- Email: [email protected]
NF-κB-mediated protective signaling in mice
European Journal of Pharmacology 697 (2012) 117–125
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Lithium exacerbates hepatic ischemia/reperfusion injury by inhibiting GSK-3b/NF-kB-mediated protective signaling in mice Yongxiang Xia a,b,1, Jianhua Rao a,b,1, Aihua Yao a,b, Feng Zhang a,b, Guoqiang Li a,b,n, Xuehao Wang a,b,n, Ling Lu a,b,n a b
Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing 210029, China
a r t i c l e i n f o
abstract
Article history: Received 7 May 2012 Received in revised form 5 September 2012 Accepted 17 September 2012 Available online 7 October 2012
Lithium (an inhibitor of GSK-3b activity) has beneficial effects on ischemia/reperfusion (I/R) injury in the central nervous system, heart and kidney. However, the role of lithium in hepatic I/R injury is unknown. The aim of this study was to assess the effects of lithium on hepatic I/R injury in a mouse model of partial hepatic I/R. Previous studies showed that lithium chloride (LiCl) can phosphorylate residue Ser9, inhibit GSK-3b activity, and improve I/R injury in other organs. In the present study, mice were pretreated with either vehicle or LiCl, which had similar effects on GSK-3b activity. Surprisingly, treatment with LiCl significantly exacerbated hepatic I/R injury, which was determined by serological and histological analyses. Acute and chronic LiCl treatment caused serious damage in hepatic I/R injury, including increased apoptosis and oxidative stress. To gain insight into the mechanism involved in this damage, the activity of nuclear factor-kB (NF-kB) (GSK-3b can regulate the transcriptional complex of NF-kB) was analyzed, which revealed that LiCl treatment significantly down-regulated the activity of NF-kB. The NF-kB-mediated protective genes were then further evaluated, including anti-apoptotic genes (RAF2, cIAP 2, Bfl-1 and cFLIP) and the antioxidant gene MnSOD. The expression of these protective genes was obviously suppressed compared with the vehicle group. Taken together, these findings show that lithium exacerbates hepatic I/R injury by suppressing the expression of GSK-3b/ NF-kB-mediated protective genes. & 2012 Elsevier B.V. All rights reserved.
Keywords: Lithium Glycogen synthase kinase-3 beta (GSK-3b) Nuclear factor-kB Apoptosis Manganese superoxide dismutase (MnSOD)
1. Introduction Hepatic ischemia/reperfusion (I/R) injury is an adverse phenomenon, which damages hepatocytes and liver sinusoidal endothelial cells in the ischemic liver. Contradictorily, this damage is aggravated following the reestablishment of blood flow. This occurs in various clinical settings, such as liver transplantation, resection of hepatic tumors, trauma, circulatory shock and other insults (Thurman et al., 1988; McCord, 1985; Jaeschke, 2003). Hepatic I/R injury is a major cause of prime graft non-function after liver transplantation, and may critically compromise the function of the remaining liver after major hepatic resection (Burroughs et al., 2006). Although the pharmaceutical effects of different types of drugs and molecules in the prevention of hepatic I/R injury have been evaluated, the outcomes of these drug therapies, such as lithium, were not comparable.
n Corresponding authors at: First Affiliated Hospital of Nanjing Medical University, Liver Transplantation Center, Guangzhou road 300, Nanjng 210029, China. Tel.: þ 86 25 83718836 6476; fax: þ 86 25 83672106. E-mail address: [email protected] (L. Lu). 1 These authors contributed equally to this work.
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.09.009
Lithium has been extensively used as a mood stabilizer in the treatment of bipolar disorder for decades. Interestingly, lithium has been reported to have a potent neuroprotective effect in brain I/R injury. Xu reported that chronic treatment with lithium at a low dose exhibited neuroprotection in transient focal cerebral ischemia by reducing apoptotic death (Xu et al., 2003). Subsequently, Ren M demonstrated that post-insult treatment with lithium reduces brain damage and facilitates neurological recovery in a rat I/R model (Ren et al., 2003). Other investigators have suggested that pretreatment with lithium chloride, before ischemia or immediately on reperfusion, can phosphorylate and inhibit GSK-3b activity and subsequently improve the recovery of postischemic function, and reduce the infarct size in myocardial I/R injury (Barillas et al., 2007; Faghihi et al., 2008; Kaga et al., 2006; Mozaffari, Schaffer (2008)). Lithium also reduced kidney I/R injury (Talab et al., 2010). The putative mechanisms involved in lithiuminduced neuroprotection are complex, and may include inhibition of GSK-3b (Barillas et al., 2007), stimulation of heat shock protein70 (Xu et al., 2006) and activation of the extracellular regulated protein kinases (ERK) signaling pathway (Yan et al., 2007). Furthermore, inhibition of GSK-3b has also been confirmed in the cardio- and reno-protection of lithium treatment in mouse I/R
118
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
injury. GSK-3b has been reported to be involved in cell membrane-to-nucleus signaling, including transcription factor activation, cytoskeletal organization, growth, survival and apoptosis (Frame and Cohen, 2001; Kockeritz et al., 2006). To date, however, no data are available on the effects of lithium on hepatic I/R injury. This study was designed to elucidate the potential roles and mechanisms of acute and chronic lithium treatment on hepatic I/R injury.
2. Materials and methods 2.1. Animals Male C57BL/6 mice weighing 22–25 g were purchased from the Animal Center of Nanjing University (Nanjing, China). The mice were kept under constant environmental conditions with a 12 h light–dark cycle, and with free access to standard rodent diet and water. The mice were fasted before surgery. The experiments were carried out in accordance with the guidelines approved by the Institutional Animal Care and Use Committee at the Nanjing Medical University (Protocol Number NJMU08-092). 2.2. Experimental design and surgical procedure The mice were randomly divided into the following experimental groups: (1) Sham group (n¼12), mice were subjected to the surgical procedure with the omission of vascular occlusion. (2) Vehicle group, mice were subjected to 90 min of ischemia followed by 1 h, 3 h, 6 h, or 24 h of reperfusion; mice were treated with saline 30 min before ischemia (n¼6 per point). (3) Acute LiCl group, all mice underwent the I/R procedure, similar to the vehicle group, but were treated with LiCl (42.3 mg/kg, i.v.; Sigma, St. Louis, MO, USA) 30 min before I/R surgery (n¼6 per point). (4) Chronic LiCl group, all mice underwent the I/R procedure, similar to the vehicle group, but were treated with LiCl (42.3 mg/kg, i.p. qd) for one week before I/R surgery (n¼6 per point). Mice were anesthetized by intraperitoneal injection of 50–60 mg sodium pentobarbital per kilogram. A midline laparotomy incision was performed, extending from the xiphisternum to the pubis, to expose the liver. Ischemia of the left and median liver lobes was induced by the placement of an atraumatic bulldog clamp across the pedicles of the left portal vein, hepatic artery and bile duct for 90 min, as described previously (Abe et al., 2009), which led to segmental (70%) hepatic ischemia. Mice were sacrificed at different periods after reperfusion to obtain liver tissue and blood samples.
TUNEL using a commercially available kit (In situ cell death detection kit, Roche-Boehringer Mannheim, Germany). 2.5. GSK-3b and caspase-3 activity GSK-3b activity was measured in liver tissues 1 h after reperfusion. Frozen liver tissues were homogenized and immunoprecipitated using anti-GSK-3b antibody (Sigma). The activity of the enzyme was assessed by a kinase assay using [32P]-ATP and glycogen synthase peptide-2 (Sigma) as a substrate, as described by Haq et al., 2000). Caspase-3 activity was determined in liver tissues 6 h after reperfusion. Frozen samples of ischemic tissues were homogenized using a polytron homogenizer and centrifuged at 16,000 g for 20 min. The activity was measured using an assay kit (Calbiochem) according to the manufacturer’s instructions, as described by Ke et al. (2009). 2.6. Liver tissue homogenate malondialdehyde (MDA) assay The liver tissues homogenized in 100 mmol/L Tris–HCl buffer were centrifuged at 10,000 g for 20 min. Total protein concentration was determined by the Coomassie blue method. MDA was measured using kits according to the manufacturer’s instructions (Jiancheng Biotechnology, Nanjing, China). The results were expressed in nmol MDA per milligram of tissue homogenate 2.7. Western blot analysis Proteins were extracted from liver tissues subjected to ischemia, and their concentrations were determined by the Bradford assay (Bio-Rad, CA, USA). Nuclear protein extracts were prepared according to the manufacturer’s protocol (NE-PER Nuclear and cytoplasmic extraction reagents; Thermo Scientific, IL, USA). 30 mg of the protein sample was resolved by SDS-PAGE and transferred to nitrocellulose membranes (Sunshine Biotechnology, China). These membranes were blocked in (5% wt/vol) skim milk powder-Trisbuffered saline (TBS) with 0.1% Tween 20 (TBS-T) at 4 1C overnight. Membranes were then incubated with antibodies to Ser9-phosphoGSK-3b, GSK-3b, MnSOD, GAPDH, Ser32/36-phospho-IkB-a (Cell Signaling Technology, Danvers, MA, USA), p65 and IkB-a (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following three washes with TBS-T, the membranes were then incubated for 1 h at room temperature with goat-anti-rabbit peroxidase-conjugated secondary antibody (Santa Cruz). The final results were obtained by exposure to Kodak autoradiography film (Kodak XAR film). The results were visualized via a chemiluminescent detection system (ECL Substrate Western blot detection system, Pierce, IL, USA).
2.3. Serum ALT 2.8. Quantitative real-time PCR Blood samples for alanine aminotransferase (ALT) assessment were obtained after 6 h of reperfusion, and analyzed using a serum analyzer (Hitachi 7600-10, Hitachi High-Technologies Corporation, Japan). 2.4. Hematoxylin and eosin (HE), immunohistochemistry (IHC), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Liver tissues were removed and fixed in 4% paraformaldehyde overnight at 4 1C and then processed, the tissues were cut into 5 mm thick sections and stained with hematoxylin and eosin (HE) using standard techniques. The sections were placed on slides and stained immunohistochemically for b-catenin (Cell Signaling Technology), p65 (Santa Cruz Biotechnology) using techniques described previously (Sun et al., 2005). To determine apoptotic cell injury in liver tissue induced by I/R, sections were stained by
Reverse transcription reactions were performed using the Super-Script First-Strand Synthesis System (Invitrogen, CA, USA). To determine the relative number of cDNA molecules in the reverse transcribed samples, real-time PCR analyses were performed using the Light-Cycler system (Roche, Indianapolis, IN, USA). PCR was performed according to the procedure previously described (Jiang et al., 2010). PCR was performed using 10 ml 2x Master Mix SYBR Green I (Takara, Japan), 0.25 mM of each 50 and 30 primer, and 2 ml samples or H2O to a final volume of 20 ml. Samples were denatured at 94 1C for 5 min. Amplification and fluorescence determination were carried out in 3 steps: denaturation at 94 1C for 10 s, annealing at 60 1C for 15 s, extension at 72 1C for 20 s; and at the end of extension, detection of SYBR green fluorescence, which reflects the amount of doublestranded DNA. The amplification cycle number was 35. To discriminate specific from nonspecific cDNA products, a melting
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
curve was obtained at the end of each run. Products were denatured at 95 1C for 3 s, and the temperature was then decreased to 58 1C for 15 s and raised slowly from 58 1C to 95 1C using a temperature transition rate of 0.1 1C/sec. Data were normalized with GAPDH levels in the samples. Primers were designed by Oligo 6.0. Primer sets (sense sequence and antisense sequence, respectively) for the following genes were: Bfl-1 forward, 50 -GGC TGA GCA CTA CCT TCA GTA-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 ; c-IAP2 forward, 50 -TCA GTG ACC TCG TTA TAG GCT T-30 , reverse, 50 -TCA CAC ACG TCA AAT GTT GGA A30 ; TRAF2 forward, 50 -AGA GAG TAG TTC GGC CTT TCC-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 ; c-FLIP forward, 50 -GGA TTA CAA GGG ATT ACA CAG GC-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 ; MnSOD forward, 50 -CAG ACC TGC CTT ACG ACT ATG G-30 , reverse, 50 -CTC GGT GGC GTT GAG ATT GTT-30 ; GAPDH forward, 50 -GGT CAC CAG GGC TGC CAT TTG-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 .
