Accepted Article
Enkhbold Ch, et al
Original Article
Dysfunction of liver regeneration in aged liver after partial hepatectomy 1
Chinbold Enkhbold, MD, Yuji Morine, MD, FACS, Tohru Utsunomiya, MD, FACS, Satoru Imura, MD, FACS, Tetsuya Ikemoto, MD, FACS, Yusuke Arakawa, MD, Yu Saito, MD, Shinichiro Yamada MD, Daichi Ishikawa, MD, Mitsuo Shimada MD, FACS, Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan
Correspondence to: Yuji Morine M.D. FACS. Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan. Phone: +81-88-633-9276
Fax: +81-88-631-9698 E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12930
This article is protected by copyright. All rights reserved.
1
Accepted Article
Enkhbold Ch, et al
2
Abstract Background. A remarkable feature of the liver is its regenerative capacity following partial hepatectomy. However, the regenerative capacity of many organs and tissues loses its
natural ability to divide with aging. In this study, we investigated the association of aging with endoplasmic reticulum stress, the cell cycle, autophagy and apoptosis-related genes
during liver regeneration after hepatectomy. Methods. Balb/c 4-week and 40-week-old male mice were subjected to 70% hepatectomy. Animals were sacrificed at 24, 48, and 72 h after hepatectomy. Immunohistochemical stainings for proliferating cell nuclear antigen, LC3, Atg5, and caspase-3 were used to quantify protein expression. Real-time reverse transcriptionpolymerase chain reaction was used to detect p16, CHOP, LC3, Atg5, hepatocyte growth factor, cMet, cyclin D1, cyclin A2, and caspase-3 expression. Results. After hepatectomy, old group showed a lower survival rate and significantly lower expression of hepatocyte growth factor, cMet, cyclin D1, cyclin A2, proliferating cell nuclear antigen labeling index, and SMP30 compared with young group. The liver
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al
weight/body weight ratio was significantly lower at 48 h and 72 h after hepatectomy, and was accompanied by markedly elevated levels of the liver cell injury markers, LC3 and caspase-3. Immunohistochemical results showed that LC3, Atg5 and caspase-3 protein expression were higher in old group than in young group. Conclusion. These results revealed that impaired liver regeneration was due to aging,
which was expressed by decreased cell cycle and increased autophagy and apoptosis. Therefore, understanding the molecular basis for aged liver regeneration might provide a new therapeutic option for old patients.
Keywords Aging, liver regeneration, partial hepatectomy, ER stress, autophagy.
This article is protected by copyright. All rights reserved.
3
Accepted Article
Enkhbold Ch, et al
Introduction The liver is one of the most complex and unique organs, which is able to regenerate itself in response to injury and after surgical resection.1,2 With increased life expectancy, the number of elderly patients who require surgical resection and transplantation in hospitals is expected to increase. The most dramatic effect of aging on the liver is its delayed and reduced proliferation after loss of mass from surgical resection or chemical injury. The aged liver does not tolerate pathological stresses well, and is linked to many pathological conditions and functional decline.3-5 Liver cell proliferation is tightly regulated by a series of cell signaling pathways and cascades that are activated after resection. Aging is associated with replicative senescence and p16 levels increase with aging in most mammalian tissues. Studies have shown that decreased proliferation of stem cells is because of enhanced expression of p16, confirming the relationship between p16 expression and stem cell regeneration.6,7 p16 binds to CDK4/6 (cyclin-dependent kinase) inhibiting its kinase activity, thereby preventing retinoblastoma phosphorylation.8 SMP30 is also an age-associated protein. The expression of SMP30 is decreased with aging, and
This article is protected by copyright. All rights reserved.
4
Accepted Article
Enkhbold Ch, et al
one of its functions is the protection of cells against injuries by enhancement of membrane calcium pump activity.9 Insufficient Ca+2 regulation can cause an aged organism to become more vulnerable to cellular dysfunction, thereby contributing to agerelated functional deficits.10 Endoplasmic reticulum (ER) stress is known as a potent trigger of macroautophagy.11 In case of unresolved and sustained ER stress, the unfolded protein response may also trigger apoptosis, mainly through the action of CHOP.12 Sustained levels of CHOP and its target gene are considered an important switch between cell adaptation and death in
response to ER stress.13 ER stress is a potent inducer of autophagy,14 and autophagosome formation is associated with posttranslational modification of the microtubule-associated protein LC3. Autophagy is a cellular process, which mediates the degradation of intracellular components in autolysosomes, thus contributing to maintenance of cellular homeostasis, intracellular clearance of damaged structures, and adaptation to environmental challenges.15 A decrease in autophagic activity with age, described in almost all of the
This article is protected by copyright. All rights reserved.
5
Accepted Article
Enkhbold Ch, et al
model organisms that have been analyzed, has been proposed to contribute to agedependent accumulation of damaged intracellular components, which leads to altered cellular homeostasis and loss of function in aging.16 The relationship of ER stress, autophagy and apoptosis is complex, with either ER stress or autophagy having a protective and apoptotic role.17 Hepatocyte growth factor (HGF)/Met activation is crucial for liver regeneration, and provide an early and sustained signal for hepatocyte proliferation.18,19 HGF is an initiator of liver regeneration, and it can cause massive hepatic enlargement and DNA synthesis in hepatocytes when injected into mice.20 On the other hand, apoptosis, a type of
programmed cell death, occurs under physiological and pathological conditions. Apoptosis and cellular proliferation are complimentary, and account for the maintenance, growth, or degradation of a tissue. The regulation of apoptosis is important during liver regeneration.21,22 Apoptosis involves the activation of catabolic proteases in signaling cascades to destroy cellular components when a cell is under stress or suffers from irreversible damage.23
This article is protected by copyright. All rights reserved.
6
Accepted Article
Enkhbold Ch, et al
To the best of our knowledge, this is the first study to demonstrate the relations among aging, autophagy, apoptosis and cell cycle genes after partial hepatectomy in mice. In this study, we provide the details and summarized the gene expressions related to aging, autophagy, apoptosis, ER stress and cell cycle genes following partial hepatectomy.
