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Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain
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Caffeic acid Attenuates rat liver reperfusion injury through Sirt3-dependent regulation of mitochondrial respiratory chain Hong-Na Mu, Quan Li, Chun-Shui Pan, Yu-Ying Liu, Li Yan, Bai-He Hu, Kai Sun, Xin Chang, XinRong Zhao, Jing-Yu Fan, Jing-Yan Han
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Received date: 30 December 2014 Revised date: 21 April 2015 Accepted date: 29 April 2015 Cite this article as: Hong-Na Mu, Quan Li, Chun-Shui Pan, Yu-Ying Liu, Li Yan, Bai-He Hu, Kai Sun, Xin Chang, Xin-Rong Zhao, Jing-Yu Fan, Jing-Yan Han, Caffeic acid Attenuates rat liver reperfusion injury through Sirt3-dependent regulation of mitochondrial respiratory chain, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2015.04.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Caffeic Acid Attenuates Rat Liver Reperfusion Injury through Sirt3-Dependent Regulation of Mitochondrial Respiratory Chain
Hong-Na Mu1,2,3, Quan Li2,3, Chun-Shui Pan2,3, Yu-Ying Liu2,3, Li Yan2,3, Bai-He Hu 2,3, Kai Sun2,3, Xin Chang2,3, Xin-Rong Zhao2,3, Jing-Yu Fan2,3 and Jing-Yan Han1,2,3
1
Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing, China
2
Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China
3
Key Laboratory of Stasis and Phlegm of State Administration of Traditional Chinese Medicine, Beijing, China
E-mail: Hong-Na Mu: [email protected] Quan Li: [email protected] Chun-Shui Pan: [email protected] Yu-Ying Liu: [email protected] Li Yan: [email protected] Bai-He Hu: [email protected] Kai Sun: [email protected]
Xin Chang: [email protected] Xin-Rong Zhao: [email protected] Jing-Yu Fan: [email protected] Jing-Yan Han: [email protected]
Running Title: Caffeic acid and liver reperfusion injury
Address correspondence to: Jing-Yan Han, M.D, Ph.D. Professor and Chairman Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, People’s Republic of China Phone: 86-10-8280-2862 Fax: 86-10-8280-2996 E-mail: [email protected]
Abstract
Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remain elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects liver I/R injury through regulating Sirt3 and mitochondrial respiratory chain (MRC) is unclear. This study investigatedthe effect of CA on liver I/R injury, microcirculatory disturbance and potential mechanism, particularly focusing on Sirt3 dependent MRC. The liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min reperfusion. CA (15 mg/kg/h) was continuously infused via femoral vein staring from 30 min before ischemia. After I/R, Sirt3 expression and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant were provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down expression, and upregulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and mitochondrial respiratory chain.
Keywords: Superoxide anion; Liver microcirculatory disturbance; Leukocytes adherence; Apoptosis; Salvia miltiorrhiza
Abbreviations:CA, Caffeic acid; I/R, ischemia and reperfusion; MRC, mitochondrial respiratory chain; NDUFA9, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9; ROS, reactive oxygen species; SDHA, succinate dehydrogenase complex, subunit A, flavoprotein variant; Sirt3, sirtuin 3.
Introduction
Liver ischemia and reperfusion (I/R) injury occurs in multiple clinical events, such as liver transplantation, liver resection, trauma, and shock [1-3]. Many pathogenic factors have been demonstrated to be involved in regulating liver I/R injury, among which reactive oxygen species (ROS) acts as a pivotal player [4, 5]. Excessive ROS leads to peroxidation of DNA, protein, and lipids [6]. Moreover, massive ROS release activates the redox-sensitive transcription factors, such as nuclear factor kappa B (NF-țB), which further gives rise to augmented expression of adhesion molecules, leukocyte infiltration and even cell death [7, 8]. Impaired mitochondria is one of the major sources to generate ROS [9, 10]. During liver I/R, protons leak from mitochondrial respiratory chain (MRC), react with molecular oxygen and generate superoxide resulting in serious liver injury [11, 12]. Besides Complex I and III, Complex II of MRC is also involved in the generation of ROS during hypoxia [13]. Accordingly, blocking mitochondria resourced ROS production may be therapeutic manoeuver to ameliorate I/R-induced liver injury. Recently, the sirtuin family protein is emerging as a potential regulator of oxidative stress [14, 15]. Sirt3 is a major mitochondrial NAD+-dependent deacetylase of sirtuin family and sensitive to the cellular energy state. Sirt3 is reported to regulate the acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9) and succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), and further affect the activity of Complex I and Complex II, regulating
mitochondrial oxidative stress [16-18]. Several proteins have been identified as Sirt3 targets in response to I/R, such as isocitrate dehydrogenase 2, manganese superoxide dismutase (MnSOD), and Complex I [19]. Decreased Sirt3 may increase the susceptibility of cardiac-derived cells and adult hearts to I/R injury and contribute to a greater level of I/R injury in the aged heart [19]. Since mitochondria and metabolism are sensitive targets for I/R injury, the role of mitochondrial sirtuins in protection of I/R injury attracts increasing interest. However, whether Sirt3 is involved in liver I/R injury and the relationship between regulation of Sirt3 and protection of liver I/R injury remain unclear. Caffeic acid (CA), also known as 3, 4-dihydroxycinnamic acid, is a single phenolic acid derived from Salvia miltiorrhiza [20]. Accumulating evidence has confirmed that CA has potent antioxidant activities [21], able to attenuate intestine and brain I/R injury [22, 23], reduce intrastriatal quinolinic acid-induced brain oxidative damage by improving mitochondrial Complex enzymes and antioxidase activities [24]. However, it is so far unknown the effect of CA on liver I/R injury. The aim of this study was to explore the effects of CA on liver I/R injury and the underlying mechanism, paying special attention to its influence on Sirt3, mitochondrial Complex activity and oxidative stress.
Material and methods
Animals: Male Sprague-Dawley (SD) rats weighing 200 ± 20 g were obtained from Animals Center of Peking University Health Science Center (Certificate no. SCXK (Jing) 2006-0008). Animals were raised at a temperature of 20 ± 2 °C with 12-hour light/dark cycles, and fed with standard rat chow and water. All experimental procedures involving animals were approved by Peking University Biomedical Ethics Committee Experimental Animal Ethics Branch (LA2010-001), complying with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, 1996).
Surgical protocols and experimental groups: After anesthetization with intraperitoneal injection of pentobarbital sodium (50 mg/kg), the femoral vein was cannulated for drug infusion. Total hepatic ischemia and reperfusion (I/R) was induced as described previously [25]. Briefly, the rats were subjected to ischemia by clamping portal area vessels for 30 min followed by removing the clamp allowing for reperfusion for 120 min. In another sets of studies, rats were observed for 24 h following reperfusion to determine survival rate. The CA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in normal saline (NS) and used at a dose based on a previous in vivo study. All intravenous infusions were performed at a constant rate of 1.8 ml/h. Rats were randomly allocated into 4 groups, 6 animals each, as follows:
NS+sham group: Rats underwent an identical surgery but without occlusion of portal area vessels, and received continuous intravenous infusion of NS. CA+sham group: Rats underwent an identical surgery as rats in sham groups, and received a continuous infusion of CA (15 mg/kg/h, i.v.) for 180 min via a femoral vein catheter starting from 30 min before ischemia. NS+I/R group: Rats were subjected to 30 min ischemia and 120 min reperfusion, and received continuous intravenous infusion of NS. CA+I/R group: Rats were subjected to I/R, and received continuous intravenous infusion of CA (15 mg/kg/h) for 180 min starting from 30 min before I/R.
Observation of liver microcirculation: Rats were anesthetized and placed on an observation board in lateral position, and then left femoral vein was cannulated. The liver was placed on an adjustable Plexiglas microscope stage within a thermo-controlled (37 °C) observation box and carefully handled to minimize the influence of respiratory movements. The left lateral lobe of liver was observed under an inverted intravital microscope (TE2000-E, Nikon, Tokyo, Japan) assisted by a 3CCD colour camera (JK-TU53H, Toshiba, Tokyo, Japan). The venules ranging from 20 to 45 ȝm in diameter and 200 ȝm in length without adherent leukocytes were selected for observation. The dynamics of hepatic microcirculation was recorded using a DVD recorder (DVR-R25, Malata, Xiamen, China) [26]. To access the sinusoidal perfusion, the number of hepatic sinusoids with red blood cells (RBCs) flowing through in the hepatic terminal portal venule and terminal
hepatic venule regions was scored on the DVD replay. The result was presented as the ratio of the value determined at 30, 60, 90 and 120 min after reperfusion to the baseline (before ischemia) [27]. The velocity of RBCs in the venules was recorded at a rate of 500 frames/s using a high-speed video camera system (Fastcam-ultima APX, Photron, San Diego, CA, USA), and from which the recordings were replayed at a rate of 25 frames/s. RBCs velocity in the venules was determined with Image-Pro Plus software at baseline (before ischemia), 30, 60, 90 and 120 min after reperfusion [28]. To evaluate the leukocytes rolling and adhesion, 0.4 mL fluorescence tracer Rhodamine 6G (0.5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was administrated via the left femoral vein for selective staining of leukocytes in vivo [27]. The venules were observed under an irradiation at wavelength of 543 nm. The adherent leukocytes were identified as cells that attached to the venular walls for more than 10 s. The leukocytes that stayed at the same position for less than 10 s were designated as rolling leukocytes [29].
