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Investigation of the effect of safranal and crocin pre-treatment on hepatic injury induced by infrarenal aortic occlusion
Biomedicine & Pharmacotherapy 83 (2016) 160–166
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Investigation of the effect of safranal and crocin pre-treatment on hepatic injury induced by infrarenal aortic occlusion Ziya Taner Ozkececia,* , Yucel Gonulb , Yasemin Yukselc , Afra Karavelioglud, Kamil Tunaye, Yusuf Gulsarib , Onder Cartillib , Omer Hazmanf , Ahmet Bala a
Department of General Surgery, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey Department of Anatomy, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey Department of Histology and Embryology, Zekai Tahir Burak Women’s Health and Research Hospital, Ankara, 06260, Turkey d Department of Pediatric Surgery, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey e Department of Emergency Medicine, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey f Department of Chemistry, Biochemistry Division, Faculty of Science and Arts, Afyon Kocatepe University, Afyonkarahisar 03200, Turkey b c
A R T I C L E I N F O
Article history: Received 6 April 2016 Received in revised form 8 May 2016 Accepted 14 June 2016 Keywords: Ischemia-reperfusion Liver injury Apoptosis Bax Blc-2 Caspase-3
A B S T R A C T
Ischemia-reperfusion (IR) injury of the liver is an unresolved problem that occurs during certain surgical approaches, including hepatic, cardiac and aortic operations. In this study we aimed to investigate whether crocin and safranal had protective effects on liver IR injury induced in an infrarenal aortic clamping (IRAC) model. Male Wistar-Albino rats (n = 32) were divided into four groups with 8 animals each as follows: Sham, IR, IR + crocin, and IR + safranal. The infrarenal aorta (IRA) was clamped for 60 min for the ischemic period and allowed to reperfuse for 120 min. Blood and tissue samples were collected for biochemical, histological and immunohistological analysis. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were found to be significantly higher in the IR group than the sham group (respectively; p = 0.015, p < 0.001). There were significant differences between the IR group and the IR + crocin group or the IR + safranal group in AST levels (respectively; p = 0.02, p < 0.001). ALT showed a significant decrease in the IR + crocin group compared to the IR group (p < 0.05). We also observed histopathological changes among the groups. Bax and Caspase-3 expression in the IR group was remarkably higher than in the other groups. Caspase-3 and Bax expression in the IR + crocin and the IR + safranal groups were significantly lower than in the IR group. Nevertheless, there were no significant differences in BCL2 expression among the groups. IRAC is a cause of IR injury in the liver. This study showed that crocin and safranal have protective effects on IR induced liver injury. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Remote organ injury caused by infrarenal aortic clamping (IRAC), which can be encountered during surgical interventions related to the abdominal aorta, is a complex process affecting the lower extremities as well as remote organs such as the heart, liver, kidney, and lungs [1–3]. Reperfusion of acutely ischemic abdominal organs causes local and distant organ damage and may contribute to high morbidity and mortality [4]. Several factors have been suggested to play a role in the pathophysiology of ischemia-reperfusion (IR) injury, such as free
* Corresponding author at: Department of General Surgery, Faculty of Medicine, Afyon Kocatepe University, Ali Cetinkaya Campus, Izmir Road 8. Km, 03200 Afyonkarahisar, Turkey. E-mail address: [email protected] (Z.T. Ozkececi). http://dx.doi.org/10.1016/j.biopha.2016.06.027 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
oxygen radicals, polymorphonuclear leukocytes, the complement system and endothelial cells. These are cellular and humoral mechanisms that have complex relationships with each other. While the amount of oxygen, the decrease in energy production and synthesis of the antioxidant enzymes, toxic substances, ions such as Ca and Na, pro-inflammatory cytokines and leukocyte adhesion molecules increase during ischemia. This makes cells relatively prone to damage during reperfusion. When blood flow resumes to ischemic tissue, cells are exposed to large amounts of oxygen, which is a source of radical oxygen species in the previously damaged tissue. However, internal antioxidant mechanisms are insufficient during IR injury. This is why many drugs have been used to reduce the effects of hepatic IR injury [5–8]. Nevertheless, studies on this subject are as yet insufficient to reach a definitive result.
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Bcl-2 is protective against both apoptotic cell death and multiple cellular events. It was reported that apoptosis can be prevented by a decreased ratio of Bax/Bcl-2 [9]. Intracellular Bcl-2 to Bax ratio is of paramount importance and may give a clue to whether a cell would progress to apoptosis. If Bax is greater, then the cell begins to progress towards apoptosis. However, apoptosis will be inhibited when Bcl-2 is greater [2]. Various substrates like Bax, Bak and Caspase trigger the apoptotic process; however Bcl-2, Bcl-xL or Mcl-1 family proteins generally act as antiapoptotic mediators, which may cause a delay or inhibition in apoptotic enzyme release. Moreover, overexpression of antiapoptotic proteins provide a survival benefit in cells treated with chemotherapeutic agents [10]. As shown in rat models, liver [8,11] and kidney [2] remote organ injury mediated IRAC can be attenuated using different protective agents. Our primary goal is to minimize damage after IRAC. Therefore, we investigated crocin and safranal as strong protective agents of liver IR induced by IRAC. Crocus sativus L., cultivated in Southwest Asia, Spain, France, Italy, Turkey and Iran, is a flowering plant used in traditional medicine. Saffron, derived from the flower of Crocus sativus L., contains at least 150 chemicals. Crocin and safranal, which are the main components of saffron, are known to have anti-inflammatory, anticonvulsant, antitussive, antioxidant, anxiolytic and antidepressant effects [12]. In our previous studies, we showed that crocin has anti-inflammatory, antiapoptotic, antioxidant and protective effects on acute kidney injury following IRAC [2] and brain injury following four vessels oclusion [13]. As far as we know, the effects of crocin and safranal on liver IR injury mediated by IRAC have not been studied thus far. In this study we therefore aimed to investigate whether crocin and safranal had protective and anti-apoptotic effects on IRAC induced liver IR injury using hematoxylin and eosin staining and Caspase-3, Bax, and Bcl-2 immunohistochemical staining. 2. Material and methods Study protocols and experimental procedures were approved by the Ethics and Animal Welfare Committee of Afyon Kocatepe University and the experiments were performed according to the Principles and Guidelines for Experimental Animals issued by The National Health and Medical Research Council and Guide for the Care and Use of Laboratory Animals (NIH issue no. 85–23, 1985 revised) prepared by The National Institutes of Health. 2.1. Animals Thirty two male Wistar-Albino rats, weighing 250–300 g, were included in the study. During the experimental procedure, the animals were kept under standard laboratory conditions including 24–26 C room temperature, 50–60% humidity, 12 h of light and night cycle and were allowed access to food and water ad libitum. The animals were fasted 12 h before the experiment. 2.2. Study groups The 32 rats were randomly divided into four groups comprising 8 rats in each group as follows: Group I (Sham, n = 8): Except for aorta clamping, procedures for laparotomy and dissection of the infrarenal aorta (IRA) were performed the same as in the other groups for equivalent operational time and stress. After closing the abdominal incision, the animals were kept under observation for 3 h to simulate the IR interval in the other groups. No drugs were administered to the rats.
