J Physiol Biochem DOI 10.1007/s13105-014-0335-2
ORIGINAL PAPER
Vitamin D ameliorates hepatic ischemic/reperfusion injury in rats Ansam Aly Seif & Doaa Mohamed Abdelwahed
Received: 6 October 2013 / Accepted: 2 April 2014 # University of Navarra 2014
Abstract Vitamin D, most commonly associated with the growth and remodeling of bone, has been shown to ameliorate ischemia/reperfusion injury (IRI) in some tissues, yet its underlying mechanism remains elusive. This study was designed to examine the protective effect of vitamin D, if any, against hepatic IRI in rats and the underlying mechanism involved. Adult female Wistar rats were randomly divided into control, sham-operated (sham), ischemia/reperfusion (I/R), and ischemicreperfused vitamin D-treated (vit D) groups. Rats in the I/R and vit D groups were subjected to partial (70 %) hepatic ischemia for 45 min, followed by 1 h of reperfusion. Vitamin D was given to rats orally in a dose of 500 IU/kg daily for 2 weeks before being subjected to I/R. Markers of liver damage, oxidative stress, inflammation and apoptosis were evaluated. Hepatic morphology was also examined. Vit D-treated rats had significantly lower serum levels of alanine aminotransferase, aspartate aminotransferase, and γ glutamyl transferase compared to rats in the I/R group. Also, vit D-treated rats showed a significant decrease in malondialdehyde, interleukin-1 beta, interleukin-6, tumor necrosis factor-α, nuclear factor κB, B cell leukemia/lymphoma 2-associated X protein, cytochrome c, and caspase-3 levels, with higher levels of glutathione peroxidase and B cell lymphoma 2 protein levels in liver tissues compared to I/R rats. Histological examination showed less damaged liver tissues with A. A. Seif (*) : D. M. Abdelwahed Faculty of Medicine, Ain Shams University, Cairo, Egypt e-mail: [email protected]
amelioration of apoptotic signs in the vit D group compared to the I/R group. In conclusion, vitamin D supplementation ameliorates hepatic IRI mostly by alleviating the inflammatory-apoptotic response mediated by the oxidative reperfusion injury insult. Keywords Vitamin D . Ischemia–reperfusion . Liver . Oxidative stress . Inflammation . Apoptosis
Introduction Ischemia–reperfusion injury (IRI) is a key contributing factor in liver dysfunction and failure being a major complication of hemorrhagic shock, resection, and transplantation [30]. It is a dynamic process that involves the two interrelated phases of local ischemic insult and inflammation-mediated reperfusion injury [30]. The redox balance, which is pivotal for normal function and integrity of tissues, is dysregulated during I/R, leading to accumulation of reactive oxygen species (ROS). Formation of ROS and oxidant stress is the disease mechanism most commonly invoked in hepatic IRI [9]. Apoptosis is also a central mechanism of IRI to the liver [13] since the coupling of inflammation and apoptosis in specialized cells and tissues is deemed fundamental in the pathogenesis of inflammatory diseases, including liver IRI [16]. Several therapeutic strategies, such as ischemic preconditioning, intermittent or selective pedicle clamping, and pharmacological interventions, have been explored to reduce morbidity caused by hepatic IRI and the
A.A. Seif, D.M. Abdelwahed
surgical stress response [15], yet it remains a serious complication in clinical practice despite several attempts to solve the problem [9]. While vitamin D is most commonly associated with the growth and remodeling of bone, recent studies have identified a much broader spectrum of activity. It is now recognized that vitamin D, and particularly its active form 1, 25 (OH)2D3, (1,25 dihydroxy vitamin D3), is an important hormone playing a crucial role in human homeostasis [24]. It is implicated in a wide range of cell functions such as differentiation, proliferation, and apoptosis [17]. Vitamin D deficiency has been associated to energetic metabolic changes, oxidative stress, fibrosis, and apoptosis [2]. Vitamin D treatment also promotes the regenerative process in injured muscle, thus representing a promising therapy to optimize recovery after injury [23]. Moreover, vitamin D signaling promotes cardioprotection after myocardial infarction through anti-inflammatory, anti-fibrotic, and antiapoptotic mechanisms [3], whereas modulation of the vitamin D pathway might be a supportive hepatocellular carcinoma therapy [10]. Pretreatment with vitamin D3 has also been shown to ameliorate IRI in the lung and muscle [20], apoptotic IRI in the kidney [22], as well as brain damage in ischemic cerebral vascular disease [26]. To our knowledge, the effect of vitamin D on liver IRI has not been studied. Based on the aforementioned data, this study is meant to elucidate any protective role and possible mechanisms for vitamin D on hepatic IRI.
