruption of liver blood flow and is exacerbated by the return of blood flow due to a complex set of metabolic and histopathological events, causing liver dysfunction and failure. This type of injury occurs during surgery for liver trauma, large tumor excision, and liver transplant and is associated with high morbidity and mortality.^1-3

Hepatic ischemia-reperfusion injury has been extensively studied, but the topic remains largely unclear. Many factors and pathways have been implicated in the process of HIRI, including adenosine triphosphate (ATP) depletion by anaerobic glycolysis, intracellular acidosis, intracellular calcium overload, reactive oxygen species (ROS) production, mitoc­hondrial dysfunction, microcirculation disturbance, complement activation, apoptosis, liver Kupfer cell (KC) and neutrophil activation, and cytokine, chemokine, and nitric oxide release.^4-14 During HIRI, many cells are involved, including hepatocytes, liver KCs, sinusoidal endothelial cells (SECs), hepatic stellate cells, neutrophils, and thrombocytes.^9-13

Microcirculatory dysfunction is one of the most important mechanisms in HIRI.^5-8,10,11,13 In the early stages of hepatic ischemia-reperfusion, secondary to ATP reduction, the swelling of KCs and SECs decreases capillary diameter and blood flow.^6-8 This is followed by vasoconstriction as a result of endothelin and nitric oxide imbalance and direct endothelial damage from ROS production and expression of adhesion molecules with accumulation of platelets and leucocytes.^7,8 In the late stages, microcirculation is further disrupted by leukocyte adhesion to the endothelium and platelet aggregation.^10,11,13,14

L-theanine (γ-glutamylethylamide) is an amino acid derived from nonprotein tea (Camellia sinensis).^14-17 It has antitumor, antimicrobial, antioxidant, neuroprotective, and hepatoprotective effects, therefore resulting in extensive research.^18-23 Its hepatoprotective effects have been attributed to its antioxidant and anti-apoptotic effects.^18,19,24 The effect of L-theanine on microcirculation in HIRI has not yet been investigated. Therefore, in this study, we aimed to examine the effects of L-theanine on the extent of hepatic cell injury, functional capillary density (FCD), serum liver function parameters, and inflammatory and oxidative stress markers in liver tissues in an HIRI rat model.

Materials and Methods

Animals
Thirty-two male Sprague Dawley rats weighing 250 to 300 g were used. The rats were housed in rooms maintained at 21 °C and a 12:12-h light-dark cycle. Animals were fasted overnight before the experiment but had free access to water. Care was provided in accordance with Karadeniz Technical University Animal Experiments Local Ethics Committee, Trabzon, Turkey (number 2018/324, date May 12, 2018).

Anesthesia was induced by an intraperitoneal injection of 50 mg/kg ketamine (Ketalar; Eczacibasi, Istanbul, Turkey). The rats were randomized into 4 experimental groups: sham + saline, L-theanine + saline, HIRI +saline, and HIRI + L-theanine.

Rats to be administered with L-theanine were given it intraperitoneally at 150 mg/kg with 1 cm³ of saline (Sigma-Aldrich Chemie GmbH; number A2129) at the same time in the morning, at 3 days before the experiment.24 On the day of the experiment, the same dose was repeated 15 minutes before ischemia and at the end of ischemia.

Hepatic ischemia-reperfusion injury was induced under ketamine anesthesia. After laparotomy, liver median and left lateral lobe vessels were closed with a small bulldog clamp for 60 minutes.^9,25 This way, 70% of the liver was exposed to ischemia. Reperfusion was achieved by opening the clamp 1 hour later, and this continued for 4 hours. This method is defined as 60 minutes of 70% HIRI and 4 hours of reperfusion.^9,25

