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Propofol alleviates liver oxidative stress via activating Nrf2 pathway Mian Ge,1 Weifeng Yao,1 Yanling Wang, Dongdong Yuan, Xinjin Chi, Gangjian Luo,2 and Ziqing Hei*,2 Department of Anesthesiology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
article info
abstract
Article history:
Background: Nuclear factor-E2erelated factor 2 (Nrf2)emediated antioxidant response is the
Received 21 October 2014
main protective system of graft-liver against ischemiaereperfusion injury after liver
Received in revised form
transplantation. Propofol is considered to confer protective effects on different organs;
9 January 2015
thus, we explored the possibility that whether propofol could attenuate graft-liver injury in
Accepted 11 March 2015
a rat autologous orthotopic liver transplantation (AOLT) model and mechanisms were
Available online xxx
associated with activation of Nrf2 pathway. Methods: SpragueeDawley rats were randomly divided into four groups: sham-operated
Keywords:
group, saline-treated AOLT group, low-dose propofol intervention group, and high-dose
Liver transplantation
propofol intervention group. Liver injury was determined, and concentration of hydroxyl
Oxidative stress
free radical (OH), superoxide anion (O 2 ), and malondialdehyde in the liver tissue were
Propofol
detected. The expression of Keap1, Nrf2, HO-1, and NQO1 were explored by Western
Nuclear factor E2erelated factor 2
blotting, and also the change of Nrf2 and keap1 was assessed by immunofluorescence. Results: Compared with sham group, pathologic damage of graft-livers was in a timedependent manner, accompanied with the increased level of oxidative stress in the AOLT group, and nuclear Nrf2 expression and its downstream antioxidant enzyme, HO-1 and NQO1, were also increased in this group. However, in propofol pretreatment groups especially in the high-dose group, the pathologic score was significantly decreased, accompanied with a lower level of OH, O 2 , and malondialdehyde than that of the AOLT group. The change of oxidative stress might be related to the Nrf2 pathway, evidenced as the elevation of protein expression level of NQO1, HO-1, and nuclear Nrf2. Conclusions: Protective effects of propofol against liver transplantationeinduced graft-liver injury may be related with Keap1-Nrf2 signal pathway activation. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Liver ischemiaereperfusion injury (IRI) is a major cause of graft dysfunction or nonfunction and results in a high mortality after liver transplantation [1]. Various kinds of mechanisms interact with each other and lead to the complex
pathophysiological processes of hepatic IRI. As reported, reactive oxygen species (ROS)emediated oxidative stress plays a central role in the early stage of IRI [2e6], and antioxidant enzyme activation induced by antioxidants has been shown to attenuate liver IRI [7,8], but still, specific strategies are lacking.
* Corresponding author. Department of Anesthesiology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China. Q2 Tel./fax: þ86 20 85252297. E-mail address: [email protected] (Z. Hei). 1 M.G. and W.Y. contributed equally to this work. 2 Z.H. and G.L. are co-corresponding authors. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.03.016
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Reducing ROS levels or enhancing antioxidant capacity is 131 always considered to be the primary strategy to prevent 132 oxidative damage [9]. Recently, it has been demonstrated that 133 nuclear factor E2erelated factor 2 (Nrf2), a kind of transcrip134 tion factor, could activate different genes encoding various 135 136 antioxidant enzymes and phase II detoxification enzymes, 137 which enabled responses to oxidative stress and ROS scav138 Q3 enging [10,11]. Nrf2 is normally sequestered in the cytoplasm 139 by Kelch-like ECH-associated protein 1 (Keap1), which is 140 considered to be a negative regulator of Nrf2 [12]. Oxidants 141 and other stimuli inactivate Keap1, resulting in the stabiliza142 tion of Nrf2 and its translocation into the nucleus, where Nrf2 143 activates cytoprotective target genes by binding antioxidant 144 response elements [13]. However, whether Nrf2 nuclear 145 translocation could protect against graft-liver injury after liver 146 transplantation has not been reported. 147 148 Nrf2 activation has been confirmed to protect different 149 organs against IRI, including livers [14], brain [15], intestine 150 [16], and hearts [17]; thus, Nrf2 is considered to be a potential 151 strategy for preventing oxidative damage. Propofol, a widely 152 used intravenous anesthetic, has marked antioxidant capac153 ity to attenuate organ IRI effectively [18e22]; however, 154 whether propofol could ameliorate liver IRI after liver trans155 plantation remains unknown, and the interaction between 156 propofol and Nrf2 antioxidant pathway was still unclear. 157 To explore protective effects of propofol on IRI and its relative 158 159 mechanisms, the rat autologous orthotopic liver transplantation 160 (AOLT) model was used to recapitulate liver transplantation 161 in vivo. We focus on the dynamic alterations of Nrf2 expression 162 during and after liver transplantation and confirmed that 163 propofol-ameliorated liver oxidative injury followed AOLT via 164 Keap1 downregulation and Nrf2 upregulation. 165 166 167 2. Materials and methods 168 169 2.1. Animals 170 171 172 Sixty-three healthy male SpragueeDawley rats, aged 8e10 wk 173 and weighing 220e280 g, were provided by the Medical 174 Experimental Animal Center of Guangdong Province. This 175 study was approved by the Institutional Animal Care and Use 176 Committee and Animal Ethics Committee of Sun Yat-sen 177 University in Guangzhou, China, and followed the “Guide for 178 the Care and Use of Laboratory Animals” (National Institutes 179 of Health publication 86-23 revised 1985) guidelines for the 180 treatment of animals. 181 182 183 2.2. AOLT model and experimental groups 184 185 The AOLT model was described in our previous study [23] and 186 consists of primary surgical procedures, such as the dissoci187 ation of vessels and ligaments, the blockade of liver vessels, 188 liver infusion with cold preservation solution, vascular off189 clamping, and hepatic reperfusion. The entire anhepatic 190 phase was controlled within 20 1 min for a high mortality 191 rate of rats when cold ischemia time was longer than 25 min 192 in our preliminary study. The sham group underwent 193 194 abdominal surgery and liver dissection under anesthesia 195 without cold perfusion and reperfusion. The AOLT model rats
were subjected to the typical pathophysiological hepatic IRI processes during liver transplantation.