2.9. Electrophoretic mobility shift assays (EMSAs) To determine NF-kB activation, we performed EMSA, as described (Chen et al., 2007). EMSAs were performed using a Biotin Gel Shift Kit (Pierce, Rockford, IL, USA). The probe was labeled with biotin at the 30 -end, and the sequence was: 50 -TTG TTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GCG TGG-30 (boldface indicates NF-kB-binding sites).
2.10. Mouse hepatocyte isolation Mouse hepatocytes were isolated using a two-step in situ collagenase perfusion procedure as previously described (Hatano and Brenner (2001)). The livers from the C57BL/6 mice were perfused in situ through the portal vein with EGTA buffer (0.5 mM EGTA, 137 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 0.65 mM MgSO4, and 10.07 mM HEPES at pH 7.4) at a flow rate of 5 ml/ min for 10 min, followed by collagenase buffer (67 mM NaCl, 6.7 mM KCl, 4.76 mM CaCl2, 0.035% collagenase type II, and 10.07 mM HEPES at pH 7.6) at a flow rate of 5 ml/min for 15 min. After centrifugation, the hepatocytes were collected and seeded in DMEM containing 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin.
2.11. Hepatocyte apoptosis assays To determine the effects of LiCl on hepatocyte apoptosis induced by H2O2 in vitro, hepatocytes were treated with different doses of LiCl for 24 h as indicated, with or without the addition of 100 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK), and then incubated with 200 mm H2O2 for 6 h. Cells were harvested and washed twice with ice-cold PBS. Specific binding of FITC-annexin V and staining with PE-propidium iodide (PI) were performed using an apoptosis detection kit (BD, USA), according to the manufacturer’s instructions. The cells were then analyzed using flow cytometry.
2.12. Statistical analysis All data are expressed as mean 7standard deviation (S.D) and analyzed with the SPSS statistical package. Differences between groups were analyzed using a one-way analysis of variance with subsequent Student–Newman–Keuls test. Differences were considered statistically significant at a probability level of Po0.05.
119
3. Results 3.1. LiCl inhibited GSK-3b activity and exacerbated hepatic I/R injury GSK-3b activity was assessed by Western-blot detection of the phosphorylated residue Ser9 on GSK-3b. Compared with the sham control, the I/R group had a higher level of phosph-GSK-3b. Interestingly, both acute and chronic LiCl treatment significantly increased phosph-GSK-3b levels after liver I/R, while the total GSK-3b remained unchanged (Fig. 1A). In addition, our results showed that the inhibitory effect was not significantly different between the acute and chronic treatment. To further confirm LiClmediated alteration of GSK-3b activity, we next performed an immune complex kinase assay. As observed with Western blotting, hepatic I/R resulted in lower GSK-3b activity compared to the sham group, these effects were further enhanced by acute and chronic treatment with LiCl (Fig. 1B). These results indicate that hepatic I/R induced phosphorylation of GSK-3b and inhibited GSK-3b activity; these effects were augmented by LiCl treatment. The extent of hepatic damage was assessed by histology and serum ALT. Surprisingly, the role of LiCl on hepatic I/R injury was different from its protective effect on the cardiovascular system, urinary system and central nervous system. Hematoxylin-eosin (HE) staining was carried out on mouse ischemic liver lobes after 6 h of reperfusion (Fig. 1C). Mouse livers treated with vehicle showed hepatocellular necrosis and vacuolization. Indeed, the morphologic changes demonstrated that LiCl treatment significantly exacerbated liver damage. Furthermore, the histology findings were reflected by the serum ALT assay, which showed that LiCl treatment significantly increased serum ALT level (acute LiCl 1368072470 U/l, chronic LiCl 1417072950 U/l) compared with the vehicle treatment group (8100 7450 U/l) (P o0.01 for each case) (Fig. 1D). Thus, our results demonstrated that LiCl treatment exacerbated hepatic I/R injury. 3.2. LiCl inhibited hepatic I/R-induced NF-kB activation Previous studies (Hoeflich et al., 2000), in addition to our findings, showed that LiCl promoted hepatocyte injury, and indicated that GSK-3b may be linked to regulation of NF-kB activation. Therefore, we examined the effect of GSK-3b inhibition on the activation of NF-kB using EMSA. Our results showed that NF-kB activation was significantly decreased after acute and chronic LiCl treatment, although hepatic I/R stimulated NF-kB DNA binding activity (Fig. 2A). These results suggest that LiCl affects NF-kB signaling by regulating GSK-3b activity during hepatic I/R. To further confirm the impact of LiCl on the suppression of NF-kB signaling, we analyzed the nuclear import of p65 (Fig. 2B). Nuclear accumulation of p65 was much lower in LiCl pretreated mice compared with the I/R group, which was further confirmed by immunohistochemical staining (2173% for acute LiCl versus 81710% for vehicle; 1972.5% for chronic LiCl versus 81710% for vehicle, each Po0.01) (Fig. 2C). Translocation of NF-kB to the nucleus requires proteolytic degradation of IkB-a (Ghosh, Karin (2002)), thus, we then carried out Western-blots to detect degradation of IkB-a. Fig. 2B shows that hepatic I/R induced IkB-a degradation was partially inhibited by acute and chronic LiCl treatment. The proteolytic degradation of IkBa requires phosphorylation at serine residues 32 and 36 (Ghosh and Karin, 2002). To further assess the degradation of IkB-a, we assessed the IkB-a phosphorylation level, which indicated that I/R-induced IkB-a phosphorylation was significantly suppressed (Fig. 2B). 3.3. LiCl increased hepatocellular apoptosis in vivo and in vitro To analyze the relationship between aggravated damage by LiCl treatment and suppression of NF-kB activation, we next
120
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
A
B
Fig. 1. LiCl inhibited GSK-3b activity and exacerbated hepatic I/R injury. (A) Effects of acute and chronic LiCl administration on GSK-3 phosphorylation of ischemia liver tissues at 1 h after reperfusion was detected by Western blot. Each immunoblot is from a single experiment and is representative of three separate experiments. Densitometric analysis of the bands is expressed as the relative OD of GSK-3 phosphorylation at Ser9, corrected for the corresponding total GSK-3 content, and normalized using the sham band. Data are means7 S.D of three separate experiments. nPo 0.05 vs vehicle. OD indicates optical density. (B) GSK-3 activity in ischemia liver tissues at 1 h after reperfusion. Results were the mean 7S.D values of 6 mice. nPo 0.05 vs vehicle. (C) Liver histopathology at 6 h after reperfusion (400 ). Sham: Normal hepatic architecture was observed in sham-operated mice. Vehicle: apparent hemorrhage and hepatocellular necrosis could be detected. Acute and chronic LiCl treatment: significantly more hepatocellular necrosis was apparent. (D) Liver injury was measured by serum levels of ALT, at 6 h after reperfusion following 90 min of ischemia. Results were the mean 7S.D values of 6 mice, nP o 0.01 vs the vehicle.
examined the expression of NF-kB-mediated anti-apoptotic genes (TRAF2, cIAP 2, Bfl-1 and cFLIP) in liver tissue after 3 h of reperfusion by real-time PCR. The results showed that transcription of these anti-apoptotic genes was significantly inhibited by acute and chronic LiCl treatment (Fig. 3A). In parallel with the expression of anti-apoptotic genes, the activity of caspase-3 showed a consistent increase after LiCl treatment in ischemic livers (vehicle 2.170.4, acute LiCl 3.270.7, chronic LiCl 3.670.9) (Fig. 3B). The TUNEL assay also indicated that hepatocellular apoptosis was significantly increased in LiCl treated liver compared with vehicle control (vehicle-control 5.271.1%, acute LiCl 15.472.9%, and chronic LiCl 16.7 73.3%) (Fig. 3C). To further determine the effects of LiCl on hepatocellular apoptosis in hepatic I/R injury, we imitated the hepatic I/R injury model in vitro using H2O2 stimulated hepatocytes. The results showed that LiCl treatment significantly enhanced hepatocellular
apoptosis induced by H2O2 (Fig. 3D). In addition, to evaluate whether this apoptosis was caspase-dependent, the caspase inhibitor, z-vad-fmk, was used on the hepatocyte culture, which showed that the LiCl-induced increase in hepatocellular apoptosis is partially caspase dependent (Fig. 3D).
3.4. LiCl increased ROS production in hepatic I/R injury Reactive oxygen species (ROS) injury is an important cause of hepatic I/R injury. We further investigated the related factors, ROS and MnSOD. MnSOD, the NF-kB-mediated mitochondrial SOD isoform, is a key antioxidant enzyme in the antioxidant system. We assessed the levels of mRNA and protein coding for MnSOD by real-time PCR and Western blots. As shown in Fig. 4, MnSOD mRNA expression was significantly reduced following treatment
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
121
Fig. 2. LiCl decreased I/R-induced NF-kB activation. (A) Nuclear extracts from liver tissues at 1 h, 3 h and 6 h after reperfusion were subjected to electrophoretic mobility shift assay. The NF-kB activity was quantified by image analysis of autoradiograms. Results were the mean 7 S.D values of 6 mice. nP o0.05, acute LiCl treatmen vs vehicle; # P o 0.05, chronic LiCl treatment vs vehicle. (B) At 1 h, 3 h and 6 h after reperfusion, nuclear extract of ischemic liver tissues was prepared and Western blot analysis using anti-p65 antibody was performed. Total extracts were prepared and Western blot analysis using anti-IkB-a, phospho-ser-32/36-IkB-a and p65 antibody was performed. (C) At 3 h of reperfusion, nuclear accumulation of p65 was assessed by immunohistochmistry (400 ). The number of nuclear p65-positive cells was quantified from six fields at high-power magnification. nPo 0.05 vs vehicle.
with LiCl at 3 h after I/R (Fig. 4A), and the corresponding change in protein levels was confirmed by Western blots (Fig. 4B). Malondialdehyde (MDA), an index of lipid peroxidation, directly reflects the levels of ROS. To detect the levels of ROS, we next
measured MDA content. The results indicated that MDA levels were significantly increased in ischemic liver following acute and chronic LiCl treatment (vehicle 118 714, acute LiCl 276725, and chronic LiCl 256 726) (Fig. 4C).