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Group II (IR, n = 8): After laparotomy, the IRA was dissected carefully and clamped to create ischemia for 60 min and the clamp was removed to apply reperfusion for two hours. Group III (IR + Crocin, n = 8): 100 mg/kg crocin (Sigma-Aldrich Chemical Co., St Louis, USA) was injected intraperitoneally 30 min before laparotomy [14]. The IRA was clamped for 60 min to create IR, achieved by opening the clamp for two hours. Group IV (IR + Safranal, n = 8): 100 mg/kg safranal (SigmaAldrich Chemical Co., St Louis, USA) [15] was administered intraperitoneally 30 min before the surgical procedure. After applying 60 min of ischemia by blocking IRA blood flow, reperfusion was supplied by opening the aortic clamp for two hours. 2.3. Surgical intervention Animals were fasted for 12 h before the surgical procedure. After intramuscular injection of 5 mg/kg Xylazine (Rompun, Bayer, Turkey) for premedication, 40 mg/kg ketamine (Ketalar, ParkeDavis, Eczacibasi, Turkey) was used for anesthesia. Rats were fixed to the operating table from four extremities to prevent movement. A median laparotomy was performed after shaving, washing with povidone-iodine solution and covering the abdomen under sterile conditions. The intestines were covered with warm, wet gas compresses to avoid heat and fluid loss after deviating to the right. The abdominal aorta was explored with retroperitoneal dissection and turned 1 cm below the renal vein and 1 cm above the iliac bifurcation. After administering 150 U/kg heparin intraperitoneally and waiting 5 min for anticoagulation, the IRA was obstructed for 60 min using atraumatic microvascular clamps (Vascu-Statt II, midi straight 1001-532; Scanlan Int., St. Paul, MN, USA). Disappearance of distal aortic pulsation verified occlusion of the IRA and determination of convincing pulsation in the femoral region with Doppler ultrasonography (BT-200 Vascular Doppler HI-dop, 7th Fl., A Bldg., Woolim Lions Valley 5-cha, 302, Korea) confirmed reperfusion after removing the clamps. Before removing the clamps for reperfusion, 1 mg/kg intraperitoneal protamine sulphate was given to the rats to neutralize the effect of heparin. Following hemostasis, the compresses covering the intestines were removed and the intestines were placed into the abdomen. Fluid resuscitation was performed intraperitoneally with 10 cc of saline and the abdominal incision was closed with a sterile dressing. Rats were sacrificed to collect tissue/blood samples after reperfusion for 120 min. Crocin and safranal were prepared just before application to the rats. Crocin was dissolved in physiological saline at a dose of 100 mg/kg. Safranal was emulsified in physiological saline at a dose of 100 mg/kg before being administered to the experimental animals intraperitoneally. While 100 mg/ kg crocin was injected intraperitoneally in group III, 100 mg/kg safranal was administered intraperitoneally in group IV 30 min before laparotomy. No drugs were administered in groups I and II. 2.4. Collection of blood and tissue samples Blood samples (3 ml) were taken from the vena cava inferior for biochemical analysis and centrifuged. Serum samples obtained in this manner were preserved at 70 C until biochemical analysis. In addition, the right lobe of the liver was removed for histopathological and immunohistochemical assays. 2.5. Biochemical analysis Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed in an autoanalyser (Cobas 6000, Roche, Switzerland) as indicators of liver ischemia.
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2.6. Histopathological evaluation 2.6.1. Tissue preparation and H&E staining Tissue samples taken from the rats were fixed in 10% neutral buffered formaldehyde solution. After formalin fixation, routine tissue processing steps were performed. Tissues samples were embedded in paraffin blocks. Sections of 5 mm thickness were cut from the blocks using a microtome. Slides were then stained with hematoxylin and eosin (H&E) and examined with a light microscope (E600, Nikon, Japan) for histopathological alterations of liver tissue. Hepatocyte morphology (destruction of cell shape and core structure, cytoplasmic shrinkage and darkening), increase of Kupffer cells-sinusoidal infiltration and congestion were evaluated semiquantitatively and scored as follows: negative (0), mild (1), moderate (2), severe (3) and excessive changes (4). Scores were calculated for each group [16]. 2.6.2. Immunohistochemical assay Sections placed on adhesive slides were stained with Caspase-3 (at 1:100 dilution, 1197P1306J, Neomarkers, Fremont, CA), Bax (at 1:100 dilution, PU3470708, Biogenex, USA) and Bcl-2 primary antibodies (NU541-UC, Biogenex) using the UltraVision HRP polymer detection system (Thermo Fisher Scientific Inc./Lab Vision, Fremont, CA, USA) according to the manufacturer’s protocol. For this purpose, slides were initially deparaffinized, rehydrated and placed in distilled water. Slides were then boiled in 10 mM sodium citrate buffer for 25 min in a microwave oven for surface epitope retrieval. The samples were then placed in 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Nonspecific binding was blocked using Ultra V block for 5 min at room temperature. Sections were then treated with Caspase-3, Bax and Bcl-2 primary antibodies and incubated overnight at 4 C. After rinsing with PBS, the sections were treated with biotin-labeled and streptavidin conjugated secondary antibodies, respectively. Finally, AEC peroxidase substrate (Labvision Corp, Fremont, CA) was implemented and the reaction was colored. The sections obtained by this procedure were counterstained with Mayer’s hematoxylin and the slides were covered with coverslips using aqueous adhesive. Finally, slides were examined under the light microscope at 200 magnification and Caspase-3, Bax and Bcl-2 positive stained cells were counted in six randomly chosen areas with Image Analysis Software (NIS Elements Nikon, Japan). H score was used to calculate immunopositive cells. 2.7. Statistical analysis The data obtained were analyzed using SPSS for Windows 15.0 software package (SPSS Inc., Chicago, Illinois, USA). Results of descriptive statistics were expressed as mean standard deviation. Continuous variables were compared among the groups with Kruskal–Wallis tests. Mann–Whitney U test was used to determine differences between the two groups when Kruskal Wallis was significant. P values less than 0.05 were considered statistically significant. 3. Results 3.1. The effects of crocin and safranal on liver function in liver IR injury Biochemical results are shown in Table 1. Serum levels of ALT and AST were found to be significantly higher in the IR group compared to the SHAM group (respectively; p = 0.004, p < 0.001). ALT and AST levels were significantly decreased in the IR + safranal group compared with the IR group (respectively; p = 0.004, p < 0.001). However, ALT levels did not significantly decrease in the crocin + IR group compared with the IR group while AST levels
Table 1 Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Group2 (IR) n=8
Group3 (IR + Crosin) n=8
Group4 p (IR + Safranal) n=8
Variables
Group1 (SHAM) n=8
AST U\L ALT U\L
116 25.6* 356.4 48.3*,z,1 253.7 25.81 183.5 15.2z 22.9 3.4* 50.1 13.2*,z 30.5 18.21 23 8z,1
<0.001 0.015
Data are mean standard deviation (Kruskal Wallis test was used). On each line, the difference between the means with the same markers are significant (p < 0.05) *,z,1 p < 0.05, Mann Whitney U test was used to compare two groups.
