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p53 signal pathway
Biochemical and Biophysical Research Communications 469 (2016) 1034e1040
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Suppressing Cyclooxygenase-2 Prevents nonalcoholic and inhibits apoptosis of hepatocytes that are involved in the Akt/p53 signal pathway Jialing Wu 1, Chong Chen 1, Xi Hu, Xianbin Cai, Yinghong Guan, Hui Hu, Qinjia Wang, Xiaofeng Chen, Bozhi Cai, Xubin Jing* Department of Gastroenterology, The First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, 515041, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 December 2015 Accepted 21 December 2015 Available online 23 December 2015
Cyclooxygenase-2 (COX-2) can exert pro-inflammatory effects in nonalcoholic steatohepatitis (NASH). The aim of this study was to determine if the inhibition of COX-2 attenuates hepatocyte apoptosis in steatohepatitis and to examine the underlying molecular mechanism. Male wild type C57BL6/J mice and COX-2 knock out (COX-2/) mice were fed a methionine choline deficient (MCD) diet for 3 weeks. The wild type mice were also treated with celecoxib or a combination of celecoxib and a Akt specific inhibitor, miltefosine (MTF). After that, liver histology, serum alanine aminotransferase (ALT) levels, hepatic triglyceride (TG) levels, hepatocyte apoptosis, phosphorylated Akt (Ser473, pAkt) and p53 protein levels in mice livers were assessed. Celecoxib attenuated the severity of liver steatohepatitis and reduced the number of apoptotic cells, accompanied by increasing the activity of Akt and decreasing expression of p53. On the contrary, MTF can abrogate the effects of celecoxib on anti-apoptosis and antisteatohepatitis. Moreover, the effects on the COX-2/ mice that were fed the MCD diet were similar to that for celecoxib. The findings suggested that suppressing COX-2 can improve steatohepatitis by inhibiting hepatocyte apoptosis in mice via regulating the Akt/p53 pathway. Celecoxib treatment may be a favorable treatment option for NASH. © 2015 Elsevier Inc. All rights reserved.
Keywords: COX-2 Celecoxib Nonalcoholic steatohepatitis Apoptosis Akt p53
1. Introduction Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders ranging from steatosis to nonalcoholic steatohepatitis (NASH) to cirrhosis [1]. NAFLD is in the process of becoming the most common liver disease worldwide. The prevalence of NAFLD in the general population of Western developed countries and Asia is 20e30% and approximately 15%, respectively [2,3]. According to an epidemiologic survey, nearly 20% of patients with NASH may progress to end-stage liver disease such as cirrhosis and hepatocellular carcinomas, whereas individuals with simple steatosis rarely develop into the advanced stages of this disease [4,5]. This distinction highlights the fact that NASH is a key phase of intervention for preventing the development of cirrhosis and hepatocellular carcinomas and even liver failure.
* Corresponding author. E-mail address: [email protected] (X. Jing). 1 These two authors are co-first authors and contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.12.096 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Cyclooxygenase-2 (COX-2) is the key inducible enzyme in eicosanoid metabolism, which enhances and participates in an inflammatory response, acting as a pro-inflammatory mediator [6]. Findings reported in recent studies suggest that the expression of COX-2 in NASH liver models is significantly higher than in normal liver tissue and is correlated with the severity of steatohepatitis [6e9]. Celecoxib, a selective COX-2 inhibitor, has been shown to reduce hepatic steatosis and inflammation in both a methionine choline deficient (MCD) diet and a high-fat diet (HFD) induced NASH [6,8]. Based on these previous studies, we hypothesize that COX-2 plays a vital role in the progression of NASH. Emerging evidence suggests that hepatocyte apoptosis may be a key component of the “second hit” involved in the progression of NASH, and is correlated with inflammation and fibrosis [10,11]. During the second hit, hepatocytes suffer from oxidative stress and lipid peroxidation, resulting in hepatocyte apoptosis [10]. Feldstein AE et al. reported that hepatocyte apoptosis was a prominent pathologic feature of human NASH [11,12]. It is therefore possible that pharmacologic agents that reduce the number of apoptosis
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cells in the liver may attenuate NASH. Recently, Cheng Q et al. used a COX-2 inhibitor to show that COX-2 indirectly promotes hepatocellular apoptosis in the pathogenesis of NASH [9]. However, the role and underlying mechanism of COX-2 in hepatocyte apoptosis remains poorly understood. Akt, also termed protein kinase B, is a major cell-signaling molecule that regulates apoptosis. It has been reported that the activity of Akt is significantly decreased by more than 50% in MCD diet-fed induced NASH mice compared with mice that were fed a control diet [13]. In addition, experiments indicated that the activation of the PI3K/Akt pathway leads to the phosphorylation of MDM2, and the subsequent inactivation of p53 through the ubiquitin-proteasome system, thus regulating apoptosis [14,15]. Previously reported data showed that the expression of p53 was increased in both mice and human cases of NASH, and that there was a positive correlation between p53 expression and the stage of liver steatosis in human liver samples [16,17]. Based on the above findings, we hypothesize that the Akt/p53 pathway is likely involved in the progression of NASH. However, the potential interaction between Akt/p53 pathway and the suppression of COX-2 in NASH has never been examined and remains to be elucidated. In this study, we report on an investigation of the role and the mechanisms responsible for COX-2 regulated hepatocyte apoptosis in MCD induced NASH models by using the pharmacological inhibitor celecoxib and COX-2 knockout mice. The findings indicate that the inhibition of COX-2 attenuates hepatocyte apoptosis and the progression of NASH. In addition, the Akt/p53 pathway appears to be involved in the anti-apoptotic effects that are observed in NASH. 2. Materials and methods 2.1. Animal treatments Male 9- to 10-week-old C57BL/6J mice were obtained from Vital River (Beijing, China). The COX-2 heterozygous knockout breeding pairs (COX-2±, mixed P129 and B6 back ground) were kindly provided by Professor Yingbi Zhou (Shantou University College of Medicine, Shantou, China). The COX-2/ were produced by the crossbreeding of male and female COX-2± mice. Genotyping was performed by CR of tail biopsies, as previously described [18,19]. Feeding mice an MCD diet is a classical method for investigating NASH, as it can induce NASH and exhibit similar histological features to human NASH [10]. Mice were divided into five groups: MCD group-mice fed MCD (cat. no. A02082002B; Research Diets) diet only, CON group-mice fed control diet (MCD diet supplemented with L-methionine [3 g/kg] and choline chloride [2 g/kg]), MCD þ CEL group-mice fed MCD diet and received intraperitoneal injections of celecoxib (20 mg/kg/day; Pfizer Pharmaceuticals LLC), MCD þ CEL þ MTF group-mice fed MCD diet co-treatment with celecoxib (20 mg/kg/day) and miltefosine (50 mg/kg/day; S3056 Selleck), MCD (KO) group-COX-2/ mice fed MCD diet without any administration. After 3 weeks, all mice were anesthetized and sacrificed after a 12 h period of fasting. Blood samples were collected for biochemical assays. The liver was removed and sampled for histological study, biochemical assays and Western blot analysis. The animal experiments were approved by the Ethics Committee for Animal Experimentation of the Shantou University Medical College and conform to the National Institutes of Health guide for the care and use of Laboratory animals.