122
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
Fig. 3. LiCl increased hepatocellular apoptosis in vivo and in vitro. (A) At 3 h after reperfusion, expression of Traf-2, cIAP2, cFlip and Bfl1 were detected by real-time PCR. nn P o 0.01, nP o 0.05, acute LiCl treatment vs vehicle. ##Po 0.01, #P o 0.05 chronic LiCl treatment vs vehicle. (B) At 6 h after reperfusion, the activity of caspase-3 was measured. nP o 0.05 vs vehicle. (C) Apoptotic cells were quantified in six high-power fields (400 ), and expressed as percentages of apoptotic cells among total cells. Results were the mean 7 S.D values of 6 mice, nPo 0.05 vs vehicle. (D): Effects of different dose LiCl on hepatocytes apoptosis induced by H2O2 in vitro, nnP o 0.01, nPo 0.05, LiCl treatment vs without LiCl treatment; effects of Z-VAD-FMK on LiCl on hepatocytes apoptosis induced by H2O2, nP o0.05, Z-VAD-FMK treatment vs without Z-VAD-FMK treatment.
4. Discussion Lithium is an established drug used in the treatment of manic depressive disorders. Recently, in vivo and in vitro studies have suggested that lithium is involved in oxidative stress, growth, survival and apoptosis. Moreover, lithium has robust protective effects in ischemic organ injury, including the brain, heart and
kidney. Therefore, our initial thoughts were to evaluate the protective effect of lithium during hepatic I/R injury. However, surprisingly, our studies revealed a damaging effect due to lithium during hepatic I/R injury. Lithium is a nonspecific inhibitor (Phiel and Klein, 2001), but has been used extensively to study cardioprotection by inhibiting GSK-3b activity (Haq et al., 2000; Klein and Melton, 1996). Our
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
123
Fig. 4. LiCl increased ROS injury in hepatic I/R injury. (A) At 3 h after reperfusion, expression of MnSOD mRNA was measured by real-time PCR. Results were the mean 7 S.D values of 6 mice. nPo 0.05 vs vehicle. (B) At 3 h after reperfusion, expression of MnSOD was determined by Western blot. Acute and chronic LiCl treatment significantly decreases MnSOD than vehicle group. (C) At 6 h after reperfusion, the levels of liver tissue MDA were determined. Results were the mean 7 S.D values of 6 mice. nP o0.05 vs vehicle.
study also confirmed that both acute and chronic LiCl treatment phosphorylated and inhibited GSK-3b activity. A previous report showed that phosphorylation and inactivation of GSK-3b contributed to hepatocyte apoptosis and severe ischemic injury in liver grafts during cold preservation (Li et al., 2003, 2005). However, warm I/R injury might have a different mechanism to that of cold I/R injury. In agreement with these studies, our results showed that inhibition of GSK-3b using LiCl exacerbated liver damage following I/R. Although these data are different from previously published reports, which showed that inhibition of GSK-3b activity resulted in hepatoprotection using GSK-3 specific inhibitors in liver I/R injury (Ren et al., 2011), our findings, at least partially, revealed the functional role of LiCl in hepatic I/R injury. The molecular mechanisms of LiCl in hepatic I/R injury are complex. It has been demonstrated that lithium treatment inhibited translocation of NF-kB and sensitized fibroblasts to TNF-amediated apoptosis (Hoeflich et al., 2000). Likewise, inhibition of GSK-3b activity by lithium down-regulated NF-kB-dependent gene transcription induced by TNF-a and sensitized hepatocytes to TNF-a-mediated apoptosis (Schwabe and Brenner, 2002). Consistent with these studies, our EMSA assay demonstrated inhibition of GSK-3b activity leading to suppression of NF-kB signaling after LiCl treatment in ischemic livers, which was similar to the conclusion drawn from GSK-3b-knockout mice (Hoeflich et al., 2000). Furthermore, we found that inhibition of GSK-3b activity suppressed phosphorylation and degradation of IkB-a, disturbed cytosolic-nuclear translocation of p65 after I/R, and finally suppressed NF-kB activation. Our results were consistent with previous findings by Takada et al. (2004), who demonstrated that TNF-a-induced IkB-a phosphorylation, IkBa degradation and NF-kB activation were all suppressed in GSK-3b ¨ gene deleted fibroblasts. Gotsche et al. (2008) also showed that GSK-3b inhibition reduced NF-kB translocation and binding activity due to impaired degradation of IkB-a in primary mouse hepatocytes. Therefore, we conclude that inhibition of GSK-3b activity enhanced the stability of IkB-a and inhibited NF-kB activity after LiCl treatment in hepatic I/R injury. Hepatocyte death after hepatic I/R is likely to be a combination of apoptotic and necrotic pathways, known as ‘‘necrapoptosis‘‘
(Gujral et al., 2001). Fig. 1C indicates that lithium enhanced apoptosis and necrosis of hepatocytes induced by hepatic I/R. We then analyzed the effects of lithium on apoptosis of hepatocytes after hepatic I/R by TUNEL staining and caspase-3 activity, which indicated that LiCl treatment enhanced hepatocellular apoptosis after hepatic I/R (Fig. 3B and C). In addition, to further determine the effects of LiCl on hepatocellular apoptosis in hepatic I/R injury, we imitated the hepatic I/R injury model in vitro using H2O2-induced hepatocellular apoptosis, which demonstrated that LiCl treatment significantly increased apoptosis of hepatocytes induced by H2O2 in vitro (Fig. 3D). Other reports have already shown that activation of NF-kB has a protective effect after hepatic I/R, which can up-regulate expression of anti-apoptotic genes and prevent the accumulation of damaging ROS (Huber et al., 2009; Kuboki et al., 2009). Our results demonstrated that inactivation of GSK-3b resulted in inhibition of NF-kB activation and the expression of anti-apoptotic genes (TRAF2, cIAP2, Bfl-1 and cFLIP) after lithium pretreatment followed by I/R. These results were supported by recent reports which demonstrated that GSK-3b inhibition induces glioma cell apoptosis through c-Myc, NF-kB and glucose regulation (Kotliarova et al., 2008). In addition, our findings confirmed the hypothesis of Li et al. (2005), who suggested that inhibition of GSK-3b by the PI3K/Akt pathway might account for cell apoptosis by dysregulation of the transcription of NF-kB and energy depletion during hepatic I/R. Since PI3K/Akt is found upstream of GSK-3, it is possible that GSK-3 activity is modulated by LiCl via PI3K/Akt signaling (Li et al., 2005). Indeed, Akt acts as an anti-apoptotic signaling molecule, which can inhibit caspasemediated cell death through phosphorylation of the antiapoptotic molecule-associated death promoter, and direct phosphorylation of caspase protease (Mullonkal and Toledo-Pereyra, 2007). In addition, AKT can promote survival by phosphorylation of Bad, whereas the dephosphorylation of Bad by calcineurin was a response to calcium overload (Wang et al., 1999). Phosphorylation of AKT and downstream GSK-3 provides cytoprotection by inhibiting the mitochondrial permeability transition pore (mPTP) opening (Zhang et al., 2011), suggesting that inhibition of GSK-3 activity by LiCl might directly regulate mitochondria membrane
124
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
LiCl I/R Hepatocyte
P GSK3β
TRAF2 cIAP2 Bfl-1 cFLIP
P
PI3K AKT
P PIP3
NF-κB MnSOD ROS
anti-apoptosis
Ca2+
Bad
P
Survival
to enhanced hepatic I/R injury by impairing the ability of hepatic anti-apoptotic genes and enhancing ROS production (Fig. 5). Lithium treatment inhibits GSK-3b activity leading to inactivation of NF-kB in hepatic I/R injury. Although our current study did not assess whether LiCl affects other signaling pathways, such as heat shock protein-70, ERK, and PI3K/Akt, these findings might explain the functional role and potential mechanism of lithium in inhibiting GSK-3/NF-kB protective signaling in hepatic I/R injury. Considering that lithium is a nonspecific inhibitor of GSK-3b, other mechanisms may be involved in the procedure. Further studies on whether lithium affects other targets during hepatic I/R are necessary.
Caspase-3 apopostis
Fig. 5. The effects of LiCl on hepatocellular apoptosis in hepatic I/R injury. GSK-3: glycogen synthase kinase-3; PI3K: phosphatidylinositol 3-kinase; AKT: Serine/ threonine kinase; TRAT2: TNF receptor-associated factor 2; Bfl-1: Bcl-2-related protein A1; cFLIP: cellular flice like inhibitory protein; cIAP2: cellular inhibitor of apoptosis 2; PIP3: phosphatidylinositol (3,4,5)-trisphosphate; solid arrow: inhibitory modification; hollow arrow: stimulatory modification.
potential change. Caspase-3 is an effecter molecule of apoptosis, which can be induced by many harmful factors, including hypoxia, stress, energy depletion, and calcium overload. Activation of caspase3 in mitochondria may be required for the effect of LiCl on liver damage triggered by I/R. In this study, our results showed that caspase-3 activity in liver was significantly increased by LiCl treatment after liver I/R. Taken together, the liver may be more sensitive to I/R injury due to multiple signaling after lithium treatment. It is possible that other hepatic impairment factors may be involved in the I/R process after lithium treatment. Specifically, the transport pathway, and the location of LiCl concentration might be critical in the modulation of GSK-3 activity in the liver in a concentration-dependent manner. We compared the effect of therapeutic concentrations of LiCl in an in vivo model. Although lacking a specific receptor in vivo, both acute and chronic LiCl treatment exacerbated liver damage following I/R, which suggested that the ischemic liver is sensitive to LiCl. More importantly, it has been shown that ROS play an important role in I/R-induced liver damage. Indeed, ROS may initiate the cascade of cell injury, necro/ apoptosis, and subsequent inflammatory infiltration (Crenesse et al., 2000; Liang et al., 2009). Conversely, MnSOD is a critical enzyme in cellular protection against ROS generation, and is considered to be a decisive player against hepatic I/R injury (Rao et al., 2010). Decreased levels of MnSOD implied that hepatic damage was more severe when hepatic GSK-3b was inhibited. Recent studies have shown that hepatic I/R-induced MnSOD expression is mediated through the activation of NF-kB (Llacuna et al., 2009). In the present study, the levels of hepatic MnSOD were significantly reduced after acute and chronic LiCl pretreatment followed by I/R. These findings may be correlated with inactivation of NF-kB. Furthermore, the levels of MDA, which directly reflect ROS production (Rao et al., 2010), were significantly increased after lithium treatment, suggesting that inactivation of NF-kB signaling induced by GSK-3b inhibition could suppress the expression of MnSOD, up-regulate MDA, and enhance ROS production. The outcome further suggested that acute and chronic treatment with lithium exacerbated hepatic I/R injury through up-regulation of ROS production via GSK-3b/NF-kB signaling.
5. Conclusions In conclusion, we demonstrated that lithium suppressed activation of NF-kB and NF-kB-mediated protective genes leading
Acknowledgements This work was supported by grants from National Eleventh Five-support sub-subject research of China (Grant numbers: 2008BAI60B02); Natural Science Foundation of China (Grant numbers: 81100270, 81070380, 30872390); Natural Science Foundation of Jiangsu province (Grant numbers: BK2009439, BZ2011041, ZX05 200904, WS2011106). First Innovation Team Foundation of Jiangsu province Hospital (for Sun BC). The authors would like to appreciate Shouyu Wang (Nanjing Medical University) for excellent technical assistance.