Liver blood flow determination: Liver blood flow (LBF) was measured by using a Laser-Doppler Perfusion Imager (PeriScan PIM3 System; PERIMED, Stockholm, Sweden) equipped with a computer at baseline and 120 min after reperfusion. For this purpose, liver was exposed and a computer-controlled optical scanner directed a low-powered He–Ne laser beam over the exposed liver. The scanner head was positioned in parallel to the surface of liver at
a distance of 18 cm. At each measuring site, the beam illuminated the tissue to a depth of 0.5 mm. A color-coded image to denote specific relative perfusion level was displayed on a video monitor, and all images were evaluated with the software of LDPIwin 3.1. The magnitude of liver blood flow was represented by different colors, with blue to red denoting low to high. A rectangle region (1.5 cm × 1.5 cm, the center of the left lateral lobe) of interest that included the main branch of the microcirculatory network was outlined on each image and used to calculate the area-averaged flux. The data were presented as a mean flux from the measured region of interest in perfusion units [28].
CD11b and CD18 expression on neutrophils: The expression of CD11b and CD18 in neutrophils was accessed after 120 min reperfusion with a flow cytometer (FACS Calibur, BD, Franklin Lakes, NJ, USA) as described previously [30].
Histology and immunohistochemistry: Hematoxylin and eosin (HE) staining and immunohistochemistry of myeloperoxidase (MPO) (Thermo Scientific, Fremont, CA, USA) were performed using standard procedures. For HE statistics, six fields of each animal were selected, and scored according to Suzuki score [31]. Sections for immunofluorescence staining were incubated with antibodies targeting intracellular adhesion molecule-1 (ICAM-1) and E-selectin (Santa Cruz, Santa Cruz, CA, USA) followed by incubation with Dylight
488-labeled rabbit anti-goat IgG (KPL, Gaithersburg, MD, USA). Hoechst 33342 (Invitrogen, St. Louis, MO, USA) was applied to stain nuclei. The sections were photographed under a laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany). For double staining of F-actin and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL), sections were stained with rhodamine phalloidine (R415)(life technologies, Carlsbad, CA, USA) and a cell death detection kit (Fluorescein dUTP Kit) (Roche, Indianapolis, IN, USA), according to the manufacture’s instruction. Then the nuclei were labeled with Hoechest33342. Six fields were selected and observed with a Laser Scanning Confocal Microscope. The number of the TUNEL-positive hepatocytes in the six fields was counted, and the average was calculated and expressed as cell number per field. The TUNEL-positive leukocytes and sinusoidal endothelial cells weren’t calculated.
Ultrastructure examination: After 120 min reperfusion, the rat liver (n=3 for each group) was perfused for 40 min with a fixative made of 4% paraformaldehyde and 2% glutaraldehyde (Ted Pella, Redding, CA, USA) in 0.1 mol/L phosphate buffer at a speed of 3 mL/min. For transmission electron microscopy, a coronal slice approximate 1 mm thick was taken. The slice was stored in freshly prepared 3% glutaraldehyde overnight at 4 °C. After rinsing with 0.1 mol/L phosphate buffer for 3 times, the tissue block was post-fixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer for 2 h at 4 °C. The samples were dehydrated and then embedded in Epon 812 (SPI-CHEM, Westchester, PA, USA).
Ultra-thin sections of liver were stained with uranium acetate and lead citrate and examined in a transmission electron microscope (JEM-1400 Plus, JEOL, Tokyo, Japan). For scanning electron microscopy, the samples were cut into blocks and further fixed in the freshly prepared glutaraldehyde for 2 h, rinsed with 0.1 mol/L phosphate buffer, and then post-fixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer for 2 h. The specimens were processed as routing and examined under a scanning electron microscope (JSM-5600LV, JEOL, Tokyo, Japan).
Enzyme-linked immunosorbent assay: Enzyme-linked immunosorbent assay (ELISA) was conducted using specific kit indicated, respectively, to determine the levels of plasma Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) activity(Sigma-Aldrich, St. Louis, MO, USA), and liver tissue MPO, methylenedioxyamphetamine (MDA)(Huanya Biomedicine Technology, Beijing, China), Hydrogen Peroxide (H2O2), 8-hydroxydeoxyguanosine (8-OHdG) (Cell Biolabs, San Diego, CA, USA), superoxide dismutase (SOD), catalase activity (CAT), Glutathione Reductase (GR), Glutathione Peroxidase (GPx) (Cayman Chemical company, Ann Arbor, MI, USA), Caspase 8, Complex I, II, IV, V activity and NAD/NADH (Abcam, Cambridge, MA, USA). For 8-OHdG detection, the DNA of liver tissue was extracted using a commercial kit (QIAGEN, Hilden, Germany). Mitochondria were isolated from liver tissues with mitochondria isolation kit for tissue (Abcam, Cambridge, MA, USA) following the manufacturer’s instructions. Sirt3 activity of mitochondrial extracts of
liver tissue was assessed using the Sirt3 direct fluorescent screening assay kit (Cayman Chemical company, Ann Arbor, MI, USA).
Immunoprecipitation and western blot analysis: Total protein was extracted using a protein extraction kit (Applygen Technologies, Beijing, China). The concentration of protein was determined with a BCA protein assay kit (Applygen Technologies, Beijing, China). For immunoprecipitation, total protein (1 mg) was incubated with antibodies against SDHA and NDUFA9 (Cell Signaling Technology, Beverly, MA, USA) or normal rabbit IgG (Santa Cruz, Santa Cruz, CA, USA) and immunoprecipitated with Protein A Sepharose CL-4B
(GE
Healthcare Life Sciences, London, England) followed by intensive washing. Then the acetylation of SDHA and NDUFA9 was detected by conventional Western blot. Western blot analysis was performed routinely, with primary antibodies against ICAM-1, MnSOD, Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), Cleaved Caspase-9, Cleaved Caspase-3, SDHA, NDUFA9, Acetylated-Lysine, Sirt3 and ȕ-actin (Cell Signaling Technology, Beverly, MA, USA). The bands were detected using ECL detection kit (Applygen Technologies, Beijing, China). For quantification, band intensity was assessed by densitometry and expressed as mean area density using the Quantity One image analyzer software (Bio-Rad, Richmond, CA, USA).
Surface plasmon resonance:
Surface plasmon resonance (SPR) can detect bio-molecules association and dissociation without label. It is highly sensitive and monitor molecular interactions in real-time. The information of interactive model, dynamics constant and so on can be acquired from SPR sensorgram. SPR is a powerful tool of biomolecular interaction analysis, and it has been widely applied in life science. The detection principle relies on an electron charge density wave phenomenon that arises at the surface of a metallic film when light is reflected at the film under specific conditions. Molecular adsorption/desorption events are messured as a change in the refractive index at the metal film surface [32]. Carboxymethylated 5 (CM5) sensor chip (GE Healthcare Life Sciences, London, England) was docked into the Biacore T200 (Biacore, GE Healthcare, Sweden), and prepared as previously reported [33]. Human Sirt3 full length protein (Abcam, Cambridge, MA, USA) was immobilized on CM5 sensor chip by injecting 40 ȝl of Sirt3 (15 ȝg/ȝl in 10 mM sodium acetate, pH 4.5) at the rate of 5 ȝl/min. CA was prepared as a 200 ȝM solution in running buffer before the experiment, and two fold diluted by running buffer into 200 ȝM 25 ȝM
12.5 ȝM 6.25 ȝM
100 ȝM 50 ȝM
3.125 ȝM 1.5625 ȝM before injection. Analytes were
injected at 30 ȝl/min over Sirt3 and control sensor chip. 90 ȝl of each concentration of CA solutions was injected and set 300 s as the dissociation time. No regeneration solution was required because all CA solutions were removed from the surface. Samples were injected from low to high concentration to eliminate the artifacts in the data from adsorption carry over on the instrument flow. Equilibrium dissociation constants (KD) was calculated by fitting a 1:1 Langmuir model using the Biacore
T200 evaluation software v2.0 (Biacore, GE Healthcare, Sweden) [33, 34].
Statistical analysis: All data were expressed as mean ± SEM. Statistics was undertaken with SPSS 15.0 software (SPSS Inc, Chicago, IL, USA). Survival time was compared by Kaplan–Meier log-rank test. Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test or using two-way ANOVA (perfused sinusoids, RBCs velocity, rolling leukocytes and adhered leukocytes and LBF) followed by Bonferroni for multiple comparisons. P value less than 0.05 was considered statistically significant.
Results
CA improves survival and attenuates liver injury after reperfusion. For survival assessment, rats were observed for 24 h following reperfusion. All sham-operated rats survived the entire observation period. The rats in I/R group displayed a 35% survival by 24 h, which was improved significantly by treatment with CA (p=0.045) (Fig. 1A). ALT and AST are well known as markers of liver injury. After I/R, ALT and AST activity were significantly increased, whereas pretreatment with CA prevented these alterations (Fig. 1B and C). We further applied HE staining to evaluate the liver injury. As shown in Fig. 1D and E, the livers of rats in the sham groups had normal hepatic architecture. After I/R, animals showed severe liver damage, with extensive sinusoidal congestion, hepatocyte necrosis and disarrangement. Obviously, rats treated with CA displayed a significantly less liver injury. Taken together, these results suggest that CA exerts protective effect on I/R-induced liver injury.
CA attennates I/R induced abnormalities in vessel perfusion. The number of perfused sinusoids in the hepatic terminal portal venule (Fig. 2A and C) and terminal hepatic venule (Fig. 2B and D) areas was decreased after I/R compared with sham-operated groups, while CA attenuated this decrease. RBCs velocity was decreased in the hepatic terminal portal venules and terminal hepatic venules after I/R, while treatment with CA attenuated this decrease (Fig. 2E and F). As shown in Fig.
2G and H, LBF was decreased obviously in I/R group, in contrast with the sham groups. However, pretreatment with CA significantly attenuated I/R-induced decrease in LBF.The results above imply that CA improves I/R-reduced vessels perfusion.