2.3.
Experimental groups
Initially, 35 male SpragueeDawley rats were randomized into a sham-operated group (sham group, n ¼ 7), a 4 h after AOLT group (4-h group, n ¼ 7), an 8 h after AOLT group (8-h group, n ¼ 7), a 16 h after AOLT group (16-h group, n ¼ 7), and a 24 h after AOLT group (24-h group, n ¼ 7). In the following experiment, 28 rats were divided into a shamoperated group (sham group, n ¼ 7), a saline-treated AOLT group (AOLT group, n ¼ 7), a low-dose propofol intervention AOLT group (L-Pro þ AOLT group, n ¼ 7), and a high-dose propofol intervention AOLT group (H-Pro þ AOLT group, n ¼ 7). The sham and AOLT groups received 3 mL of normal saline via intraperitoneal injections for 3 consecutive days before the experiment, and the LPro þ AOLT and H-Pro þ AOLT groups were pretreated with propofol (Diprivan, 1% propofol, CG411; AstraZeneca, Caponago, Italy) intraperitoneally at a dose or rate of 50 mg/kg (low dose, sedative effect [24]) or 100 mg/kg (high dose, anesthetic effect [25]) daily for 3 d before AOLT.
2.4.
Histologic assessment
Liver tissues were collected immediately after the rats were euthanized. The liver tissues were then fixed with 10% para- Q4 formaldehyde (pH 7.0; Sigma) for 48 h and embedded in paraffin. After hematoxylineeosin staining, liver sections (5 mm) were analyzed microscopically for pathology. Ten randomly selected images were captured in a blinded manner from each slide using a light microscope. The severity of IRI was graded based on Suzuki criteria [26].
2.5.
Biochemical analysis of serum samples
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum were measured with the test kits from SigmaeAldrich (St. Louis, MO) [27].
2.6. Superoxide anion (O 2 ), hydroxyl radical ($OH) and malondialdehyde assays Spectrophotometer methods were used to determine O 2 and $OH levels. The superoxide anion radical scavenging ability was measured by the method of the active oxygen generation of xanthineexanthine oxidase and detected at 550 nm with the spectrophotometer [28]. Determining the $OH suppression of the propofol was conducted according to the Fenton reaction principle. The procedure was performed using a $OH assay kit, according to the manufacturer’s instructions [29]. Malondialdehyde (MDA) content was detected using the thiobarbituric acid method. All procedures were performed according to the manufacturer’s instructions (Nanjing KeyGen Biotech. Co., Ltd, Nanjing, China).
2.7.
Immunofluorescence staining
Frozen sections of liver tissue were washed three times with phosphate-buffered saline (PBS). After fixing with PBS
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containing 5% bovine serum albumin and 0.3% Triton X-100 for 1 h, tissue sections were incubated with anti-Nrf2 (1:150) or anti-Keap1 (1:100) antibodies at 4 C overnight. After washing with PBS, the slides were incubated with a fluorescently labeled secondary antibody (1:100) for 1 h at 25 C. The cover slips were then washed again and mounted using a mounting medium (Applygen Technologies, Beijing. No. 01210) and observed using a fluorescent microscope (DM LB2; Leica Microsystems, Germany).
2.8.