References Abe, Y., Hines, I.N., Zibari, G., Pavlick, K., Gray, L., Kitagawa, Y., Grisham, M.B., 2009. Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo. Free Radical Biol. Med. 46, 1–7. Barillas, R., Friehs, I., Cao-Danh, H., Martinez, J.F., del Nido, P.J., 2007. Inhibition of glycogen synthase kinase-3beta improves tolerance to ischemia in hypertrophied hearts. Ann. Thorac. Surg. 84, 126–133. Burroughs, A.K., Sabin, C.A., Rolles, K., Delvart, V., Karam, V., Buckels, J., O’Grady, J.G., Castaing, D., Klempnauer, J., Jamieson, N., Neuhaus, P., Lerut, J., de Ville, de Goyet, J., Pollard, S., Salizzoni, M., Rogiers, X., Muhlbacher, F., Garcia Valdecasas, J.C., Broelsch, C., Jaeck, D., Berenguer, J., Gonzalez, E.M., Adam, R., European Liver Transplant Association, 2006. 3-month and 12-month mortality after first liver transplant in adults in Europe: predictive models for outcome. Lancet 367, 225–232. Chen, R., Qiu, W., Liu, Z., Cao, X., Zhu, T., Li, A., Wei, Q., Zhou, J., 2007. Identification of JWA as a novel functional gene responsive to environmental oxidative stress induced by benzo[a]pyrene and hydrogen peroxide. Free Radical Biol. Med. 42, 1704–1714. Crenesse, D., Schmid-Alliana, A., Hornoy, J., Rossi, B., Gugenheim, J., 2000. Hypoxiareoxygenation differentially stimulates stress-activated protein kinases in primary-cultured rat hepatocytes. Transplant. Int. 13, S597–S599. Faghihi, M., Mirershadi, F., Dehpour, A.R., Bazargan, M., 2008. Preconditioning with acute and chronic lithium administration reduces ischemia/reperfusion injury mediated by cyclooxygenase not nitric oxide synthase pathway in isolated rat heart. Eur. J. Pharmacol. 597, 57–63. Frame, S., Cohen, P., 2001. GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359 (Pt 1), 1–16. Ghosh, S., Karin, M., 2002. Missing pieces in the NF-kappaB puzzle. Cell 109, S81–S96. ¨ Gotsche, l.F., Kern, C., Lang, S., Sparna, T., Markmann, C., Schwager, J., McNelly, S., ¨ von Weizsacker, F., Laufer, S., Hecht, A., Merfort, I., 2008. Inhibition of GSK3 differentially modulates NF-kappaB, CREB, AP-1 and beta-catenin signaling in hepatocytes, but fails to promote TNF-alpha-induced apoptosis. Exp. Cell. Res. 314, 1351–1366. Gujral, J.S., Bucci, T.J., Farhood, A., Jaeschke, H., 2001. Mechanism of cell death during warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis? Hepatology 33, 397–405. Haq, S., Choukroun, G., Kang, Z.B., 2000. Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol. 151, 117–130. Hatano, E., Brenner, D.A., 2001. Akt protects mouse hepatocytes from TNF-alphaand Fas-mediated apoptosis through NK-kappa B activation. Am. J. Physiol. Gastrointest. Liver Physiol. 281, G1357–G1368. Hoeflich, K.P., Luo, J., Rubie, E.A., Tsao, M.S., Jin, O., Woodgett, J.R., 2000. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 406, 86–90. Huber, N., Sakai, N., Eismann, T., Shin, T., Kuboki, S., Blanchard, J., Schuster, R., Edwards, M.J., Wong, H.R., Lentsch, A.B., 2009. Age-related decrease in proteasome expression contributes to defective nuclear factor-kappaB activation during hepatic ischemia/reperfusion. Hepatology 49, 1718–1728.
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
Jaeschke, H., 2003. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am. J. Physiol. Gastrointest. Liver Physiol. 284, G15–G26. Jiang, R., Xia, Y., Li, J., Deng, L., Zhao, L., Shi, J., Wang, X., Sun, B., 2010. High expression levels of IKKalpha and IKKbeta are necessary for the malignant properties of liver cancer. Int. J. Cancer 126, 1263–1274. Kaga, S., Zhan, L., Altaf, E., Maulik, N., 2006. Glycogen synthase kinase-3beta/betacatenin promotes angiogenic and anti-apoptotic signaling through the induction of VEGF, Bcl-2 and survivin expression in rat ischemic preconditioned myocardium. J. Mol. Cell. Cardiol. 40, 138–147. Ke, B., Shen, X.D., Gao, F., Qiao, B., Ji, H., Busuttil, R.W., Volk, H.D., KupiecWeglinski, J.W., 2009. Small interfering RNA targeting heme oxygenase-1 (HO-1) reinforces liver apoptosis induced by ischemia-reperfusion injury in mice: HO-1 is necessary for cytoprotection. Hum. Gene Ther. 20, 1133–1142. Klein, P.S., Melton, D.A., 1996. A molecular mechanism for the effect of lithium on development. Proc. Nat. Acad. Sci. U.S.A. 93, 8455–8459. Kockeritz, L., Doble, B., Patel, S., Woodgett, J.R., 2006. Glycogen synthase kinase3—an overview of an over-achieving protein kinase. Curr. Drug. Targets 7, 1377–1388. Kotliarova, S., Pastorino, S., Kovell, L.C., Kot liarov, Y., Song, H., Zhang, W., Bailey, R., Maric, D., Zenklusen, J.C., Lee, J., Fine, H.A., 2008. Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation. Cancer Res. 68, 6643–6651. Kuboki, S., Sakai, N., Clarke, C., Schuster, R., Blanchard, J., Edwards, M.J., Lentsch, A.B., 2009. The peptidyl-prolyl isomerase, Pin1, facilitates NF-kappaB binding in hepatocytes and protects against hepatic ischemia/reperfusion injury. J. Hepatol. 51, 296–306. Li, X.L., Man, K., Liu, Y.F., Lee, T.K., Tsui, S.H., Lau, C.K., Lo, C.M., Fan, S.T., 2003. Insulin in University of Wisconsin solution exacerbates the ischemic injury and decreases the graft survival rate in rat liver transplantation. Transplantation 76, 44–49. Li, X.L., Man, K., Ng, K.T., Sun, C.K., Lo, C.M., Fan, S.T., 2005. The influence of phosphatidylinositol 3-kinase/Akt pathway on the ischemic injury during rat liver graft preservation. Am. J. Transplant. 5, 1264–1275. Liang, H.L., Hilton, G., Mortensen, J., Regner, K., Johnson, C.P., Nilakantan, V., 2009. MnTMPyP, a cell-permeant SOD mimetic, reduces oxidative stress and apoptosis following renal ischemia-reperfusion. Am. J. Physiol. Renal. Physiol. 296, F266–F276. Llacuna, L., Marı´, M., Lluis, J.M., Garcı´a-Ruiz, C., Ferna´ndez-Checa, J.C., Morales, A., 2009. Reactive oxygen species mediate liver injury through parenchymal nuclear factor-kappaB inactivation in prolonged ischemia/reperfusion. Am. J. Pathol. 174, 1776–1785. McCord, J.M., 1985. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312, 159–163. Mozaffari, M.S., Schaffer, S.W., 2008. Effect of pressure overload on cardioprotection of mitochondrial KATP channels and GSK-3beta: interaction with the MPT pore. Am. J. Hypertens. 21, 570–575. Mullonkal, C.J., Toledo-Pereyra, L.H., 2007. Akt in ischemia and reperfusion. J. Invest. Surg. 20, 195–203.
125
Phiel, C.J., Klein, P.S., 2001. Molecular targets of lithium action. Annu. Rev. Pharmacol. Toxicol. 41, 789–813. Rao, J., Zhang, C., Wang, P., Lu, L., Zhang, F., 2010. All-trans retinoic acid alleviates hepatic ischemia/reperfusion injury by enhancing manganese superoxide dismutase in rats. Biol. Pharm. Bull. 33, 869–875. Ren, F., Duan, Z., Cheng, Q., Shen, X., Gao, F., Bai, L., Liu, J., Busuttil, R.W., KupiecWeglinski, J.W., Zhai, Y., 2011. Inhibition of glycogen synthase kinase 3 beta ameliorates liver ischemia reperfusion injury by way of an interleukin-10mediated immune regulatory mechanism. Hepatology 54, 687–696. Ren, M., Senatorov, V.V., Chen, R.W., Chuang, D.M., 2003. Postinsult treatment with lithium reduces brain damage and facilitates neurological recovery in a rat ischemia/reperfusion model. Proc. Nat. Acad. Sci. U.S.A. 100, 6210–6215. Schwabe, R.F., Brenner, D.A., 2002. Role of glycogen synthase kinase-3 in TNFalpha-induced NF-kappaB activation and apoptosis in hepatocytes. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G204–G211. Sun, B., Chen, M., Hawks, C.L., Pereira-Smith, O.M., Hornsby, P.J., 2005. The minimal set of genetic alterations required for conversion of primary human fibroblasts to cancer cells in the subrenal capsule assay. Neoplasia 7, 585–593. Takada, Y., Fang, X., Jamaluddin, M.S., Boyd, D.D., Aggarwal, B.B., 2004. Genetic deletion of glycogen synthase kinase-3beta abrogates activation of IkappaBalpha kinase, JNK, Akt, and p44/p42 MAPK but potentiates apoptosis induced by tumor necrosis factor. J. Biol. Chem. 279, 39541–39542. Talab, S.S., Emami, H., Elmi, A., Nezami, B.G., Assa, S., Deroee, A.F., Daneshmand, A., Tavangar, S.M., Dehpour, A.R., 2010. Chronic lithium treatment protects the rat kidney against ischemia/reperfusion injury: the role of nitric oxide and cyclooxygenase pathways. Eur. J. Pharmacol. 647, 171–177. Thurman, R.G., Marzi, I., Seitz, G., Thies, J., Lemasters, J.J., Zimmerman, F., 1988. Hepatic reperfusion injury following orthotopic liver transplantation in the rat. Transplantation 46, 502–506. Wang, H.G., Pathan, N., Ethell, I.M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T.F., Reed, J.C., 1999. Ca2þ -induced apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339–343. Xu, J., Culman, J., Blume, A., Brecht, S., Gohlke, P., 2003. Chronic treatment with a low dose of lithium protects the brain against ischemic injury by reducing apoptotic death. Stroke 34, 1287–1292. Xu, X.H., Zhang, H.L., Han, R., Gu, Z.L., Qin, Z.H., 2006. Enhancement of neuroprotection and heat shock protein induction by combined prostaglandin A1 and lithium in rodent models of focal ischemia. Brain Res. 1102, 154–162. Yan, X.B., Hou, H.L., Wu, L.M., Liu, J., Zhou, J.N., 2007. Lithium regulates hippocampal neurogenesis by ERK pathway and facilitates recovery of spatial learning and memory in rats after transient global cerebral ischemia. Neuropharmacology 53, 487–495. Zhang, Y., Xia, Z., La Cour, K.H., Ren, J., 2011. Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3b-mediated suppression of mitochondrial permeation pore opening. Antioxid. Redox Signal 15, 2407–2424.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Lithium exacerbates hepatic ischemia/reperfusion injury by inhibiting GSK-3b/NF-kB-mediated protective signaling in mice Yongxiang Xia a,b,1, Jianhua Rao a,b,1, Aihua Yao a,b, Feng Zhang a,b, Guoqiang Li a,b,n, Xuehao Wang a,b,n, Ling Lu a,b,n a b
Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing 210029, China
a r t i c l e i n f o
abstract
Article history: Received 7 May 2012 Received in revised form 5 September 2012 Accepted 17 September 2012 Available online 7 October 2012
Lithium (an inhibitor of GSK-3b activity) has beneficial effects on ischemia/reperfusion (I/R) injury in the central nervous system, heart and kidney. However, the role of lithium in hepatic I/R injury is unknown. The aim of this study was to assess the effects of lithium on hepatic I/R injury in a mouse model of partial hepatic I/R. Previous studies showed that lithium chloride (LiCl) can phosphorylate residue Ser9, inhibit GSK-3b activity, and improve I/R injury in other organs. In the present study, mice were pretreated with either vehicle or LiCl, which had similar effects on GSK-3b activity. Surprisingly, treatment with LiCl significantly exacerbated hepatic I/R injury, which was determined by serological and histological analyses. Acute and chronic LiCl treatment caused serious damage in hepatic I/R injury, including increased apoptosis and oxidative stress. To gain insight into the mechanism involved in this damage, the activity of nuclear factor-kB (NF-kB) (GSK-3b can regulate the transcriptional complex of NF-kB) was analyzed, which revealed that LiCl treatment significantly down-regulated the activity of NF-kB. The NF-kB-mediated protective genes were then further evaluated, including anti-apoptotic genes (RAF2, cIAP 2, Bfl-1 and cFLIP) and the antioxidant gene MnSOD. The expression of these protective genes was obviously suppressed compared with the vehicle group. Taken together, these findings show that lithium exacerbates hepatic I/R injury by suppressing the expression of GSK-3b/ NF-kB-mediated protective genes. & 2012 Elsevier B.V. All rights reserved.