CA alleviates I/R-induced leukocytes recruitment. The rolling and adhered leukocytes obviously increased after 30 min of reperfusion and persisted increasing till 120-min reperfusion in the hepatic terminal portal venules (Fig. 3 A, B and C). Similar results were observed in terminal hepatic venules (Fig. 3 D, E and F) and sinusoids regions (Fig. 3 G and H). However, CA treatment reduced the enhancement in rolling and adhered leukocytes induced by I/R in all the areas examined. The expression of the adhesion molecule CD11b and CD18 on neutrophils is presented as mean fluorescence intensity in Fig. 3I. I/R increased the fluorescence intensity of CD11b significantly compared with sham groups. The expression of CD18 increased as well after I/R but without significance. The increase in the expression of both CD11b and CD18 was significantly attenuated by pretreatment with CA. In addition, as shown in Figure 3J and K, CA pretreatment protected against the upregulation of ICAM-1 and E-selectin in I/R rats. In accord with the above results, western blot analysis revealed evident increase of ICAM-1 in I/R group, whereas CA administration effectively inhibited the increase (Fig. 3L).
CA reduces I/R-induced leukocyte infiltration. The scanning electron microscope revealed no inflammatory cells in sham-operated groups (Fig. 4A, a1-a6), while abundant inflammatory cells and erythrocytes in hepatic sinusoids (Fig. 4A, a8), even thrombus in terminal hepatic venules (Fig. 4A, a9), but few in terminal portal venules (Fig. 4A, a7) after reperfusion. However, CA-treated rats demonstrated few inflammatory cells and erythrocytes (Fig. 4A, a10-a12), indicating that CA may affect I/R-induced leukocytes recruitment. As a marker enzyme of neutrophils, MPO expression in liver tissue was assessed by immunohistochemistry and ELISA assay. The MPO-positive staining cells were detected mainly in hepatic sinusoids (Fig. 4B
b8-1 and b8-2) and terminal hepatic
venules areas (Fig. 4B b9-1 and b9-2) in the liver of I/R rats, whereas few were detected in sham groups (Fig. 4B
b1-1~b6-2). CA treatment group displayed less
positive cells (Fig. 4B b10-1~b12-2). In agreement with the immunohistochemistry results, ELISA results of liver tissue MPO confirmed that CA treatment weakened MPO expression in comparison with I/R rats (Fig. 4C).
CA blunts I/R-induced NF-țB pathway activation and inflammatory cytokines release. Immunofluorescence staining and Western blot result demonstrated that significantly more NF-țB p65 located in nuclei in the I/R groupcompared to sham-operated groups, however, which was blunted by CA administration (Supplementary Fig. 1). Furthermore, the levels of TNF-Į, IL-1ȕ and MCP-1 in liver tissue as well as in
plasma were significantly up-regulated 2 hours after reperfusion. CA administration blocked I/R-induced up-regulation of TNF-Į, IL-1ȕ and MCP-1, suggesting that CA can negatively affect TNF-Į, IL-1ȕ and MCP-1 secretion (Supplementary Fig. 2).
CA inhibits I/R-induced hepatocyte apoptosis. The double staining of F-actin and TUNEL revealed that the positive nuclei were rarely observed in sham groups, in contrast, increased TUNEL-positive nuclei were detected in I/R injured-livers, which was protected against by pretreatment with CA (Fig. 5A and B). Moreover, several apoptosis-regulated proteins were assessed by western blot. Following I/R, the level of Bcl-2 was decreased, while the levels of Cleaved Caspase-9 and Cleaved Caspase-3 were increased (Fig. 5C, D and F, G and H). Notably, CA significantly inhibited the I/R-induced decrease in Bcl-2 expression, and the increase in Cleaved Caspase-9 and Cleaved Caspase-3 expression. A similar pattern of change was observed for Caspase-8 activity, and it was stimulated in I/R rats (Fig. 5E). The expression of Bax showed no apparent difference among the four groups.
CA reduces oxidative stress induced by I/R. H2O2 production, was measured. MDA, an indicator of cellular lipid peroxidation, and 8-OHdG, an oxidative DNA damage marker, were assessed by ELISA to evaluate oxidative injury. The results showed that H2O2, MDA and 8-OHdG increased significantly after I/R whichwere protected by CA pretreatment (Fig. 6A-C).
The expression of MnSOD, SOD and GPx and CAT activity in liver tissue decreased significantly in I/R rats in comparison with that in sham-operated rats, while CA pretreatment almost completely prevented these changes (Fig. 6D-G). There was no significant difference among the four groups at 120 min of reperfusion in the expression of GR in liver tissue (Fig. 6H).
CA mitigates I/R-induced changes in mitochondrial Complexes activity and subunits acetylation. Electron microscopy showed that the hepatocytes in sham-operated rats exhibited normal structure with oval nuclei, regular plasma and nuclear membrane, and oval to round shaped mitochondria with homogenously electron dense matrix and well organized cristae. Obviously, ischemia-reperfusion caused a serious hepatocyte damage, as shown by cell extraction, nucleus pycnosis, mitochondria edema and fragmentation of cristae. The hepatocyte damage was less pronounced in the CA-pretreated group (Fig. 7A). Then we tested with ELISA the activities of MRC Complex I, II, IV and V in liver tissues of different groups. We found that activities of Complex I, II, IV and V were suppressed to varying degrees in I/R group compared with sham groups. Of notice, CA administration significantly restored MRC Complexes activities in I/R rats (Fig. 7B-E). In addition, western blot showed that the expression of SDHA was decreased after I/R, which was recovered by CA treatment (Fig. 7F). Moreover, acetylation of the
SDHA and NDUFA9 was pronouncedly upregulated by I/R compared to that in sham groups, which was remarkably inhibited by pretreatment of CA (Fig. 7G and I). The expression of NDUFA9 showed no significant difference among the four groups (Fig. 7H).
CA binds to and activates Sirt3. After I/R, the expression level of Sirt3 in liver was diminished, while CA treatment restored the level of Sirt3 significantly (Fig. 8A). We determined Sirt3 activity in mitochondrial extracts of liver tissue by ELISA, and found that Sirt3 activity was suppressed after I/R, while CA treatment obviously promoted Sirt3 activity (Fig. 8B). Sirt3 is a NAD+-dependent enzyme, so we detected the change in NAD/NADH and found that NAD/NADH was depressed after I/R, as shown in Fig. 8C, and CA could remarkably enhance its expression. The results above proved that CA influenced the acetylation of downstream substrates of Sirt3, including SDHA and NDUFA9. Surface plasmon resonance (SPR) was further conducted to determine the binding capacity of CA to Sirt3. As shown in Figure 8D and Supplementary Figure 3, CA was able to bind to Sirt3 in a dose-dependent manner. The equilibrium dissociation constants (KD) of CA binding to Sirt3 was 7.931×10-5(M).
Discussion
The present study demonstrated that CA attenuated I/R-induced liver microcirculatory disturbance, inflammation, hepatocytes apoptosis and liver injury. More importantly, CA was revealed able to protect liver from oxidative insults, such as elevation of H2O2, MDA and 8-OHdG, as well as to attenuate I/R-elicited down-regulation of Sirt3 expression and activity, up-regulation of NDUFA9 and SDHA acetylation and decreased MRC Complexes activities. Finally, by SPR, we found that CA could bind to Sirt3. Microcirculatory disturbance acts as an important part in I/R. During the ischemic period, vascular hypoxia can cause increased vascular permeability. After reperfusion, complement system activation, leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation further aggravate microvascular dysfunction. Disturbances of the hepatic microcirculation may lead to no-reflow phenomenon with release of proinflammatory cytokines, sinusoidal plugging of neutrophils, oxidative stress, and hypoxic cell injury and parenchymal failure in I/R [34, 35]. It is reported that inhibition liver microcirculatory disturbance could help to protect the liver against reperfusion damage [34]. In this article, we found that CA attenuated microcirculatory disturbance and I/R injuryin rat, suggesting CA as an option for prophylaxis of I/R-induced liver injury in clinic. The dosage of CA used in our experiments was based on a pilot study, which showed that CA given at 15 mg/kg/h improved hepatic microcirculation and LBF most (Supplementary Fig.8). The total
salvianolic acids injection is injected continuously for the treatment of ischemic stroke in clinic in China. As one of the salvianolic acids, CA was administrated continuously as well in the present study. In addition, that CA was continuously infused was to keep CA at a constant concentration because the half-life of CA is very short. Furthermore, CA administration started from 30 min before ischemia in our experiment was a consideration of clinic translation of the results obtained. Ischemia and reperfusion injury is very common in liver resection and transplantation. To protect against liver I/R injury in such conditions, CA given before surgery may reach an effective concentration during surgery and is feasible in clinic. Oxidative stress plays a central role in I/R injury, and mitochondria is a major source of ROS.Complex I, II and III are all involved in ROS generation [13, 36, 37], thus, any impairment of which trigger I/R injury. For example, decreased Complex I activity and increased ROS generation occurred in hepatic I/R [38]. And Complex II activity impairment augmented the overall magnitude of oxidative injury in the post-ischemic heart [37]. Dysfunction of Complex II-III has been observed in ischemic injury following orthotopic liver transplantation [39]. Therapies that increase Complexes activities may provide protection against I/R injury. To this end, anti-anginal drug ranolazine was reported to improve Complex I structure and function, melatonin had strong protective effect against oxidative alterations to Complex I and III, which are associated with protection of I/R myocardial damage [40, 41].An important finding of the present study is that CA plays a role in restoration of MRC activities following hepatic I/R, suggesting implication of MRC in CA action.