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Protein expression of Nrf2, Keap1, HO-1, and NQO1 in the nuclear and cytosolic fractions was detected using Western blotting. Nuclear and cytoplasmic proteins were separated according to the protocol described in the Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). Protein concentrations were measured using the BCA method (KeyGen BioTech, China). The primary antibodies used were monoclonal rabbit anti-mouse Keap1 (1:1000 dilution; Millipore Company), polyclonal rabbit anti-mouse Nrf2 (1:250 dilution; Santa Cruz Biotechnology), anti-HO-1 (1:250 dilution; Santa Cruz Biotechnology), anti-NQO1 NQO1 (1:250 dilution; Santa Cruz Biotechnology), antieb-actin (1:2000 dilution; Santa Cruz Biotechnology), and anti-H2A (1:1000 dilution; Santa Cruz Biotechnology). The secondary antibody used was goat antirabbit HRP-conjugated IgG (1:2000 dilution; Boster, China). The optical density values of the bands were normalized to those of b-actin or H2A.
2.9.
Q10
Western blotting
Data analyses and statistics
The data were expressed as the mean standard deviation or as the median (range) where appropriate. Comparisons of more than two groups were performed using a one-way analysis of variance followed by LSD multiple-comparison
3
tests. P < 0.05 was selected to indicate statistically significant differences.
3.
Results
3.1. Dynamic AOLT-induced pathologic changes in the liver and biochemical assessment As depicted in Figure 1, the sinusoids and hepatocytes were normal in the sham-operated group (Fig. 1A). Most severe hepatic injury occurred at 4 and 8 h after reperfusion, which manifested as impaired sinusoids and hepatocytes with edema and congestion, obstructed sinusoids, infiltration of inflammatory cells, and necrosis and vacuolation (Fig. 1B and C). After 16 h of reperfusion, pathologic injuries were alleviated significantly (Fig. 1D). Even after 24 h of reperfusion, only slight congestion and sinusoid obstruction were observed, and a small quantity of hepatocytes showed necrosis and vacuolation (Fig. 1E). These results suggested that hepatic IRI that followed AOLT was obvious at the early stage after reperfusion, and then resolved gradually, which correlated with changes in the Suzuki score (Fig. 1F). Serum AST and ALT levels, two indexes of hepatocellular injury, began to increase significantly at 4 h after reperfusion, peaked at 8 h, and then gradually declined, indicating that the most serious liver damage occurred at 8 h after AOLT (Fig. 1G and H).
3.2. Changes in OH, O 2 , and MDA levels in the liver after AOLT OH and O 2 levels reflect ROS levels, and MDA levels were used to determine the extent of oxidative injury in the liver tissue. As shown in Figure 2, OH and O 2 levels increased significantly at 4, 8, and 16 h after AOLT but returned to near
Fig. 1 e AOLT-induced dynamic pathologic changes in the liver visualized using HE staining (3200) and serum biochemical changes. Images AeE are representative of the sham group (Sham) and the experimental groups 4 h after AOLT (4-h group), 8 h after AOLT (8-h group), 16 h after AOLT (16-h group), and 24 h after AOLT (24-h group), respectively; image F shows Suzuki histologic grading of IRI at each time point after AOLT. Images G and H show the serum biochemical changes of AST O and ALT. *P < 0.05 versus sham group; P < 0.05 versus 24-h group. (Color version of figure available online.) 5.2.0 DTD YJSRE13195_proof 1 April 2015 11:32 pm ce
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Fig. 2 e Changes in liver OH, OL 2 , and MDA levels after AOLT. AeC show changes in hydroxyl radical (OH), superoxide anion (OL 2 ), and MDA levels over time. Sham: sham-operated group; 4 h: 4 h after AOLT; 8 h: 8 h after AOLT; 16 h: 16 h after AOLT; 24 h: 24 h after AOLT. *P < 0.05 versus sham group. the baseline at 24 h after AOLT (Fig. 2A and B). MDA levels showed a similar pattern to OH and O 2 levels (Fig. 2C).
3.3. Dynamic alteration in Nrf2 and HO-1 expression after liver IRI Nrf2 is the key protein regulating downstream antioxidant expression, and its nuclear levels were always used to determine Nrf2 nuclear translocation. Figure 3A shows that Nrf2 expression increased from 8 h after reperfusion (P < 0.05 versus sham group) and peaked at 24 h. A similar tendency was observed for HO-1 expression, which is a downstream Nrf2 antioxidant. HO-1 expression increased gradually from 8e24 h after reperfusion (P < 0.05 versus sham group), increased dramatically at 16 h after AOLT compared with that at 4 h (P < 0.05 versus 4-h group), and peaked at 24 h (P < 0.05 versus 4- and 8-h groups; Fig. 3B).