Keywords: Lithium Glycogen synthase kinase-3 beta (GSK-3b) Nuclear factor-kB Apoptosis Manganese superoxide dismutase (MnSOD)
1. Introduction Hepatic ischemia/reperfusion (I/R) injury is an adverse phenomenon, which damages hepatocytes and liver sinusoidal endothelial cells in the ischemic liver. Contradictorily, this damage is aggravated following the reestablishment of blood flow. This occurs in various clinical settings, such as liver transplantation, resection of hepatic tumors, trauma, circulatory shock and other insults (Thurman et al., 1988; McCord, 1985; Jaeschke, 2003). Hepatic I/R injury is a major cause of prime graft non-function after liver transplantation, and may critically compromise the function of the remaining liver after major hepatic resection (Burroughs et al., 2006). Although the pharmaceutical effects of different types of drugs and molecules in the prevention of hepatic I/R injury have been evaluated, the outcomes of these drug therapies, such as lithium, were not comparable.
n Corresponding authors at: First Affiliated Hospital of Nanjing Medical University, Liver Transplantation Center, Guangzhou road 300, Nanjng 210029, China. Tel.: þ 86 25 83718836 6476; fax: þ 86 25 83672106. E-mail address: [email protected] (L. Lu). 1 These authors contributed equally to this work.
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.09.009
Lithium has been extensively used as a mood stabilizer in the treatment of bipolar disorder for decades. Interestingly, lithium has been reported to have a potent neuroprotective effect in brain I/R injury. Xu reported that chronic treatment with lithium at a low dose exhibited neuroprotection in transient focal cerebral ischemia by reducing apoptotic death (Xu et al., 2003). Subsequently, Ren M demonstrated that post-insult treatment with lithium reduces brain damage and facilitates neurological recovery in a rat I/R model (Ren et al., 2003). Other investigators have suggested that pretreatment with lithium chloride, before ischemia or immediately on reperfusion, can phosphorylate and inhibit GSK-3b activity and subsequently improve the recovery of postischemic function, and reduce the infarct size in myocardial I/R injury (Barillas et al., 2007; Faghihi et al., 2008; Kaga et al., 2006; Mozaffari, Schaffer (2008)). Lithium also reduced kidney I/R injury (Talab et al., 2010). The putative mechanisms involved in lithiuminduced neuroprotection are complex, and may include inhibition of GSK-3b (Barillas et al., 2007), stimulation of heat shock protein70 (Xu et al., 2006) and activation of the extracellular regulated protein kinases (ERK) signaling pathway (Yan et al., 2007). Furthermore, inhibition of GSK-3b has also been confirmed in the cardio- and reno-protection of lithium treatment in mouse I/R
118
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
injury. GSK-3b has been reported to be involved in cell membrane-to-nucleus signaling, including transcription factor activation, cytoskeletal organization, growth, survival and apoptosis (Frame and Cohen, 2001; Kockeritz et al., 2006). To date, however, no data are available on the effects of lithium on hepatic I/R injury. This study was designed to elucidate the potential roles and mechanisms of acute and chronic lithium treatment on hepatic I/R injury.
2. Materials and methods 2.1. Animals Male C57BL/6 mice weighing 22–25 g were purchased from the Animal Center of Nanjing University (Nanjing, China). The mice were kept under constant environmental conditions with a 12 h light–dark cycle, and with free access to standard rodent diet and water. The mice were fasted before surgery. The experiments were carried out in accordance with the guidelines approved by the Institutional Animal Care and Use Committee at the Nanjing Medical University (Protocol Number NJMU08-092). 2.2. Experimental design and surgical procedure The mice were randomly divided into the following experimental groups: (1) Sham group (n¼12), mice were subjected to the surgical procedure with the omission of vascular occlusion. (2) Vehicle group, mice were subjected to 90 min of ischemia followed by 1 h, 3 h, 6 h, or 24 h of reperfusion; mice were treated with saline 30 min before ischemia (n¼6 per point). (3) Acute LiCl group, all mice underwent the I/R procedure, similar to the vehicle group, but were treated with LiCl (42.3 mg/kg, i.v.; Sigma, St. Louis, MO, USA) 30 min before I/R surgery (n¼6 per point). (4) Chronic LiCl group, all mice underwent the I/R procedure, similar to the vehicle group, but were treated with LiCl (42.3 mg/kg, i.p. qd) for one week before I/R surgery (n¼6 per point). Mice were anesthetized by intraperitoneal injection of 50–60 mg sodium pentobarbital per kilogram. A midline laparotomy incision was performed, extending from the xiphisternum to the pubis, to expose the liver. Ischemia of the left and median liver lobes was induced by the placement of an atraumatic bulldog clamp across the pedicles of the left portal vein, hepatic artery and bile duct for 90 min, as described previously (Abe et al., 2009), which led to segmental (70%) hepatic ischemia. Mice were sacrificed at different periods after reperfusion to obtain liver tissue and blood samples.
TUNEL using a commercially available kit (In situ cell death detection kit, Roche-Boehringer Mannheim, Germany). 2.5. GSK-3b and caspase-3 activity GSK-3b activity was measured in liver tissues 1 h after reperfusion. Frozen liver tissues were homogenized and immunoprecipitated using anti-GSK-3b antibody (Sigma). The activity of the enzyme was assessed by a kinase assay using [32P]-ATP and glycogen synthase peptide-2 (Sigma) as a substrate, as described by Haq et al., 2000). Caspase-3 activity was determined in liver tissues 6 h after reperfusion. Frozen samples of ischemic tissues were homogenized using a polytron homogenizer and centrifuged at 16,000 g for 20 min. The activity was measured using an assay kit (Calbiochem) according to the manufacturer’s instructions, as described by Ke et al. (2009). 2.6. Liver tissue homogenate malondialdehyde (MDA) assay The liver tissues homogenized in 100 mmol/L Tris–HCl buffer were centrifuged at 10,000 g for 20 min. Total protein concentration was determined by the Coomassie blue method. MDA was measured using kits according to the manufacturer’s instructions (Jiancheng Biotechnology, Nanjing, China). The results were expressed in nmol MDA per milligram of tissue homogenate 2.7. Western blot analysis Proteins were extracted from liver tissues subjected to ischemia, and their concentrations were determined by the Bradford assay (Bio-Rad, CA, USA). Nuclear protein extracts were prepared according to the manufacturer’s protocol (NE-PER Nuclear and cytoplasmic extraction reagents; Thermo Scientific, IL, USA). 30 mg of the protein sample was resolved by SDS-PAGE and transferred to nitrocellulose membranes (Sunshine Biotechnology, China). These membranes were blocked in (5% wt/vol) skim milk powder-Trisbuffered saline (TBS) with 0.1% Tween 20 (TBS-T) at 4 1C overnight. Membranes were then incubated with antibodies to Ser9-phosphoGSK-3b, GSK-3b, MnSOD, GAPDH, Ser32/36-phospho-IkB-a (Cell Signaling Technology, Danvers, MA, USA), p65 and IkB-a (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following three washes with TBS-T, the membranes were then incubated for 1 h at room temperature with goat-anti-rabbit peroxidase-conjugated secondary antibody (Santa Cruz). The final results were obtained by exposure to Kodak autoradiography film (Kodak XAR film). The results were visualized via a chemiluminescent detection system (ECL Substrate Western blot detection system, Pierce, IL, USA).
2.3. Serum ALT 2.8. Quantitative real-time PCR Blood samples for alanine aminotransferase (ALT) assessment were obtained after 6 h of reperfusion, and analyzed using a serum analyzer (Hitachi 7600-10, Hitachi High-Technologies Corporation, Japan). 2.4. Hematoxylin and eosin (HE), immunohistochemistry (IHC), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Liver tissues were removed and fixed in 4% paraformaldehyde overnight at 4 1C and then processed, the tissues were cut into 5 mm thick sections and stained with hematoxylin and eosin (HE) using standard techniques. The sections were placed on slides and stained immunohistochemically for b-catenin (Cell Signaling Technology), p65 (Santa Cruz Biotechnology) using techniques described previously (Sun et al., 2005). To determine apoptotic cell injury in liver tissue induced by I/R, sections were stained by
Reverse transcription reactions were performed using the Super-Script First-Strand Synthesis System (Invitrogen, CA, USA). To determine the relative number of cDNA molecules in the reverse transcribed samples, real-time PCR analyses were performed using the Light-Cycler system (Roche, Indianapolis, IN, USA). PCR was performed according to the procedure previously described (Jiang et al., 2010). PCR was performed using 10 ml 2x Master Mix SYBR Green I (Takara, Japan), 0.25 mM of each 50 and 30 primer, and 2 ml samples or H2O to a final volume of 20 ml. Samples were denatured at 94 1C for 5 min. Amplification and fluorescence determination were carried out in 3 steps: denaturation at 94 1C for 10 s, annealing at 60 1C for 15 s, extension at 72 1C for 20 s; and at the end of extension, detection of SYBR green fluorescence, which reflects the amount of doublestranded DNA. The amplification cycle number was 35. To discriminate specific from nonspecific cDNA products, a melting
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
curve was obtained at the end of each run. Products were denatured at 95 1C for 3 s, and the temperature was then decreased to 58 1C for 15 s and raised slowly from 58 1C to 95 1C using a temperature transition rate of 0.1 1C/sec. Data were normalized with GAPDH levels in the samples. Primers were designed by Oligo 6.0. Primer sets (sense sequence and antisense sequence, respectively) for the following genes were: Bfl-1 forward, 50 -GGC TGA GCA CTA CCT TCA GTA-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 ; c-IAP2 forward, 50 -TCA GTG ACC TCG TTA TAG GCT T-30 , reverse, 50 -TCA CAC ACG TCA AAT GTT GGA A30 ; TRAF2 forward, 50 -AGA GAG TAG TTC GGC CTT TCC-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 ; c-FLIP forward, 50 -GGA TTA CAA GGG ATT ACA CAG GC-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 ; MnSOD forward, 50 -CAG ACC TGC CTT ACG ACT ATG G-30 , reverse, 50 -CTC GGT GGC GTT GAG ATT GTT-30 ; GAPDH forward, 50 -GGT CAC CAG GGC TGC CAT TTG-30 , reverse, 50 -CTG GTA CTC CAT ACA CTG GCT-30 .