As free radicals scavengers, antioxidant enzymes contribute profoundly to I/R injury. Endogenous antioxidant enzymes, such as SOD, CAT and GPx work together to reduce free radicals and minimize ROS-induced injury [42]. The maintenance of the mitochondrial antioxidant status has been recognized as an important determinant of mitochondrial ROS levels. And upregulation of endogenous antioxidant enzymes exerts protective effects on liver I/R injury [43, 44]. CA and its derivative, caffeic acid phenethyl ester, have been reported to protect mouse brain and liver mitochondria against anoxia–reoxygenation injury mainly due to their antioxidative activities [45]. Caffeic acid phenethyl ester has been shown to exert beneficial effects on I/R injury in rat skin flaps via increasing SOD, GPx enzyme activities [46]. In accord with these previous reports, CA blunted the decrease in MnSOD, SOD, GPx and CAT activity after liver I/R, suggesting antioxidant enzyme as one of the targets for CA to attenuate oxidative injury in liver I/R. Most interestingly, our results revealed CA was able to improve the decrease of Sirt3 expression and activity following liver I/R. Sirt3 belongs to Sirtuins family, and locates in the mitochondria [16, 47]. Sirt3 deacetylates several metabolic and respiratory enzymes highlighting a role in regulating mitochondrial functions [48]. Sirt3 was shown to play an important role in cardiac I/R injury and deacetylates several proteins regulating the response to I/R. Sirt3-deficient rats are more susceptible to cardiac I/R injury, implying increased Sirt3 may protect from I/R injury [19, 32]. It has been reported that Sirt3 directly deacetylates NDUFA9 [16], SDHA [17], ATP synthase ȕ [49] and regulates Complexes activity [50] modulating ROS
production. However, the protective effect of Sirt3 against ischemic processes in liver has not yet been demonstrated. In the present study, we demonstrated that Sirt3 expression and activity was down regulated in liver after I/R, while CA obviously provoked Sirt3 expression and activity, prevented the acetylation of SDHA and NDUFA9 and enhanced MRC activity in liver following I/R, highlighting Sirt3 as the direct target for CA to exert beneficial role in hepatic I/R injury (Supplementary Fig. 13). This argument is further strengthened by the finding that CA is able to bind to Sirt3. However, some issues remain to be addressed. (1) To validate the significance of CA binding to Sirt3 in liver I/R injury, experiment needs to be conducted to knock down Sirt3; (2) More study is required to preclude the involvement of other signaling pathway in CA effect on I/R injury.
Conclusions
Taken together, CA attenuated liver I/R injury and microcirculatory disturbance which is related to inhibition of oxidative stress, possibly by action on Sirt3, reducing acetylation of NDUFA9 and SDHA and improving MRC activities.
Conflict of interest The authors declare no competing financial interest.
Financial Support This work was supported by the Production of New Medicine Program of Ministry of Science and Technology of China [2013ZX09402202].
Acknowledgements The authors thank Li-Jun Wang and Ge Fu for excellent technical assistance.
References
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Figure Legends
Fig. 1. Effects of CA on 24-hour survival rate, ALT, AST and HE. (A) Rats of four groups were monitored for 24 h survival (n=20). (B) Plasma levels of ALT activity. (C) Plasma levels of AST activity. Data are mean ± SEM (n=8). (D) Liver samples in four groups stained by HE. d1, d4, d7 and d10 show portal areas, d2, d5, d8 and d11 show hepatic sinusoids, d3, d6, d9 and d12 show terminal hepatic venules, Bar = 50 ȝm. (E) Liver injury was quantitatively evaluated by Suzuki score. Data are mean ± SEM (n=6). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 2. Effects of CA on number of perfused sinusoids and RBC velocity in microvessles and liver blood flow. (A) and (D) Representative images of sinusoids in terminal portal venule and terminal hepatic venule areas. (B) and (E) Quantitative evaluation of perfused sinusoids in terminal portal venules and terminal hepatic venules, respectively. (C) and (F) Quantitative evaluation of RBCs velocity in terminal portal venules and terminal hepatic venules, respectively. (G) Representative color images of liver blood flow acquired by Laser–Doppler Perfusion Imager. (H) Quantification of the liver blood flow. Arrows in (A) and (B) indicate the sinusoids with no flow. Bar = 50 ȝm. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 3. Effects of CA on leukocyte rolling, adherence and adhesion molecule expression. (A) Representative images of leukocytes adhered to terminal portal venules. (B) Quantitative evaluation of rolling leukocytes in terminal portal venules. (C) Quantitative evaluation of adhered leukocytes in terminal portal venules. (D) Representative images of leukocytes adhered to terminal hepatic venules. (E) Quantitative evaluation of rolling leukocytes in terminal hepatic venules. (F) Quantitative evaluation of adhered leukocytes in terminal hepatic venules. (G) Representative images of leukocytes adhered to sinusoids area. (H) Quantitative evaluation of leukocytes adhered to sinusoids area. Arrows in (A), (D) and (G) indicate adhered leukocytes. Bar = 50 ȝm. Data are mean ± SEM (n=8). (I) The expressions of adhesion molecules CD11b and CD18 on rat neutrophils presented as fluorescence
intensity
of
FITC.
(J)
and
(K)
Representative
images
of
immunofluorescence staining for ICAM-1 and E-selectin, respectively. ICAM-1 and E-selectin are stained green, and nuclei are identified by Hoechst staining in bluecolor. Bar = 50 ȝm. (L) ICAM-1 expression in different groups determined by Western blot. All band intensities were calculated based on the results from 3 independent experiments. All protein intensities were normalized to ߚ-actin. Data are mean ± SEM (n=4). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 4. Ultrastructure of vessels and effects of CA on MPO expression. (A) Scanning electron microscopic images of the vessels in different groups, wherein
arrows indicate adhered leukocytes. a1, a4, a7 and a10 show terminal portal venules. a2, a5, a8 and a11 show hepatic sinusoids. a3, a6, a9 and a12 show terminal hepatic venules. Bar = 5 ȝm. (B) Representative images of immunohistochemistry staining for MPO in different groups, in which arrows indicate MPO-positive leukocytes. The area within the rectangle in each picture is enlarged and presented aside correspondingly. b1-1, b1-2, b4-1, b4-2, b7-1, b7-2, b10-1 and b10-2 show portal areas. b2-1, b2-2, b5-1, b5-2, b8-1, b8-2, b11-1 and b11-2 show hepatic sinusoids. b3-1, b3-2, b6-1, b6-2, b9-1, b9-2, b12-1 and b12-2 show terminal hepatic venules. Bar = 50 ȝm. (C) ELISA analysis of MPO in liver tissue from different groups. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 5. Effects of CA on I/R-induced hepatocyte apoptosis and apoptosis-related protein expressions. (A) The representative photographs of double staining of F-actin and TUNEL, wherein nuclei are stained with blue color, F-actin with red, and TUNEL-positive nuclei green, Bar=50 ȝm. H: hepatocyte; L: leukocyte; S: sinusoidal endothelial cell. a1-a8 show hepatic sinusoids, a9 and a10 show portal areas of NS+I/R group, a11 and a12 show terminal hepatic venules of NS+I/R group. (B) Quantitative analysis of apoptosis cells in various groups. n=6. (C) The expressions of Bax and Bcl-2 determined by Western blot. (F), (G) and (H) Cleaved Caspase-9 and Cleaved Caspase-3 determined by Western blot. (D) The quantitative analysis of the ratio of Bcl-2/Bax. (G) The quantitative analysis of Cleaved Caspase-9. (H) The quantitative
analysis of Cleaved Caspase-3. All the quantifications were undertaken based on the data of 4 animals and normalized to ȕ-actin. All band intensities were calculated based on the results from 3 independent experiments. (E) ELISA analysis results of liver tissue Caspase-8 activity from various groups. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 6. Effects of CA on liver antioxidant enzymes expression, ROS production and oxidative injuryafter I/R. (A) H2O2 production. (B) MDA production. (C) 8-OHdG production. (E) SOD expression. (F) CAT activity. (G) GR expression. (H) GRx expression. The results above were detected by ELISA in liver tissue. Data are mean ± SEM (n=8) (D) Western blot result of MnSOD. The blot image is a representative of 4 animals in a group. The densitometry is an averaged result for the 4 animals and normalized to ȕ-actin. *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 7. Effects of CA on I/R-induced mitochondria injury, Complexes inhibition and subunits acetylation in liver. (A) Ultrastructure of hepatocyte. a1-1, a2-1, a3-1 and a4-1 show hepatocytes at low magnification, Bar=5 ȝm. a1-2, a2-2, a3-2 and a4-2 show mitochondria at high magnification, Bar=1 ȝm. ELISA analysis was performed to determine (B) Complex I Activity, (C) Complex II activity, (D) Complex IV activity and (E) ATP synthase activity in liver tissue. Data are mean ± SEM (n=8). Western blot was conducted for
assessment of (F) SDHA expression, (G) SDHA acetylation, (H) NDUFA9expression and (I) NDUFA9 acetylation in liver tissue. The blot image is a representative of 4 animals in a group. The densitometry is an averaged result for the 4 animals and normalized to ȕ-actin. Data are mean ± SEM (n=4). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 8. CA interacts with Sirt3. (A) Western blot analysis of Sirt3 expression in liver tissue of different groups. The blot image is a representative of 4 animals in a group. The densitometry is an averaged result for the 4 animals and normalized to ȕ-actin. (B) Sirt3 activity was detected by ELISA in liver tissue. Data are mean ± SEM (n=8). (C) NAD/NADH of liver tissue detected by ELISA. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group. (D) The affinity of CA and Sirt3 tested by SPR. Shown are the representative sensorgrams obtained from the injections of CA at concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 50 and 200 ȝM (curves from bottom to top) using SPR.
Highlights 1. Caffeic acid (CA) protected rat liver from ischemia and reperfusion (I/R) injury. 2. CA prevented I/R induced decrease of Sirt3 expression and activity. 3. CA reduced acetylation of SDHA and NDUFA9 and enhanced MRC activity.