Methods Ethics statement and animal treatment The experiments were performed under guidelines of the National Institutes of Health and the Institutional Animal Use and Care Committee at the University of Tokushima. BALB/c 20 old (40 weeks) mice and 20 young (4 weeks) male mice were used. The mice were kept in a 12:12-hour light/dark cycle, at room temperature and humidity, and had unlimited access to water and commercial feed. Animals were anesthetized by inhalation of isoflurane (2% v/v; Butler Schein, OH). Median laparotomy and the left lateral and median lobes (70% of the liver) were resected. Sterile ligatures were used to isolate both lobes simultaneously as close as possible to the inferior vena cava. The mice were
This article is protected by copyright. All rights reserved.
7
Accepted Article
Enkhbold Ch, et al
sacrificed at 24, 48, and 72 h after hepatectomy (n=5 at each time point).
Immunohistochemistry Tissue specimens were fixed in neutral buffered formalin and then embedded in paraffin. The 4- µm paraffin sections were deparaffinized in xylene and rehydrated in graded alcohol series. Endogenous peroxidase was inhibited using 0.3% H2O2 in methanol. The sections were heated in a microwave oven (in citrate buffer 10 mM, pH 6.0) for 20 min for epitope retrieval. The primary antibodies were against proliferating cell nuclear antigen (PCNA) (1:4000; Abcam), caspase-3 (1:200; Cell Signaling), LC3 (1:50; Abcam), and Atg5 (1:100; Abcam). The slides were incubated with anti-mouse/rabbit biotinylated bridging antibodies (dilution: 1/200, DAKO) for 60 min. The sections were counterstained with Mayer’s hematoxylin. Stained tissue sections were reviewed and scored for the intensity and proportion of staining on five randomly selected fields under an optical microscope. The intensity of staining was scored from 0 to 3 (0, without staining; 1, weak; 2, moderate; and 3, strong). The proportion of staining was scored from
This article is protected by copyright. All rights reserved.
8
Accepted Article
Enkhbold Ch, et al
0 to 4 (0–5%, not stained; 5–25% score=1; 25–50% score=2; 50–75% score=3; 75–100%, score=4). PCNA antigen expression levels were determined based on staining of nuclei in five randomly selected fields for each group and time point.
RNA extraction and real-time reverse transcription-polymerase chain reaction (RTPCR)
Total RNA was isolated from the livers using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. A total of 10 µg of RNA was used as a template for the synthesis of cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science). The following TaqMan (Applied Biosystems) probe and primer sets were purchased and used: HGF (Mm01135193), cMet (Mm01156972), p16 (Mm00494449), SMP30 (Mm00485711), CHOP (Mm01135937), Atg5 (Mm00504340), LC3 (Mm00458725), caspase-3 (Mm01195084), cyclin D1 (Mm00432359), and cyclin A2 (Mm00438063). The results were normalized to the level of mRNA for human glyceraldehyde-3-phosphate dehydrogenase.
This article is protected by copyright. All rights reserved.
9
Accepted Article
Enkhbold Ch, et al 10
Western blot analysis For western blot analysis, ~50 mg of liver samples were homogenized. The supernatant was collected, and the protein concentration was determined using the BCA method (BCA kit 23227). Samples were heated for 5 min under reducing conditions and loaded onto sodium dodecyl sulfate-polyacrylamide gels. Proteins from the gels were then transferred onto PVDF membranes. Electroblots were blocked in Tris-buffered NaClTween (TBST) containing 3% bovine serum albumin at room temperature. Western blot analysis was then conducted using specific antibodies for LC3A/B (Cell Signaling Technology, 4108). Blots were incubated with antibodies of interest (in TBST buffer and 2% bovine serum albumin) and agitated 1 hour at room temperature. Following washing with TBST, the blots were incubated with horseradish peroxidase-labeled anti-rabbit antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) in BSA for 1 h at room temperature. Immunoreactivity was detected by enhanced chemiluminescence.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 11
Measurements in blood Blood samples were collected via the inferior hepatic vein and at designated time points before sacrifice. Samples were centrifuged at 3000 rpm for 5 min. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and lactate dehydrogenase (LDH) were measured.
Statistical analysis
Statistical analysis was performed using JMP 8.0.1 statistical software (SAS, NC, USA). Statistical assessment for significance was determined using the ANOVA. All variables are presented as the mean ± standard deviation (SD). P values less than 0.05 were considered significant (*), and less than 0.01 were considered highly significant (**).
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 12
Results Survival and LW/BW ratio Survival after hepatectomy was 100% in the young group and 60% in the old group (Fig. 1A, P=0.04). To evaluate liver regeneration after partial hepatectomy, the LW/BW ratio was analyzed (Fig. 1B). The LW/BW ratio gradually recovered to basal preoperative levels after partial hepatectomy. The LW/BW ratio in the old group was significantly lower at 48 h (2.7±0.003 vs 3.3±0.004, P=0.02) and 72 h (2.9±0.001 vs 3.9±0.003, P=0.002) compared with that in the young group.
Liver function test LDH levels in the old group were significantly elevated at 24 h (12 394±7934 vs 2102±1459, P=0.02), 48 h (4838±2689 vs 1071±493, P=0.01), and 72 h (2136±1149 vs 603±244, P=0.01; Fig. 2A) compared with those in the young group. ALT levels in the old group were significantly elevated at 48 h (2762±1446 vs 622±463, P=0.01) and 72 h (1116±588 vs 124±90, P=0.005; Fig. 2B) compared with those in the young group. AST
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 13
levels in the old group were significantly elevated at 48 h (2729±1741 vs 531±369, P=0.001) and 72 h (1280±690 vs 213±134, P=0.01; Fig 2C) compared with those in the young group. Total bilirubin levels were markedly higher in the old group than those in the young group at 72 h (1.122±0.86 vs 0.094±0.05, P=0.02; Fig. 2D). After hepatectomy, consistent with increased death of hepatocytes and inflammation in the liver, serum levels of ALT, AST, total bilirubin, and LDH were elevated in the old group at each time point. These results suggested that high levels of total bilirubin, ALT, AST, and LDH in the serum reflected more liver cell injury and death in the old group.