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triglycerides (TG) were measured using the triglyceride E-test kit (F001-2; Nanjing Jiancheng, China). Prostaglandin E2 (PGE2) levels in liver were measured by an ELISA kit (DRE30055; panera, France). 2.3. Morphological studies The liver samples were fixed in 10% formalin, then sliced into 4to 6-mm pieces, dehydrated in ethanol, embedded in paraffin wax, sectioned (5 mm thick), and stained with hematoxylin and eosin. Frozen sections of the liver were stained with Oil Red O (Sigma) to determine hepatic lipid accumulation. Tissues were sliced (10 mm thick) and stained in 60% of the oil red O stock solution (0.5 g oil red O in 100 mL isopropanol) for 30 min in 60 C oven. Tissues were briefly washed in 60% isopropanol and then rinsed in distilled water. Hematoxylin counterstaining was employed for microscopic observation and photography. 2.4. TUNEL assay Apoptosis was measured using a TUNEL assay kit (Nanjing KeyGen Biotech Co., Ltd.). Sections were dewaxed in xylene and rehydrated through a graded series of alcohols to water, digested with proteinase-K for 30 min. Select a section randomly used for positive control and incubate with DNase I for 30 min at 37 C to induce DNA strand breaks, prior to labeling procedure. Then all sections were incubated with equilibration buffer incorporation of dUTP-digoxigenin in the presence of working-strength TdT for 60 min in a humidified chamber at 37 C, after which the sections were washed in three changes of PBS. Anti-digoxigenin-fluorescein conjugate (FITC) was applied and the sections were incubated for 30 min protected from light. Sections were washed with four changes of PBS, and 40 ,6-diamidino-2-phenylindole(DAPI)(Roche, Shanghai, China) was used for counterstaining. Sections were viewed using a fluorescence microscope (Nikon90 Ni-E). The number of total and TUNEL-stained nuclei were counted manually (magnification 200.), and the apoptotic index was calculated (number of TUNEL-positive nuclei/number of DAPI-positive nuclei). 2.5. Western blot analysis Total and phosphorylated Akt, p53 and GAPDH proteins were assayed by Western immunoblots. Liver tissues were lysed in a RIPA buffer (Bocai Company, Shanghai, China) supplemented with a mixture of protease and phosphatase inhibitors (10 ml), NaF (10 ml of 100 mM), sodium orthovanadate (10 ml of 100 mM) and PMSF (10 ml of 100 mM). For Western blot analysis, the protein samples with an equal amount were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corporation, Billerica, Mass, USA). The membranes were then blocked with 10% non-fat dried milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. The membranes were next incubated with primary antibodies overnight at 4 C. In the next day, the membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma) for 2 h at room temperature. The peroxidase activity was detected by enhanced chemiluminescence (ECL; Pierce, Rockford, USA) and exposed to x-ray film (Kodak Company, Guangzhou, China). The positive protein bands were quantitated by laser densitometry. The antibodies used for Western blotting included antibodies against mouse phospho-Akt (Ser473, p-Akt), total-Akt, p53 (Cell Signaling) and GAPDH (Bioworld Technology, Inc).
2.2. Biochemical assays 2.6. Statistical analysis Serum alanine aminotransferase (ALT) levels were measured using the ALT Test Kit (C009-2; Nanjing Jiancheng, China), liver
Data were expressed as the mean ± SEM and analyses were
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performed using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA). Data were analyzed using paired t-tests for two groups. Results were considered to be statistically significant at P < 0.05.
resulted in increased serum ALT levels, while celecoxib resulted in a reduction, consistent with hepatic steatosis (Fig. 1D). This indicates that the inhibition of COX-2 alleviated the hepatic lipid accumulation and hepatic injury in MCD-fed mice.