Graphical Abstract (for review)
Figure
Figure
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Caffeic acid Attenuates rat liver reperfusion injury through Sirt3-dependent regulation of mitochondrial respiratory chain Hong-Na Mu, Quan Li, Chun-Shui Pan, Yu-Ying Liu, Li Yan, Bai-He Hu, Kai Sun, Xin Chang, XinRong Zhao, Jing-Yu Fan, Jing-Yan Han
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Received date: 30 December 2014 Revised date: 21 April 2015 Accepted date: 29 April 2015 Cite this article as: Hong-Na Mu, Quan Li, Chun-Shui Pan, Yu-Ying Liu, Li Yan, Bai-He Hu, Kai Sun, Xin Chang, Xin-Rong Zhao, Jing-Yu Fan, Jing-Yan Han, Caffeic acid Attenuates rat liver reperfusion injury through Sirt3-dependent regulation of mitochondrial respiratory chain, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2015.04.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Caffeic Acid Attenuates Rat Liver Reperfusion Injury through Sirt3-Dependent Regulation of Mitochondrial Respiratory Chain
Hong-Na Mu1,2,3, Quan Li2,3, Chun-Shui Pan2,3, Yu-Ying Liu2,3, Li Yan2,3, Bai-He Hu 2,3, Kai Sun2,3, Xin Chang2,3, Xin-Rong Zhao2,3, Jing-Yu Fan2,3 and Jing-Yan Han1,2,3
1
Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing, China
2
Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China
3
Key Laboratory of Stasis and Phlegm of State Administration of Traditional Chinese Medicine, Beijing, China
E-mail: Hong-Na Mu: [email protected] Quan Li: [email protected] Chun-Shui Pan: [email protected] Yu-Ying Liu: [email protected] Li Yan: [email protected] Bai-He Hu: [email protected] Kai Sun: [email protected]
Xin Chang: [email protected] Xin-Rong Zhao: [email protected] Jing-Yu Fan: [email protected] Jing-Yan Han: [email protected]
Running Title: Caffeic acid and liver reperfusion injury
Address correspondence to: Jing-Yan Han, M.D, Ph.D. Professor and Chairman Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, People’s Republic of China Phone: 86-10-8280-2862 Fax: 86-10-8280-2996 E-mail: [email protected]
Abstract
Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remain elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects liver I/R injury through regulating Sirt3 and mitochondrial respiratory chain (MRC) is unclear. This study investigatedthe effect of CA on liver I/R injury, microcirculatory disturbance and potential mechanism, particularly focusing on Sirt3 dependent MRC. The liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min reperfusion. CA (15 mg/kg/h) was continuously infused via femoral vein staring from 30 min before ischemia. After I/R, Sirt3 expression and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant were provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down expression, and upregulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and mitochondrial respiratory chain.
Keywords: Superoxide anion; Liver microcirculatory disturbance; Leukocytes adherence; Apoptosis; Salvia miltiorrhiza
Abbreviations:CA, Caffeic acid; I/R, ischemia and reperfusion; MRC, mitochondrial respiratory chain; NDUFA9, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9; ROS, reactive oxygen species; SDHA, succinate dehydrogenase complex, subunit A, flavoprotein variant; Sirt3, sirtuin 3.
Introduction
Liver ischemia and reperfusion (I/R) injury occurs in multiple clinical events, such as liver transplantation, liver resection, trauma, and shock [1-3]. Many pathogenic factors have been demonstrated to be involved in regulating liver I/R injury, among which reactive oxygen species (ROS) acts as a pivotal player [4, 5]. Excessive ROS leads to peroxidation of DNA, protein, and lipids [6]. Moreover, massive ROS release activates the redox-sensitive transcription factors, such as nuclear factor kappa B (NF-țB), which further gives rise to augmented expression of adhesion molecules, leukocyte infiltration and even cell death [7, 8]. Impaired mitochondria is one of the major sources to generate ROS [9, 10]. During liver I/R, protons leak from mitochondrial respiratory chain (MRC), react with molecular oxygen and generate superoxide resulting in serious liver injury [11, 12]. Besides Complex I and III, Complex II of MRC is also involved in the generation of ROS during hypoxia [13]. Accordingly, blocking mitochondria resourced ROS production may be therapeutic manoeuver to ameliorate I/R-induced liver injury. Recently, the sirtuin family protein is emerging as a potential regulator of oxidative stress [14, 15]. Sirt3 is a major mitochondrial NAD+-dependent deacetylase of sirtuin family and sensitive to the cellular energy state. Sirt3 is reported to regulate the acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9) and succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), and further affect the activity of Complex I and Complex II, regulating
mitochondrial oxidative stress [16-18]. Several proteins have been identified as Sirt3 targets in response to I/R, such as isocitrate dehydrogenase 2, manganese superoxide dismutase (MnSOD), and Complex I [19]. Decreased Sirt3 may increase the susceptibility of cardiac-derived cells and adult hearts to I/R injury and contribute to a greater level of I/R injury in the aged heart [19]. Since mitochondria and metabolism are sensitive targets for I/R injury, the role of mitochondrial sirtuins in protection of I/R injury attracts increasing interest. However, whether Sirt3 is involved in liver I/R injury and the relationship between regulation of Sirt3 and protection of liver I/R injury remain unclear. Caffeic acid (CA), also known as 3, 4-dihydroxycinnamic acid, is a single phenolic acid derived from Salvia miltiorrhiza [20]. Accumulating evidence has confirmed that CA has potent antioxidant activities [21], able to attenuate intestine and brain I/R injury [22, 23], reduce intrastriatal quinolinic acid-induced brain oxidative damage by improving mitochondrial Complex enzymes and antioxidase activities [24]. However, it is so far unknown the effect of CA on liver I/R injury. The aim of this study was to explore the effects of CA on liver I/R injury and the underlying mechanism, paying special attention to its influence on Sirt3, mitochondrial Complex activity and oxidative stress.
Material and methods
Animals: Male Sprague-Dawley (SD) rats weighing 200 ± 20 g were obtained from Animals Center of Peking University Health Science Center (Certificate no. SCXK (Jing) 2006-0008). Animals were raised at a temperature of 20 ± 2 °C with 12-hour light/dark cycles, and fed with standard rat chow and water. All experimental procedures involving animals were approved by Peking University Biomedical Ethics Committee Experimental Animal Ethics Branch (LA2010-001), complying with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, 1996).
Surgical protocols and experimental groups: After anesthetization with intraperitoneal injection of pentobarbital sodium (50 mg/kg), the femoral vein was cannulated for drug infusion. Total hepatic ischemia and reperfusion (I/R) was induced as described previously [25]. Briefly, the rats were subjected to ischemia by clamping portal area vessels for 30 min followed by removing the clamp allowing for reperfusion for 120 min. In another sets of studies, rats were observed for 24 h following reperfusion to determine survival rate. The CA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in normal saline (NS) and used at a dose based on a previous in vivo study. All intravenous infusions were performed at a constant rate of 1.8 ml/h. Rats were randomly allocated into 4 groups, 6 animals each, as follows:
NS+sham group: Rats underwent an identical surgery but without occlusion of portal area vessels, and received continuous intravenous infusion of NS. CA+sham group: Rats underwent an identical surgery as rats in sham groups, and received a continuous infusion of CA (15 mg/kg/h, i.v.) for 180 min via a femoral vein catheter starting from 30 min before ischemia. NS+I/R group: Rats were subjected to 30 min ischemia and 120 min reperfusion, and received continuous intravenous infusion of NS. CA+I/R group: Rats were subjected to I/R, and received continuous intravenous infusion of CA (15 mg/kg/h) for 180 min starting from 30 min before I/R.
Observation of liver microcirculation: Rats were anesthetized and placed on an observation board in lateral position, and then left femoral vein was cannulated. The liver was placed on an adjustable Plexiglas microscope stage within a thermo-controlled (37 °C) observation box and carefully handled to minimize the influence of respiratory movements. The left lateral lobe of liver was observed under an inverted intravital microscope (TE2000-E, Nikon, Tokyo, Japan) assisted by a 3CCD colour camera (JK-TU53H, Toshiba, Tokyo, Japan). The venules ranging from 20 to 45 ȝm in diameter and 200 ȝm in length without adherent leukocytes were selected for observation. The dynamics of hepatic microcirculation was recorded using a DVD recorder (DVR-R25, Malata, Xiamen, China) [26]. To access the sinusoidal perfusion, the number of hepatic sinusoids with red blood cells (RBCs) flowing through in the hepatic terminal portal venule and terminal
hepatic venule regions was scored on the DVD replay. The result was presented as the ratio of the value determined at 30, 60, 90 and 120 min after reperfusion to the baseline (before ischemia) [27]. The velocity of RBCs in the venules was recorded at a rate of 500 frames/s using a high-speed video camera system (Fastcam-ultima APX, Photron, San Diego, CA, USA), and from which the recordings were replayed at a rate of 25 frames/s. RBCs velocity in the venules was determined with Image-Pro Plus software at baseline (before ischemia), 30, 60, 90 and 120 min after reperfusion [28]. To evaluate the leukocytes rolling and adhesion, 0.4 mL fluorescence tracer Rhodamine 6G (0.5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was administrated via the left femoral vein for selective staining of leukocytes in vivo [27]. The venules were observed under an irradiation at wavelength of 543 nm. The adherent leukocytes were identified as cells that attached to the venular walls for more than 10 s. The leukocytes that stayed at the same position for less than 10 s were designated as rolling leukocytes [29].