Evaluation of proliferative gene expression after partial hepatectomy Analysis of HGF and cMet expression by PCR showed that their expression was higher in the young group compared with the old group at all of the time points (Fig. 3A). HGF expression levels in the young group were significantly higher at 24 h (0.906±0.31 vs 0.315±0.028, P=0.003), and cMet expression levels in the young group were significantly
higher at 48 h (0.872±0.29 vs 0.544±0.21, P=0.04; Fig. 3B) compared with those in the
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 14
old group. Cyclin A2 in the young group was significantly elevated at 24 h (2.644±1.74 vs 0.288±0.244, P=0.01), 48 h (1.79±0.51 vs 0.418±0.227, P=0.006), and 72 h
(0.820±0.35 vs 0.146±0.17, P=0.005; Fig. 3C) compared with that in the old group. Cyclin D1 in the young group was significantly elevated at 48 h (1.060±0.51 vs 0.380±0.31, P=0.03; Fig 3D) compared with that in the old group. To evaluate
proliferation of hepatocytes, immunohistochemistry for PCNA was performed. There was significantly more PCNA staining in the young group compared with that in the old group at 24 h (71% vs 42% P=0.001), 48 h (79% vs 61% P=0.002), and 72 h (89% vs 63%
P=0.007) (PCNA-positive nuclei and varying staining intensity are shown in Fig. 4A-C). These data indicated that expression of the proliferative genes HGF and cMet was significantly reduced at all of the time points in the old group compared with the young group.
Evaluation of aging related to ER stress gene expression SMP30 expression in the young group was significantly higher at 24 h (3.30±2.19 vs
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 15
0.94±0.37, P=0.04), 48 h (4.66±3.06 vs 0.442±0.309, P=0.01), and 72 h (5.834±3.93 vs 0.164±0.02, P=0.01) compared with that in the old group (Fig. 5A). Expression levels of
p16 were significantly higher in the old group compared with the young group at 48 h (1.53±0.78 vs 0.343±0.108, P=0.01) and 72 h (1.372±0.41 vs 0.476±0.26, P=0.003; Fig 5B). Expression levels of p16 progressively increased after partial hepatectomy in the old group. Lower SMP30 expression and higher p16 expression were found in the old group compared with the young group after hepatectomy at all of the time points. These results indicated that hepatocytes were more vulnerable and there was more cell cycle arrest in the old group compared with the young group. We also examined CHOP expression, which is the main apoptotic pathway of ER stress. According to PCR analysis, we found higher CHOP expression and a progressive increase in CHOP expression in the old group compared with the young group at all of the time points. However, significant differences were not found at each time point 24 h (0.36±0.23 vs 0.32±0.21, P=0.18), 48 h (0.55±0.44 vs 0.25±0.11, P=0.80), and 72 h
(1.26±0.43 vs 0.78±0.31, P=0.08).
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 16
Evaluation of autophagy and apoptosis
After partial hepatectomy, autophagy-related LC3 and Atg5 protein expressions were markedly higher in the old group. We did not found significant difference in LC3 at 24 h (0.86±0.12 vs 0.65±0.25, P=0.09), 48 h (1.59±0.31 vs 2.06±1.03, P=0.35), and 72 h
(0.93±0.13 vs 0.74±0.19, P=0.06) and Atg5 mRNA expression at 24 h (0.81±0.13 vs
0.57±0.31, P=0.14), 48 h (1.02±0.45 vs 0.82±0.05, P=0.36), and 72 h (2.26±0.86 vs 2.53±0.39, P=0.53) between the old and young groups. Immunohistochemically, LC3 was strongly positive in the cytoplasm in the old group (score=5, Fig. 6A). However, LC3 was weakly stained in the young group (score=2, Fig. 6B). Also, western blot analysis result showed higher LC3 expression in the old group (Fig. 4D). Additionally, Atg5 protein was highly expressed in the old group (score=5, Fig. 6C, D). Caspase-3 mRNA expression in the old group was significantly higher at 48 h (1.886±0.23 vs 1.260±0.61, P=0.04) and 72 h (2.456±0.77 vs 1.352±0.24, P=0.01; Fig 5C) compared with that in the young group. Immunohistochemically, caspase-3 was more
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 17
strongly stained in the old group (score=6) compared with the young group (score=1, Fig. 6E, F). The intensity of caspase-3 protein expression increased with prolonged exposure time in the old group, whereas that of the young group remained constant. These results indicated that autophagy- and apoptosis-related markers were activated after hepatectomy in the old group.