significantly reduced in the crocin + IR group compared to the IR group (respectively; p = 0.15, p = 0.03). In addition, there were significant differences between the IR + crocin group and the IR + safranal group in ALT levels. 3.2. The effects of crocin and safranal on liver IR damage 3.2.1. H&E staining results Images from the experimental groups stained with H&E are shown in Fig. 1 and scores are shown in Table 2. In the evaluation of liver slides, the SHAM group showed normal morphology in hepatocyte and sinusoidal structure (Fig. 1a). The IR group showed destruction of hepatocyte structure with cytoplasmic darkening and shrinkage. In addition, in the IR group, sinusoidal dilatation and congestion were prominent compared to SHAM. It was observed that there was an increase in the number of inflammatory cells in the sinusoidal area and Kupffer cells in the IR injury group (Fig. 1b). The IR + crocin treated group showed less deterioration in hepatocyte structure than the IR group. The sinusoidal structure was partially protected with crocin treatment, and inflammatory cell migration and Kupffer cells were less than in the IR group (Fig. 1c). The IR + safranal treated group demonstrated better histopathological scores than the IR and IR + crocin treated group (Fig. 1d). Hepatocyte structure was more protected from ischemic injury and also sinusoidal dilatation and inflammatory cell migration was reduced by safranal treatment compared to the IR group. 3.2.2. Immunohistochemistry results 3.2.2.1. The effects of crocin and safranal on Caspase-3 expression in liver IR injury. Caspase-3 immunohistochemistry images are shown in Fig. 2 and results are shown in Table 3. In the SHAM group, a small amount of Caspase-3 staining of hepatocytes was observed (Fig. 2a). IR injury remarkably increased the number of Caspase-3 positive cells. This increase was statistically significant when compared to the SHAM group (p = 0.004) (Fig. 2b). There was a statistically significant reduction in Caspase-3 positive stained cells in the crocin (Fig. 2c) and safranal (Fig. 2d) treated groups when compared to the IR group. When crocin and safranal treated groups were compared to each other, safranal reduced Caspase-3 expression to a greater degree than crocin. 3.2.2.2. The effects of crocin and safranal on bax expression in liver IR injury. Bax immunohistochemistry images are shown in Fig. 3 and the results are shown in Table 3. In sections of the SHAM group, some hepatocytes showed positive staining with Bax antibody (Fig. 3a). Ischemia-reperfusion injury demonstrated a significant increase in the number of cells stained with Bax compared to SHAM (p = 0.004) (Fig. 3b). Crocin administration significantly decreased the number of Bax positive cells compared to the IR group (p = 0.025) (Fig. 3c). However, this decrease was significantly higher than the SHAM group (p = 0.04). The number of Bax expressing cells significantly decreased in the safranal treated group compared to the IR group (p = 0.004) (Fig. 3d). This decrease
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Fig. 1. Representative images of liver samples stained with H&E (X200, scalebar = 50 mm). Destructive hepatocytes were defined by the presence of cytoplasmic shrinkage, eosinophilic cytoplasm and dark nuclei. Bold arrows indicate destructive hepatocytes undergoing apoptosis–necrosis and hollow arrows indicate sinusoidal dilatation. (a) SHAM group showing normal hepatocyte structure (b) IR group showing increase in destruction of hepatocyte morphology, sinusoidal dilatation and increase in Kupffer cells (c) crocin treated IR group showing less destruction in hepatocytes (d) safranal treated IR group showing less destruction in hepatocytes and decreased sinusoidal dilatation.
Table 2 Histopathological damage scores of the experimental groups. Histopathological damage parameters
Group 1 (SHAM)
Group 2 (IR)
-Hepatocyte morphology -Sinusoidal dilatation and Congestion -Infiltration
0.37 0.51* 0.25 0.46* 0.37 0.51*
2.25 0.46 2.87 0.83 2.50 0.53
z,*,1 z,*,1 *,1
Group 3 (IR+Crocin)
Group 4 (IR+Safranal)
P value
1.37 0.51z 1.87 0.64z,p 1.87 0.64
1.12 0.831 0.87 0.641, p 1.00 0.751
<0.001 <0.001 <0.001
-Group 1: control, group 2: I/R, group 3: I/R + crocin, group 4: I/R + safranal. -Data are mean standard deviation. Kruskal Wallis test was used for comparison among groups, p < 0,001. On each line, the difference between the means with the same markers are significant (p < 0.05). Mann Whitney U test was used for pairwise comparasions. *,z,1 p < 0.05
Fig. 2. Representative images of Caspase-3 immunostaining of liver samples (X200, scale bar = 50 mm). Arrows refer to Caspase-3 positive staining of hepatocytes (a) in the SHAM group, a few hepatocytes show Caspase-3 positive staining around the central vein (b) in the IR group an increased number of Caspase-3 positive cells are observed (c) Caspase-3 positivity is visible in the crocin treated group after ischemia but the number of Caspase-3 positive cells is lower than in the IR group (d) the safranal treated group shows lower Caspase-3 positivity than the IR group.
Table 3 Values of expression of Caspase-3, Bax and Bcl-2 with H score in liver tissue.
Caspase-3 Expression Bax Expression Bcl-2 Expression
Group 1 (SHAM)
Group 2 (IR)
Group 3 (IR + Crocin)
Group 4 (IR + Safranal)
P value
87.66 15.01* 59.66 17.14*,# 88.00 22.39
242.00 57.77*,#,& 171.16 49.99*,m,& 71.50 17.55
123.66 21.29#,a 114.16 21.88#,m,a 96.16 10.26
94.16 8.65&,a 79.33 12.51&,a 105.00 41.51
<0.001 <0.001 0.17
Data are mean standard deviation (Kruskal Wallis test was used). On each line, the difference between the means with the same markers are significant (p < 0.05). Mann Whitney U test was used for pairwise comparisons *,#,&,a,m p < 0.05.
Fig. 3. Representative images of Bax immunostaining of liver samples (X200, scale bar = 50 mm). Arrows refer to Bax positive staining (a) in the SHAM group, some hepatocytes show Bax positive staining (b) Bax expression prominently increased in the IR group (c) Bax expression is lower in the crocin treated group than in the IR group (d) Bax expression is lower in the safranal treated group than in the IR group.