3.4. Propofol-attenuated liver injury and oxidative stress induced by hepatic IRI in AOLT rats We selected the time point of 8 h after reperfusion for the following intervention studies, for pathologic changes were
the most obvious at that time. Rats were pretreated with propofol intraperitoneally at low and high doses, and particularly in the high-dose group, propofol ameliorated hepatic IRI markedly, evidenced as minimal pathologic changes with less sinusoidal congestion, cytoplasmic vacuolization, and hepatocellular necrosis (Fig. 4AeD), as well as reversed the elevated levels of plasma AST and ALT (Fig. 4C and D). In the low-dose propofol group, there was minor congestion, obstruction, and ballooning, and 200 antioxidants that contributed to the majority of important endogenous antioxidant systems [34]. Keap1, a master regulator of intracellular redox homeostasis, has been shown to interact with Nrf2 [12]. As mentioned previously, Nrf2 is normally sequestered in the cytoplasm by Keap1, which prevents Nrf2 nuclear translocation and induces Nrf2 ubiquitin-mediated degradation. Oxidative or other stimuli enhance Keap1 degradation, and Nrf2 activation results in nuclear translocation and activation of genes encoding various antioxidant enzymes that protect cells from oxidative damage. Thus, the molecular mechanism has been confirmed by in vivo and in vitro experiments using genetic approaches including gene knockout and gene silencing [10]. Tanaka et al. [35] demonstrated that transient middle cerebral artery occlusion induced a progressive decrease in Keap1 expression in ischemic brains relative to Nrf2 and its downstream antioxidants. However, thus far, studies of Nrf2 expression after liver transplantation have been lacking. As shown in our results, during24 h after AOLT, a dynamic process of pathologic and oxidative injury occurs in which the injury is worse during the early stages and that gradually resolves or improves; this process occurs parallel to dynamic changes in nuclear levels of Nrf2. We suggest that Nrf2 and its antioxidant effects play a crucial role in preventing hepatic IRI
induced by AOLT and that it promotes hepatic repair. It is critical to identify effective methods that alleviate hepatic IRI. This study shows that targeting Nrf2 is a feasible strategy for early hepatic protection against IRI. Propofol is a commonly used sedative [18,19] and has been shown to promote the antioxidant capacity of various antioxidants and attenuates heart, brain, liver, and renal IRI [20e22]. However, mechanisms of propofol on its antioxidant activity remain unclear. In this study, we focused on the influence of propofol pretreatment on nuclear Nrf2 expression during hepatic IRI induced by AOLT. Immunofluorescence assays and Western blotting results showed that propofol pretreatment promoted Keap1 degradation and nuclear translocation of Nrf2. Consistent with these results, ROS levels decreased and oxidative damage was ameliorated. HO-1 and NQO1 are regarded as antioxidants that act downstream of Nrf2 [34]. In previous studies, it had been demonstrated that NQO1 expression was mediated by Nrf2 [36,37], and more importantly, HO-1 expression upregulation is always considered to be the evidence for the activation of Nrf2 signaling pathway [38]. NQO1 facilitates scavenging of ROS generated by mitochondria [39e42], and HO-1 exerts multiple regulatory functions during oxidative stress [43e47]. Our study revealed that propofol enhanced antioxidant capacity by upregulating HO-1 and NQO1 through Nrf2 activation. Based on these results and those of other studies, propofol enhances the nuclear translocation of Nrf2 and upregulates antioxidant expression, which alleviates hepatic IRI. In summary, our findings document that propofol ameliorated whole liver IRI in AOLT via downregulation of Keap1 and Nrf2 nuclear translocation. However, the optimal
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time for propofol administration requires further investigation. For the future studies, we will use more different methods to get the direct and specific evidence for the conclusion that the role of propofol was Nrf2 dependent, such as Nrf2 inhibitors, Nrf2 KO mice or Nrf2-siRNA on cells.
Acknowledgment This work was supported by grants from the National Natural Science Foundation of China (No. 81372090) and the Planned Science and Technology Project of Guangzhou (No. 2013B021800181 and 2013B051000035). The authors acknowledge the language help from Nature Publishing Group Language Editing. Authors’ contribution: M.G. and W.Y. performed the experiment and wrote the draft, Y.W. analyzed the data, D.Y. and X.C. finished the immunofluorescence staining, and G.L. and Z.H. designed and revised the article.
Disclosure The authors have no conflicts of interests, financial, or otherwise related to the publication of this study or its findings.