For rats in the sham + saline group (n = 8), 1 cm³ saline was given intraperitoneally 15 minutes before the experiment and then rats underwent anesthesia (50 mg/kg ketamine), laparotomy, and exposure of the portal triad without hepatic ischemia in a 60-minute period; rats were given 1 cm³ saline intraperitoneally again. Laparotomy was closed; 4 hours later, the rats were again anesthetized by ketamine, and relaparotomy was performed. The liver was exposed on an adjustable stage. The orthogonal polarization imaging video microscope (Cytoscan A/R; Cytometrics) was attached to the moveable shaft, and microcirculation was recorded in 6 different capillary regions of the left lateral or central lobe for at least 20 seconds.^26,27 The images were stored in audio-video interleave format on a computer (Sony VGN-FW 230 J/H). Thereafter, blood samples were taken from the abdominal aorta for measurements of serum concentrations of tumor necrosis factor alpha (TNF-α) and activities of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and gamma-glutamyltransferase (GGT). The liver from the left lateral or central lobe was retrieved and was divided into 2 parts: one for histologic examination and the other for measurements of myeloperoxidase, malondialdehyde, oxide glutathione (GSSG), and reduced glutathione (GSH) activities. The excised liver tissues were frozen in liquid nitrogen and stored frozen at -80 °C until thawing for measurement of tissue enzyme activities. Liver tissues taken for histopathology were fixed in 10% formalin solution until examination and stored.

For the sham+ L-theanine group (n = 8), L-theanine (150 mg/kg) was administered intraperitoneally 15 minutes before laparotomy and 60 minutes after laparotomy.19 The other procedures were the same as described for the sham + saline group.

Rats in the HIRI + saline group (n = 8) were treated according to the protocol for the sham + saline group after the induction of HIRI. For the HIRI + L-theanine group (n = 8), HIRI was induced and L-theanine was administered as in the sham+ L-theanine group.

Analysis of functional capillary density
Orthogonal polarization imaging has been suggested for recording and quantifying changes in microcirculation.^26-28 The technique uses optical filtration of polarized light absorbed by hemoglobin so that the red blood cells appear dark. The recorded images were analyzed by software using the MAS image analysis system. This system was developed at the Academic Medical Center, University of Amsterdam (Amersterdam, the Netherlands) by Dr. Iwan Dobbe, Professor Can Ince, and Dr. K. R Mathura. Functional capillary density, which was identified as the best parameter for the measurement of microcirculation, was defined by Messmer and colleagues²⁶ as the length of red blood cell-perfused capillaries (in mm) per observation area (in mm2). Therefore, we selected FCD as the parameter for measurement of microcirculation.

Analysis of biochemical parameters
The serum activities of ALT, GGT, and LDH were measured by an autoanalyzer (Beckman Coulter AU5800). Serum TNF-α levels (in ng/L) were determined by the commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit (catalog no: E0764Ra; Bioassay Technology Laboratory).

Tissue-associated myeloperoxidase activity (in U/mg protein) was assessed by a modification of the method described by Schierwagen and colleagues.²⁸ Lipid peroxidation in liver tissues was assessed by measuring the concentration of malondialdehyde (expressed as nmol/mg protein) using a colorimetric reaction with thiobarbituric acid by modification of the method described by Buege and Aust.²⁹ Tissue inducible nitric oxide synthase (iNOS) levels were measured using the commercial sandwich ELISA kit (catalog no. E0008Ge, Bioassay Technology Laboratory), with results given in nanograms per milligram of protein. Protein concentrations in liver tissue were measured by the method of Lowry and colleagues.³⁰ Liver GSH levels (in nmol/mg protein) were measured by a commercial colorimetric assay kit (catalog no. E-BC-K030, lot: MNFVKV4XP1, Elabscience) and a GSSG commercial kit (catalog no. E1264Ra, Bioassay Technology Laboratory). Ratios of GSH-to-GSSG were also evaluated.

Morphologic examination
For pathological examination, hepatic tissue was fixed in 10% buffered formalin, sectioned, and then stained with hematoxylin and eosin. Two pathology experts in liver pathology conducted the histopathologic evaluation. Each was blinded to both the induction techniques and the additional drugs given. Sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration were assessed using a scoring system from 0 (no pathologic changes) to 5 (severe congestion/ballooning degeneration, as well as >60% lobular necrosis ), as previously described by Suzuki and colleagues.³¹ The H score for hypoxia-inducible factor 1-alpha (HIF-1α) was detected by immunohistochemical stain (anti-HIF-1α antibody ab114977, Abcam). Histologic slides were digitally imaged for immunohistochemical scoring under ×100 magnification using the Nikon Eclipse Ni-U and the Nikon NIS-Elements D microscope imaging software version 4.0. Digital images were evaluated using the H-score method with minor modifications.^32,33

Statistical analyses
Results are presented as means ± SEM. Enzyme activities, histopathologic results, FCD, and H scores were analyzed by Kruskal Wallis and Mann Whitney U test with Bonferroni correction, and differences were considered significant at P < .05.