Discussion The phenomenon of liver regeneration after partial hepatectomy is complex. In this study, we investigated aged liver regeneration and focused on the aging, cell cycle, autophagyand apoptosis-related genes that are crucial for liver regeneration. We first demonstrated that liver regeneration was impaired in the liver of aged mice after partial hepatectomy, and this was characterized by reduced DNA synthesis in hepatocytes, and increased apoptosis and autophagy. Survival in the old group was worse than that in the young group after partial hepatectomy and the LW/BW ratio was also lower in the old group. Liver function test
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 18
results showed that markers of liver injury, such as ALT, AST, total bilirubin, and LDH, were significantly higher in the old group. These results possibly reflect the extent of hepatocyte injury. HGF is a potent inducer of DNA synthesis, and HGF and its receptor cMet are important growth factors for liver regeneration. HGF has been shown to be indispensable and important for liver regeneration.19 Consistent with this finding, we found that HGF and cMet expressions were lower in the old group at all of the time points after partial hepatectomy. Senescent cells express p16 ink4a, a cyclin dependent kinase inhibitor and tumor suppressor, which enforces growth arrest by activating retinoblastoma.24 This pathway
contributes to impaired cellular regeneration of an aging organism and acts as a marker in this process. In our study, p16 expression in the old group was upregulated after partial
hepatectomy, and its downstream target CDK/cyclin D1 was lower in the old group than in the young group. This result indicated that the cell cycle mechanism of the liver progressively decreased in the old group, and thereafter, cell cycle arrest mechanisms
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 19
were increased. Autophagy, a homeostatic, catabolic degradation process, prevents the accumulation of damaged proteins or organisms. Autophagy has cytoprotective and cytocidal effects, and is induced by ER stress, and is a downstream mediator of ER stress-related cell death. Excessive autophagy might promote cell death and intense degranulation may be the turning point that commits to a damaged cell death process rather than a cell protective process.17,25 Many studies have suggested that autophagy may mediates cell death, and autophagy has been classified as one of three morphological forms of cell death together with apoptosis and necrosis.26 However, other investigations have suggested an alternative function for macroautophagy as a critical pathway for cell survival.27 In our study, after partial hepatectomy, autophagy-related LC3 and Atg5 expressions were increased with the ER stress marker CHOP in the old group at all of the time points. CHOP is a proapoptotic transcription factor, which is expressed at low levels under physiological conditions, but is strongly induced during severe ER stress.28 Therefore, in the current study, partial hepatectomy was an excessive stress for the aged liver, and
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 20
resulted in an increase in those markers. Increased autophagy might be triggered by accumulation of damaged mitochondria and other organelles, intracellular components and unfolded proteins in aged liver. In such cases, dying cells activate autophagy to degrade by own lysosomes, that is an alternative way of cell death pathway.23,29 Cell cycle arrest, autophagy, and senescence are closely linked with conserved biological processes, such that autophagy induces senescence, and conversely, senescence induces autophagy. Cell lines overexpressing p16 are clearly more sensitive to the induction of senescence and autophagy, especially under conditions of stress. 30 SMP30 has an antiapoptotic function, which prevents cells from various injuries occurring during their lifespan. In SMP30-KO mice, hepatocytes are more susceptible to apoptosis. 31 Our study showed that SMP30 expression was lower in the old group than in the young group at all of the time points after partial hepatectomy. Alterations in Ca+2 homeostasis can negatively affect mitochondrial Ca+2 signaling. Therefore, Ca+2 is a second messenger that can initiate the intrinsic apoptotic pathway, which is responsible for releasing proapoptotic proteins from the intermembrane space to the cytoplasm. It
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 21
activates the conversion of procaspase-9 to caspase-9, which then cleaves procaspase-3 and activated caspase-3, causing the release of caspase-activated DNAse.32-35 In our study, caspase-3, which is one of the major indicators of apoptosis, was significantly higher in the old group at all of the time points. This finding suggested that antiapoptotic function was decreased in old liver regeneration after hepatectomy. All of our results demonstrated that ER stress, apoptosis, and autophagy are important for regulating liver regeneration. Liver regeneration is affected by age and aging, and cell cycle-related genes are affected by this process. In conclusion, regulation of these genes is required for liver regeneration to proceed normally. Further studies are needed to address the potential differences in old liver regeneration, and the implications of these differences. It is reasonable to assume that blocking and activating specific signaling pathways and genes will more likely lead to liver regeneration stimulus. Therefore, understanding the molecular basis for aging liver regeneration will not only help define the impaired conditions, but it will also hopefully lead to new therapeutic options for old patients.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 22
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research (C) (21462091) and Scientific Research (B) (23390324), from the Japan Society for the Promotion of Science.
References
1. Fausto N, Campbell JS, Riehle KJ. Liver Regeneration. Hepatology 2006; 43: 45-53. 2. Michalopoulos GK. Liver Regeneration. J Cell Physiol 2007; 231: 286-300. 3. Suzuki A, Sakaguchi T, Inaba K, Suzuki S, Konno H. Impact of cell cycle disruption on impaired hepatic regeneration in aged livers with ischemic insult. J Surg Res 2012; 173: 267-277.
4. Sanchez-Hidalgo JM, Naranjo A, Ciria R, Ranchal I, Aguilar-Melero P. Ferrin G, et al. Impact of age on liver regeneration response to injury after partial hepatectomy in a rat model. J Surg Res 2012; 175: 1-9.
5. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young systematic environment. Nature 2005;
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 23
433: 760-764.
6. Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, et al. Ink4a/Arf expression is a biomarker of aging. J Clin Invest 2004; 114: 1299-1307.
7. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003; 425: 962-967.
8. Rayess H, Wang MB, Srivatsan ES. Cellular senescence and tumor suppressor gene p16. Int J Cancer 2012; 130: 1715-1725.
9. Kim WY, Sharpless NE. The regulation of INK4/ARF in cancer and aging. Cell 2006; 127: 265-275.
10. Gil J, Peters G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 2006; 7: 667-677.
11. Bernales S, McDonald KL, Walter P. Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLos Biol 2006; 4: e423.
12. Lin JH, Walter P, Yen TS. Endoplasmic reticulum stress in disease pathogenesis. Annu
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 24
Rev Pathol 2008; 3: 399-425.
13. Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y, Jungreis R, et al. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic retilculum. Genes Dev 2004; 18: 3066-3077.
14. Rutkowski DT, Arnold SM, Miller CN, Wu J, Li J, Gunnison KM, et al. Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and
proteins. PLos Biol 2006; 4: e374.
15. Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, et al. Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 2006; 26: 9220-9231.
16. Cuervo AM, Bergamini E, Brunk UT, Droge W, Ffrench M, Terman A. Autophagy and aging: the importance of maintaining “clean” cells. Autophagy 2005; 1: 131-140.
17. Zhang J, Morris MW Jr, Dorsett-Martin WA, Drake LC, Anderson CD. Autophagy is involved in endoplasmic reticulum stress-induced cell death of rat hepatocytes. J Surg Res 2013; 183: 929-935.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 25
18. Zarnegar R, DeFrances MC, Kost DP, Lindroos P, Michalopoulos GK. Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy. Biochem Biophys Res Commun 1991; 177: 559-565.
19. Nejak-Bowen K, Orr A, Bowen WC Jr, Michalopoulos GK. Conditional genetic elimination of hepatocyte growth factor in mice compromises liver regeneration after partial hepatectomy. PLoS One 2013; 8: e59836.
20. Patijn GA, Lieber A, Schowalter DB, Schwall R, Kay MA. Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice. Hepatology 1998; 28: 707-716.
21. Weber A, Boger R, Vick B, Urbanik T, Haybaeck J, Zoller S, et al. Hepatocyte-specific deletion of the antiapoptotic protein myeloid cell leukemia-1 triggers proliferation and hepatocarcinogenesis in mice. Hepatology 2010; 51: 1226-1236.
22. Xiong Y, Fang JH, Yun JP, Yang J, Zhang Y, Jia WH, et al. Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology 2010; 51: 836-845.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 26
23. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007; 8: 741-752.