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impairment in the activity of NAD(P)H: quinone oxidoreductase 1 and g glutamyl cysteine ligase: studies in mitochondria isolated from rat kidney and in LLC-PK1 cells. Toxicol Lett 2010;199:80. Gang GT, Kim YH, Noh JR, et al. Protective role of NAD(P) H:quinone oxidoreductase 1 (NQO1) in cisplatin-induced nephrotoxicity. Toxicol Lett 2013;221:165. Lamberti MJ, Vittar NB, da Silva Fde C, Ferreira VF, Rivarola VA. Synergistic enhancement of antitumor effect of b-Lapachone by photodynamic induction of quinone oxidoreductase (NQO1). Phytomedicine 2013;20:1007. Meyer ML, Potts-Kant EN, Ghio AJ, et al. NAD(P)H quinone oxidoreductase 1 regulates neutrophil elastase-induced mucous cell metaplasia. Am J Physiol Lung Cell Mol Physiol 2012;303:L181. Yun N, Cho HI, Lee SM. Impaired autophagy contributes to hepatocellular damage during ischemia/reperfusion: heme oxygenase-1 as a possible regulator. Free Radic Biol Med 2013;68C:168. Katori M, Busuttil RW, Kupiec-Weglinski JW. Heme oxygenase-1 system in organ transplantation. Transplantation 2002;74:905. Gozzelino R, Jeney V, Soares MP. Mechanisms of cell protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol 2010;50:323. Zhao Y, Zhang L, Qiao Y, et al. Heme oxygenase-1 prevents cardiac dysfunction in streptozotocin-diabetic mice by reducing inflammation, oxidative stress, apoptosis and enhancing autophagy. PLoS One 2013;8:e75927. Haines DD, Lekli I, Teissier P, Bak I, Tosaki A. Role of haeme oxygenase-1 in resolution of oxidative stressrelated pathologies: focus on cardiovascular, lung, neurological and kidney disorders. Acta Physiol (oxf) 2012; 204:487.
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Propofol alleviates liver oxidative stress via activating Nrf2 pathway Mian Ge,1 Weifeng Yao,1 Yanling Wang, Dongdong Yuan, Xinjin Chi, Gangjian Luo,2 and Ziqing Hei*,2 Department of Anesthesiology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
article info
abstract
Article history:
Background: Nuclear factor-E2erelated factor 2 (Nrf2)emediated antioxidant response is the
Received 21 October 2014
main protective system of graft-liver against ischemiaereperfusion injury after liver
Received in revised form
transplantation. Propofol is considered to confer protective effects on different organs;
9 January 2015
thus, we explored the possibility that whether propofol could attenuate graft-liver injury in
Accepted 11 March 2015
a rat autologous orthotopic liver transplantation (AOLT) model and mechanisms were
Available online xxx
associated with activation of Nrf2 pathway. Methods: SpragueeDawley rats were randomly divided into four groups: sham-operated
Keywords:
group, saline-treated AOLT group, low-dose propofol intervention group, and high-dose
Liver transplantation
propofol intervention group. Liver injury was determined, and concentration of hydroxyl
Oxidative stress
free radical (OH), superoxide anion (O 2 ), and malondialdehyde in the liver tissue were
Propofol
detected. The expression of Keap1, Nrf2, HO-1, and NQO1 were explored by Western
Nuclear factor E2erelated factor 2
blotting, and also the change of Nrf2 and keap1 was assessed by immunofluorescence. Results: Compared with sham group, pathologic damage of graft-livers was in a timedependent manner, accompanied with the increased level of oxidative stress in the AOLT group, and nuclear Nrf2 expression and its downstream antioxidant enzyme, HO-1 and NQO1, were also increased in this group. However, in propofol pretreatment groups especially in the high-dose group, the pathologic score was significantly decreased, accompanied with a lower level of OH, O 2 , and malondialdehyde than that of the AOLT group. The change of oxidative stress might be related to the Nrf2 pathway, evidenced as the elevation of protein expression level of NQO1, HO-1, and nuclear Nrf2. Conclusions: Protective effects of propofol against liver transplantationeinduced graft-liver injury may be related with Keap1-Nrf2 signal pathway activation. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Liver ischemiaereperfusion injury (IRI) is a major cause of graft dysfunction or nonfunction and results in a high mortality after liver transplantation [1]. Various kinds of mechanisms interact with each other and lead to the complex
pathophysiological processes of hepatic IRI. As reported, reactive oxygen species (ROS)emediated oxidative stress plays a central role in the early stage of IRI [2e6], and antioxidant enzyme activation induced by antioxidants has been shown to attenuate liver IRI [7,8], but still, specific strategies are lacking.