3. Results 3.1. Celecoxib alleviates hepatic lipid accumulation and hepatic injury in MCD-fed mice Celecoxib, a highly selective inhibitor of COX-2, was used to evaluate the effect of COX-2 in our investigation. First, to confirm the pharmacological effects of celecoxib, PGE2 which is the main product of COX-2 was detected by means of an ELISA kit. As shown in Fig. 1A, celecoxib dramatically inhibited the PGE2 levels in MCDfed mice. Next, we evaluated the effect of COX-2 on hepatic lipid accumulation. HE and Oil Red O stain were used to observe the morphology of the mice livers after MCD feeding with or without celecoxib. Mice that were fed the MCD diet developed prominent macrovesicular lipid droplets in the liver compared with the control group, while the celecoxib treatment significantly attenuated the accumulation of lipids in the livers of MCD diet-fed mice (Fig. 1B). Consistent with the Oil Red O staining, the hepatic TG content was significantly reduced in the MCD þ CEL group compared to the MCD group (Fig. 1C). Moreover, the MCD diet
3.2. Inhibition of COX-2 reduced hepatocyte apoptosis and the effect on Akt and p53 in MCD-fed mice To determine the effect of COX-2 on hepatocyte apoptosis in NASH mice models, we performed TUNEL assays on the liver tissues of the experimental mice. Compared with the control group, the MCD diet-fed treatment induced a significant increase in the number of TUNEL-positive cells as well as the apoptotic index (Fig. 2A and B). Furthermore, when the COX-2 inhibitor celecoxib was added, the apoptotic index decreased markedly. (Fig. 2A and B). Moreover, to further explore the molecular mechanisms, we analyzed the expression of Akt and p53. The levels of phosphorylated Akt (Ser473) were significantly lower and p53 levels were markedly higher in the MCD group compared to control group. However, celecoxib largely reversed the expression of Akt and p53 in MCD group. (Fig. 2CeE). This indicates that COX-2 mediated hepatocyte apoptosis may be regulated via the Akt/p53 pathway.
Fig. 1. Effects of the MCD diet and treatment with celecoxib on hepatic triglyceride levels and serum ALT. C57BL/6J mice were fed the control diet, MCD diet, or MCD diet with celecoxib (20 mg/kg/day) treatment for 3 weeks. (A) PGE2 levels in mice livers in the indicated groups. (B) HE-stained and oil red O-stained sections of liver samples are representative of the indicated groups. (Original magnification 200) (C) Hepatic triglyceride levels in the indicated groups. (D) Serum ALT levels in the indicated groups. Values are means ± SEM (n ¼ 46 mice/group). *P < 0.05 compared with MCD mice. Abbreviations: MCD, methionine and choline deficient; TG, triglyceride; ALT, alanine aminotransferase; CON, control; CEL, celecoxib.
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Fig. 2. Effects of celecoxib on hepatocyte apoptosis, Akt activation and p53 expression in MCD-diet fed mice livers with celecoxib treatment. (A) TUNEL-stained sections of liver samples are representative of the indicated groups (magnification 400). (B) Hepatic apoptotic index in the indicated groups. Quantification of hepatic apoptotic index as determined by TUNEL-positive nuclei/total nuclei, and the number of total and TUNEL-stained nuclei were counted manually (magnification 200). (C) Activation of Akt and the expression of p53 were determined by Western blotting in mice fed the control diet, MCD, or MCD with celecoxib (20 mg/kg/day) treatment for 3 weeks. (D and E) Bands were quantified; Graphs represented were the gray value ratio of phosphorylated Akt to total Akt and p53 protein to GAPDH. Values are means ± SEM (n ¼ 46 mice/group). *P < 0.05 compared with control mice; #P < 0.05 compared with MCD mice. Abbreviations: MCD, methionine and choline deficient; CON, control; CEL, celecoxib.
3.3. Miltefosine abrogates the preventative effects of celecoxib on steatohepatitis and hepatocyte apoptosis involved in the Akt/p53 pathway in MCD-fed mice To further investigate whether the Akt/p53 pathway is involved in the regulating of hepatocyte apoptosis in NASH, miltefosine, a specific Akt inhibitor, was administrated. Our results show that miltefosine could abrogate the preventive effect of celecoxib on hepatic TG accumulation (Fig. 3A and B). The TUNEL-positive cells and the serum ALT levels were significantly increased after adding miltefosine compared with the celecoxib treatment in MCD-fed mice (Fig. 3CeE). Moreover, As expected, the suppression of Akt induced by miltefosine and the expression of p53 protein was correspondingly increased (Fig. 3FeH). Taken together, these data show that miltefosine diminished the anti-apoptosis and antisteatohepatitis effect of celecoxib in MCD-fed mice and that the Akt/p53 pathway may be one of the molecular targets of celecoxib in the treatment of NASH. 3.4. Genetic deficiency of COX-2 ameliorates steatohepatitis and hepatocyte apoptosis involved in Akt/p53 pathway in MCD-fed mice To further verify the significant role of COX-2 in NASH, we used COX-2- deficient mice with an MCD diet-fed in this study. Our data showed that both the hepatic lipid accumulation and the number of apoptotic hepatocytes were significantly regressed in the MCD (KO) group mice (Fig. 4AeD). We next detected the activation of Akt and p53 protein levels in COX-2 KO mice livers. Consistent with our study of celecoxib drug therapy, the activation of Akt appeared to