2.9. Electrophoretic mobility shift assays (EMSAs) To determine NF-kB activation, we performed EMSA, as described (Chen et al., 2007). EMSAs were performed using a Biotin Gel Shift Kit (Pierce, Rockford, IL, USA). The probe was labeled with biotin at the 30 -end, and the sequence was: 50 -TTG TTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GCG TGG-30 (boldface indicates NF-kB-binding sites).
2.10. Mouse hepatocyte isolation Mouse hepatocytes were isolated using a two-step in situ collagenase perfusion procedure as previously described (Hatano and Brenner (2001)). The livers from the C57BL/6 mice were perfused in situ through the portal vein with EGTA buffer (0.5 mM EGTA, 137 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 0.65 mM MgSO4, and 10.07 mM HEPES at pH 7.4) at a flow rate of 5 ml/ min for 10 min, followed by collagenase buffer (67 mM NaCl, 6.7 mM KCl, 4.76 mM CaCl2, 0.035% collagenase type II, and 10.07 mM HEPES at pH 7.6) at a flow rate of 5 ml/min for 15 min. After centrifugation, the hepatocytes were collected and seeded in DMEM containing 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin.
2.11. Hepatocyte apoptosis assays To determine the effects of LiCl on hepatocyte apoptosis induced by H2O2 in vitro, hepatocytes were treated with different doses of LiCl for 24 h as indicated, with or without the addition of 100 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK), and then incubated with 200 mm H2O2 for 6 h. Cells were harvested and washed twice with ice-cold PBS. Specific binding of FITC-annexin V and staining with PE-propidium iodide (PI) were performed using an apoptosis detection kit (BD, USA), according to the manufacturer’s instructions. The cells were then analyzed using flow cytometry.
2.12. Statistical analysis All data are expressed as mean 7standard deviation (S.D) and analyzed with the SPSS statistical package. Differences between groups were analyzed using a one-way analysis of variance with subsequent Student–Newman–Keuls test. Differences were considered statistically significant at a probability level of Po0.05.
119
3. Results 3.1. LiCl inhibited GSK-3b activity and exacerbated hepatic I/R injury GSK-3b activity was assessed by Western-blot detection of the phosphorylated residue Ser9 on GSK-3b. Compared with the sham control, the I/R group had a higher level of phosph-GSK-3b. Interestingly, both acute and chronic LiCl treatment significantly increased phosph-GSK-3b levels after liver I/R, while the total GSK-3b remained unchanged (Fig. 1A). In addition, our results showed that the inhibitory effect was not significantly different between the acute and chronic treatment. To further confirm LiClmediated alteration of GSK-3b activity, we next performed an immune complex kinase assay. As observed with Western blotting, hepatic I/R resulted in lower GSK-3b activity compared to the sham group, these effects were further enhanced by acute and chronic treatment with LiCl (Fig. 1B). These results indicate that hepatic I/R induced phosphorylation of GSK-3b and inhibited GSK-3b activity; these effects were augmented by LiCl treatment. The extent of hepatic damage was assessed by histology and serum ALT. Surprisingly, the role of LiCl on hepatic I/R injury was different from its protective effect on the cardiovascular system, urinary system and central nervous system. Hematoxylin-eosin (HE) staining was carried out on mouse ischemic liver lobes after 6 h of reperfusion (Fig. 1C). Mouse livers treated with vehicle showed hepatocellular necrosis and vacuolization. Indeed, the morphologic changes demonstrated that LiCl treatment significantly exacerbated liver damage. Furthermore, the histology findings were reflected by the serum ALT assay, which showed that LiCl treatment significantly increased serum ALT level (acute LiCl 1368072470 U/l, chronic LiCl 1417072950 U/l) compared with the vehicle treatment group (8100 7450 U/l) (P o0.01 for each case) (Fig. 1D). Thus, our results demonstrated that LiCl treatment exacerbated hepatic I/R injury. 3.2. LiCl inhibited hepatic I/R-induced NF-kB activation Previous studies (Hoeflich et al., 2000), in addition to our findings, showed that LiCl promoted hepatocyte injury, and indicated that GSK-3b may be linked to regulation of NF-kB activation. Therefore, we examined the effect of GSK-3b inhibition on the activation of NF-kB using EMSA. Our results showed that NF-kB activation was significantly decreased after acute and chronic LiCl treatment, although hepatic I/R stimulated NF-kB DNA binding activity (Fig. 2A). These results suggest that LiCl affects NF-kB signaling by regulating GSK-3b activity during hepatic I/R. To further confirm the impact of LiCl on the suppression of NF-kB signaling, we analyzed the nuclear import of p65 (Fig. 2B). Nuclear accumulation of p65 was much lower in LiCl pretreated mice compared with the I/R group, which was further confirmed by immunohistochemical staining (2173% for acute LiCl versus 81710% for vehicle; 1972.5% for chronic LiCl versus 81710% for vehicle, each Po0.01) (Fig. 2C). Translocation of NF-kB to the nucleus requires proteolytic degradation of IkB-a (Ghosh, Karin (2002)), thus, we then carried out Western-blots to detect degradation of IkB-a. Fig. 2B shows that hepatic I/R induced IkB-a degradation was partially inhibited by acute and chronic LiCl treatment. The proteolytic degradation of IkBa requires phosphorylation at serine residues 32 and 36 (Ghosh and Karin, 2002). To further assess the degradation of IkB-a, we assessed the IkB-a phosphorylation level, which indicated that I/R-induced IkB-a phosphorylation was significantly suppressed (Fig. 2B). 3.3. LiCl increased hepatocellular apoptosis in vivo and in vitro To analyze the relationship between aggravated damage by LiCl treatment and suppression of NF-kB activation, we next
120
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
A
B
Fig. 1. LiCl inhibited GSK-3b activity and exacerbated hepatic I/R injury. (A) Effects of acute and chronic LiCl administration on GSK-3 phosphorylation of ischemia liver tissues at 1 h after reperfusion was detected by Western blot. Each immunoblot is from a single experiment and is representative of three separate experiments. Densitometric analysis of the bands is expressed as the relative OD of GSK-3 phosphorylation at Ser9, corrected for the corresponding total GSK-3 content, and normalized using the sham band. Data are means7 S.D of three separate experiments. nPo 0.05 vs vehicle. OD indicates optical density. (B) GSK-3 activity in ischemia liver tissues at 1 h after reperfusion. Results were the mean 7S.D values of 6 mice. nPo 0.05 vs vehicle. (C) Liver histopathology at 6 h after reperfusion (400 ). Sham: Normal hepatic architecture was observed in sham-operated mice. Vehicle: apparent hemorrhage and hepatocellular necrosis could be detected. Acute and chronic LiCl treatment: significantly more hepatocellular necrosis was apparent. (D) Liver injury was measured by serum levels of ALT, at 6 h after reperfusion following 90 min of ischemia. Results were the mean 7S.D values of 6 mice, nP o 0.01 vs the vehicle.
examined the expression of NF-kB-mediated anti-apoptotic genes (TRAF2, cIAP 2, Bfl-1 and cFLIP) in liver tissue after 3 h of reperfusion by real-time PCR. The results showed that transcription of these anti-apoptotic genes was significantly inhibited by acute and chronic LiCl treatment (Fig. 3A). In parallel with the expression of anti-apoptotic genes, the activity of caspase-3 showed a consistent increase after LiCl treatment in ischemic livers (vehicle 2.170.4, acute LiCl 3.270.7, chronic LiCl 3.670.9) (Fig. 3B). The TUNEL assay also indicated that hepatocellular apoptosis was significantly increased in LiCl treated liver compared with vehicle control (vehicle-control 5.271.1%, acute LiCl 15.472.9%, and chronic LiCl 16.7 73.3%) (Fig. 3C). To further determine the effects of LiCl on hepatocellular apoptosis in hepatic I/R injury, we imitated the hepatic I/R injury model in vitro using H2O2 stimulated hepatocytes. The results showed that LiCl treatment significantly enhanced hepatocellular
apoptosis induced by H2O2 (Fig. 3D). In addition, to evaluate whether this apoptosis was caspase-dependent, the caspase inhibitor, z-vad-fmk, was used on the hepatocyte culture, which showed that the LiCl-induced increase in hepatocellular apoptosis is partially caspase dependent (Fig. 3D).
3.4. LiCl increased ROS production in hepatic I/R injury Reactive oxygen species (ROS) injury is an important cause of hepatic I/R injury. We further investigated the related factors, ROS and MnSOD. MnSOD, the NF-kB-mediated mitochondrial SOD isoform, is a key antioxidant enzyme in the antioxidant system. We assessed the levels of mRNA and protein coding for MnSOD by real-time PCR and Western blots. As shown in Fig. 4, MnSOD mRNA expression was significantly reduced following treatment
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
121
Fig. 2. LiCl decreased I/R-induced NF-kB activation. (A) Nuclear extracts from liver tissues at 1 h, 3 h and 6 h after reperfusion were subjected to electrophoretic mobility shift assay. The NF-kB activity was quantified by image analysis of autoradiograms. Results were the mean 7 S.D values of 6 mice. nP o0.05, acute LiCl treatmen vs vehicle; # P o 0.05, chronic LiCl treatment vs vehicle. (B) At 1 h, 3 h and 6 h after reperfusion, nuclear extract of ischemic liver tissues was prepared and Western blot analysis using anti-p65 antibody was performed. Total extracts were prepared and Western blot analysis using anti-IkB-a, phospho-ser-32/36-IkB-a and p65 antibody was performed. (C) At 3 h of reperfusion, nuclear accumulation of p65 was assessed by immunohistochmistry (400 ). The number of nuclear p65-positive cells was quantified from six fields at high-power magnification. nPo 0.05 vs vehicle.
with LiCl at 3 h after I/R (Fig. 4A), and the corresponding change in protein levels was confirmed by Western blots (Fig. 4B). Malondialdehyde (MDA), an index of lipid peroxidation, directly reflects the levels of ROS. To detect the levels of ROS, we next
measured MDA content. The results indicated that MDA levels were significantly increased in ischemic liver following acute and chronic LiCl treatment (vehicle 118 714, acute LiCl 276725, and chronic LiCl 256 726) (Fig. 4C).