Results

The induction of HIRI resulted in a significantly elevated effect on the activity of serum ALT, LDH, GGT, and TNF-α and tissue myeloperoxidase, malondialdehyde, iNOS, and GSSG, as well as a decreasing effect on the tissue activity of GSH and the GSH-to-GSSG ratio (Table 1). The use of L-theanine improved these changes.

Functional capillary density was 37.35 ± 0.28 mm/mm2 in the sham + saline group and significantly lower at 33.22 ± 0.22 mm/mm2 in the HIRI + saline group after the induction of HIRI; FCD was elevated after administration of L-theanine in the HIRI + L-theanine group to 36.78 ± 0.25 mm/mm2 (P < .05; Table 1).

The histologic examination showed that the HIRI + saline group had greater congestion, necrosis, and balloning degeneration compared with the other groups (Table 2). L-theanine in the HIRI + L-theanine group improved these changes (Table 2).

H score for HIF-1α immunostaining was 61.66 ± 9.45 in the sham + saline group, 75.5 ± 8.76 in the sham + L-theanine group, 226.33 ± 5.66 in HIRI + saline group, and 142.33 ±16.9 in the HIRI + L-theanine group. The increase in the HIRI + saline group was significant compared with that shown in the other groups (P < .01; Table 1). L-theanine resulted in a significantly decreased H score (P < .05; Table 1).

Discussion

Our results indicated that HIRI resulted in significant increases in the serum activities of ALT, LDH, GGT, and TNF-α; tissue levels of iNOS, myeloperoxidase, malondialdehyde, and GSSG; and H score for HIF-1α in the liver. There were also significant decreases in the tissue activity of GSH in the liver, the ratio of GSH/GSSG, and FCD. In addition, more congestion and lobular necrosis were observed in histologic examination. Administration of L-theanine improved these changes.

We chose ischemia of 70% of the liver for 1 hour and reperfusion for 4 hours as the experimental model. This model is one of the most used methods and does not cause death.^9,25

L-theanine, a glutamate derivative, is a unique nonprotein amino acid present in the leaves of Camellia sinensis.^15-17 Many studies have shown its anti-inflammatory, antitumor antioxidant, hepatoprotective, and neuroprotective effects.^15-24 It affects through the nuclear factor kappa B pathway.^16,18 It can affect glutamate and glutamine metabolism.^15-17 Considering these beneficial effects, L-theanine may have a protective effect against the progression of the HIRI process.

Serum ALT values indicate liver function and extent of liver damage. In our study, similar to the literature, HIRI induction caused significantly increased ALT levels and necrosis.^4,6,9,25 There has been considerable controversy over the mode of hepatocyte cell death after HIRI.^{7,8,10,11,13,14} The 2 forms of hepatocyte cell death, as apoptosis and necrosis, differ greatly in mechanism and morphology. Cell shrinkage, chromatin density increase, nuclear fragmentation, and apoptotic body formation are among the hallmarks of apoptosis. On the other hand, necrosis can be defined morphologically by swelling of the mitochondria and cells by disruption of cell membrane integrity and vacuolization.¹¹ Apoptosis and necrosis usually occur together, and the main cause of both is mitochondrial dysfunction.^10,11,13,14 The severity of stimulation determines whether the event will result in apoptosis or necrosis. Low-dose ROS or ATP deficiency can lead to apoptosis, whereas high-dose ROS or too little ATP can cause necrosis. There is considerable work in the literature on HIRI and apoptosis.^7,8,10-14 These studies have shown that apoptotic pathways are activated by HIRI, but most of the final cell death is necrosis. Unfortunately, we could not study apoptosis because of economic reasons.

Oxidant stress due to ROS production is one of the important pathogenetic mechanisms of HIRI.^4,6,10-14 In cells without oxygen deficiency, there is a limited amount of ROS production in the mitochondria, and this cell is eliminated by the antioxidant system inside the cell. Upon reperfusion, hepatic ischemia occurs with high production of ROS that causes oxidative stress and overwhelms the antioxidant system.^{4,6,8,10,11,13} This is supported by the increased tissue myeloperoxidase, malondialdehyde, and GSSG values and the low GSH-to-GSSG ratio shown in our HIRI + saline group. The main sources are mitochondrial metabolism, xanthine oxidase/reductase, and NADPH oxidase.¹³ Reactive oxygen species destroys proteins, lipids, DNA, and endothelium and additionally can damage endothelial cells, thus compromising the microvasculature.^{4,6,8,10,11,13} Another effect of ROS is the activation of KCs.^{4,6,8,10,11,13} The increased antioxidant capacity by using L-theanine in our present study showed its beneficial effects in the liver.