24. Sharpless NE. Ink4a/ Arf links senescence and aging. Exp Gerontol 2004; 39: 1751-1759. 25. Passegue E, Wagner EF. JunB suppresses cell proliferation by transcriptional activation of p16 (INK4a) expression. EMBO J 2000; 19: 2969-2979.
26. Ito M, Nakagawa H, Okada T, Miyazaki S, Matsuo S. ER-stress caused by accumulated intracistinal granules activates autophagy through a different signal pathway from unfolded protein response in exocrine pancreas cells of rats exposed to fluoride. Arch Toxicol 2009; 83: 151-159.
27. Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 2008; 9: 1004-1010.
28. Bhogal RH, Weston CJ, Curbishley SM, Adams DH, Afford SC. A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress. Autophagy 2012; 8: 545-558.
29. Levine B, Yuan J. Autophagy in cell death: an innocent convict?. J Clin Invest 2005; 115:
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 27
2679-2688.
30. Capparelli C, Chiavarina B, Whitaker-Menezes D, Pestell TG, Pestell RG, Hulit J, et al. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neoangiogenesis. Cell cycle 2012; 11: 3599-3610.
31. Galehdar Z, Swan P, Fuerth B, Callaghan SM, Park DS, Cregan SP. Neuronal apoptosis induced by endoplasmic reticulum stress is regulated by ATF4-CHOP-mediated induction of the Bcl-2 homology 3-only member PUMA. J Neurosci 2010; 30: 16938-16948.
32. Chandra D, Bratton SB, Person MD, Tian Y, Martin AG, Ayres M, et al. Intracellular nucleodites act as critical prosurvival factors by binding to cytochrome C and inhibiting
apoptosome. Cell 2006; 125: 1333-1346.
33. Saelens X, Festjens N, Vande Walle L, van Gurp M, van Loo G, Vandenabeele P. Toxic proteins released from mitochondria in cell death. Oncogene 2004; 23: 2861-2874.
34. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 2003; 4: 552-565.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 28
35. Enari M, Sakahira H, Yokohama H, Okawa K, Iwamatsu A, Nagata S. A caspase activated DNAse when released from mitochondria. Nature 1998; 391: 43-50.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 29
Figure Legends Figure 1. A.
Survival in the young group was 100% after surgery and old group survival was
60%.
B.
Liver weight/body weight (LW/BW) ratio in both group after partial hepatectomy.
LW/BW ratio was significantly low at 48h and 72h in old group.
Figure 2. A. LDH were significantly elevated at 24h and 72h in old group.
B. Serum level of ALT was significantly elevated at 48h and 72h in old group. C. AST was markedly elevated at 48h and 72h in old group. D. Total bilirubin level of old group was increased markedly at 72h.
Figure 3. A.
Level of HGF was lower in old group at all-time points but significantly
decreased at 24h.
B.
Met was significantly lower at 48h in old group.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 30
C.
CyclinA2 was significantly decreased at 24h, 48h and 72h in old group.
D.
CyclinD1 was found significant at 48h.
Figure 4. A.
PCNA was significantly well stained in the young group compared with old
group at 24h, 48h and 72.
B.
PCNA positive nuclei in young group at 72h (×200).
C.
PCNA positive nuclei in old group at 72h (×200).
D.
Western blot analysis for LC3-I and LC3-II expression at 24h, 48h and 72h.
Figure 5. A. SMP30 was significantly higher at 24h, at 48h and 72h in young group. B. p16 expression level were significantly higher in old group at 48h and 72h. C. Caspase3 was higher in old group and significantly at 48h and 72h.
Figure 6. A.
LC3 protein expression in old group at 48h after hepatectomy (×200).
B.
LC3 protein expression in young group at 48h after hepatectomy (×200).
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 31
C.
Atg5 protein expression in old group at 72h after hepatectomy (×200).
D.
Atg5 protein expression in young group at 72h after hepatectomy (×200)
E.
Caspase3 protein expression in old group at 72h after hepatectomy (×200)
F. Caspase3 protein expression in young group at 72h after hepatectomy (×200)
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 32
Figure 1
Young Old
A
Survival after hepatectomy
B
LW/ BW Ratio
% 100 Young (n=20)
*
**
48h
72h
4.0
p=0.04
50
3.0 Old (n=20) 2.0 0
0 0
2
4
6 Days
8
10
This article is protected by copyright. All rights reserved.
24h
Accepted Article
Enkhbold Ch, et al 33
Figure 2
Young
LDH (U/L)
A 25000
*
20000
**
B **
Old
ALT (U/L) 7000 6000
**
**
48h
72h
5000
15000
4000 3000
10000
2000 5000 0
1000
24h
48h
AST (U/L)
C
0
72h
Total Bilirubin (U/L)
D
14000
24h
8
12000
**
10000
**
6
*
8000 4
6000 4000
2
2000 0
24h
48h
72h
This article is protected by copyright. All rights reserved.
0
24h
48h
72h
Accepted Article
Enkhbold Ch, et al 34
Figure 3
Young
HGF B
**
1.2 1.0 0.8 0.6 0.4 0.2 0
24h
48h
Relative mRNA expression
5
** **
4
**
3 2 1 0
24h
48h
72h
This article is protected by copyright. All rights reserved.
*
1.2 1.0 0.8 0.6 0.4 0.2
24h
48h
72h
Cyclin D1
D
6
1.4
0
72h
Cyclin A2
C
1.6 Relative mRNA expression
Relative mRNA expression
1.4
2.5 Relative mRNA expression
A
Old
Met
*
2.0 1.5 1.0 0.5 0
24h
48h
72h
Accepted Article
Enkhbold Ch, et al 35
Figure 4
Young Old
PCNA
A 100
Labeling index
90
**
**
B **
80 70 60 50 40 30
200µm
C
D LC3-I 15kDa
LC3-II GAPDH 24h 48h 72h 24h 48h 72h
Young 200µm
This article is protected by copyright. All rights reserved.