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was not significantly higher than the SHAM group (p = 0.06). When crocin and safranal treated groups were compared to each other, there was a significant difference in Bax expression (p = 0.025). 3.2.2.3. The effects of crocin and safranal on Bcl-2 expression in liver IR injury. Bcl-2 immunohistochemistry images are shown in Fig. 4 and the results are shown in Table 3. The SHAM group demonstrated positive Bcl-2 expression (Fig. 4a). There was a non-significant decrease in Bcl-2 expression in the IR group (p > 0.05) (Fig. 4b). Crocin (Fig. 4c) and safranal (Fig. 4d) administrations non-significantly increased Bcl-2 expression. There was no significant difference in Bcl-2 expression among all groups (p > 0.05). Finally, we observed that IR injury significantly increased proapoptotic Bax and Caspase-3 expression in liver tissue, which was reduced by treatment with crocin and safranal. Antiapoptotic Bcl-2 expression was decreased in the IR group while crocin and safranal treatments mildly increased Bcl-2 expression. 4. Discussion In the present study, we have demonstrated that crocin and safranal can decrease hepatic IR damage induced by IRAC during abdominal surgery intervention. Moreover, it can also be said that crocin and safranal: 1) improve liver function, such as AST and ALT levels. 2) attenuate apoptosis of liver tissue via these agents’ antiapoptotic effect on Caspase-3, Bax and Bcl-2 expression, which are altered during the liver IR process and 3) decrease negative morphological changes seen in the liver, such as destruction of hepatocyte structure with cytoplasmic darkening, eosinophilia and shrinkage, sinusoidal dilatation and congestion, increase in the number of inflammatory cells in sinusoidal areas and Kupffer cells. These improvements may be attributed to the potential antiapoptotic and protective effects of crocin and safranal. Moreover, we showed that the effects of safranal seemed greater than those of crocin. Reperfusion of tissues and organs that occurs following prolonged ischemia gives rise to IR injury via various mechanisms [17]. IR injury occurs through two different pathophysiological mechanisms. The first is that ischemia leads to the production of reactive oxygen species and anaerobic glycolysis. The second mechanism occurs in response to reperfusion. It includes proinflammatory cytokine activation, polymorphonuclear cell activation, cell death through apoptosis and necrosis, microcirculation damage and oxidative stress. IR injury in the liver is a common pathology, since trauma, liver resection, liver transplantation, hemorrhage and shock can all cause IR injury and in some cases can be fatal [17,18]. In addition, the effect of IR injury on distant organs and tissues is also another major problem. Therefore, efforts to prevent these damages have been the focus of attention of various research studies.
Apoptosis is programmed cell death and consists of gene activation, protease activation, and nuclear DNA degradation [19]. Caspases are endoproteases that play an important role in regulating inflammation and cell death. While Caspase -3, -6, -7, -8 and -9 are responsible for apoptosis in mammalian cells, Caspase -1, -4, 5, and -12 in humans and Caspase -1, -11, and -12 in mice are involved in inflammation [20]. Caspase-3 is a primary mediator of apoptotic cell death [19]. The Bcl-2 protein family is another important group of proteins involved in cell death. This family includes Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1, which support survival of cells. In other words, they are anti-apoptotic while other members such as Bax, Bak and Bok are pro-apoptotic [21]. The Bcl2 protein family mainly regulates the release of mitochondrial inter-membrane space proteins, which activate caspases [22]. The anti-apoptotic Bcl-2 proteins are responsible for maintaining the integrity of the outer mitochondrial membrane. However, the proapoptotic Bcl-2 proteins increase mitochondrial outer membrane permeability and stimulate apoptosis [21,22]. In the present study, we evaluated IRAC induced liver IR injury, and the hepatoprotective and anti-apoptotic effects of safranal and crocin via Caspase-3, Bax and Bcl-2 immunohistochemical staining. We observed that IRAC significantly increased the pro-apoptotic proteins Bax and Caspase-3, which are involved in the irreversible step of apoptosis in liver tissue. These markers were reduced by treatment with crocin and safranal. In addition, anti-apoptotic Bcl2 expression decreased in the IR group. Crocin and safranal mildly increased Bcl-2 expression. Lari et al. suggested that the hepatotoxic effect of diazinon decreases with crocin treatment [23]. They showed that diazinon stimulates apoptosis by means of activation of Caspases-9 and -3 and by increasing the Bax/Bcl-2 ratio. Crocin weakened the activation of caspases and decreased the Bax/Bcl-2 ratio. Our findings also support the results of this study. Saffron is a spice that is obtained by drying the plant Crocus sativus, widely used to give color and smell to food in some cultures [24]. Saffron and its components, which include safranal and crocin, have strong antioxidant properties [25–27]. Recent studies reported that saffron and its components have a protective effect against some toxic substances in various tissues such as liver [28,29]. Sun et al. investigated the effect of crocin on hepatocyte injury caused by cisplatin [30]. They showed that crocin reduces hepatic focal necrosis caused by cisplatin. In addition, crocin diminished the levels of phospho-p38 mitogen-activated protein kinase, tumor protein 53 (p53) and cleaved Caspase-3. Bandegi et al. [6] suggested that saffron and its active product crocin have a protective role against damage in the brain, liver and kidney caused by chronic stress. Consistent with the literature, we also showed by biochemical, histopathological and immunohistochemical methods that IR injury occurring in the liver can be reduced by crocin and safranal treatment. We have suggested that crocin and safranal may prevent apoptosis of liver tissue caused by IRAC and their
Fig. 4. Representative images of Bcl-2 immunostaining of liver samples (X200, scale bar = 50 mm). Arrows refer to Bcl-2 positive staining (a) Bcl-2 positivity is observed in some hepatocytes in the SHAM group (b) Bcl-2 expression is mildly decreased in the IR group (c) Bcl-2 expression is mildly higher in the crocin treated group than in the IR group (d) Bcl-2 expression is slightly prominent in the safranal treated group compared to the others.
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hepatoprotective effects may be due to an anti-apoptotic effect on Caspase-3, Bax and Bcl-2 expression. We also observed that IR caused significant histopathological changes in the liver. These changes included destruction of hepatocyte structure with cytoplasmic darkening, eosinophilia and shrinkage, sinusoidal dilatation and congestion, increase in the number of inflammatory cells in sinusoidal areas and Kupffer cells. However, less histopathological changes were observed in the safranal and crocin treated groups. In light of these findings, IR injury resulted in deterioration of hepatocyte morphology and sinusoidal structure but crocin and safranal treatments decreased the damage parameters and partially protected the liver from IR induced liver injury. We showed that ALT and AST increased in the IR group compared with SHAM group. In previous studies with the IRAC model conducted by Cure et al. [11,31], both AST and ALT levels were increased in the IR group when compared with a sham group. This finding was in accordance with the literature. In hepatic IR models with various durations, increased levels of AST, ALT can be considerably lowered after administration of various therapeutic agents [7,32,33]. Similarly, we have observed that AST and ALT levels significantly decreased via crocin and safranal administration. 5. Limitations of the current study There are some limitations of our study. The first limitation of the present study was that we examined apoptotic mechanisms but we did not measure inflammatory cytokines or oxidative stress markers. Therefore, we do not know the effect of safranal and crocin on these factors. Secondly, we used crocin and safranal separately, but we did not investigate their additive impact. There is a need for further research investigating the additive effects of safranal in this rat model. Other limitations include the brief monitoring period, as well as the fact that there was no hemodynamic monitoring of the animals during our experiments. In addition, we studied a single dose of crocin, safranal and IR duration, thus we can only speculate about the effect of different dosing regimens and IR durations. 6. Conclusion We believe that our results may contribute to understanding the mechanism underlying liver injury induced by IRAC and thereby could represent an important contribution to current abdominal surgery practice. The current study would be the first to evaluate the effects of crocin and safranal pre-treatment on liver damage induced by IRAC. Reperfusion following IRAC may give rise to IR injury in the liver. Data established in the current study may suggest that one of the damage mechanisms is increased apoptosis and that crocin and safranal, by reducing apoptosis, protect the liver against IR injury. This effect seems to be somewhat greater with safranal compared to crocin. With these agents, postoperative complications related to IRAC could be minimized and post-operative hospitalization and treatment costs could be decreased, while enhancing the healing process. However, before clinical application of crocin and safranal, more comprehensive studies using different dose regimens and time intervals are required to fully achieve this goal. Conflict of interest The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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Available online at