increase, and, adversely, the expression of the p53 protein was decreased in COX-2 KO mice compared with MCD-fed mice (Fig. 4EeG). Taken together, these data demonstrated that the suppression of COX-2 halts the progression of NASH and hepatocyte apoptosis associated with the Akt/p53 pathway. 4. Discussion Several important discoveries are reported in the present study. First, we observed that the suppression of COX-2, not only ameliorated hepatic steatosis, but also reduced the number of hepatocyte apoptosis in MCD diet induced NASH models. In this study, we adopted COX-2 knockout mice for the first time to confirm the vital role of COX-2 on hepatocyte apoptosis in the progression of NASH. Furthermore, we also report evidence to show that the Akt/ p53 pathway is involved in the anti-apoptotic effects in NASH. Therefore, COX-2 may become a effective target for the treatment and prevention of NASH. It is known that lipid accumulation and inflammation in hepatic tissue play an important role in the development of NASH. Celecoxib, an inhibitor of COX-2 is recognized to play an antiinflammatory role in the treatment of NASH. However, Cheng Q et al. reported that a higher expression of COX-2 is accompanied by more hepatocyte apoptosis in NASH [9]. In an animal model of NASH, hepatocyte apoptosis was detected early after the initiation of an MCD diet [20]. Both the intrinsic (triggered by cellular stress) and the extrinsic (induced by death receptors) apoptosis pathways are involved in the pathogenesis of NASH [21,22]. Accordingly, inhibition of hepatocyte apoptosis appears to be a potential target for
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Fig. 3. Effects of a miltefosine co-treatment with celecoxib on steatohepatitis, hepatocyte apoptosis, Akt activation and p53 expression in the liver of MCD-diet fed mice. Mice were fed the control diet, an MCD diet, an MCD diet with celecoxib (20 mg/kg/day) treatment, or an MCD diet co-treatment with celecoxib and miltefosine (50 mg/kg/day) for 3 weeks. (A) HE-stained and oil red O-stained sections of liver samples are representative of the indicated groups. (Original magnification 200) (B) Hepatic triglyceride levels in the indicated groups. (C) TUNEL-stained sections of liver samples are representative of the indicated groups (magnification 400). (D) Hepatic apoptotic index in the indicated groups. (E) Serum ALT levels in the indicated groups. (F) phosphorylated Akt and p53 protein levels were determined by Western blotting in mice fed the control diet, MCD, MCD with celecoxib (20 mg/kg/day) treatment, or an MCD diet and treatment with celecoxib and miltefosine (50 mg/kg/day) for 3 weeks. (G and H) Bands were quantified, Graphs represent the gray value ratio of phosphorylated Akt to total Akt and p53 protein to GAPDH. Data are expressed as the means ± SME (n ¼ 46 mice/group). *P < 0.05, compared with control group; #P < 0.05 compared with MCD mice; **P < 0.05, compared with MCD þ CEL group. Abbreviations: MCD, methionine and choline deficient; CON, control; CEL, celecoxib; MTF, miltefosine.
therapy regarding NASH. In the present study, irrespective of whether it involved pharmacological effects or genetic deletion, the inhibition of COX-2 significantly decreased the number of apoptosis cells. This suggests that inhibiting COX-2 not only plays an antiinflammatory role, but also exerts an anti-apoptosis effect in the treatment of NASH. At the molecular level, the PI3K/Akt pathway plays an important role in the progression of NASH. It indicates that the regulation of the PI3K/Akt pathway in hepatocytes is associated with the progression of NASH, including oxidative stress, insulin resistance and metabolic dysfunction [23]. The activation of Akt provides cells with a survival signal allows them to withstand apoptotic stimuli [24,25]. p53, which acts downstream from Akt, is another significant apoptosis regulator. Several studies have shown that activation of the p53 pathway is involved in hepatocyte apoptosis in NASH and the expression of p53 is positively correlated with the stage of
liver steatosis [17,20]. Consistent with previous studies, our data show that the levels of pAkt were significantly reduced and the expression of p53 was markedly elevated in experimental NASH, and that this was responsible for the observed hepatocyte apoptosis, whereas these were reversed by the COX-2 inhibitor celecoxib. These results demonstrate that the effects of celecoxib in attenuating hepatocyte apoptosis likely involves regulating the Akt/ p53 signaling pathway. However, Lu S et al. have shown that celecoxib decreases fat accumulation through a COX-2-independent pathway, as the PGE2 levels in HFD-fed mice liver remained the same with or without celecoxib treatment [26]. Considering that celecoxib is not only a COX-2 inhibitor, there may be other multiple mechanisms involved in the treatment of NASH. Therefore, to confirm the role of COX-2, we used COX-2/ mice for an analysis of NASH for the first time. Interestingly, COX-2/ mice that were fed an MCD diet also
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Fig. 4. The effect of a genetic deficiency of COX-2 on steatohepatitis, hepatocyte apoptosis, Akt activation and p53 expression in the liver of MCD-diet fed mice. Wild type mice were fed the control diet, an MCD diet, an MCD diet with celecoxib (20 mg/kg/day), and COX-2/ mice were fed an MCD diet for 3 weeks. (A) HE-stained and oil red O-stained sections of liver samples are representative of the indicated groups. (Original magnification 200) (B) Hepatic triglyceride levels in the indicated groups. (C) TUNEL-stained sections of liver samples are representative of the indicated groups (magnification 400). (D) Hepatic apoptotic index in the indicated groups. (E) phosphorylated Akt and p53 protein levels were determined by Western *blotting in wild type mice that had been fed the control diet, MCD diet, MCD with celecoxib (20 mg/kg/day) treatment, and COX-2/ mice fed with MCD diet for 3 weeks. (F and G) Bands were quantified, Graphs represent the gray value ratio of phosphorylated Akt to total Akt and p53 protein to GAPDH. Data are expressed as the means ± SME (n ¼ 35 mice/group). *P < 0.05, compared with control group; #P > 0.05 compared with MCD (KO) group; **P < 0.05, compared with MCD group. Abbreviations: MCD, methionine and choline deficient; CON, control; CEL, celecoxib; KO, COX-2 knock out.