Old
Accepted Article
Enkhbold Ch, et al 36
Figure 5
Young
SMP30 B
Relative mRNA expression
14 12
*
**
10
**
8 6 4 2 0
24h
48h
72h
Caspase3 C
**
Relative mRNA expression
3.5 3.0
*
2.5 2.0 1.5 1.0 0.5 0
24h
48h
72h
This article is protected by copyright. All rights reserved.
3.0 Relative mRNA expression
A
Old
p16 **
**
48h
72h
2.5 2.0 1.5 1.0 0.5 0
24h
Accepted Article
Enkhbold Ch, et al 37
Figure 6
A
B
200µm
D
C
200µm
E
200µm
This article is protected by copyright. All rights reserved.
200µm
F
200µm
200µm
Enkhbold Ch, et al
Original Article
Dysfunction of liver regeneration in aged liver after partial hepatectomy 1
Chinbold Enkhbold, MD, Yuji Morine, MD, FACS, Tohru Utsunomiya, MD, FACS, Satoru Imura, MD, FACS, Tetsuya Ikemoto, MD, FACS, Yusuke Arakawa, MD, Yu Saito, MD, Shinichiro Yamada MD, Daichi Ishikawa, MD, Mitsuo Shimada MD, FACS, Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan
Correspondence to: Yuji Morine M.D. FACS. Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan. Phone: +81-88-633-9276
Fax: +81-88-631-9698 E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12930
This article is protected by copyright. All rights reserved.
1
Accepted Article
Enkhbold Ch, et al
2
Abstract Background. A remarkable feature of the liver is its regenerative capacity following partial hepatectomy. However, the regenerative capacity of many organs and tissues loses its
natural ability to divide with aging. In this study, we investigated the association of aging with endoplasmic reticulum stress, the cell cycle, autophagy and apoptosis-related genes
during liver regeneration after hepatectomy. Methods. Balb/c 4-week and 40-week-old male mice were subjected to 70% hepatectomy. Animals were sacrificed at 24, 48, and 72 h after hepatectomy. Immunohistochemical stainings for proliferating cell nuclear antigen, LC3, Atg5, and caspase-3 were used to quantify protein expression. Real-time reverse transcriptionpolymerase chain reaction was used to detect p16, CHOP, LC3, Atg5, hepatocyte growth factor, cMet, cyclin D1, cyclin A2, and caspase-3 expression. Results. After hepatectomy, old group showed a lower survival rate and significantly lower expression of hepatocyte growth factor, cMet, cyclin D1, cyclin A2, proliferating cell nuclear antigen labeling index, and SMP30 compared with young group. The liver
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al
weight/body weight ratio was significantly lower at 48 h and 72 h after hepatectomy, and was accompanied by markedly elevated levels of the liver cell injury markers, LC3 and caspase-3. Immunohistochemical results showed that LC3, Atg5 and caspase-3 protein expression were higher in old group than in young group. Conclusion. These results revealed that impaired liver regeneration was due to aging,
which was expressed by decreased cell cycle and increased autophagy and apoptosis. Therefore, understanding the molecular basis for aged liver regeneration might provide a new therapeutic option for old patients.
Keywords Aging, liver regeneration, partial hepatectomy, ER stress, autophagy.
This article is protected by copyright. All rights reserved.
3
Accepted Article
Enkhbold Ch, et al
Introduction The liver is one of the most complex and unique organs, which is able to regenerate itself in response to injury and after surgical resection.1,2 With increased life expectancy, the number of elderly patients who require surgical resection and transplantation in hospitals is expected to increase. The most dramatic effect of aging on the liver is its delayed and reduced proliferation after loss of mass from surgical resection or chemical injury. The aged liver does not tolerate pathological stresses well, and is linked to many pathological conditions and functional decline.3-5 Liver cell proliferation is tightly regulated by a series of cell signaling pathways and cascades that are activated after resection. Aging is associated with replicative senescence and p16 levels increase with aging in most mammalian tissues. Studies have shown that decreased proliferation of stem cells is because of enhanced expression of p16, confirming the relationship between p16 expression and stem cell regeneration.6,7 p16 binds to CDK4/6 (cyclin-dependent kinase) inhibiting its kinase activity, thereby preventing retinoblastoma phosphorylation.8 SMP30 is also an age-associated protein. The expression of SMP30 is decreased with aging, and
This article is protected by copyright. All rights reserved.
4
Accepted Article
Enkhbold Ch, et al
one of its functions is the protection of cells against injuries by enhancement of membrane calcium pump activity.9 Insufficient Ca+2 regulation can cause an aged organism to become more vulnerable to cellular dysfunction, thereby contributing to agerelated functional deficits.10 Endoplasmic reticulum (ER) stress is known as a potent trigger of macroautophagy.11 In case of unresolved and sustained ER stress, the unfolded protein response may also trigger apoptosis, mainly through the action of CHOP.12 Sustained levels of CHOP and its target gene are considered an important switch between cell adaptation and death in
response to ER stress.13 ER stress is a potent inducer of autophagy,14 and autophagosome formation is associated with posttranslational modification of the microtubule-associated protein LC3. Autophagy is a cellular process, which mediates the degradation of intracellular components in autolysosomes, thus contributing to maintenance of cellular homeostasis, intracellular clearance of damaged structures, and adaptation to environmental challenges.15 A decrease in autophagic activity with age, described in almost all of the
This article is protected by copyright. All rights reserved.
5
Accepted Article
Enkhbold Ch, et al
model organisms that have been analyzed, has been proposed to contribute to agedependent accumulation of damaged intracellular components, which leads to altered cellular homeostasis and loss of function in aging.16 The relationship of ER stress, autophagy and apoptosis is complex, with either ER stress or autophagy having a protective and apoptotic role.17 Hepatocyte growth factor (HGF)/Met activation is crucial for liver regeneration, and provide an early and sustained signal for hepatocyte proliferation.18,19 HGF is an initiator of liver regeneration, and it can cause massive hepatic enlargement and DNA synthesis in hepatocytes when injected into mice.20 On the other hand, apoptosis, a type of
programmed cell death, occurs under physiological and pathological conditions. Apoptosis and cellular proliferation are complimentary, and account for the maintenance, growth, or degradation of a tissue. The regulation of apoptosis is important during liver regeneration.21,22 Apoptosis involves the activation of catabolic proteases in signaling cascades to destroy cellular components when a cell is under stress or suffers from irreversible damage.23
This article is protected by copyright. All rights reserved.