Microcirculation disturbance is another important pathogenetic factor during HIRI.^{4,6-8,10,11,13} Microcircu-lation of the liver can be measured by diffuse reflectance spectroscopy, intravital microscopy, multiple indicator dilution technique, and orthogonal polarization imaging.^26-28 We measured hepatic microcirculation by orthogonal polarization imaging in this study. In our study, the induction of HIRI resulted in a decrease in FCD. Many authors have reported similar results using intravital microscopy, diffuse reflectance spectroscopy, or orthogonal polarization imaging during HIRI.^{10,11,13,28-30} Three factors play an important role in the occurrence of microcirculatory insufficiency during HIRI. First, during the hepatic ischemia period, ATP depletion in the cell decreases, which causes swelling of KCs and SECs with an increase in cell membrane permeability. This swelling creates a narrowing of the sinosoid diameter.^4,6,8,9Second, excessive ROS during reperfusion leads to damage in SEC and KC activation. Activation of KCs causes cytokine and chemokine release, leukocyte chemotaxis and activation, and expression of adhesion molecules in the endothelium, which is followed by leukocyte adhesion and platelet aggregation on the SECs.^4,6,8-11,13 As a result, microcirculation is reduced. The third mechanism in microcirculation disruption is the imbalance between nitric oxide and endothelin. The HIRI balance is disturbed in favor of the endothelin, and vasoconstriction occurs.^{4,6,8,10,11,13} In our study, L-theanine may have corrected FCD by reducing the effect of ROS as an antioxidant. We found no study similar to ours so far in the literature.

Nitric oxide plays a key role during HIRI. Nitric oxide acts as a vasodilator to sinusoids through hepatic stellate cells and also inhibits proinflammatory cytokine release, platelet aggregation, leukocyte adhesion, and apoptosis.^7,8,11,13 The effects of nitric oxide are dose dependent. Although it has a vasodilator effect on sinosoids in low doses, it has the opposite effect in excessive doses.^7,8,13 In our study, we observed an increase in iNOS levels; however, administration of L-theanine decreased it, which may explain the improvement in microcirculation.

One of the most researched substances in medicine in the past decade is HIF-1α, which functions as the oxygen sensor of the cell. Although it does not react at normal oxygen pressures, its release increases at low oxygen pressures and ensures the release of protective substances from the nucleus.^32-35 In our study, we found a significant increase in HIF-1α in the HIRI + saline group. Similar results have been found in the literature.^34,35 Administration of L-theanine reduced the HIF-1α in the HIRI + L-theanine group. We suggest that the cell remains in deep hypoxia, and the situation improves with the administration of L-theanine.^32-35 However, in these previous studies, HIF-1α was studied in different time periods,^34,35 and we only observed our results at 4 hours of reperfusion.

Tumor necrosis factor α is an important cytokine released from hepatic KCs during HIRI.^4,6,8-11,13 This cytokine stimulates leukocyte chemotaxis and activation and the expression of adhesion molecules from the endothelium.^{4,6,8,10,11,13,17} It also has direct toxic effects on hepatocytes and SECs.^4,10,11,13 In our study, TNF-α concentration was found to be significantly increased in the HIRI + saline group but was decreased with L-theanine administration. Activation of KCs occurs through ROS. The antioxidant effect of L-theanine explains the reducing effect of L-theanine on TNF-α levels.¹³ In addition, results from Malkoç and colleagues supported our findings.¹⁹

The limitations of our study are that different reperfusion times and apoptosis could not be investigated due to budget constraints.

Conclusions

We found that administration of L-theanine helped to prevent the detrimental effects of HIRI. This was indicated by decreased lipid peroxidation, iNOS level, TNF-α concentration, and HIF-1α level and by restoration of FCD, histologic changes, and cellular antioxidant levels. L-theanine exerts these effects with its antioxidant and anti-inflammatory properties. Additional studies are needed to confirm the preventive or reducing effects of L-theanine on HIRI.

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