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Investigation of the effect of safranal and crocin pre-treatment on hepatic injury induced by infrarenal aortic occlusion Ziya Taner Ozkececia,* , Yucel Gonulb , Yasemin Yukselc , Afra Karavelioglud, Kamil Tunaye, Yusuf Gulsarib , Onder Cartillib , Omer Hazmanf , Ahmet Bala a
Department of General Surgery, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey Department of Anatomy, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey Department of Histology and Embryology, Zekai Tahir Burak Women’s Health and Research Hospital, Ankara, 06260, Turkey d Department of Pediatric Surgery, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey e Department of Emergency Medicine, Faculty of Medicine, Afyon Kocatepe University, Afyon, 03200, Turkey f Department of Chemistry, Biochemistry Division, Faculty of Science and Arts, Afyon Kocatepe University, Afyonkarahisar 03200, Turkey b c
A R T I C L E I N F O
Article history: Received 6 April 2016 Received in revised form 8 May 2016 Accepted 14 June 2016 Keywords: Ischemia-reperfusion Liver injury Apoptosis Bax Blc-2 Caspase-3
A B S T R A C T
Ischemia-reperfusion (IR) injury of the liver is an unresolved problem that occurs during certain surgical approaches, including hepatic, cardiac and aortic operations. In this study we aimed to investigate whether crocin and safranal had protective effects on liver IR injury induced in an infrarenal aortic clamping (IRAC) model. Male Wistar-Albino rats (n = 32) were divided into four groups with 8 animals each as follows: Sham, IR, IR + crocin, and IR + safranal. The infrarenal aorta (IRA) was clamped for 60 min for the ischemic period and allowed to reperfuse for 120 min. Blood and tissue samples were collected for biochemical, histological and immunohistological analysis. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were found to be significantly higher in the IR group than the sham group (respectively; p = 0.015, p < 0.001). There were significant differences between the IR group and the IR + crocin group or the IR + safranal group in AST levels (respectively; p = 0.02, p < 0.001). ALT showed a significant decrease in the IR + crocin group compared to the IR group (p < 0.05). We also observed histopathological changes among the groups. Bax and Caspase-3 expression in the IR group was remarkably higher than in the other groups. Caspase-3 and Bax expression in the IR + crocin and the IR + safranal groups were significantly lower than in the IR group. Nevertheless, there were no significant differences in BCL2 expression among the groups. IRAC is a cause of IR injury in the liver. This study showed that crocin and safranal have protective effects on IR induced liver injury. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Remote organ injury caused by infrarenal aortic clamping (IRAC), which can be encountered during surgical interventions related to the abdominal aorta, is a complex process affecting the lower extremities as well as remote organs such as the heart, liver, kidney, and lungs [1–3]. Reperfusion of acutely ischemic abdominal organs causes local and distant organ damage and may contribute to high morbidity and mortality [4]. Several factors have been suggested to play a role in the pathophysiology of ischemia-reperfusion (IR) injury, such as free
* Corresponding author at: Department of General Surgery, Faculty of Medicine, Afyon Kocatepe University, Ali Cetinkaya Campus, Izmir Road 8. Km, 03200 Afyonkarahisar, Turkey. E-mail address: [email protected] (Z.T. Ozkececi). http://dx.doi.org/10.1016/j.biopha.2016.06.027 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
oxygen radicals, polymorphonuclear leukocytes, the complement system and endothelial cells. These are cellular and humoral mechanisms that have complex relationships with each other. While the amount of oxygen, the decrease in energy production and synthesis of the antioxidant enzymes, toxic substances, ions such as Ca and Na, pro-inflammatory cytokines and leukocyte adhesion molecules increase during ischemia. This makes cells relatively prone to damage during reperfusion. When blood flow resumes to ischemic tissue, cells are exposed to large amounts of oxygen, which is a source of radical oxygen species in the previously damaged tissue. However, internal antioxidant mechanisms are insufficient during IR injury. This is why many drugs have been used to reduce the effects of hepatic IR injury [5–8]. Nevertheless, studies on this subject are as yet insufficient to reach a definitive result.
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Bcl-2 is protective against both apoptotic cell death and multiple cellular events. It was reported that apoptosis can be prevented by a decreased ratio of Bax/Bcl-2 [9]. Intracellular Bcl-2 to Bax ratio is of paramount importance and may give a clue to whether a cell would progress to apoptosis. If Bax is greater, then the cell begins to progress towards apoptosis. However, apoptosis will be inhibited when Bcl-2 is greater [2]. Various substrates like Bax, Bak and Caspase trigger the apoptotic process; however Bcl-2, Bcl-xL or Mcl-1 family proteins generally act as antiapoptotic mediators, which may cause a delay or inhibition in apoptotic enzyme release. Moreover, overexpression of antiapoptotic proteins provide a survival benefit in cells treated with chemotherapeutic agents [10]. As shown in rat models, liver [8,11] and kidney [2] remote organ injury mediated IRAC can be attenuated using different protective agents. Our primary goal is to minimize damage after IRAC. Therefore, we investigated crocin and safranal as strong protective agents of liver IR induced by IRAC. Crocus sativus L., cultivated in Southwest Asia, Spain, France, Italy, Turkey and Iran, is a flowering plant used in traditional medicine. Saffron, derived from the flower of Crocus sativus L., contains at least 150 chemicals. Crocin and safranal, which are the main components of saffron, are known to have anti-inflammatory, anticonvulsant, antitussive, antioxidant, anxiolytic and antidepressant effects [12]. In our previous studies, we showed that crocin has anti-inflammatory, antiapoptotic, antioxidant and protective effects on acute kidney injury following IRAC [2] and brain injury following four vessels oclusion [13]. As far as we know, the effects of crocin and safranal on liver IR injury mediated by IRAC have not been studied thus far. In this study we therefore aimed to investigate whether crocin and safranal had protective and anti-apoptotic effects on IRAC induced liver IR injury using hematoxylin and eosin staining and Caspase-3, Bax, and Bcl-2 immunohistochemical staining. 2. Material and methods Study protocols and experimental procedures were approved by the Ethics and Animal Welfare Committee of Afyon Kocatepe University and the experiments were performed according to the Principles and Guidelines for Experimental Animals issued by The National Health and Medical Research Council and Guide for the Care and Use of Laboratory Animals (NIH issue no. 85–23, 1985 revised) prepared by The National Institutes of Health. 2.1. Animals Thirty two male Wistar-Albino rats, weighing 250–300 g, were included in the study. During the experimental procedure, the animals were kept under standard laboratory conditions including 24–26 C room temperature, 50–60% humidity, 12 h of light and night cycle and were allowed access to food and water ad libitum. The animals were fasted 12 h before the experiment. 2.2. Study groups The 32 rats were randomly divided into four groups comprising 8 rats in each group as follows: Group I (Sham, n = 8): Except for aorta clamping, procedures for laparotomy and dissection of the infrarenal aorta (IRA) were performed the same as in the other groups for equivalent operational time and stress. After closing the abdominal incision, the animals were kept under observation for 3 h to simulate the IR interval in the other groups. No drugs were administered to the rats.