6
Accepted Article
Enkhbold Ch, et al
To the best of our knowledge, this is the first study to demonstrate the relations among aging, autophagy, apoptosis and cell cycle genes after partial hepatectomy in mice. In this study, we provide the details and summarized the gene expressions related to aging, autophagy, apoptosis, ER stress and cell cycle genes following partial hepatectomy.
Methods Ethics statement and animal treatment The experiments were performed under guidelines of the National Institutes of Health and the Institutional Animal Use and Care Committee at the University of Tokushima. BALB/c 20 old (40 weeks) mice and 20 young (4 weeks) male mice were used. The mice were kept in a 12:12-hour light/dark cycle, at room temperature and humidity, and had unlimited access to water and commercial feed. Animals were anesthetized by inhalation of isoflurane (2% v/v; Butler Schein, OH). Median laparotomy and the left lateral and median lobes (70% of the liver) were resected. Sterile ligatures were used to isolate both lobes simultaneously as close as possible to the inferior vena cava. The mice were
This article is protected by copyright. All rights reserved.
7
Accepted Article
Enkhbold Ch, et al
sacrificed at 24, 48, and 72 h after hepatectomy (n=5 at each time point).
Immunohistochemistry Tissue specimens were fixed in neutral buffered formalin and then embedded in paraffin. The 4- µm paraffin sections were deparaffinized in xylene and rehydrated in graded alcohol series. Endogenous peroxidase was inhibited using 0.3% H2O2 in methanol. The sections were heated in a microwave oven (in citrate buffer 10 mM, pH 6.0) for 20 min for epitope retrieval. The primary antibodies were against proliferating cell nuclear antigen (PCNA) (1:4000; Abcam), caspase-3 (1:200; Cell Signaling), LC3 (1:50; Abcam), and Atg5 (1:100; Abcam). The slides were incubated with anti-mouse/rabbit biotinylated bridging antibodies (dilution: 1/200, DAKO) for 60 min. The sections were counterstained with Mayer’s hematoxylin. Stained tissue sections were reviewed and scored for the intensity and proportion of staining on five randomly selected fields under an optical microscope. The intensity of staining was scored from 0 to 3 (0, without staining; 1, weak; 2, moderate; and 3, strong). The proportion of staining was scored from
This article is protected by copyright. All rights reserved.
8
Accepted Article
Enkhbold Ch, et al
0 to 4 (0–5%, not stained; 5–25% score=1; 25–50% score=2; 50–75% score=3; 75–100%, score=4). PCNA antigen expression levels were determined based on staining of nuclei in five randomly selected fields for each group and time point.
RNA extraction and real-time reverse transcription-polymerase chain reaction (RTPCR)
Total RNA was isolated from the livers using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. A total of 10 µg of RNA was used as a template for the synthesis of cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science). The following TaqMan (Applied Biosystems) probe and primer sets were purchased and used: HGF (Mm01135193), cMet (Mm01156972), p16 (Mm00494449), SMP30 (Mm00485711), CHOP (Mm01135937), Atg5 (Mm00504340), LC3 (Mm00458725), caspase-3 (Mm01195084), cyclin D1 (Mm00432359), and cyclin A2 (Mm00438063). The results were normalized to the level of mRNA for human glyceraldehyde-3-phosphate dehydrogenase.
This article is protected by copyright. All rights reserved.
9
Accepted Article
Enkhbold Ch, et al 10
Western blot analysis For western blot analysis, ~50 mg of liver samples were homogenized. The supernatant was collected, and the protein concentration was determined using the BCA method (BCA kit 23227). Samples were heated for 5 min under reducing conditions and loaded onto sodium dodecyl sulfate-polyacrylamide gels. Proteins from the gels were then transferred onto PVDF membranes. Electroblots were blocked in Tris-buffered NaClTween (TBST) containing 3% bovine serum albumin at room temperature. Western blot analysis was then conducted using specific antibodies for LC3A/B (Cell Signaling Technology, 4108). Blots were incubated with antibodies of interest (in TBST buffer and 2% bovine serum albumin) and agitated 1 hour at room temperature. Following washing with TBST, the blots were incubated with horseradish peroxidase-labeled anti-rabbit antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) in BSA for 1 h at room temperature. Immunoreactivity was detected by enhanced chemiluminescence.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 11
Measurements in blood Blood samples were collected via the inferior hepatic vein and at designated time points before sacrifice. Samples were centrifuged at 3000 rpm for 5 min. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and lactate dehydrogenase (LDH) were measured.
Statistical analysis
Statistical analysis was performed using JMP 8.0.1 statistical software (SAS, NC, USA). Statistical assessment for significance was determined using the ANOVA. All variables are presented as the mean ± standard deviation (SD). P values less than 0.05 were considered significant (*), and less than 0.01 were considered highly significant (**).
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 12
Results Survival and LW/BW ratio Survival after hepatectomy was 100% in the young group and 60% in the old group (Fig. 1A, P=0.04). To evaluate liver regeneration after partial hepatectomy, the LW/BW ratio was analyzed (Fig. 1B). The LW/BW ratio gradually recovered to basal preoperative levels after partial hepatectomy. The LW/BW ratio in the old group was significantly lower at 48 h (2.7±0.003 vs 3.3±0.004, P=0.02) and 72 h (2.9±0.001 vs 3.9±0.003, P=0.002) compared with that in the young group.
Liver function test LDH levels in the old group were significantly elevated at 24 h (12 394±7934 vs 2102±1459, P=0.02), 48 h (4838±2689 vs 1071±493, P=0.01), and 72 h (2136±1149 vs 603±244, P=0.01; Fig. 2A) compared with those in the young group. ALT levels in the old group were significantly elevated at 48 h (2762±1446 vs 622±463, P=0.01) and 72 h (1116±588 vs 124±90, P=0.005; Fig. 2B) compared with those in the young group. AST
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 13
levels in the old group were significantly elevated at 48 h (2729±1741 vs 531±369, P=0.001) and 72 h (1280±690 vs 213±134, P=0.01; Fig 2C) compared with those in the young group. Total bilirubin levels were markedly higher in the old group than those in the young group at 72 h (1.122±0.86 vs 0.094±0.05, P=0.02; Fig. 2D). After hepatectomy, consistent with increased death of hepatocytes and inflammation in the liver, serum levels of ALT, AST, total bilirubin, and LDH were elevated in the old group at each time point. These results suggested that high levels of total bilirubin, ALT, AST, and LDH in the serum reflected more liver cell injury and death in the old group.