Liver blood flow determination: Liver blood flow (LBF) was measured by using a Laser-Doppler Perfusion Imager (PeriScan PIM3 System; PERIMED, Stockholm, Sweden) equipped with a computer at baseline and 120 min after reperfusion. For this purpose, liver was exposed and a computer-controlled optical scanner directed a low-powered He–Ne laser beam over the exposed liver. The scanner head was positioned in parallel to the surface of liver at
a distance of 18 cm. At each measuring site, the beam illuminated the tissue to a depth of 0.5 mm. A color-coded image to denote specific relative perfusion level was displayed on a video monitor, and all images were evaluated with the software of LDPIwin 3.1. The magnitude of liver blood flow was represented by different colors, with blue to red denoting low to high. A rectangle region (1.5 cm × 1.5 cm, the center of the left lateral lobe) of interest that included the main branch of the microcirculatory network was outlined on each image and used to calculate the area-averaged flux. The data were presented as a mean flux from the measured region of interest in perfusion units [28].
CD11b and CD18 expression on neutrophils: The expression of CD11b and CD18 in neutrophils was accessed after 120 min reperfusion with a flow cytometer (FACS Calibur, BD, Franklin Lakes, NJ, USA) as described previously [30].
Histology and immunohistochemistry: Hematoxylin and eosin (HE) staining and immunohistochemistry of myeloperoxidase (MPO) (Thermo Scientific, Fremont, CA, USA) were performed using standard procedures. For HE statistics, six fields of each animal were selected, and scored according to Suzuki score [31]. Sections for immunofluorescence staining were incubated with antibodies targeting intracellular adhesion molecule-1 (ICAM-1) and E-selectin (Santa Cruz, Santa Cruz, CA, USA) followed by incubation with Dylight
488-labeled rabbit anti-goat IgG (KPL, Gaithersburg, MD, USA). Hoechst 33342 (Invitrogen, St. Louis, MO, USA) was applied to stain nuclei. The sections were photographed under a laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany). For double staining of F-actin and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL), sections were stained with rhodamine phalloidine (R415)(life technologies, Carlsbad, CA, USA) and a cell death detection kit (Fluorescein dUTP Kit) (Roche, Indianapolis, IN, USA), according to the manufacture’s instruction. Then the nuclei were labeled with Hoechest33342. Six fields were selected and observed with a Laser Scanning Confocal Microscope. The number of the TUNEL-positive hepatocytes in the six fields was counted, and the average was calculated and expressed as cell number per field. The TUNEL-positive leukocytes and sinusoidal endothelial cells weren’t calculated.
Ultrastructure examination: After 120 min reperfusion, the rat liver (n=3 for each group) was perfused for 40 min with a fixative made of 4% paraformaldehyde and 2% glutaraldehyde (Ted Pella, Redding, CA, USA) in 0.1 mol/L phosphate buffer at a speed of 3 mL/min. For transmission electron microscopy, a coronal slice approximate 1 mm thick was taken. The slice was stored in freshly prepared 3% glutaraldehyde overnight at 4 °C. After rinsing with 0.1 mol/L phosphate buffer for 3 times, the tissue block was post-fixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer for 2 h at 4 °C. The samples were dehydrated and then embedded in Epon 812 (SPI-CHEM, Westchester, PA, USA).
Ultra-thin sections of liver were stained with uranium acetate and lead citrate and examined in a transmission electron microscope (JEM-1400 Plus, JEOL, Tokyo, Japan). For scanning electron microscopy, the samples were cut into blocks and further fixed in the freshly prepared glutaraldehyde for 2 h, rinsed with 0.1 mol/L phosphate buffer, and then post-fixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer for 2 h. The specimens were processed as routing and examined under a scanning electron microscope (JSM-5600LV, JEOL, Tokyo, Japan).
Enzyme-linked immunosorbent assay: Enzyme-linked immunosorbent assay (ELISA) was conducted using specific kit indicated, respectively, to determine the levels of plasma Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) activity(Sigma-Aldrich, St. Louis, MO, USA), and liver tissue MPO, methylenedioxyamphetamine (MDA)(Huanya Biomedicine Technology, Beijing, China), Hydrogen Peroxide (H2O2), 8-hydroxydeoxyguanosine (8-OHdG) (Cell Biolabs, San Diego, CA, USA), superoxide dismutase (SOD), catalase activity (CAT), Glutathione Reductase (GR), Glutathione Peroxidase (GPx) (Cayman Chemical company, Ann Arbor, MI, USA), Caspase 8, Complex I, II, IV, V activity and NAD/NADH (Abcam, Cambridge, MA, USA). For 8-OHdG detection, the DNA of liver tissue was extracted using a commercial kit (QIAGEN, Hilden, Germany). Mitochondria were isolated from liver tissues with mitochondria isolation kit for tissue (Abcam, Cambridge, MA, USA) following the manufacturer’s instructions. Sirt3 activity of mitochondrial extracts of
liver tissue was assessed using the Sirt3 direct fluorescent screening assay kit (Cayman Chemical company, Ann Arbor, MI, USA).
Immunoprecipitation and western blot analysis: Total protein was extracted using a protein extraction kit (Applygen Technologies, Beijing, China). The concentration of protein was determined with a BCA protein assay kit (Applygen Technologies, Beijing, China). For immunoprecipitation, total protein (1 mg) was incubated with antibodies against SDHA and NDUFA9 (Cell Signaling Technology, Beverly, MA, USA) or normal rabbit IgG (Santa Cruz, Santa Cruz, CA, USA) and immunoprecipitated with Protein A Sepharose CL-4B
(GE
Healthcare Life Sciences, London, England) followed by intensive washing. Then the acetylation of SDHA and NDUFA9 was detected by conventional Western blot. Western blot analysis was performed routinely, with primary antibodies against ICAM-1, MnSOD, Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), Cleaved Caspase-9, Cleaved Caspase-3, SDHA, NDUFA9, Acetylated-Lysine, Sirt3 and ȕ-actin (Cell Signaling Technology, Beverly, MA, USA). The bands were detected using ECL detection kit (Applygen Technologies, Beijing, China). For quantification, band intensity was assessed by densitometry and expressed as mean area density using the Quantity One image analyzer software (Bio-Rad, Richmond, CA, USA).
Surface plasmon resonance:
Surface plasmon resonance (SPR) can detect bio-molecules association and dissociation without label. It is highly sensitive and monitor molecular interactions in real-time. The information of interactive model, dynamics constant and so on can be acquired from SPR sensorgram. SPR is a powerful tool of biomolecular interaction analysis, and it has been widely applied in life science. The detection principle relies on an electron charge density wave phenomenon that arises at the surface of a metallic film when light is reflected at the film under specific conditions. Molecular adsorption/desorption events are messured as a change in the refractive index at the metal film surface [32]. Carboxymethylated 5 (CM5) sensor chip (GE Healthcare Life Sciences, London, England) was docked into the Biacore T200 (Biacore, GE Healthcare, Sweden), and prepared as previously reported [33]. Human Sirt3 full length protein (Abcam, Cambridge, MA, USA) was immobilized on CM5 sensor chip by injecting 40 ȝl of Sirt3 (15 ȝg/ȝl in 10 mM sodium acetate, pH 4.5) at the rate of 5 ȝl/min. CA was prepared as a 200 ȝM solution in running buffer before the experiment, and two fold diluted by running buffer into 200 ȝM 25 ȝM
12.5 ȝM 6.25 ȝM
100 ȝM 50 ȝM
3.125 ȝM 1.5625 ȝM before injection. Analytes were
injected at 30 ȝl/min over Sirt3 and control sensor chip. 90 ȝl of each concentration of CA solutions was injected and set 300 s as the dissociation time. No regeneration solution was required because all CA solutions were removed from the surface. Samples were injected from low to high concentration to eliminate the artifacts in the data from adsorption carry over on the instrument flow. Equilibrium dissociation constants (KD) was calculated by fitting a 1:1 Langmuir model using the Biacore
T200 evaluation software v2.0 (Biacore, GE Healthcare, Sweden) [33, 34].
Statistical analysis: All data were expressed as mean ± SEM. Statistics was undertaken with SPSS 15.0 software (SPSS Inc, Chicago, IL, USA). Survival time was compared by Kaplan–Meier log-rank test. Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test or using two-way ANOVA (perfused sinusoids, RBCs velocity, rolling leukocytes and adhered leukocytes and LBF) followed by Bonferroni for multiple comparisons. P value less than 0.05 was considered statistically significant.
Results
CA improves survival and attenuates liver injury after reperfusion. For survival assessment, rats were observed for 24 h following reperfusion. All sham-operated rats survived the entire observation period. The rats in I/R group displayed a 35% survival by 24 h, which was improved significantly by treatment with CA (p=0.045) (Fig. 1A). ALT and AST are well known as markers of liver injury. After I/R, ALT and AST activity were significantly increased, whereas pretreatment with CA prevented these alterations (Fig. 1B and C). We further applied HE staining to evaluate the liver injury. As shown in Fig. 1D and E, the livers of rats in the sham groups had normal hepatic architecture. After I/R, animals showed severe liver damage, with extensive sinusoidal congestion, hepatocyte necrosis and disarrangement. Obviously, rats treated with CA displayed a significantly less liver injury. Taken together, these results suggest that CA exerts protective effect on I/R-induced liver injury.
CA attennates I/R induced abnormalities in vessel perfusion. The number of perfused sinusoids in the hepatic terminal portal venule (Fig. 2A and C) and terminal hepatic venule (Fig. 2B and D) areas was decreased after I/R compared with sham-operated groups, while CA attenuated this decrease. RBCs velocity was decreased in the hepatic terminal portal venules and terminal hepatic venules after I/R, while treatment with CA attenuated this decrease (Fig. 2E and F). As shown in Fig.
2G and H, LBF was decreased obviously in I/R group, in contrast with the sham groups. However, pretreatment with CA significantly attenuated I/R-induced decrease in LBF.The results above imply that CA improves I/R-reduced vessels perfusion.