Material and methods The present study was conducted in the Physiology Department, Faculty of Medicine, Ain Shams University and approved by FMASU, REC, Cairo, Egypt, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. This study was performed on 32 female Wistar rats weighing 150–200 g. Rats were maintained in the Physiology Department animal house under standard conditions of boarding. They were fed standard laboratory chow with free access to water. Rats were randomly allocated into four groups: control (n = 8), shamoperated (sham) (n = 8), ischemic-reperfused nontreated (I/R) (n=8), and ischemic-reperfused vitamin D-treated (vit D) (n=8) which received vitamin D (cholecalciferol) orally in a dose of 500 IU/kg daily for 2 weeks before being subjected to I/R. Cholecalciferol
(Vidrop, Medical Union Pharmaceuticals, Abu-Sultan, Ismailia, Egypt) was in the form of oral drops 2,800 IU/ ml where each drop contains 100 IU of vitamin D.
Experimental procedures The present study used segmental (70 %) hepatic ischemia model [7]. On the day of sacrifice, rats were weighed and anesthetized by ketamine (50 mg/kg/ i.p.) (ROTEXMEDICA, TRITTAU, Germany) and xylazine (2 mg/kg/i.m.) (Smithkline Beecham, L.L.C., for NOVARTIS PHARMA, Egypt,). Midline laparotomy was performed. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were occluded with an atraumatic bulldog clamp for 45 min. The clamp was then removed for 60 min reperfusion. This method of partial hepatic ischemia allows for blood flow (portal decompression) through right and caudate lobes and so prevents mesenteric venous congestion. Blood samples were collected from the abdominal aorta. Liver tissue samples from the left and median liver lobes were also harvested for analysis. Blood samples collected after reperfusion were centrifuged to obtain serum. Both tissue and serum samples were stored at −80 °C until the biochemical assay. Parts from the left and median liver lobes were processed for histological examination. In the I/R and vit D groups, rats were subjected to ischemia and reperfusion as described above. In the sham group, rats were given anesthesia and subjected to laparotomy as well as exposure of the portal triad without hepatic ischemia.
Biochemical examination Serum ALT and AST Serum levels of alanine aminotransferase (ALT), also known as serum glutamate pyruvate transaminase (SGPT), and aspartate aminotransferase (AST), also known as serum glutamic oxalacetic transaminase (SGOT), were assessed by a colorimetric, endpoint procedure (TECO DIAGNOSTICS, USA).
Vitamin D ameliorates hepatic injury in rats
Serum γGT
Tissue cytochrome c
γ-Glutamyl transferase (γGT) serum levels were assessed by colorimetric enzymatic assay using Xpress Bio Life Science Products, USA.
Cytochrome c was determined in liver homogenates using rat cytochrome-C, Cyt-C ELISA kit (EIAabTM). Tissue Bax
Tissue MDA Malondialdehyde (MDA) was estimated in liver homogenates using the OxiSelect™ TBARS Assay kit (MDA Quantitation) (CELL BIOLABS, Inc.).
B cell leukemia/lymphoma 2 (Bcl2)-associated X protein (BAX) was determined in liver homogenates using Bax ELISA kit (EIAabTM). Tissue Bcl 2
Tissue GPx Glutathione peroxidase (GPx) was measured in liver homogenates using the rat glutathione peroxidase, GSH-Px ELISA Kit, (EIAabTM).
B cell leukemia/lymphoma 2 (Bcl2) was determined in liver homogenates using the enzyme-linked immunosorbent assay kit for B cell leukemia/lymphoma 2 (Bcl2), organism: Bos taurus; bovine (cattle) (Uscn, Life Science Inc.).
Tissue IL-1β Interleukin-1beta (IL-1β) was measured in liver homogenates using RayBio® Rat IL-1beta ELISA kit (RayBiotech, Inc.). Tissue IL-6 Interleukin-6 (IL-6) was measured in liver homogenates using Rat IL-6 ELISA kit (Immuno-Biological Laboratories, Inc.) (IBL-America).
Histological examination Histological examination of the liver Liver specimens from the left and median liver lobes were fixed in 10 % buffered neutral paraformaldehyde solution, processed, and embedded in paraffin. Thin paraffin sections (5 μm) were stained by H&E [4].
Tissue TNF-α
Statistical analysis
Tumor necrosis factor-alpha (TNF-α) in the liver homogenates was measured by the RayBio® Rat TNFalpha ELISA kit (RayBiotech, Inc.).
Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version 16 for windows was used for the statistical evaluation of the results. All data were expressed as mean±SEM. Statistical significance for data was determined using a one-way analysis of variance (ANOVA) with post hoc test; significance calculated by least significance difference (LSD) test to find inter-group significance. The level of significance was accepted as p
ORIGINAL PAPER
Vitamin D ameliorates hepatic ischemic/reperfusion injury in rats Ansam Aly Seif & Doaa Mohamed Abdelwahed
Received: 6 October 2013 / Accepted: 2 April 2014 # University of Navarra 2014
Abstract Vitamin D, most commonly associated with the growth and remodeling of bone, has been shown to ameliorate ischemia/reperfusion injury (IRI) in some tissues, yet its underlying mechanism remains elusive. This study was designed to examine the protective effect of vitamin D, if any, against hepatic IRI in rats and the underlying mechanism involved. Adult female Wistar rats were randomly divided into control, sham-operated (sham), ischemia/reperfusion (I/R), and ischemicreperfused vitamin D-treated (vit D) groups. Rats in the I/R and vit D groups were subjected to partial (70 %) hepatic ischemia for 45 min, followed by 1 h of reperfusion. Vitamin D was given to rats orally in a dose of 500 IU/kg daily for 2 weeks before being subjected to I/R. Markers of liver damage, oxidative stress, inflammation and apoptosis were evaluated. Hepatic morphology was also examined. Vit D-treated rats had significantly lower serum levels of alanine aminotransferase, aspartate aminotransferase, and γ glutamyl transferase compared to rats in the I/R group. Also, vit D-treated rats showed a significant decrease in malondialdehyde, interleukin-1 beta, interleukin-6, tumor necrosis factor-α, nuclear factor κB, B cell leukemia/lymphoma 2-associated X protein, cytochrome c, and caspase-3 levels, with higher levels of glutathione peroxidase and B cell lymphoma 2 protein levels in liver tissues compared to I/R rats. Histological examination showed less damaged liver tissues with A. A. Seif (*) : D. M. Abdelwahed Faculty of Medicine, Ain Shams University, Cairo, Egypt e-mail: [email protected]
amelioration of apoptotic signs in the vit D group compared to the I/R group. In conclusion, vitamin D supplementation ameliorates hepatic IRI mostly by alleviating the inflammatory-apoptotic response mediated by the oxidative reperfusion injury insult. Keywords Vitamin D . Ischemia–reperfusion . Liver . Oxidative stress . Inflammation . Apoptosis
Introduction Ischemia–reperfusion injury (IRI) is a key contributing factor in liver dysfunction and failure being a major complication of hemorrhagic shock, resection, and transplantation [30]. It is a dynamic process that involves the two interrelated phases of local ischemic insult and inflammation-mediated reperfusion injury [30]. The redox balance, which is pivotal for normal function and integrity of tissues, is dysregulated during I/R, leading to accumulation of reactive oxygen species (ROS). Formation of ROS and oxidant stress is the disease mechanism most commonly invoked in hepatic IRI [9]. Apoptosis is also a central mechanism of IRI to the liver [13] since the coupling of inflammation and apoptosis in specialized cells and tissues is deemed fundamental in the pathogenesis of inflammatory diseases, including liver IRI [16]. Several therapeutic strategies, such as ischemic preconditioning, intermittent or selective pedicle clamping, and pharmacological interventions, have been explored to reduce morbidity caused by hepatic IRI and the
A.A. Seif, D.M. Abdelwahed
surgical stress response [15], yet it remains a serious complication in clinical practice despite several attempts to solve the problem [9]. While vitamin D is most commonly associated with the growth and remodeling of bone, recent studies have identified a much broader spectrum of activity. It is now recognized that vitamin D, and particularly its active form 1, 25 (OH)2D3, (1,25 dihydroxy vitamin D3), is an important hormone playing a crucial role in human homeostasis [24]. It is implicated in a wide range of cell functions such as differentiation, proliferation, and apoptosis [17]. Vitamin D deficiency has been associated to energetic metabolic changes, oxidative stress, fibrosis, and apoptosis [2]. Vitamin D treatment also promotes the regenerative process in injured muscle, thus representing a promising therapy to optimize recovery after injury [23]. Moreover, vitamin D signaling promotes cardioprotection after myocardial infarction through anti-inflammatory, anti-fibrotic, and antiapoptotic mechanisms [3], whereas modulation of the vitamin D pathway might be a supportive hepatocellular carcinoma therapy [10]. Pretreatment with vitamin D3 has also been shown to ameliorate IRI in the lung and muscle [20], apoptotic IRI in the kidney [22], as well as brain damage in ischemic cerebral vascular disease [26]. To our knowledge, the effect of vitamin D on liver IRI has not been studied. Based on the aforementioned data, this study is meant to elucidate any protective role and possible mechanisms for vitamin D on hepatic IRI.