showed an ameliorated hepatic steatosis and hepatocyte apoptosis via regulating the Akt/p53 signaling pathway, an effect that is similar to the effects of celecoxib. From our current data, we conclude that COX-2 plays an important role in hepatocyte apoptosis during the progression of NASH. NASH is a chronic progressive disease. During the pathogenesis of NASH, hepatocyte apoptosis, inflammation and hepatic steatosis are coexist and all are highly interrelated and mutually reinforcing [27,28]. The anti-inflammation effect of inhibiting COX-2 is generally accepted, whereas we observed that a COX-2 inhibitor ameliorated hepatic steatosis and hepatocytes apoptosis via regulating the Akt/p53 signaling pathway in MCD diet induced NASH models. In a word, the inhibition of COX-2 breaks the vicious circle and inhibits the progression of NASH, thus making COX-2 a
potential target for the treatment and prevention of NASH. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments This study was supported in part by a grant from the National Natural Science Foundation of China (grants number: 81172263). References [1] D.G. Tiniakos, M.B. Vos, E.M. Brunt, Nonalcoholic fatty liver disease: pathology
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Suppressing Cyclooxygenase-2 Prevents nonalcoholic and inhibits apoptosis of hepatocytes that are involved in the Akt/p53 signal pathway Jialing Wu 1, Chong Chen 1, Xi Hu, Xianbin Cai, Yinghong Guan, Hui Hu, Qinjia Wang, Xiaofeng Chen, Bozhi Cai, Xubin Jing* Department of Gastroenterology, The First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, 515041, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 December 2015 Accepted 21 December 2015 Available online 23 December 2015
Cyclooxygenase-2 (COX-2) can exert pro-inflammatory effects in nonalcoholic steatohepatitis (NASH). The aim of this study was to determine if the inhibition of COX-2 attenuates hepatocyte apoptosis in steatohepatitis and to examine the underlying molecular mechanism. Male wild type C57BL6/J mice and COX-2 knock out (COX-2/) mice were fed a methionine choline deficient (MCD) diet for 3 weeks. The wild type mice were also treated with celecoxib or a combination of celecoxib and a Akt specific inhibitor, miltefosine (MTF). After that, liver histology, serum alanine aminotransferase (ALT) levels, hepatic triglyceride (TG) levels, hepatocyte apoptosis, phosphorylated Akt (Ser473, pAkt) and p53 protein levels in mice livers were assessed. Celecoxib attenuated the severity of liver steatohepatitis and reduced the number of apoptotic cells, accompanied by increasing the activity of Akt and decreasing expression of p53. On the contrary, MTF can abrogate the effects of celecoxib on anti-apoptosis and antisteatohepatitis. Moreover, the effects on the COX-2/ mice that were fed the MCD diet were similar to that for celecoxib. The findings suggested that suppressing COX-2 can improve steatohepatitis by inhibiting hepatocyte apoptosis in mice via regulating the Akt/p53 pathway. Celecoxib treatment may be a favorable treatment option for NASH. © 2015 Elsevier Inc. All rights reserved.
Keywords: COX-2 Celecoxib Nonalcoholic steatohepatitis Apoptosis Akt p53
1. Introduction Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders ranging from steatosis to nonalcoholic steatohepatitis (NASH) to cirrhosis [1]. NAFLD is in the process of becoming the most common liver disease worldwide. The prevalence of NAFLD in the general population of Western developed countries and Asia is 20e30% and approximately 15%, respectively [2,3]. According to an epidemiologic survey, nearly 20% of patients with NASH may progress to end-stage liver disease such as cirrhosis and hepatocellular carcinomas, whereas individuals with simple steatosis rarely develop into the advanced stages of this disease [4,5]. This distinction highlights the fact that NASH is a key phase of intervention for preventing the development of cirrhosis and hepatocellular carcinomas and even liver failure.
* Corresponding author. E-mail address: [email protected] (X. Jing). 1 These two authors are co-first authors and contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.12.096 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Cyclooxygenase-2 (COX-2) is the key inducible enzyme in eicosanoid metabolism, which enhances and participates in an inflammatory response, acting as a pro-inflammatory mediator [6]. Findings reported in recent studies suggest that the expression of COX-2 in NASH liver models is significantly higher than in normal liver tissue and is correlated with the severity of steatohepatitis [6e9]. Celecoxib, a selective COX-2 inhibitor, has been shown to reduce hepatic steatosis and inflammation in both a methionine choline deficient (MCD) diet and a high-fat diet (HFD) induced NASH [6,8]. Based on these previous studies, we hypothesize that COX-2 plays a vital role in the progression of NASH. Emerging evidence suggests that hepatocyte apoptosis may be a key component of the “second hit” involved in the progression of NASH, and is correlated with inflammation and fibrosis [10,11]. During the second hit, hepatocytes suffer from oxidative stress and lipid peroxidation, resulting in hepatocyte apoptosis [10]. Feldstein AE et al. reported that hepatocyte apoptosis was a prominent pathologic feature of human NASH [11,12]. It is therefore possible that pharmacologic agents that reduce the number of apoptosis
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cells in the liver may attenuate NASH. Recently, Cheng Q et al. used a COX-2 inhibitor to show that COX-2 indirectly promotes hepatocellular apoptosis in the pathogenesis of NASH [9]. However, the role and underlying mechanism of COX-2 in hepatocyte apoptosis remains poorly understood. Akt, also termed protein kinase B, is a major cell-signaling molecule that regulates apoptosis. It has been reported that the activity of Akt is significantly decreased by more than 50% in MCD diet-fed induced NASH mice compared with mice that were fed a control diet [13]. In addition, experiments indicated that the activation of the PI3K/Akt pathway leads to the phosphorylation of MDM2, and the subsequent inactivation of p53 through the ubiquitin-proteasome system, thus regulating apoptosis [14,15]. Previously reported data showed that the expression of p53 was increased in both mice and human cases of NASH, and that there was a positive correlation between p53 expression and the stage of liver steatosis in human liver samples [16,17]. Based on the above findings, we hypothesize that the Akt/p53 pathway is likely involved in the progression of NASH. However, the potential interaction between Akt/p53 pathway and the suppression of COX-2 in NASH has never been examined and remains to be elucidated. In this study, we report on an investigation of the role and the mechanisms responsible for COX-2 regulated hepatocyte apoptosis in MCD induced NASH models by using the pharmacological inhibitor celecoxib and COX-2 knockout mice. The findings indicate that the inhibition of COX-2 attenuates hepatocyte apoptosis and the progression of NASH. In addition, the Akt/p53 pathway appears to be involved in the anti-apoptotic effects that are observed in NASH. 2. Materials and methods 2.1. Animal treatments Male 9- to 10-week-old C57BL/6J mice were obtained from Vital River (Beijing, China). The COX-2 heterozygous knockout breeding pairs (COX-2±, mixed P129 and B6 back ground) were kindly provided by Professor Yingbi Zhou (Shantou University College of Medicine, Shantou, China). The COX-2/ were produced by the crossbreeding of male and female COX-2± mice. Genotyping was performed by CR of tail biopsies, as previously described [18,19]. Feeding mice an MCD diet is a classical method for investigating NASH, as it can induce NASH and exhibit similar histological features to human NASH [10]. Mice were divided into five groups: MCD group-mice fed MCD (cat. no. A02082002B; Research Diets) diet only, CON group-mice fed control diet (MCD diet supplemented with L-methionine [3 g/kg] and choline chloride [2 g/kg]), MCD þ CEL group-mice fed MCD diet and received intraperitoneal injections of celecoxib (20 mg/kg/day; Pfizer Pharmaceuticals LLC), MCD þ CEL þ MTF group-mice fed MCD diet co-treatment with celecoxib (20 mg/kg/day) and miltefosine (50 mg/kg/day; S3056 Selleck), MCD (KO) group-COX-2/ mice fed MCD diet without any administration. After 3 weeks, all mice were anesthetized and sacrificed after a 12 h period of fasting. Blood samples were collected for biochemical assays. The liver was removed and sampled for histological study, biochemical assays and Western blot analysis. The animal experiments were approved by the Ethics Committee for Animal Experimentation of the Shantou University Medical College and conform to the National Institutes of Health guide for the care and use of Laboratory animals.