CA alleviates I/R-induced leukocytes recruitment. The rolling and adhered leukocytes obviously increased after 30 min of reperfusion and persisted increasing till 120-min reperfusion in the hepatic terminal portal venules (Fig. 3 A, B and C). Similar results were observed in terminal hepatic venules (Fig. 3 D, E and F) and sinusoids regions (Fig. 3 G and H). However, CA treatment reduced the enhancement in rolling and adhered leukocytes induced by I/R in all the areas examined. The expression of the adhesion molecule CD11b and CD18 on neutrophils is presented as mean fluorescence intensity in Fig. 3I. I/R increased the fluorescence intensity of CD11b significantly compared with sham groups. The expression of CD18 increased as well after I/R but without significance. The increase in the expression of both CD11b and CD18 was significantly attenuated by pretreatment with CA. In addition, as shown in Figure 3J and K, CA pretreatment protected against the upregulation of ICAM-1 and E-selectin in I/R rats. In accord with the above results, western blot analysis revealed evident increase of ICAM-1 in I/R group, whereas CA administration effectively inhibited the increase (Fig. 3L).
CA reduces I/R-induced leukocyte infiltration. The scanning electron microscope revealed no inflammatory cells in sham-operated groups (Fig. 4A, a1-a6), while abundant inflammatory cells and erythrocytes in hepatic sinusoids (Fig. 4A, a8), even thrombus in terminal hepatic venules (Fig. 4A, a9), but few in terminal portal venules (Fig. 4A, a7) after reperfusion. However, CA-treated rats demonstrated few inflammatory cells and erythrocytes (Fig. 4A, a10-a12), indicating that CA may affect I/R-induced leukocytes recruitment. As a marker enzyme of neutrophils, MPO expression in liver tissue was assessed by immunohistochemistry and ELISA assay. The MPO-positive staining cells were detected mainly in hepatic sinusoids (Fig. 4B
b8-1 and b8-2) and terminal hepatic
venules areas (Fig. 4B b9-1 and b9-2) in the liver of I/R rats, whereas few were detected in sham groups (Fig. 4B
b1-1~b6-2). CA treatment group displayed less
positive cells (Fig. 4B b10-1~b12-2). In agreement with the immunohistochemistry results, ELISA results of liver tissue MPO confirmed that CA treatment weakened MPO expression in comparison with I/R rats (Fig. 4C).
CA blunts I/R-induced NF-țB pathway activation and inflammatory cytokines release. Immunofluorescence staining and Western blot result demonstrated that significantly more NF-țB p65 located in nuclei in the I/R groupcompared to sham-operated groups, however, which was blunted by CA administration (Supplementary Fig. 1). Furthermore, the levels of TNF-Į, IL-1ȕ and MCP-1 in liver tissue as well as in
plasma were significantly up-regulated 2 hours after reperfusion. CA administration blocked I/R-induced up-regulation of TNF-Į, IL-1ȕ and MCP-1, suggesting that CA can negatively affect TNF-Į, IL-1ȕ and MCP-1 secretion (Supplementary Fig. 2).
CA inhibits I/R-induced hepatocyte apoptosis. The double staining of F-actin and TUNEL revealed that the positive nuclei were rarely observed in sham groups, in contrast, increased TUNEL-positive nuclei were detected in I/R injured-livers, which was protected against by pretreatment with CA (Fig. 5A and B). Moreover, several apoptosis-regulated proteins were assessed by western blot. Following I/R, the level of Bcl-2 was decreased, while the levels of Cleaved Caspase-9 and Cleaved Caspase-3 were increased (Fig. 5C, D and F, G and H). Notably, CA significantly inhibited the I/R-induced decrease in Bcl-2 expression, and the increase in Cleaved Caspase-9 and Cleaved Caspase-3 expression. A similar pattern of change was observed for Caspase-8 activity, and it was stimulated in I/R rats (Fig. 5E). The expression of Bax showed no apparent difference among the four groups.
CA reduces oxidative stress induced by I/R. H2O2 production, was measured. MDA, an indicator of cellular lipid peroxidation, and 8-OHdG, an oxidative DNA damage marker, were assessed by ELISA to evaluate oxidative injury. The results showed that H2O2, MDA and 8-OHdG increased significantly after I/R whichwere protected by CA pretreatment (Fig. 6A-C).
The expression of MnSOD, SOD and GPx and CAT activity in liver tissue decreased significantly in I/R rats in comparison with that in sham-operated rats, while CA pretreatment almost completely prevented these changes (Fig. 6D-G). There was no significant difference among the four groups at 120 min of reperfusion in the expression of GR in liver tissue (Fig. 6H).
CA mitigates I/R-induced changes in mitochondrial Complexes activity and subunits acetylation. Electron microscopy showed that the hepatocytes in sham-operated rats exhibited normal structure with oval nuclei, regular plasma and nuclear membrane, and oval to round shaped mitochondria with homogenously electron dense matrix and well organized cristae. Obviously, ischemia-reperfusion caused a serious hepatocyte damage, as shown by cell extraction, nucleus pycnosis, mitochondria edema and fragmentation of cristae. The hepatocyte damage was less pronounced in the CA-pretreated group (Fig. 7A). Then we tested with ELISA the activities of MRC Complex I, II, IV and V in liver tissues of different groups. We found that activities of Complex I, II, IV and V were suppressed to varying degrees in I/R group compared with sham groups. Of notice, CA administration significantly restored MRC Complexes activities in I/R rats (Fig. 7B-E). In addition, western blot showed that the expression of SDHA was decreased after I/R, which was recovered by CA treatment (Fig. 7F). Moreover, acetylation of the
SDHA and NDUFA9 was pronouncedly upregulated by I/R compared to that in sham groups, which was remarkably inhibited by pretreatment of CA (Fig. 7G and I). The expression of NDUFA9 showed no significant difference among the four groups (Fig. 7H).
CA binds to and activates Sirt3. After I/R, the expression level of Sirt3 in liver was diminished, while CA treatment restored the level of Sirt3 significantly (Fig. 8A). We determined Sirt3 activity in mitochondrial extracts of liver tissue by ELISA, and found that Sirt3 activity was suppressed after I/R, while CA treatment obviously promoted Sirt3 activity (Fig. 8B). Sirt3 is a NAD+-dependent enzyme, so we detected the change in NAD/NADH and found that NAD/NADH was depressed after I/R, as shown in Fig. 8C, and CA could remarkably enhance its expression. The results above proved that CA influenced the acetylation of downstream substrates of Sirt3, including SDHA and NDUFA9. Surface plasmon resonance (SPR) was further conducted to determine the binding capacity of CA to Sirt3. As shown in Figure 8D and Supplementary Figure 3, CA was able to bind to Sirt3 in a dose-dependent manner. The equilibrium dissociation constants (KD) of CA binding to Sirt3 was 7.931×10-5(M).
Discussion
The present study demonstrated that CA attenuated I/R-induced liver microcirculatory disturbance, inflammation, hepatocytes apoptosis and liver injury. More importantly, CA was revealed able to protect liver from oxidative insults, such as elevation of H2O2, MDA and 8-OHdG, as well as to attenuate I/R-elicited down-regulation of Sirt3 expression and activity, up-regulation of NDUFA9 and SDHA acetylation and decreased MRC Complexes activities. Finally, by SPR, we found that CA could bind to Sirt3. Microcirculatory disturbance acts as an important part in I/R. During the ischemic period, vascular hypoxia can cause increased vascular permeability. After reperfusion, complement system activation, leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation further aggravate microvascular dysfunction. Disturbances of the hepatic microcirculation may lead to no-reflow phenomenon with release of proinflammatory cytokines, sinusoidal plugging of neutrophils, oxidative stress, and hypoxic cell injury and parenchymal failure in I/R [34, 35]. It is reported that inhibition liver microcirculatory disturbance could help to protect the liver against reperfusion damage [34]. In this article, we found that CA attenuated microcirculatory disturbance and I/R injuryin rat, suggesting CA as an option for prophylaxis of I/R-induced liver injury in clinic. The dosage of CA used in our experiments was based on a pilot study, which showed that CA given at 15 mg/kg/h improved hepatic microcirculation and LBF most (Supplementary Fig.8). The total
salvianolic acids injection is injected continuously for the treatment of ischemic stroke in clinic in China. As one of the salvianolic acids, CA was administrated continuously as well in the present study. In addition, that CA was continuously infused was to keep CA at a constant concentration because the half-life of CA is very short. Furthermore, CA administration started from 30 min before ischemia in our experiment was a consideration of clinic translation of the results obtained. Ischemia and reperfusion injury is very common in liver resection and transplantation. To protect against liver I/R injury in such conditions, CA given before surgery may reach an effective concentration during surgery and is feasible in clinic. Oxidative stress plays a central role in I/R injury, and mitochondria is a major source of ROS.Complex I, II and III are all involved in ROS generation [13, 36, 37], thus, any impairment of which trigger I/R injury. For example, decreased Complex I activity and increased ROS generation occurred in hepatic I/R [38]. And Complex II activity impairment augmented the overall magnitude of oxidative injury in the post-ischemic heart [37]. Dysfunction of Complex II-III has been observed in ischemic injury following orthotopic liver transplantation [39]. Therapies that increase Complexes activities may provide protection against I/R injury. To this end, anti-anginal drug ranolazine was reported to improve Complex I structure and function, melatonin had strong protective effect against oxidative alterations to Complex I and III, which are associated with protection of I/R myocardial damage [40, 41].An important finding of the present study is that CA plays a role in restoration of MRC activities following hepatic I/R, suggesting implication of MRC in CA action.