* Corresponding author. Department of Anesthesiology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China. Q2 Tel./fax: þ86 20 85252297. E-mail address: [email protected] (Z. Hei). 1 M.G. and W.Y. contributed equally to this work. 2 Z.H. and G.L. are co-corresponding authors. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.03.016
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Reducing ROS levels or enhancing antioxidant capacity is 131 always considered to be the primary strategy to prevent 132 oxidative damage [9]. Recently, it has been demonstrated that 133 nuclear factor E2erelated factor 2 (Nrf2), a kind of transcrip134 tion factor, could activate different genes encoding various 135 136 antioxidant enzymes and phase II detoxification enzymes, 137 which enabled responses to oxidative stress and ROS scav138 Q3 enging [10,11]. Nrf2 is normally sequestered in the cytoplasm 139 by Kelch-like ECH-associated protein 1 (Keap1), which is 140 considered to be a negative regulator of Nrf2 [12]. Oxidants 141 and other stimuli inactivate Keap1, resulting in the stabiliza142 tion of Nrf2 and its translocation into the nucleus, where Nrf2 143 activates cytoprotective target genes by binding antioxidant 144 response elements [13]. However, whether Nrf2 nuclear 145 translocation could protect against graft-liver injury after liver 146 transplantation has not been reported. 147 148 Nrf2 activation has been confirmed to protect different 149 organs against IRI, including livers [14], brain [15], intestine 150 [16], and hearts [17]; thus, Nrf2 is considered to be a potential 151 strategy for preventing oxidative damage. Propofol, a widely 152 used intravenous anesthetic, has marked antioxidant capac153 ity to attenuate organ IRI effectively [18e22]; however, 154 whether propofol could ameliorate liver IRI after liver trans155 plantation remains unknown, and the interaction between 156 propofol and Nrf2 antioxidant pathway was still unclear. 157 To explore protective effects of propofol on IRI and its relative 158 159 mechanisms, the rat autologous orthotopic liver transplantation 160 (AOLT) model was used to recapitulate liver transplantation 161 in vivo. We focus on the dynamic alterations of Nrf2 expression 162 during and after liver transplantation and confirmed that 163 propofol-ameliorated liver oxidative injury followed AOLT via 164 Keap1 downregulation and Nrf2 upregulation. 165 166 167 2. Materials and methods 168 169 2.1. Animals 170 171 172 Sixty-three healthy male SpragueeDawley rats, aged 8e10 wk 173 and weighing 220e280 g, were provided by the Medical 174 Experimental Animal Center of Guangdong Province. This 175 study was approved by the Institutional Animal Care and Use 176 Committee and Animal Ethics Committee of Sun Yat-sen 177 University in Guangzhou, China, and followed the “Guide for 178 the Care and Use of Laboratory Animals” (National Institutes 179 of Health publication 86-23 revised 1985) guidelines for the 180 treatment of animals. 181 182 183 2.2. AOLT model and experimental groups 184 185 The AOLT model was described in our previous study [23] and 186 consists of primary surgical procedures, such as the dissoci187 ation of vessels and ligaments, the blockade of liver vessels, 188 liver infusion with cold preservation solution, vascular off189 clamping, and hepatic reperfusion. The entire anhepatic 190 phase was controlled within 20 1 min for a high mortality 191 rate of rats when cold ischemia time was longer than 25 min 192 in our preliminary study. The sham group underwent 193 194 abdominal surgery and liver dissection under anesthesia 195 without cold perfusion and reperfusion. The AOLT model rats
were subjected to the typical pathophysiological hepatic IRI processes during liver transplantation.
2.3.
Experimental groups
Initially, 35 male SpragueeDawley rats were randomized into a sham-operated group (sham group, n ¼ 7), a 4 h after AOLT group (4-h group, n ¼ 7), an 8 h after AOLT group (8-h group, n ¼ 7), a 16 h after AOLT group (16-h group, n ¼ 7), and a 24 h after AOLT group (24-h group, n ¼ 7). In the following experiment, 28 rats were divided into a shamoperated group (sham group, n ¼ 7), a saline-treated AOLT group (AOLT group, n ¼ 7), a low-dose propofol intervention AOLT group (L-Pro þ AOLT group, n ¼ 7), and a high-dose propofol intervention AOLT group (H-Pro þ AOLT group, n ¼ 7). The sham and AOLT groups received 3 mL of normal saline via intraperitoneal injections for 3 consecutive days before the experiment, and the LPro þ AOLT and H-Pro þ AOLT groups were pretreated with propofol (Diprivan, 1% propofol, CG411; AstraZeneca, Caponago, Italy) intraperitoneally at a dose or rate of 50 mg/kg (low dose, sedative effect [24]) or 100 mg/kg (high dose, anesthetic effect [25]) daily for 3 d before AOLT.
2.4.
Histologic assessment
Liver tissues were collected immediately after the rats were euthanized. The liver tissues were then fixed with 10% para- Q4 formaldehyde (pH 7.0; Sigma) for 48 h and embedded in paraffin. After hematoxylineeosin staining, liver sections (5 mm) were analyzed microscopically for pathology. Ten randomly selected images were captured in a blinded manner from each slide using a light microscope. The severity of IRI was graded based on Suzuki criteria [26].
2.5.
Biochemical analysis of serum samples
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum were measured with the test kits from SigmaeAldrich (St. Louis, MO) [27].
2.6. Superoxide anion (O 2 ), hydroxyl radical ($OH) and malondialdehyde assays Spectrophotometer methods were used to determine O 2 and $OH levels. The superoxide anion radical scavenging ability was measured by the method of the active oxygen generation of xanthineexanthine oxidase and detected at 550 nm with the spectrophotometer [28]. Determining the $OH suppression of the propofol was conducted according to the Fenton reaction principle. The procedure was performed using a $OH assay kit, according to the manufacturer’s instructions [29]. Malondialdehyde (MDA) content was detected using the thiobarbituric acid method. All procedures were performed according to the manufacturer’s instructions (Nanjing KeyGen Biotech. Co., Ltd, Nanjing, China).
2.7.