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Group II (IR, n = 8): After laparotomy, the IRA was dissected carefully and clamped to create ischemia for 60 min and the clamp was removed to apply reperfusion for two hours. Group III (IR + Crocin, n = 8): 100 mg/kg crocin (Sigma-Aldrich Chemical Co., St Louis, USA) was injected intraperitoneally 30 min before laparotomy [14]. The IRA was clamped for 60 min to create IR, achieved by opening the clamp for two hours. Group IV (IR + Safranal, n = 8): 100 mg/kg safranal (SigmaAldrich Chemical Co., St Louis, USA) [15] was administered intraperitoneally 30 min before the surgical procedure. After applying 60 min of ischemia by blocking IRA blood flow, reperfusion was supplied by opening the aortic clamp for two hours. 2.3. Surgical intervention Animals were fasted for 12 h before the surgical procedure. After intramuscular injection of 5 mg/kg Xylazine (Rompun, Bayer, Turkey) for premedication, 40 mg/kg ketamine (Ketalar, ParkeDavis, Eczacibasi, Turkey) was used for anesthesia. Rats were fixed to the operating table from four extremities to prevent movement. A median laparotomy was performed after shaving, washing with povidone-iodine solution and covering the abdomen under sterile conditions. The intestines were covered with warm, wet gas compresses to avoid heat and fluid loss after deviating to the right. The abdominal aorta was explored with retroperitoneal dissection and turned 1 cm below the renal vein and 1 cm above the iliac bifurcation. After administering 150 U/kg heparin intraperitoneally and waiting 5 min for anticoagulation, the IRA was obstructed for 60 min using atraumatic microvascular clamps (Vascu-Statt II, midi straight 1001-532; Scanlan Int., St. Paul, MN, USA). Disappearance of distal aortic pulsation verified occlusion of the IRA and determination of convincing pulsation in the femoral region with Doppler ultrasonography (BT-200 Vascular Doppler HI-dop, 7th Fl., A Bldg., Woolim Lions Valley 5-cha, 302, Korea) confirmed reperfusion after removing the clamps. Before removing the clamps for reperfusion, 1 mg/kg intraperitoneal protamine sulphate was given to the rats to neutralize the effect of heparin. Following hemostasis, the compresses covering the intestines were removed and the intestines were placed into the abdomen. Fluid resuscitation was performed intraperitoneally with 10 cc of saline and the abdominal incision was closed with a sterile dressing. Rats were sacrificed to collect tissue/blood samples after reperfusion for 120 min. Crocin and safranal were prepared just before application to the rats. Crocin was dissolved in physiological saline at a dose of 100 mg/kg. Safranal was emulsified in physiological saline at a dose of 100 mg/kg before being administered to the experimental animals intraperitoneally. While 100 mg/ kg crocin was injected intraperitoneally in group III, 100 mg/kg safranal was administered intraperitoneally in group IV 30 min before laparotomy. No drugs were administered in groups I and II. 2.4. Collection of blood and tissue samples Blood samples (3 ml) were taken from the vena cava inferior for biochemical analysis and centrifuged. Serum samples obtained in this manner were preserved at 70 C until biochemical analysis. In addition, the right lobe of the liver was removed for histopathological and immunohistochemical assays. 2.5. Biochemical analysis Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed in an autoanalyser (Cobas 6000, Roche, Switzerland) as indicators of liver ischemia.
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2.6. Histopathological evaluation 2.6.1. Tissue preparation and H&E staining Tissue samples taken from the rats were fixed in 10% neutral buffered formaldehyde solution. After formalin fixation, routine tissue processing steps were performed. Tissues samples were embedded in paraffin blocks. Sections of 5 mm thickness were cut from the blocks using a microtome. Slides were then stained with hematoxylin and eosin (H&E) and examined with a light microscope (E600, Nikon, Japan) for histopathological alterations of liver tissue. Hepatocyte morphology (destruction of cell shape and core structure, cytoplasmic shrinkage and darkening), increase of Kupffer cells-sinusoidal infiltration and congestion were evaluated semiquantitatively and scored as follows: negative (0), mild (1), moderate (2), severe (3) and excessive changes (4). Scores were calculated for each group [16]. 2.6.2. Immunohistochemical assay Sections placed on adhesive slides were stained with Caspase-3 (at 1:100 dilution, 1197P1306J, Neomarkers, Fremont, CA), Bax (at 1:100 dilution, PU3470708, Biogenex, USA) and Bcl-2 primary antibodies (NU541-UC, Biogenex) using the UltraVision HRP polymer detection system (Thermo Fisher Scientific Inc./Lab Vision, Fremont, CA, USA) according to the manufacturer’s protocol. For this purpose, slides were initially deparaffinized, rehydrated and placed in distilled water. Slides were then boiled in 10 mM sodium citrate buffer for 25 min in a microwave oven for surface epitope retrieval. The samples were then placed in 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Nonspecific binding was blocked using Ultra V block for 5 min at room temperature. Sections were then treated with Caspase-3, Bax and Bcl-2 primary antibodies and incubated overnight at 4 C. After rinsing with PBS, the sections were treated with biotin-labeled and streptavidin conjugated secondary antibodies, respectively. Finally, AEC peroxidase substrate (Labvision Corp, Fremont, CA) was implemented and the reaction was colored. The sections obtained by this procedure were counterstained with Mayer’s hematoxylin and the slides were covered with coverslips using aqueous adhesive. Finally, slides were examined under the light microscope at 200 magnification and Caspase-3, Bax and Bcl-2 positive stained cells were counted in six randomly chosen areas with Image Analysis Software (NIS Elements Nikon, Japan). H score was used to calculate immunopositive cells. 2.7. Statistical analysis The data obtained were analyzed using SPSS for Windows 15.0 software package (SPSS Inc., Chicago, Illinois, USA). Results of descriptive statistics were expressed as mean standard deviation. Continuous variables were compared among the groups with Kruskal–Wallis tests. Mann–Whitney U test was used to determine differences between the two groups when Kruskal Wallis was significant. P values less than 0.05 were considered statistically significant. 3. Results 3.1. The effects of crocin and safranal on liver function in liver IR injury Biochemical results are shown in Table 1. Serum levels of ALT and AST were found to be significantly higher in the IR group compared to the SHAM group (respectively; p = 0.004, p < 0.001). ALT and AST levels were significantly decreased in the IR + safranal group compared with the IR group (respectively; p = 0.004, p < 0.001). However, ALT levels did not significantly decrease in the crocin + IR group compared with the IR group while AST levels
Table 1 Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Group2 (IR) n=8
Group3 (IR + Crosin) n=8
Group4 p (IR + Safranal) n=8
Variables
Group1 (SHAM) n=8
AST U\L ALT U\L
116 25.6* 356.4 48.3*,z,1 253.7 25.81 183.5 15.2z 22.9 3.4* 50.1 13.2*,z 30.5 18.21 23 8z,1
<0.001 0.015
Data are mean standard deviation (Kruskal Wallis test was used). On each line, the difference between the means with the same markers are significant (p < 0.05) *,z,1 p < 0.05, Mann Whitney U test was used to compare two groups.