As free radicals scavengers, antioxidant enzymes contribute profoundly to I/R injury. Endogenous antioxidant enzymes, such as SOD, CAT and GPx work together to reduce free radicals and minimize ROS-induced injury [42]. The maintenance of the mitochondrial antioxidant status has been recognized as an important determinant of mitochondrial ROS levels. And upregulation of endogenous antioxidant enzymes exerts protective effects on liver I/R injury [43, 44]. CA and its derivative, caffeic acid phenethyl ester, have been reported to protect mouse brain and liver mitochondria against anoxia–reoxygenation injury mainly due to their antioxidative activities [45]. Caffeic acid phenethyl ester has been shown to exert beneficial effects on I/R injury in rat skin flaps via increasing SOD, GPx enzyme activities [46]. In accord with these previous reports, CA blunted the decrease in MnSOD, SOD, GPx and CAT activity after liver I/R, suggesting antioxidant enzyme as one of the targets for CA to attenuate oxidative injury in liver I/R. Most interestingly, our results revealed CA was able to improve the decrease of Sirt3 expression and activity following liver I/R. Sirt3 belongs to Sirtuins family, and locates in the mitochondria [16, 47]. Sirt3 deacetylates several metabolic and respiratory enzymes highlighting a role in regulating mitochondrial functions [48]. Sirt3 was shown to play an important role in cardiac I/R injury and deacetylates several proteins regulating the response to I/R. Sirt3-deficient rats are more susceptible to cardiac I/R injury, implying increased Sirt3 may protect from I/R injury [19, 32]. It has been reported that Sirt3 directly deacetylates NDUFA9 [16], SDHA [17], ATP synthase ȕ [49] and regulates Complexes activity [50] modulating ROS
production. However, the protective effect of Sirt3 against ischemic processes in liver has not yet been demonstrated. In the present study, we demonstrated that Sirt3 expression and activity was down regulated in liver after I/R, while CA obviously provoked Sirt3 expression and activity, prevented the acetylation of SDHA and NDUFA9 and enhanced MRC activity in liver following I/R, highlighting Sirt3 as the direct target for CA to exert beneficial role in hepatic I/R injury (Supplementary Fig. 13). This argument is further strengthened by the finding that CA is able to bind to Sirt3. However, some issues remain to be addressed. (1) To validate the significance of CA binding to Sirt3 in liver I/R injury, experiment needs to be conducted to knock down Sirt3; (2) More study is required to preclude the involvement of other signaling pathway in CA effect on I/R injury.
Conclusions
Taken together, CA attenuated liver I/R injury and microcirculatory disturbance which is related to inhibition of oxidative stress, possibly by action on Sirt3, reducing acetylation of NDUFA9 and SDHA and improving MRC activities.
Conflict of interest The authors declare no competing financial interest.
Financial Support This work was supported by the Production of New Medicine Program of Ministry of Science and Technology of China [2013ZX09402202].
Acknowledgements The authors thank Li-Jun Wang and Ge Fu for excellent technical assistance.
References
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Figure Legends
Fig. 1. Effects of CA on 24-hour survival rate, ALT, AST and HE. (A) Rats of four groups were monitored for 24 h survival (n=20). (B) Plasma levels of ALT activity. (C) Plasma levels of AST activity. Data are mean ± SEM (n=8). (D) Liver samples in four groups stained by HE. d1, d4, d7 and d10 show portal areas, d2, d5, d8 and d11 show hepatic sinusoids, d3, d6, d9 and d12 show terminal hepatic venules, Bar = 50 ȝm. (E) Liver injury was quantitatively evaluated by Suzuki score. Data are mean ± SEM (n=6). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 2. Effects of CA on number of perfused sinusoids and RBC velocity in microvessles and liver blood flow. (A) and (D) Representative images of sinusoids in terminal portal venule and terminal hepatic venule areas. (B) and (E) Quantitative evaluation of perfused sinusoids in terminal portal venules and terminal hepatic venules, respectively. (C) and (F) Quantitative evaluation of RBCs velocity in terminal portal venules and terminal hepatic venules, respectively. (G) Representative color images of liver blood flow acquired by Laser–Doppler Perfusion Imager. (H) Quantification of the liver blood flow. Arrows in (A) and (B) indicate the sinusoids with no flow. Bar = 50 ȝm. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 3. Effects of CA on leukocyte rolling, adherence and adhesion molecule expression. (A) Representative images of leukocytes adhered to terminal portal venules. (B) Quantitative evaluation of rolling leukocytes in terminal portal venules. (C) Quantitative evaluation of adhered leukocytes in terminal portal venules. (D) Representative images of leukocytes adhered to terminal hepatic venules. (E) Quantitative evaluation of rolling leukocytes in terminal hepatic venules. (F) Quantitative evaluation of adhered leukocytes in terminal hepatic venules. (G) Representative images of leukocytes adhered to sinusoids area. (H) Quantitative evaluation of leukocytes adhered to sinusoids area. Arrows in (A), (D) and (G) indicate adhered leukocytes. Bar = 50 ȝm. Data are mean ± SEM (n=8). (I) The expressions of adhesion molecules CD11b and CD18 on rat neutrophils presented as fluorescence
intensity
of
FITC.
(J)
and
(K)
Representative
images
of
immunofluorescence staining for ICAM-1 and E-selectin, respectively. ICAM-1 and E-selectin are stained green, and nuclei are identified by Hoechst staining in bluecolor. Bar = 50 ȝm. (L) ICAM-1 expression in different groups determined by Western blot. All band intensities were calculated based on the results from 3 independent experiments. All protein intensities were normalized to ߚ-actin. Data are mean ± SEM (n=4). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 4. Ultrastructure of vessels and effects of CA on MPO expression. (A) Scanning electron microscopic images of the vessels in different groups, wherein
arrows indicate adhered leukocytes. a1, a4, a7 and a10 show terminal portal venules. a2, a5, a8 and a11 show hepatic sinusoids. a3, a6, a9 and a12 show terminal hepatic venules. Bar = 5 ȝm. (B) Representative images of immunohistochemistry staining for MPO in different groups, in which arrows indicate MPO-positive leukocytes. The area within the rectangle in each picture is enlarged and presented aside correspondingly. b1-1, b1-2, b4-1, b4-2, b7-1, b7-2, b10-1 and b10-2 show portal areas. b2-1, b2-2, b5-1, b5-2, b8-1, b8-2, b11-1 and b11-2 show hepatic sinusoids. b3-1, b3-2, b6-1, b6-2, b9-1, b9-2, b12-1 and b12-2 show terminal hepatic venules. Bar = 50 ȝm. (C) ELISA analysis of MPO in liver tissue from different groups. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 5. Effects of CA on I/R-induced hepatocyte apoptosis and apoptosis-related protein expressions. (A) The representative photographs of double staining of F-actin and TUNEL, wherein nuclei are stained with blue color, F-actin with red, and TUNEL-positive nuclei green, Bar=50 ȝm. H: hepatocyte; L: leukocyte; S: sinusoidal endothelial cell. a1-a8 show hepatic sinusoids, a9 and a10 show portal areas of NS+I/R group, a11 and a12 show terminal hepatic venules of NS+I/R group. (B) Quantitative analysis of apoptosis cells in various groups. n=6. (C) The expressions of Bax and Bcl-2 determined by Western blot. (F), (G) and (H) Cleaved Caspase-9 and Cleaved Caspase-3 determined by Western blot. (D) The quantitative analysis of the ratio of Bcl-2/Bax. (G) The quantitative analysis of Cleaved Caspase-9. (H) The quantitative
analysis of Cleaved Caspase-3. All the quantifications were undertaken based on the data of 4 animals and normalized to ȕ-actin. All band intensities were calculated based on the results from 3 independent experiments. (E) ELISA analysis results of liver tissue Caspase-8 activity from various groups. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 6. Effects of CA on liver antioxidant enzymes expression, ROS production and oxidative injuryafter I/R. (A) H2O2 production. (B) MDA production. (C) 8-OHdG production. (E) SOD expression. (F) CAT activity. (G) GR expression. (H) GRx expression. The results above were detected by ELISA in liver tissue. Data are mean ± SEM (n=8) (D) Western blot result of MnSOD. The blot image is a representative of 4 animals in a group. The densitometry is an averaged result for the 4 animals and normalized to ȕ-actin. *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 7. Effects of CA on I/R-induced mitochondria injury, Complexes inhibition and subunits acetylation in liver. (A) Ultrastructure of hepatocyte. a1-1, a2-1, a3-1 and a4-1 show hepatocytes at low magnification, Bar=5 ȝm. a1-2, a2-2, a3-2 and a4-2 show mitochondria at high magnification, Bar=1 ȝm. ELISA analysis was performed to determine (B) Complex I Activity, (C) Complex II activity, (D) Complex IV activity and (E) ATP synthase activity in liver tissue. Data are mean ± SEM (n=8). Western blot was conducted for
assessment of (F) SDHA expression, (G) SDHA acetylation, (H) NDUFA9expression and (I) NDUFA9 acetylation in liver tissue. The blot image is a representative of 4 animals in a group. The densitometry is an averaged result for the 4 animals and normalized to ȕ-actin. Data are mean ± SEM (n=4). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group.
Fig. 8. CA interacts with Sirt3. (A) Western blot analysis of Sirt3 expression in liver tissue of different groups. The blot image is a representative of 4 animals in a group. The densitometry is an averaged result for the 4 animals and normalized to ȕ-actin. (B) Sirt3 activity was detected by ELISA in liver tissue. Data are mean ± SEM (n=8). (C) NAD/NADH of liver tissue detected by ELISA. Data are mean ± SEM (n=8). *p < 0.05 vs. NS+sham group; #p < 0.05 vs. NS+I/R group. (D) The affinity of CA and Sirt3 tested by SPR. Shown are the representative sensorgrams obtained from the injections of CA at concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 50 and 200 ȝM (curves from bottom to top) using SPR.
Highlights 1. Caffeic acid (CA) protected rat liver from ischemia and reperfusion (I/R) injury. 2. CA prevented I/R induced decrease of Sirt3 expression and activity. 3. CA reduced acetylation of SDHA and NDUFA9 and enhanced MRC activity.
Graphical Abstract (for review)
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