122
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
Fig. 3. LiCl increased hepatocellular apoptosis in vivo and in vitro. (A) At 3 h after reperfusion, expression of Traf-2, cIAP2, cFlip and Bfl1 were detected by real-time PCR. nn P o 0.01, nP o 0.05, acute LiCl treatment vs vehicle. ##Po 0.01, #P o 0.05 chronic LiCl treatment vs vehicle. (B) At 6 h after reperfusion, the activity of caspase-3 was measured. nP o 0.05 vs vehicle. (C) Apoptotic cells were quantified in six high-power fields (400 ), and expressed as percentages of apoptotic cells among total cells. Results were the mean 7 S.D values of 6 mice, nPo 0.05 vs vehicle. (D): Effects of different dose LiCl on hepatocytes apoptosis induced by H2O2 in vitro, nnP o 0.01, nPo 0.05, LiCl treatment vs without LiCl treatment; effects of Z-VAD-FMK on LiCl on hepatocytes apoptosis induced by H2O2, nP o0.05, Z-VAD-FMK treatment vs without Z-VAD-FMK treatment.
4. Discussion Lithium is an established drug used in the treatment of manic depressive disorders. Recently, in vivo and in vitro studies have suggested that lithium is involved in oxidative stress, growth, survival and apoptosis. Moreover, lithium has robust protective effects in ischemic organ injury, including the brain, heart and
kidney. Therefore, our initial thoughts were to evaluate the protective effect of lithium during hepatic I/R injury. However, surprisingly, our studies revealed a damaging effect due to lithium during hepatic I/R injury. Lithium is a nonspecific inhibitor (Phiel and Klein, 2001), but has been used extensively to study cardioprotection by inhibiting GSK-3b activity (Haq et al., 2000; Klein and Melton, 1996). Our
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
123
Fig. 4. LiCl increased ROS injury in hepatic I/R injury. (A) At 3 h after reperfusion, expression of MnSOD mRNA was measured by real-time PCR. Results were the mean 7 S.D values of 6 mice. nPo 0.05 vs vehicle. (B) At 3 h after reperfusion, expression of MnSOD was determined by Western blot. Acute and chronic LiCl treatment significantly decreases MnSOD than vehicle group. (C) At 6 h after reperfusion, the levels of liver tissue MDA were determined. Results were the mean 7 S.D values of 6 mice. nP o0.05 vs vehicle.
study also confirmed that both acute and chronic LiCl treatment phosphorylated and inhibited GSK-3b activity. A previous report showed that phosphorylation and inactivation of GSK-3b contributed to hepatocyte apoptosis and severe ischemic injury in liver grafts during cold preservation (Li et al., 2003, 2005). However, warm I/R injury might have a different mechanism to that of cold I/R injury. In agreement with these studies, our results showed that inhibition of GSK-3b using LiCl exacerbated liver damage following I/R. Although these data are different from previously published reports, which showed that inhibition of GSK-3b activity resulted in hepatoprotection using GSK-3 specific inhibitors in liver I/R injury (Ren et al., 2011), our findings, at least partially, revealed the functional role of LiCl in hepatic I/R injury. The molecular mechanisms of LiCl in hepatic I/R injury are complex. It has been demonstrated that lithium treatment inhibited translocation of NF-kB and sensitized fibroblasts to TNF-amediated apoptosis (Hoeflich et al., 2000). Likewise, inhibition of GSK-3b activity by lithium down-regulated NF-kB-dependent gene transcription induced by TNF-a and sensitized hepatocytes to TNF-a-mediated apoptosis (Schwabe and Brenner, 2002). Consistent with these studies, our EMSA assay demonstrated inhibition of GSK-3b activity leading to suppression of NF-kB signaling after LiCl treatment in ischemic livers, which was similar to the conclusion drawn from GSK-3b-knockout mice (Hoeflich et al., 2000). Furthermore, we found that inhibition of GSK-3b activity suppressed phosphorylation and degradation of IkB-a, disturbed cytosolic-nuclear translocation of p65 after I/R, and finally suppressed NF-kB activation. Our results were consistent with previous findings by Takada et al. (2004), who demonstrated that TNF-a-induced IkB-a phosphorylation, IkBa degradation and NF-kB activation were all suppressed in GSK-3b ¨ gene deleted fibroblasts. Gotsche et al. (2008) also showed that GSK-3b inhibition reduced NF-kB translocation and binding activity due to impaired degradation of IkB-a in primary mouse hepatocytes. Therefore, we conclude that inhibition of GSK-3b activity enhanced the stability of IkB-a and inhibited NF-kB activity after LiCl treatment in hepatic I/R injury. Hepatocyte death after hepatic I/R is likely to be a combination of apoptotic and necrotic pathways, known as ‘‘necrapoptosis‘‘
(Gujral et al., 2001). Fig. 1C indicates that lithium enhanced apoptosis and necrosis of hepatocytes induced by hepatic I/R. We then analyzed the effects of lithium on apoptosis of hepatocytes after hepatic I/R by TUNEL staining and caspase-3 activity, which indicated that LiCl treatment enhanced hepatocellular apoptosis after hepatic I/R (Fig. 3B and C). In addition, to further determine the effects of LiCl on hepatocellular apoptosis in hepatic I/R injury, we imitated the hepatic I/R injury model in vitro using H2O2-induced hepatocellular apoptosis, which demonstrated that LiCl treatment significantly increased apoptosis of hepatocytes induced by H2O2 in vitro (Fig. 3D). Other reports have already shown that activation of NF-kB has a protective effect after hepatic I/R, which can up-regulate expression of anti-apoptotic genes and prevent the accumulation of damaging ROS (Huber et al., 2009; Kuboki et al., 2009). Our results demonstrated that inactivation of GSK-3b resulted in inhibition of NF-kB activation and the expression of anti-apoptotic genes (TRAF2, cIAP2, Bfl-1 and cFLIP) after lithium pretreatment followed by I/R. These results were supported by recent reports which demonstrated that GSK-3b inhibition induces glioma cell apoptosis through c-Myc, NF-kB and glucose regulation (Kotliarova et al., 2008). In addition, our findings confirmed the hypothesis of Li et al. (2005), who suggested that inhibition of GSK-3b by the PI3K/Akt pathway might account for cell apoptosis by dysregulation of the transcription of NF-kB and energy depletion during hepatic I/R. Since PI3K/Akt is found upstream of GSK-3, it is possible that GSK-3 activity is modulated by LiCl via PI3K/Akt signaling (Li et al., 2005). Indeed, Akt acts as an anti-apoptotic signaling molecule, which can inhibit caspasemediated cell death through phosphorylation of the antiapoptotic molecule-associated death promoter, and direct phosphorylation of caspase protease (Mullonkal and Toledo-Pereyra, 2007). In addition, AKT can promote survival by phosphorylation of Bad, whereas the dephosphorylation of Bad by calcineurin was a response to calcium overload (Wang et al., 1999). Phosphorylation of AKT and downstream GSK-3 provides cytoprotection by inhibiting the mitochondrial permeability transition pore (mPTP) opening (Zhang et al., 2011), suggesting that inhibition of GSK-3 activity by LiCl might directly regulate mitochondria membrane
124
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
LiCl I/R Hepatocyte
P GSK3β
TRAF2 cIAP2 Bfl-1 cFLIP
P
PI3K AKT
P PIP3
NF-κB MnSOD ROS
anti-apoptosis
Ca2+
Bad
P
Survival
to enhanced hepatic I/R injury by impairing the ability of hepatic anti-apoptotic genes and enhancing ROS production (Fig. 5). Lithium treatment inhibits GSK-3b activity leading to inactivation of NF-kB in hepatic I/R injury. Although our current study did not assess whether LiCl affects other signaling pathways, such as heat shock protein-70, ERK, and PI3K/Akt, these findings might explain the functional role and potential mechanism of lithium in inhibiting GSK-3/NF-kB protective signaling in hepatic I/R injury. Considering that lithium is a nonspecific inhibitor of GSK-3b, other mechanisms may be involved in the procedure. Further studies on whether lithium affects other targets during hepatic I/R are necessary.
Caspase-3 apopostis
Fig. 5. The effects of LiCl on hepatocellular apoptosis in hepatic I/R injury. GSK-3: glycogen synthase kinase-3; PI3K: phosphatidylinositol 3-kinase; AKT: Serine/ threonine kinase; TRAT2: TNF receptor-associated factor 2; Bfl-1: Bcl-2-related protein A1; cFLIP: cellular flice like inhibitory protein; cIAP2: cellular inhibitor of apoptosis 2; PIP3: phosphatidylinositol (3,4,5)-trisphosphate; solid arrow: inhibitory modification; hollow arrow: stimulatory modification.
potential change. Caspase-3 is an effecter molecule of apoptosis, which can be induced by many harmful factors, including hypoxia, stress, energy depletion, and calcium overload. Activation of caspase3 in mitochondria may be required for the effect of LiCl on liver damage triggered by I/R. In this study, our results showed that caspase-3 activity in liver was significantly increased by LiCl treatment after liver I/R. Taken together, the liver may be more sensitive to I/R injury due to multiple signaling after lithium treatment. It is possible that other hepatic impairment factors may be involved in the I/R process after lithium treatment. Specifically, the transport pathway, and the location of LiCl concentration might be critical in the modulation of GSK-3 activity in the liver in a concentration-dependent manner. We compared the effect of therapeutic concentrations of LiCl in an in vivo model. Although lacking a specific receptor in vivo, both acute and chronic LiCl treatment exacerbated liver damage following I/R, which suggested that the ischemic liver is sensitive to LiCl. More importantly, it has been shown that ROS play an important role in I/R-induced liver damage. Indeed, ROS may initiate the cascade of cell injury, necro/ apoptosis, and subsequent inflammatory infiltration (Crenesse et al., 2000; Liang et al., 2009). Conversely, MnSOD is a critical enzyme in cellular protection against ROS generation, and is considered to be a decisive player against hepatic I/R injury (Rao et al., 2010). Decreased levels of MnSOD implied that hepatic damage was more severe when hepatic GSK-3b was inhibited. Recent studies have shown that hepatic I/R-induced MnSOD expression is mediated through the activation of NF-kB (Llacuna et al., 2009). In the present study, the levels of hepatic MnSOD were significantly reduced after acute and chronic LiCl pretreatment followed by I/R. These findings may be correlated with inactivation of NF-kB. Furthermore, the levels of MDA, which directly reflect ROS production (Rao et al., 2010), were significantly increased after lithium treatment, suggesting that inactivation of NF-kB signaling induced by GSK-3b inhibition could suppress the expression of MnSOD, up-regulate MDA, and enhance ROS production. The outcome further suggested that acute and chronic treatment with lithium exacerbated hepatic I/R injury through up-regulation of ROS production via GSK-3b/NF-kB signaling.
5. Conclusions In conclusion, we demonstrated that lithium suppressed activation of NF-kB and NF-kB-mediated protective genes leading
Acknowledgements This work was supported by grants from National Eleventh Five-support sub-subject research of China (Grant numbers: 2008BAI60B02); Natural Science Foundation of China (Grant numbers: 81100270, 81070380, 30872390); Natural Science Foundation of Jiangsu province (Grant numbers: BK2009439, BZ2011041, ZX05 200904, WS2011106). First Innovation Team Foundation of Jiangsu province Hospital (for Sun BC). The authors would like to appreciate Shouyu Wang (Nanjing Medical University) for excellent technical assistance.