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triglycerides (TG) were measured using the triglyceride E-test kit (F001-2; Nanjing Jiancheng, China). Prostaglandin E2 (PGE2) levels in liver were measured by an ELISA kit (DRE30055; panera, France). 2.3. Morphological studies The liver samples were fixed in 10% formalin, then sliced into 4to 6-mm pieces, dehydrated in ethanol, embedded in paraffin wax, sectioned (5 mm thick), and stained with hematoxylin and eosin. Frozen sections of the liver were stained with Oil Red O (Sigma) to determine hepatic lipid accumulation. Tissues were sliced (10 mm thick) and stained in 60% of the oil red O stock solution (0.5 g oil red O in 100 mL isopropanol) for 30 min in 60 C oven. Tissues were briefly washed in 60% isopropanol and then rinsed in distilled water. Hematoxylin counterstaining was employed for microscopic observation and photography. 2.4. TUNEL assay Apoptosis was measured using a TUNEL assay kit (Nanjing KeyGen Biotech Co., Ltd.). Sections were dewaxed in xylene and rehydrated through a graded series of alcohols to water, digested with proteinase-K for 30 min. Select a section randomly used for positive control and incubate with DNase I for 30 min at 37 C to induce DNA strand breaks, prior to labeling procedure. Then all sections were incubated with equilibration buffer incorporation of dUTP-digoxigenin in the presence of working-strength TdT for 60 min in a humidified chamber at 37 C, after which the sections were washed in three changes of PBS. Anti-digoxigenin-fluorescein conjugate (FITC) was applied and the sections were incubated for 30 min protected from light. Sections were washed with four changes of PBS, and 40 ,6-diamidino-2-phenylindole(DAPI)(Roche, Shanghai, China) was used for counterstaining. Sections were viewed using a fluorescence microscope (Nikon90 Ni-E). The number of total and TUNEL-stained nuclei were counted manually (magnification 200.), and the apoptotic index was calculated (number of TUNEL-positive nuclei/number of DAPI-positive nuclei). 2.5. Western blot analysis Total and phosphorylated Akt, p53 and GAPDH proteins were assayed by Western immunoblots. Liver tissues were lysed in a RIPA buffer (Bocai Company, Shanghai, China) supplemented with a mixture of protease and phosphatase inhibitors (10 ml), NaF (10 ml of 100 mM), sodium orthovanadate (10 ml of 100 mM) and PMSF (10 ml of 100 mM). For Western blot analysis, the protein samples with an equal amount were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corporation, Billerica, Mass, USA). The membranes were then blocked with 10% non-fat dried milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. The membranes were next incubated with primary antibodies overnight at 4 C. In the next day, the membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma) for 2 h at room temperature. The peroxidase activity was detected by enhanced chemiluminescence (ECL; Pierce, Rockford, USA) and exposed to x-ray film (Kodak Company, Guangzhou, China). The positive protein bands were quantitated by laser densitometry. The antibodies used for Western blotting included antibodies against mouse phospho-Akt (Ser473, p-Akt), total-Akt, p53 (Cell Signaling) and GAPDH (Bioworld Technology, Inc).
2.2. Biochemical assays 2.6. Statistical analysis Serum alanine aminotransferase (ALT) levels were measured using the ALT Test Kit (C009-2; Nanjing Jiancheng, China), liver
Data were expressed as the mean ± SEM and analyses were
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performed using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA). Data were analyzed using paired t-tests for two groups. Results were considered to be statistically significant at P < 0.05.
resulted in increased serum ALT levels, while celecoxib resulted in a reduction, consistent with hepatic steatosis (Fig. 1D). This indicates that the inhibition of COX-2 alleviated the hepatic lipid accumulation and hepatic injury in MCD-fed mice.