significantly reduced in the crocin + IR group compared to the IR group (respectively; p = 0.15, p = 0.03). In addition, there were significant differences between the IR + crocin group and the IR + safranal group in ALT levels. 3.2. The effects of crocin and safranal on liver IR damage 3.2.1. H&E staining results Images from the experimental groups stained with H&E are shown in Fig. 1 and scores are shown in Table 2. In the evaluation of liver slides, the SHAM group showed normal morphology in hepatocyte and sinusoidal structure (Fig. 1a). The IR group showed destruction of hepatocyte structure with cytoplasmic darkening and shrinkage. In addition, in the IR group, sinusoidal dilatation and congestion were prominent compared to SHAM. It was observed that there was an increase in the number of inflammatory cells in the sinusoidal area and Kupffer cells in the IR injury group (Fig. 1b). The IR + crocin treated group showed less deterioration in hepatocyte structure than the IR group. The sinusoidal structure was partially protected with crocin treatment, and inflammatory cell migration and Kupffer cells were less than in the IR group (Fig. 1c). The IR + safranal treated group demonstrated better histopathological scores than the IR and IR + crocin treated group (Fig. 1d). Hepatocyte structure was more protected from ischemic injury and also sinusoidal dilatation and inflammatory cell migration was reduced by safranal treatment compared to the IR group. 3.2.2. Immunohistochemistry results 3.2.2.1. The effects of crocin and safranal on Caspase-3 expression in liver IR injury. Caspase-3 immunohistochemistry images are shown in Fig. 2 and results are shown in Table 3. In the SHAM group, a small amount of Caspase-3 staining of hepatocytes was observed (Fig. 2a). IR injury remarkably increased the number of Caspase-3 positive cells. This increase was statistically significant when compared to the SHAM group (p = 0.004) (Fig. 2b). There was a statistically significant reduction in Caspase-3 positive stained cells in the crocin (Fig. 2c) and safranal (Fig. 2d) treated groups when compared to the IR group. When crocin and safranal treated groups were compared to each other, safranal reduced Caspase-3 expression to a greater degree than crocin. 3.2.2.2. The effects of crocin and safranal on bax expression in liver IR injury. Bax immunohistochemistry images are shown in Fig. 3 and the results are shown in Table 3. In sections of the SHAM group, some hepatocytes showed positive staining with Bax antibody (Fig. 3a). Ischemia-reperfusion injury demonstrated a significant increase in the number of cells stained with Bax compared to SHAM (p = 0.004) (Fig. 3b). Crocin administration significantly decreased the number of Bax positive cells compared to the IR group (p = 0.025) (Fig. 3c). However, this decrease was significantly higher than the SHAM group (p = 0.04). The number of Bax expressing cells significantly decreased in the safranal treated group compared to the IR group (p = 0.004) (Fig. 3d). This decrease
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Fig. 1. Representative images of liver samples stained with H&E (X200, scalebar = 50 mm). Destructive hepatocytes were defined by the presence of cytoplasmic shrinkage, eosinophilic cytoplasm and dark nuclei. Bold arrows indicate destructive hepatocytes undergoing apoptosis–necrosis and hollow arrows indicate sinusoidal dilatation. (a) SHAM group showing normal hepatocyte structure (b) IR group showing increase in destruction of hepatocyte morphology, sinusoidal dilatation and increase in Kupffer cells (c) crocin treated IR group showing less destruction in hepatocytes (d) safranal treated IR group showing less destruction in hepatocytes and decreased sinusoidal dilatation.
Table 2 Histopathological damage scores of the experimental groups. Histopathological damage parameters
Group 1 (SHAM)
Group 2 (IR)
-Hepatocyte morphology -Sinusoidal dilatation and Congestion -Infiltration
0.37 0.51* 0.25 0.46* 0.37 0.51*
2.25 0.46 2.87 0.83 2.50 0.53
z,*,1 z,*,1 *,1
Group 3 (IR+Crocin)
Group 4 (IR+Safranal)
P value
1.37 0.51z 1.87 0.64z,p 1.87 0.64
1.12 0.831 0.87 0.641, p 1.00 0.751
<0.001 <0.001 <0.001
-Group 1: control, group 2: I/R, group 3: I/R + crocin, group 4: I/R + safranal. -Data are mean standard deviation. Kruskal Wallis test was used for comparison among groups, p < 0,001. On each line, the difference between the means with the same markers are significant (p < 0.05). Mann Whitney U test was used for pairwise comparasions. *,z,1 p < 0.05
Fig. 2. Representative images of Caspase-3 immunostaining of liver samples (X200, scale bar = 50 mm). Arrows refer to Caspase-3 positive staining of hepatocytes (a) in the SHAM group, a few hepatocytes show Caspase-3 positive staining around the central vein (b) in the IR group an increased number of Caspase-3 positive cells are observed (c) Caspase-3 positivity is visible in the crocin treated group after ischemia but the number of Caspase-3 positive cells is lower than in the IR group (d) the safranal treated group shows lower Caspase-3 positivity than the IR group.
Table 3 Values of expression of Caspase-3, Bax and Bcl-2 with H score in liver tissue.
Caspase-3 Expression Bax Expression Bcl-2 Expression
Group 1 (SHAM)
Group 2 (IR)
Group 3 (IR + Crocin)
Group 4 (IR + Safranal)
P value
87.66 15.01* 59.66 17.14*,# 88.00 22.39
242.00 57.77*,#,& 171.16 49.99*,m,& 71.50 17.55
123.66 21.29#,a 114.16 21.88#,m,a 96.16 10.26
94.16 8.65&,a 79.33 12.51&,a 105.00 41.51
<0.001 <0.001 0.17
Data are mean standard deviation (Kruskal Wallis test was used). On each line, the difference between the means with the same markers are significant (p < 0.05). Mann Whitney U test was used for pairwise comparisons *,#,&,a,m p < 0.05.
Fig. 3. Representative images of Bax immunostaining of liver samples (X200, scale bar = 50 mm). Arrows refer to Bax positive staining (a) in the SHAM group, some hepatocytes show Bax positive staining (b) Bax expression prominently increased in the IR group (c) Bax expression is lower in the crocin treated group than in the IR group (d) Bax expression is lower in the safranal treated group than in the IR group.