References Abe, Y., Hines, I.N., Zibari, G., Pavlick, K., Gray, L., Kitagawa, Y., Grisham, M.B., 2009. Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo. Free Radical Biol. Med. 46, 1–7. Barillas, R., Friehs, I., Cao-Danh, H., Martinez, J.F., del Nido, P.J., 2007. Inhibition of glycogen synthase kinase-3beta improves tolerance to ischemia in hypertrophied hearts. Ann. Thorac. Surg. 84, 126–133. Burroughs, A.K., Sabin, C.A., Rolles, K., Delvart, V., Karam, V., Buckels, J., O’Grady, J.G., Castaing, D., Klempnauer, J., Jamieson, N., Neuhaus, P., Lerut, J., de Ville, de Goyet, J., Pollard, S., Salizzoni, M., Rogiers, X., Muhlbacher, F., Garcia Valdecasas, J.C., Broelsch, C., Jaeck, D., Berenguer, J., Gonzalez, E.M., Adam, R., European Liver Transplant Association, 2006. 3-month and 12-month mortality after first liver transplant in adults in Europe: predictive models for outcome. Lancet 367, 225–232. Chen, R., Qiu, W., Liu, Z., Cao, X., Zhu, T., Li, A., Wei, Q., Zhou, J., 2007. Identification of JWA as a novel functional gene responsive to environmental oxidative stress induced by benzo[a]pyrene and hydrogen peroxide. Free Radical Biol. Med. 42, 1704–1714. Crenesse, D., Schmid-Alliana, A., Hornoy, J., Rossi, B., Gugenheim, J., 2000. Hypoxiareoxygenation differentially stimulates stress-activated protein kinases in primary-cultured rat hepatocytes. Transplant. Int. 13, S597–S599. Faghihi, M., Mirershadi, F., Dehpour, A.R., Bazargan, M., 2008. Preconditioning with acute and chronic lithium administration reduces ischemia/reperfusion injury mediated by cyclooxygenase not nitric oxide synthase pathway in isolated rat heart. Eur. J. Pharmacol. 597, 57–63. Frame, S., Cohen, P., 2001. GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359 (Pt 1), 1–16. Ghosh, S., Karin, M., 2002. Missing pieces in the NF-kappaB puzzle. Cell 109, S81–S96. ¨ Gotsche, l.F., Kern, C., Lang, S., Sparna, T., Markmann, C., Schwager, J., McNelly, S., ¨ von Weizsacker, F., Laufer, S., Hecht, A., Merfort, I., 2008. Inhibition of GSK3 differentially modulates NF-kappaB, CREB, AP-1 and beta-catenin signaling in hepatocytes, but fails to promote TNF-alpha-induced apoptosis. Exp. Cell. Res. 314, 1351–1366. Gujral, J.S., Bucci, T.J., Farhood, A., Jaeschke, H., 2001. Mechanism of cell death during warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis? Hepatology 33, 397–405. Haq, S., Choukroun, G., Kang, Z.B., 2000. Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol. 151, 117–130. Hatano, E., Brenner, D.A., 2001. Akt protects mouse hepatocytes from TNF-alphaand Fas-mediated apoptosis through NK-kappa B activation. Am. J. Physiol. Gastrointest. Liver Physiol. 281, G1357–G1368. Hoeflich, K.P., Luo, J., Rubie, E.A., Tsao, M.S., Jin, O., Woodgett, J.R., 2000. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 406, 86–90. Huber, N., Sakai, N., Eismann, T., Shin, T., Kuboki, S., Blanchard, J., Schuster, R., Edwards, M.J., Wong, H.R., Lentsch, A.B., 2009. Age-related decrease in proteasome expression contributes to defective nuclear factor-kappaB activation during hepatic ischemia/reperfusion. Hepatology 49, 1718–1728.
Y. Xia et al. / European Journal of Pharmacology 697 (2012) 117–125
Jaeschke, H., 2003. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am. J. Physiol. Gastrointest. Liver Physiol. 284, G15–G26. Jiang, R., Xia, Y., Li, J., Deng, L., Zhao, L., Shi, J., Wang, X., Sun, B., 2010. High expression levels of IKKalpha and IKKbeta are necessary for the malignant properties of liver cancer. Int. J. Cancer 126, 1263–1274. Kaga, S., Zhan, L., Altaf, E., Maulik, N., 2006. Glycogen synthase kinase-3beta/betacatenin promotes angiogenic and anti-apoptotic signaling through the induction of VEGF, Bcl-2 and survivin expression in rat ischemic preconditioned myocardium. J. Mol. Cell. Cardiol. 40, 138–147. Ke, B., Shen, X.D., Gao, F., Qiao, B., Ji, H., Busuttil, R.W., Volk, H.D., KupiecWeglinski, J.W., 2009. Small interfering RNA targeting heme oxygenase-1 (HO-1) reinforces liver apoptosis induced by ischemia-reperfusion injury in mice: HO-1 is necessary for cytoprotection. Hum. Gene Ther. 20, 1133–1142. Klein, P.S., Melton, D.A., 1996. A molecular mechanism for the effect of lithium on development. Proc. Nat. Acad. Sci. U.S.A. 93, 8455–8459. Kockeritz, L., Doble, B., Patel, S., Woodgett, J.R., 2006. Glycogen synthase kinase3—an overview of an over-achieving protein kinase. Curr. Drug. Targets 7, 1377–1388. Kotliarova, S., Pastorino, S., Kovell, L.C., Kot liarov, Y., Song, H., Zhang, W., Bailey, R., Maric, D., Zenklusen, J.C., Lee, J., Fine, H.A., 2008. Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation. Cancer Res. 68, 6643–6651. Kuboki, S., Sakai, N., Clarke, C., Schuster, R., Blanchard, J., Edwards, M.J., Lentsch, A.B., 2009. The peptidyl-prolyl isomerase, Pin1, facilitates NF-kappaB binding in hepatocytes and protects against hepatic ischemia/reperfusion injury. J. Hepatol. 51, 296–306. Li, X.L., Man, K., Liu, Y.F., Lee, T.K., Tsui, S.H., Lau, C.K., Lo, C.M., Fan, S.T., 2003. Insulin in University of Wisconsin solution exacerbates the ischemic injury and decreases the graft survival rate in rat liver transplantation. Transplantation 76, 44–49. Li, X.L., Man, K., Ng, K.T., Sun, C.K., Lo, C.M., Fan, S.T., 2005. The influence of phosphatidylinositol 3-kinase/Akt pathway on the ischemic injury during rat liver graft preservation. Am. J. Transplant. 5, 1264–1275. Liang, H.L., Hilton, G., Mortensen, J., Regner, K., Johnson, C.P., Nilakantan, V., 2009. MnTMPyP, a cell-permeant SOD mimetic, reduces oxidative stress and apoptosis following renal ischemia-reperfusion. Am. J. Physiol. Renal. Physiol. 296, F266–F276. Llacuna, L., Marı´, M., Lluis, J.M., Garcı´a-Ruiz, C., Ferna´ndez-Checa, J.C., Morales, A., 2009. Reactive oxygen species mediate liver injury through parenchymal nuclear factor-kappaB inactivation in prolonged ischemia/reperfusion. Am. J. Pathol. 174, 1776–1785. McCord, J.M., 1985. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312, 159–163. Mozaffari, M.S., Schaffer, S.W., 2008. Effect of pressure overload on cardioprotection of mitochondrial KATP channels and GSK-3beta: interaction with the MPT pore. Am. J. Hypertens. 21, 570–575. Mullonkal, C.J., Toledo-Pereyra, L.H., 2007. Akt in ischemia and reperfusion. J. Invest. Surg. 20, 195–203.
125
Phiel, C.J., Klein, P.S., 2001. Molecular targets of lithium action. Annu. Rev. Pharmacol. Toxicol. 41, 789–813. Rao, J., Zhang, C., Wang, P., Lu, L., Zhang, F., 2010. All-trans retinoic acid alleviates hepatic ischemia/reperfusion injury by enhancing manganese superoxide dismutase in rats. Biol. Pharm. Bull. 33, 869–875. Ren, F., Duan, Z., Cheng, Q., Shen, X., Gao, F., Bai, L., Liu, J., Busuttil, R.W., KupiecWeglinski, J.W., Zhai, Y., 2011. Inhibition of glycogen synthase kinase 3 beta ameliorates liver ischemia reperfusion injury by way of an interleukin-10mediated immune regulatory mechanism. Hepatology 54, 687–696. Ren, M., Senatorov, V.V., Chen, R.W., Chuang, D.M., 2003. Postinsult treatment with lithium reduces brain damage and facilitates neurological recovery in a rat ischemia/reperfusion model. Proc. Nat. Acad. Sci. U.S.A. 100, 6210–6215. Schwabe, R.F., Brenner, D.A., 2002. Role of glycogen synthase kinase-3 in TNFalpha-induced NF-kappaB activation and apoptosis in hepatocytes. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G204–G211. Sun, B., Chen, M., Hawks, C.L., Pereira-Smith, O.M., Hornsby, P.J., 2005. The minimal set of genetic alterations required for conversion of primary human fibroblasts to cancer cells in the subrenal capsule assay. Neoplasia 7, 585–593. Takada, Y., Fang, X., Jamaluddin, M.S., Boyd, D.D., Aggarwal, B.B., 2004. Genetic deletion of glycogen synthase kinase-3beta abrogates activation of IkappaBalpha kinase, JNK, Akt, and p44/p42 MAPK but potentiates apoptosis induced by tumor necrosis factor. J. Biol. Chem. 279, 39541–39542. Talab, S.S., Emami, H., Elmi, A., Nezami, B.G., Assa, S., Deroee, A.F., Daneshmand, A., Tavangar, S.M., Dehpour, A.R., 2010. Chronic lithium treatment protects the rat kidney against ischemia/reperfusion injury: the role of nitric oxide and cyclooxygenase pathways. Eur. J. Pharmacol. 647, 171–177. Thurman, R.G., Marzi, I., Seitz, G., Thies, J., Lemasters, J.J., Zimmerman, F., 1988. Hepatic reperfusion injury following orthotopic liver transplantation in the rat. Transplantation 46, 502–506. Wang, H.G., Pathan, N., Ethell, I.M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T.F., Reed, J.C., 1999. Ca2þ -induced apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339–343. Xu, J., Culman, J., Blume, A., Brecht, S., Gohlke, P., 2003. Chronic treatment with a low dose of lithium protects the brain against ischemic injury by reducing apoptotic death. Stroke 34, 1287–1292. Xu, X.H., Zhang, H.L., Han, R., Gu, Z.L., Qin, Z.H., 2006. Enhancement of neuroprotection and heat shock protein induction by combined prostaglandin A1 and lithium in rodent models of focal ischemia. Brain Res. 1102, 154–162. Yan, X.B., Hou, H.L., Wu, L.M., Liu, J., Zhou, J.N., 2007. Lithium regulates hippocampal neurogenesis by ERK pathway and facilitates recovery of spatial learning and memory in rats after transient global cerebral ischemia. Neuropharmacology 53, 487–495. Zhang, Y., Xia, Z., La Cour, K.H., Ren, J., 2011. Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3b-mediated suppression of mitochondrial permeation pore opening. Antioxid. Redox Signal 15, 2407–2424.