3. Results 3.1. Celecoxib alleviates hepatic lipid accumulation and hepatic injury in MCD-fed mice Celecoxib, a highly selective inhibitor of COX-2, was used to evaluate the effect of COX-2 in our investigation. First, to confirm the pharmacological effects of celecoxib, PGE2 which is the main product of COX-2 was detected by means of an ELISA kit. As shown in Fig. 1A, celecoxib dramatically inhibited the PGE2 levels in MCDfed mice. Next, we evaluated the effect of COX-2 on hepatic lipid accumulation. HE and Oil Red O stain were used to observe the morphology of the mice livers after MCD feeding with or without celecoxib. Mice that were fed the MCD diet developed prominent macrovesicular lipid droplets in the liver compared with the control group, while the celecoxib treatment significantly attenuated the accumulation of lipids in the livers of MCD diet-fed mice (Fig. 1B). Consistent with the Oil Red O staining, the hepatic TG content was significantly reduced in the MCD þ CEL group compared to the MCD group (Fig. 1C). Moreover, the MCD diet
3.2. Inhibition of COX-2 reduced hepatocyte apoptosis and the effect on Akt and p53 in MCD-fed mice To determine the effect of COX-2 on hepatocyte apoptosis in NASH mice models, we performed TUNEL assays on the liver tissues of the experimental mice. Compared with the control group, the MCD diet-fed treatment induced a significant increase in the number of TUNEL-positive cells as well as the apoptotic index (Fig. 2A and B). Furthermore, when the COX-2 inhibitor celecoxib was added, the apoptotic index decreased markedly. (Fig. 2A and B). Moreover, to further explore the molecular mechanisms, we analyzed the expression of Akt and p53. The levels of phosphorylated Akt (Ser473) were significantly lower and p53 levels were markedly higher in the MCD group compared to control group. However, celecoxib largely reversed the expression of Akt and p53 in MCD group. (Fig. 2CeE). This indicates that COX-2 mediated hepatocyte apoptosis may be regulated via the Akt/p53 pathway.
Fig. 1. Effects of the MCD diet and treatment with celecoxib on hepatic triglyceride levels and serum ALT. C57BL/6J mice were fed the control diet, MCD diet, or MCD diet with celecoxib (20 mg/kg/day) treatment for 3 weeks. (A) PGE2 levels in mice livers in the indicated groups. (B) HE-stained and oil red O-stained sections of liver samples are representative of the indicated groups. (Original magnification 200) (C) Hepatic triglyceride levels in the indicated groups. (D) Serum ALT levels in the indicated groups. Values are means ± SEM (n ¼ 46 mice/group). *P < 0.05 compared with MCD mice. Abbreviations: MCD, methionine and choline deficient; TG, triglyceride; ALT, alanine aminotransferase; CON, control; CEL, celecoxib.
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Fig. 2. Effects of celecoxib on hepatocyte apoptosis, Akt activation and p53 expression in MCD-diet fed mice livers with celecoxib treatment. (A) TUNEL-stained sections of liver samples are representative of the indicated groups (magnification 400). (B) Hepatic apoptotic index in the indicated groups. Quantification of hepatic apoptotic index as determined by TUNEL-positive nuclei/total nuclei, and the number of total and TUNEL-stained nuclei were counted manually (magnification 200). (C) Activation of Akt and the expression of p53 were determined by Western blotting in mice fed the control diet, MCD, or MCD with celecoxib (20 mg/kg/day) treatment for 3 weeks. (D and E) Bands were quantified; Graphs represented were the gray value ratio of phosphorylated Akt to total Akt and p53 protein to GAPDH. Values are means ± SEM (n ¼ 46 mice/group). *P < 0.05 compared with control mice; #P < 0.05 compared with MCD mice. Abbreviations: MCD, methionine and choline deficient; CON, control; CEL, celecoxib.
3.3. Miltefosine abrogates the preventative effects of celecoxib on steatohepatitis and hepatocyte apoptosis involved in the Akt/p53 pathway in MCD-fed mice To further investigate whether the Akt/p53 pathway is involved in the regulating of hepatocyte apoptosis in NASH, miltefosine, a specific Akt inhibitor, was administrated. Our results show that miltefosine could abrogate the preventive effect of celecoxib on hepatic TG accumulation (Fig. 3A and B). The TUNEL-positive cells and the serum ALT levels were significantly increased after adding miltefosine compared with the celecoxib treatment in MCD-fed mice (Fig. 3CeE). Moreover, As expected, the suppression of Akt induced by miltefosine and the expression of p53 protein was correspondingly increased (Fig. 3FeH). Taken together, these data show that miltefosine diminished the anti-apoptosis and antisteatohepatitis effect of celecoxib in MCD-fed mice and that the Akt/p53 pathway may be one of the molecular targets of celecoxib in the treatment of NASH. 3.4. Genetic deficiency of COX-2 ameliorates steatohepatitis and hepatocyte apoptosis involved in Akt/p53 pathway in MCD-fed mice To further verify the significant role of COX-2 in NASH, we used COX-2- deficient mice with an MCD diet-fed in this study. Our data showed that both the hepatic lipid accumulation and the number of apoptotic hepatocytes were significantly regressed in the MCD (KO) group mice (Fig. 4AeD). We next detected the activation of Akt and p53 protein levels in COX-2 KO mice livers. Consistent with our study of celecoxib drug therapy, the activation of Akt appeared to
increase, and, adversely, the expression of the p53 protein was decreased in COX-2 KO mice compared with MCD-fed mice (Fig. 4EeG). Taken together, these data demonstrated that the suppression of COX-2 halts the progression of NASH and hepatocyte apoptosis associated with the Akt/p53 pathway. 4. Discussion Several important discoveries are reported in the present study. First, we observed that the suppression of COX-2, not only ameliorated hepatic steatosis, but also reduced the number of hepatocyte apoptosis in MCD diet induced NASH models. In this study, we adopted COX-2 knockout mice for the first time to confirm the vital role of COX-2 on hepatocyte apoptosis in the progression of NASH. Furthermore, we also report evidence to show that the Akt/ p53 pathway is involved in the anti-apoptotic effects in NASH. Therefore, COX-2 may become a effective target for the treatment and prevention of NASH. It is known that lipid accumulation and inflammation in hepatic tissue play an important role in the development of NASH. Celecoxib, an inhibitor of COX-2 is recognized to play an antiinflammatory role in the treatment of NASH. However, Cheng Q et al. reported that a higher expression of COX-2 is accompanied by more hepatocyte apoptosis in NASH [9]. In an animal model of NASH, hepatocyte apoptosis was detected early after the initiation of an MCD diet [20]. Both the intrinsic (triggered by cellular stress) and the extrinsic (induced by death receptors) apoptosis pathways are involved in the pathogenesis of NASH [21,22]. Accordingly, inhibition of hepatocyte apoptosis appears to be a potential target for