Immunofluorescence staining
Frozen sections of liver tissue were washed three times with phosphate-buffered saline (PBS). After fixing with PBS
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containing 5% bovine serum albumin and 0.3% Triton X-100 for 1 h, tissue sections were incubated with anti-Nrf2 (1:150) or anti-Keap1 (1:100) antibodies at 4 C overnight. After washing with PBS, the slides were incubated with a fluorescently labeled secondary antibody (1:100) for 1 h at 25 C. The cover slips were then washed again and mounted using a mounting medium (Applygen Technologies, Beijing. No. 01210) and observed using a fluorescent microscope (DM LB2; Leica Microsystems, Germany).
2.8.
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Protein expression of Nrf2, Keap1, HO-1, and NQO1 in the nuclear and cytosolic fractions was detected using Western blotting. Nuclear and cytoplasmic proteins were separated according to the protocol described in the Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). Protein concentrations were measured using the BCA method (KeyGen BioTech, China). The primary antibodies used were monoclonal rabbit anti-mouse Keap1 (1:1000 dilution; Millipore Company), polyclonal rabbit anti-mouse Nrf2 (1:250 dilution; Santa Cruz Biotechnology), anti-HO-1 (1:250 dilution; Santa Cruz Biotechnology), anti-NQO1 NQO1 (1:250 dilution; Santa Cruz Biotechnology), antieb-actin (1:2000 dilution; Santa Cruz Biotechnology), and anti-H2A (1:1000 dilution; Santa Cruz Biotechnology). The secondary antibody used was goat antirabbit HRP-conjugated IgG (1:2000 dilution; Boster, China). The optical density values of the bands were normalized to those of b-actin or H2A.
2.9.
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Western blotting
Data analyses and statistics
The data were expressed as the mean standard deviation or as the median (range) where appropriate. Comparisons of more than two groups were performed using a one-way analysis of variance followed by LSD multiple-comparison
3
tests. P < 0.05 was selected to indicate statistically significant differences.
3.
Results
3.1. Dynamic AOLT-induced pathologic changes in the liver and biochemical assessment As depicted in Figure 1, the sinusoids and hepatocytes were normal in the sham-operated group (Fig. 1A). Most severe hepatic injury occurred at 4 and 8 h after reperfusion, which manifested as impaired sinusoids and hepatocytes with edema and congestion, obstructed sinusoids, infiltration of inflammatory cells, and necrosis and vacuolation (Fig. 1B and C). After 16 h of reperfusion, pathologic injuries were alleviated significantly (Fig. 1D). Even after 24 h of reperfusion, only slight congestion and sinusoid obstruction were observed, and a small quantity of hepatocytes showed necrosis and vacuolation (Fig. 1E). These results suggested that hepatic IRI that followed AOLT was obvious at the early stage after reperfusion, and then resolved gradually, which correlated with changes in the Suzuki score (Fig. 1F). Serum AST and ALT levels, two indexes of hepatocellular injury, began to increase significantly at 4 h after reperfusion, peaked at 8 h, and then gradually declined, indicating that the most serious liver damage occurred at 8 h after AOLT (Fig. 1G and H).
3.2. Changes in OH, O 2 , and MDA levels in the liver after AOLT OH and O 2 levels reflect ROS levels, and MDA levels were used to determine the extent of oxidative injury in the liver tissue. As shown in Figure 2, OH and O 2 levels increased significantly at 4, 8, and 16 h after AOLT but returned to near
Fig. 1 e AOLT-induced dynamic pathologic changes in the liver visualized using HE staining (3200) and serum biochemical changes. Images AeE are representative of the sham group (Sham) and the experimental groups 4 h after AOLT (4-h group), 8 h after AOLT (8-h group), 16 h after AOLT (16-h group), and 24 h after AOLT (24-h group), respectively; image F shows Suzuki histologic grading of IRI at each time point after AOLT. Images G and H show the serum biochemical changes of AST O and ALT. *P < 0.05 versus sham group; P < 0.05 versus 24-h group. (Color version of figure available online.) 5.2.0 DTD YJSRE13195_proof 1 April 2015 11:32 pm ce
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Fig. 2 e Changes in liver OH, OL 2 , and MDA levels after AOLT. AeC show changes in hydroxyl radical (OH), superoxide anion (OL 2 ), and MDA levels over time. Sham: sham-operated group; 4 h: 4 h after AOLT; 8 h: 8 h after AOLT; 16 h: 16 h after AOLT; 24 h: 24 h after AOLT. *P < 0.05 versus sham group. the baseline at 24 h after AOLT (Fig. 2A and B). MDA levels showed a similar pattern to OH and O 2 levels (Fig. 2C).