Material and methods The present study was conducted in the Physiology Department, Faculty of Medicine, Ain Shams University and approved by FMASU, REC, Cairo, Egypt, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. This study was performed on 32 female Wistar rats weighing 150–200 g. Rats were maintained in the Physiology Department animal house under standard conditions of boarding. They were fed standard laboratory chow with free access to water. Rats were randomly allocated into four groups: control (n = 8), shamoperated (sham) (n = 8), ischemic-reperfused nontreated (I/R) (n=8), and ischemic-reperfused vitamin D-treated (vit D) (n=8) which received vitamin D (cholecalciferol) orally in a dose of 500 IU/kg daily for 2 weeks before being subjected to I/R. Cholecalciferol
(Vidrop, Medical Union Pharmaceuticals, Abu-Sultan, Ismailia, Egypt) was in the form of oral drops 2,800 IU/ ml where each drop contains 100 IU of vitamin D.
Experimental procedures The present study used segmental (70 %) hepatic ischemia model [7]. On the day of sacrifice, rats were weighed and anesthetized by ketamine (50 mg/kg/ i.p.) (ROTEXMEDICA, TRITTAU, Germany) and xylazine (2 mg/kg/i.m.) (Smithkline Beecham, L.L.C., for NOVARTIS PHARMA, Egypt,). Midline laparotomy was performed. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were occluded with an atraumatic bulldog clamp for 45 min. The clamp was then removed for 60 min reperfusion. This method of partial hepatic ischemia allows for blood flow (portal decompression) through right and caudate lobes and so prevents mesenteric venous congestion. Blood samples were collected from the abdominal aorta. Liver tissue samples from the left and median liver lobes were also harvested for analysis. Blood samples collected after reperfusion were centrifuged to obtain serum. Both tissue and serum samples were stored at −80 °C until the biochemical assay. Parts from the left and median liver lobes were processed for histological examination. In the I/R and vit D groups, rats were subjected to ischemia and reperfusion as described above. In the sham group, rats were given anesthesia and subjected to laparotomy as well as exposure of the portal triad without hepatic ischemia.
Biochemical examination Serum ALT and AST Serum levels of alanine aminotransferase (ALT), also known as serum glutamate pyruvate transaminase (SGPT), and aspartate aminotransferase (AST), also known as serum glutamic oxalacetic transaminase (SGOT), were assessed by a colorimetric, endpoint procedure (TECO DIAGNOSTICS, USA).
Vitamin D ameliorates hepatic injury in rats
Serum γGT
Tissue cytochrome c
γ-Glutamyl transferase (γGT) serum levels were assessed by colorimetric enzymatic assay using Xpress Bio Life Science Products, USA.
Cytochrome c was determined in liver homogenates using rat cytochrome-C, Cyt-C ELISA kit (EIAabTM). Tissue Bax
Tissue MDA Malondialdehyde (MDA) was estimated in liver homogenates using the OxiSelect™ TBARS Assay kit (MDA Quantitation) (CELL BIOLABS, Inc.).
B cell leukemia/lymphoma 2 (Bcl2)-associated X protein (BAX) was determined in liver homogenates using Bax ELISA kit (EIAabTM). Tissue Bcl 2
Tissue GPx Glutathione peroxidase (GPx) was measured in liver homogenates using the rat glutathione peroxidase, GSH-Px ELISA Kit, (EIAabTM).
B cell leukemia/lymphoma 2 (Bcl2) was determined in liver homogenates using the enzyme-linked immunosorbent assay kit for B cell leukemia/lymphoma 2 (Bcl2), organism: Bos taurus; bovine (cattle) (Uscn, Life Science Inc.).
Tissue IL-1β Interleukin-1beta (IL-1β) was measured in liver homogenates using RayBio® Rat IL-1beta ELISA kit (RayBiotech, Inc.). Tissue IL-6 Interleukin-6 (IL-6) was measured in liver homogenates using Rat IL-6 ELISA kit (Immuno-Biological Laboratories, Inc.) (IBL-America).
Histological examination Histological examination of the liver Liver specimens from the left and median liver lobes were fixed in 10 % buffered neutral paraformaldehyde solution, processed, and embedded in paraffin. Thin paraffin sections (5 μm) were stained by H&E [4].
Tissue TNF-α
Statistical analysis
Tumor necrosis factor-alpha (TNF-α) in the liver homogenates was measured by the RayBio® Rat TNFalpha ELISA kit (RayBiotech, Inc.).
Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version 16 for windows was used for the statistical evaluation of the results. All data were expressed as mean±SEM. Statistical significance for data was determined using a one-way analysis of variance (ANOVA) with post hoc test; significance calculated by least significance difference (LSD) test to find inter-group significance. The level of significance was accepted as p