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Fig. 3. Effects of a miltefosine co-treatment with celecoxib on steatohepatitis, hepatocyte apoptosis, Akt activation and p53 expression in the liver of MCD-diet fed mice. Mice were fed the control diet, an MCD diet, an MCD diet with celecoxib (20 mg/kg/day) treatment, or an MCD diet co-treatment with celecoxib and miltefosine (50 mg/kg/day) for 3 weeks. (A) HE-stained and oil red O-stained sections of liver samples are representative of the indicated groups. (Original magnification 200) (B) Hepatic triglyceride levels in the indicated groups. (C) TUNEL-stained sections of liver samples are representative of the indicated groups (magnification 400). (D) Hepatic apoptotic index in the indicated groups. (E) Serum ALT levels in the indicated groups. (F) phosphorylated Akt and p53 protein levels were determined by Western blotting in mice fed the control diet, MCD, MCD with celecoxib (20 mg/kg/day) treatment, or an MCD diet and treatment with celecoxib and miltefosine (50 mg/kg/day) for 3 weeks. (G and H) Bands were quantified, Graphs represent the gray value ratio of phosphorylated Akt to total Akt and p53 protein to GAPDH. Data are expressed as the means ± SME (n ¼ 46 mice/group). *P < 0.05, compared with control group; #P < 0.05 compared with MCD mice; **P < 0.05, compared with MCD þ CEL group. Abbreviations: MCD, methionine and choline deficient; CON, control; CEL, celecoxib; MTF, miltefosine.
therapy regarding NASH. In the present study, irrespective of whether it involved pharmacological effects or genetic deletion, the inhibition of COX-2 significantly decreased the number of apoptosis cells. This suggests that inhibiting COX-2 not only plays an antiinflammatory role, but also exerts an anti-apoptosis effect in the treatment of NASH. At the molecular level, the PI3K/Akt pathway plays an important role in the progression of NASH. It indicates that the regulation of the PI3K/Akt pathway in hepatocytes is associated with the progression of NASH, including oxidative stress, insulin resistance and metabolic dysfunction [23]. The activation of Akt provides cells with a survival signal allows them to withstand apoptotic stimuli [24,25]. p53, which acts downstream from Akt, is another significant apoptosis regulator. Several studies have shown that activation of the p53 pathway is involved in hepatocyte apoptosis in NASH and the expression of p53 is positively correlated with the stage of
liver steatosis [17,20]. Consistent with previous studies, our data show that the levels of pAkt were significantly reduced and the expression of p53 was markedly elevated in experimental NASH, and that this was responsible for the observed hepatocyte apoptosis, whereas these were reversed by the COX-2 inhibitor celecoxib. These results demonstrate that the effects of celecoxib in attenuating hepatocyte apoptosis likely involves regulating the Akt/ p53 signaling pathway. However, Lu S et al. have shown that celecoxib decreases fat accumulation through a COX-2-independent pathway, as the PGE2 levels in HFD-fed mice liver remained the same with or without celecoxib treatment [26]. Considering that celecoxib is not only a COX-2 inhibitor, there may be other multiple mechanisms involved in the treatment of NASH. Therefore, to confirm the role of COX-2, we used COX-2/ mice for an analysis of NASH for the first time. Interestingly, COX-2/ mice that were fed an MCD diet also
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Fig. 4. The effect of a genetic deficiency of COX-2 on steatohepatitis, hepatocyte apoptosis, Akt activation and p53 expression in the liver of MCD-diet fed mice. Wild type mice were fed the control diet, an MCD diet, an MCD diet with celecoxib (20 mg/kg/day), and COX-2/ mice were fed an MCD diet for 3 weeks. (A) HE-stained and oil red O-stained sections of liver samples are representative of the indicated groups. (Original magnification 200) (B) Hepatic triglyceride levels in the indicated groups. (C) TUNEL-stained sections of liver samples are representative of the indicated groups (magnification 400). (D) Hepatic apoptotic index in the indicated groups. (E) phosphorylated Akt and p53 protein levels were determined by Western *blotting in wild type mice that had been fed the control diet, MCD diet, MCD with celecoxib (20 mg/kg/day) treatment, and COX-2/ mice fed with MCD diet for 3 weeks. (F and G) Bands were quantified, Graphs represent the gray value ratio of phosphorylated Akt to total Akt and p53 protein to GAPDH. Data are expressed as the means ± SME (n ¼ 35 mice/group). *P < 0.05, compared with control group; #P > 0.05 compared with MCD (KO) group; **P < 0.05, compared with MCD group. Abbreviations: MCD, methionine and choline deficient; CON, control; CEL, celecoxib; KO, COX-2 knock out.
showed an ameliorated hepatic steatosis and hepatocyte apoptosis via regulating the Akt/p53 signaling pathway, an effect that is similar to the effects of celecoxib. From our current data, we conclude that COX-2 plays an important role in hepatocyte apoptosis during the progression of NASH. NASH is a chronic progressive disease. During the pathogenesis of NASH, hepatocyte apoptosis, inflammation and hepatic steatosis are coexist and all are highly interrelated and mutually reinforcing [27,28]. The anti-inflammation effect of inhibiting COX-2 is generally accepted, whereas we observed that a COX-2 inhibitor ameliorated hepatic steatosis and hepatocytes apoptosis via regulating the Akt/p53 signaling pathway in MCD diet induced NASH models. In a word, the inhibition of COX-2 breaks the vicious circle and inhibits the progression of NASH, thus making COX-2 a
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