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was not significantly higher than the SHAM group (p = 0.06). When crocin and safranal treated groups were compared to each other, there was a significant difference in Bax expression (p = 0.025). 3.2.2.3. The effects of crocin and safranal on Bcl-2 expression in liver IR injury. Bcl-2 immunohistochemistry images are shown in Fig. 4 and the results are shown in Table 3. The SHAM group demonstrated positive Bcl-2 expression (Fig. 4a). There was a non-significant decrease in Bcl-2 expression in the IR group (p > 0.05) (Fig. 4b). Crocin (Fig. 4c) and safranal (Fig. 4d) administrations non-significantly increased Bcl-2 expression. There was no significant difference in Bcl-2 expression among all groups (p > 0.05). Finally, we observed that IR injury significantly increased proapoptotic Bax and Caspase-3 expression in liver tissue, which was reduced by treatment with crocin and safranal. Antiapoptotic Bcl-2 expression was decreased in the IR group while crocin and safranal treatments mildly increased Bcl-2 expression. 4. Discussion In the present study, we have demonstrated that crocin and safranal can decrease hepatic IR damage induced by IRAC during abdominal surgery intervention. Moreover, it can also be said that crocin and safranal: 1) improve liver function, such as AST and ALT levels. 2) attenuate apoptosis of liver tissue via these agents’ antiapoptotic effect on Caspase-3, Bax and Bcl-2 expression, which are altered during the liver IR process and 3) decrease negative morphological changes seen in the liver, such as destruction of hepatocyte structure with cytoplasmic darkening, eosinophilia and shrinkage, sinusoidal dilatation and congestion, increase in the number of inflammatory cells in sinusoidal areas and Kupffer cells. These improvements may be attributed to the potential antiapoptotic and protective effects of crocin and safranal. Moreover, we showed that the effects of safranal seemed greater than those of crocin. Reperfusion of tissues and organs that occurs following prolonged ischemia gives rise to IR injury via various mechanisms [17]. IR injury occurs through two different pathophysiological mechanisms. The first is that ischemia leads to the production of reactive oxygen species and anaerobic glycolysis. The second mechanism occurs in response to reperfusion. It includes proinflammatory cytokine activation, polymorphonuclear cell activation, cell death through apoptosis and necrosis, microcirculation damage and oxidative stress. IR injury in the liver is a common pathology, since trauma, liver resection, liver transplantation, hemorrhage and shock can all cause IR injury and in some cases can be fatal [17,18]. In addition, the effect of IR injury on distant organs and tissues is also another major problem. Therefore, efforts to prevent these damages have been the focus of attention of various research studies.
Apoptosis is programmed cell death and consists of gene activation, protease activation, and nuclear DNA degradation [19]. Caspases are endoproteases that play an important role in regulating inflammation and cell death. While Caspase -3, -6, -7, -8 and -9 are responsible for apoptosis in mammalian cells, Caspase -1, -4, 5, and -12 in humans and Caspase -1, -11, and -12 in mice are involved in inflammation [20]. Caspase-3 is a primary mediator of apoptotic cell death [19]. The Bcl-2 protein family is another important group of proteins involved in cell death. This family includes Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1, which support survival of cells. In other words, they are anti-apoptotic while other members such as Bax, Bak and Bok are pro-apoptotic [21]. The Bcl2 protein family mainly regulates the release of mitochondrial inter-membrane space proteins, which activate caspases [22]. The anti-apoptotic Bcl-2 proteins are responsible for maintaining the integrity of the outer mitochondrial membrane. However, the proapoptotic Bcl-2 proteins increase mitochondrial outer membrane permeability and stimulate apoptosis [21,22]. In the present study, we evaluated IRAC induced liver IR injury, and the hepatoprotective and anti-apoptotic effects of safranal and crocin via Caspase-3, Bax and Bcl-2 immunohistochemical staining. We observed that IRAC significantly increased the pro-apoptotic proteins Bax and Caspase-3, which are involved in the irreversible step of apoptosis in liver tissue. These markers were reduced by treatment with crocin and safranal. In addition, anti-apoptotic Bcl2 expression decreased in the IR group. Crocin and safranal mildly increased Bcl-2 expression. Lari et al. suggested that the hepatotoxic effect of diazinon decreases with crocin treatment [23]. They showed that diazinon stimulates apoptosis by means of activation of Caspases-9 and -3 and by increasing the Bax/Bcl-2 ratio. Crocin weakened the activation of caspases and decreased the Bax/Bcl-2 ratio. Our findings also support the results of this study. Saffron is a spice that is obtained by drying the plant Crocus sativus, widely used to give color and smell to food in some cultures [24]. Saffron and its components, which include safranal and crocin, have strong antioxidant properties [25–27]. Recent studies reported that saffron and its components have a protective effect against some toxic substances in various tissues such as liver [28,29]. Sun et al. investigated the effect of crocin on hepatocyte injury caused by cisplatin [30]. They showed that crocin reduces hepatic focal necrosis caused by cisplatin. In addition, crocin diminished the levels of phospho-p38 mitogen-activated protein kinase, tumor protein 53 (p53) and cleaved Caspase-3. Bandegi et al. [6] suggested that saffron and its active product crocin have a protective role against damage in the brain, liver and kidney caused by chronic stress. Consistent with the literature, we also showed by biochemical, histopathological and immunohistochemical methods that IR injury occurring in the liver can be reduced by crocin and safranal treatment. We have suggested that crocin and safranal may prevent apoptosis of liver tissue caused by IRAC and their
Fig. 4. Representative images of Bcl-2 immunostaining of liver samples (X200, scale bar = 50 mm). Arrows refer to Bcl-2 positive staining (a) Bcl-2 positivity is observed in some hepatocytes in the SHAM group (b) Bcl-2 expression is mildly decreased in the IR group (c) Bcl-2 expression is mildly higher in the crocin treated group than in the IR group (d) Bcl-2 expression is slightly prominent in the safranal treated group compared to the others.
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hepatoprotective effects may be due to an anti-apoptotic effect on Caspase-3, Bax and Bcl-2 expression. We also observed that IR caused significant histopathological changes in the liver. These changes included destruction of hepatocyte structure with cytoplasmic darkening, eosinophilia and shrinkage, sinusoidal dilatation and congestion, increase in the number of inflammatory cells in sinusoidal areas and Kupffer cells. However, less histopathological changes were observed in the safranal and crocin treated groups. In light of these findings, IR injury resulted in deterioration of hepatocyte morphology and sinusoidal structure but crocin and safranal treatments decreased the damage parameters and partially protected the liver from IR induced liver injury. We showed that ALT and AST increased in the IR group compared with SHAM group. In previous studies with the IRAC model conducted by Cure et al. [11,31], both AST and ALT levels were increased in the IR group when compared with a sham group. This finding was in accordance with the literature. In hepatic IR models with various durations, increased levels of AST, ALT can be considerably lowered after administration of various therapeutic agents [7,32,33]. Similarly, we have observed that AST and ALT levels significantly decreased via crocin and safranal administration. 5. Limitations of the current study There are some limitations of our study. The first limitation of the present study was that we examined apoptotic mechanisms but we did not measure inflammatory cytokines or oxidative stress markers. Therefore, we do not know the effect of safranal and crocin on these factors. Secondly, we used crocin and safranal separately, but we did not investigate their additive impact. There is a need for further research investigating the additive effects of safranal in this rat model. Other limitations include the brief monitoring period, as well as the fact that there was no hemodynamic monitoring of the animals during our experiments. In addition, we studied a single dose of crocin, safranal and IR duration, thus we can only speculate about the effect of different dosing regimens and IR durations. 6. Conclusion We believe that our results may contribute to understanding the mechanism underlying liver injury induced by IRAC and thereby could represent an important contribution to current abdominal surgery practice. The current study would be the first to evaluate the effects of crocin and safranal pre-treatment on liver damage induced by IRAC. Reperfusion following IRAC may give rise to IR injury in the liver. Data established in the current study may suggest that one of the damage mechanisms is increased apoptosis and that crocin and safranal, by reducing apoptosis, protect the liver against IR injury. This effect seems to be somewhat greater with safranal compared to crocin. With these agents, postoperative complications related to IRAC could be minimized and post-operative hospitalization and treatment costs could be decreased, while enhancing the healing process. However, before clinical application of crocin and safranal, more comprehensive studies using different dose regimens and time intervals are required to fully achieve this goal. Conflict of interest The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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