3.3. Dynamic alteration in Nrf2 and HO-1 expression after liver IRI Nrf2 is the key protein regulating downstream antioxidant expression, and its nuclear levels were always used to determine Nrf2 nuclear translocation. Figure 3A shows that Nrf2 expression increased from 8 h after reperfusion (P < 0.05 versus sham group) and peaked at 24 h. A similar tendency was observed for HO-1 expression, which is a downstream Nrf2 antioxidant. HO-1 expression increased gradually from 8e24 h after reperfusion (P < 0.05 versus sham group), increased dramatically at 16 h after AOLT compared with that at 4 h (P < 0.05 versus 4-h group), and peaked at 24 h (P < 0.05 versus 4- and 8-h groups; Fig. 3B).
3.4. Propofol-attenuated liver injury and oxidative stress induced by hepatic IRI in AOLT rats We selected the time point of 8 h after reperfusion for the following intervention studies, for pathologic changes were
the most obvious at that time. Rats were pretreated with propofol intraperitoneally at low and high doses, and particularly in the high-dose group, propofol ameliorated hepatic IRI markedly, evidenced as minimal pathologic changes with less sinusoidal congestion, cytoplasmic vacuolization, and hepatocellular necrosis (Fig. 4AeD), as well as reversed the elevated levels of plasma AST and ALT (Fig. 4C and D). In the low-dose propofol group, there was minor congestion, obstruction, and ballooning, and 200 antioxidants that contributed to the majority of important endogenous antioxidant systems [34]. Keap1, a master regulator of intracellular redox homeostasis, has been shown to interact with Nrf2 [12]. As mentioned previously, Nrf2 is normally sequestered in the cytoplasm by Keap1, which prevents Nrf2 nuclear translocation and induces Nrf2 ubiquitin-mediated degradation. Oxidative or other stimuli enhance Keap1 degradation, and Nrf2 activation results in nuclear translocation and activation of genes encoding various antioxidant enzymes that protect cells from oxidative damage. Thus, the molecular mechanism has been confirmed by in vivo and in vitro experiments using genetic approaches including gene knockout and gene silencing [10]. Tanaka et al. [35] demonstrated that transient middle cerebral artery occlusion induced a progressive decrease in Keap1 expression in ischemic brains relative to Nrf2 and its downstream antioxidants. However, thus far, studies of Nrf2 expression after liver transplantation have been lacking. As shown in our results, during24 h after AOLT, a dynamic process of pathologic and oxidative injury occurs in which the injury is worse during the early stages and that gradually resolves or improves; this process occurs parallel to dynamic changes in nuclear levels of Nrf2. We suggest that Nrf2 and its antioxidant effects play a crucial role in preventing hepatic IRI
induced by AOLT and that it promotes hepatic repair. It is critical to identify effective methods that alleviate hepatic IRI. This study shows that targeting Nrf2 is a feasible strategy for early hepatic protection against IRI. Propofol is a commonly used sedative [18,19] and has been shown to promote the antioxidant capacity of various antioxidants and attenuates heart, brain, liver, and renal IRI [20e22]. However, mechanisms of propofol on its antioxidant activity remain unclear. In this study, we focused on the influence of propofol pretreatment on nuclear Nrf2 expression during hepatic IRI induced by AOLT. Immunofluorescence assays and Western blotting results showed that propofol pretreatment promoted Keap1 degradation and nuclear translocation of Nrf2. Consistent with these results, ROS levels decreased and oxidative damage was ameliorated. HO-1 and NQO1 are regarded as antioxidants that act downstream of Nrf2 [34]. In previous studies, it had been demonstrated that NQO1 expression was mediated by Nrf2 [36,37], and more importantly, HO-1 expression upregulation is always considered to be the evidence for the activation of Nrf2 signaling pathway [38]. NQO1 facilitates scavenging of ROS generated by mitochondria [39e42], and HO-1 exerts multiple regulatory functions during oxidative stress [43e47]. Our study revealed that propofol enhanced antioxidant capacity by upregulating HO-1 and NQO1 through Nrf2 activation. Based on these results and those of other studies, propofol enhances the nuclear translocation of Nrf2 and upregulates antioxidant expression, which alleviates hepatic IRI. In summary, our findings document that propofol ameliorated whole liver IRI in AOLT via downregulation of Keap1 and Nrf2 nuclear translocation. However, the optimal
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time for propofol administration requires further investigation. For the future studies, we will use more different methods to get the direct and specific evidence for the conclusion that the role of propofol was Nrf2 dependent, such as Nrf2 inhibitors, Nrf2 KO mice or Nrf2-siRNA on cells.
Acknowledgment This work was supported by grants from the National Natural Science Foundation of China (No. 81372090) and the Planned Science and Technology Project of Guangzhou (No. 2013B021800181 and 2013B051000035). The authors acknowledge the language help from Nature Publishing Group Language Editing. Authors’ contribution: M.G. and W.Y. performed the experiment and wrote the draft, Y.W. analyzed the data, D.Y. and X.C. finished the immunofluorescence staining, and G.L. and Z.H. designed and revised the article.
Disclosure The authors have no conflicts of interests, financial, or otherwise related to the publication of this study or its findings.
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