Evaluation of proliferative gene expression after partial hepatectomy Analysis of HGF and cMet expression by PCR showed that their expression was higher in the young group compared with the old group at all of the time points (Fig. 3A). HGF expression levels in the young group were significantly higher at 24 h (0.906±0.31 vs 0.315±0.028, P=0.003), and cMet expression levels in the young group were significantly
higher at 48 h (0.872±0.29 vs 0.544±0.21, P=0.04; Fig. 3B) compared with those in the
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 14
old group. Cyclin A2 in the young group was significantly elevated at 24 h (2.644±1.74 vs 0.288±0.244, P=0.01), 48 h (1.79±0.51 vs 0.418±0.227, P=0.006), and 72 h
(0.820±0.35 vs 0.146±0.17, P=0.005; Fig. 3C) compared with that in the old group. Cyclin D1 in the young group was significantly elevated at 48 h (1.060±0.51 vs 0.380±0.31, P=0.03; Fig 3D) compared with that in the old group. To evaluate
proliferation of hepatocytes, immunohistochemistry for PCNA was performed. There was significantly more PCNA staining in the young group compared with that in the old group at 24 h (71% vs 42% P=0.001), 48 h (79% vs 61% P=0.002), and 72 h (89% vs 63%
P=0.007) (PCNA-positive nuclei and varying staining intensity are shown in Fig. 4A-C). These data indicated that expression of the proliferative genes HGF and cMet was significantly reduced at all of the time points in the old group compared with the young group.
Evaluation of aging related to ER stress gene expression SMP30 expression in the young group was significantly higher at 24 h (3.30±2.19 vs
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 15
0.94±0.37, P=0.04), 48 h (4.66±3.06 vs 0.442±0.309, P=0.01), and 72 h (5.834±3.93 vs 0.164±0.02, P=0.01) compared with that in the old group (Fig. 5A). Expression levels of
p16 were significantly higher in the old group compared with the young group at 48 h (1.53±0.78 vs 0.343±0.108, P=0.01) and 72 h (1.372±0.41 vs 0.476±0.26, P=0.003; Fig 5B). Expression levels of p16 progressively increased after partial hepatectomy in the old group. Lower SMP30 expression and higher p16 expression were found in the old group compared with the young group after hepatectomy at all of the time points. These results indicated that hepatocytes were more vulnerable and there was more cell cycle arrest in the old group compared with the young group. We also examined CHOP expression, which is the main apoptotic pathway of ER stress. According to PCR analysis, we found higher CHOP expression and a progressive increase in CHOP expression in the old group compared with the young group at all of the time points. However, significant differences were not found at each time point 24 h (0.36±0.23 vs 0.32±0.21, P=0.18), 48 h (0.55±0.44 vs 0.25±0.11, P=0.80), and 72 h
(1.26±0.43 vs 0.78±0.31, P=0.08).
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 16
Evaluation of autophagy and apoptosis
After partial hepatectomy, autophagy-related LC3 and Atg5 protein expressions were markedly higher in the old group. We did not found significant difference in LC3 at 24 h (0.86±0.12 vs 0.65±0.25, P=0.09), 48 h (1.59±0.31 vs 2.06±1.03, P=0.35), and 72 h
(0.93±0.13 vs 0.74±0.19, P=0.06) and Atg5 mRNA expression at 24 h (0.81±0.13 vs
0.57±0.31, P=0.14), 48 h (1.02±0.45 vs 0.82±0.05, P=0.36), and 72 h (2.26±0.86 vs 2.53±0.39, P=0.53) between the old and young groups. Immunohistochemically, LC3 was strongly positive in the cytoplasm in the old group (score=5, Fig. 6A). However, LC3 was weakly stained in the young group (score=2, Fig. 6B). Also, western blot analysis result showed higher LC3 expression in the old group (Fig. 4D). Additionally, Atg5 protein was highly expressed in the old group (score=5, Fig. 6C, D). Caspase-3 mRNA expression in the old group was significantly higher at 48 h (1.886±0.23 vs 1.260±0.61, P=0.04) and 72 h (2.456±0.77 vs 1.352±0.24, P=0.01; Fig 5C) compared with that in the young group. Immunohistochemically, caspase-3 was more
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 17
strongly stained in the old group (score=6) compared with the young group (score=1, Fig. 6E, F). The intensity of caspase-3 protein expression increased with prolonged exposure time in the old group, whereas that of the young group remained constant. These results indicated that autophagy- and apoptosis-related markers were activated after hepatectomy in the old group.
Discussion The phenomenon of liver regeneration after partial hepatectomy is complex. In this study, we investigated aged liver regeneration and focused on the aging, cell cycle, autophagyand apoptosis-related genes that are crucial for liver regeneration. We first demonstrated that liver regeneration was impaired in the liver of aged mice after partial hepatectomy, and this was characterized by reduced DNA synthesis in hepatocytes, and increased apoptosis and autophagy. Survival in the old group was worse than that in the young group after partial hepatectomy and the LW/BW ratio was also lower in the old group. Liver function test
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 18
results showed that markers of liver injury, such as ALT, AST, total bilirubin, and LDH, were significantly higher in the old group. These results possibly reflect the extent of hepatocyte injury. HGF is a potent inducer of DNA synthesis, and HGF and its receptor cMet are important growth factors for liver regeneration. HGF has been shown to be indispensable and important for liver regeneration.19 Consistent with this finding, we found that HGF and cMet expressions were lower in the old group at all of the time points after partial hepatectomy. Senescent cells express p16 ink4a, a cyclin dependent kinase inhibitor and tumor suppressor, which enforces growth arrest by activating retinoblastoma.24 This pathway
contributes to impaired cellular regeneration of an aging organism and acts as a marker in this process. In our study, p16 expression in the old group was upregulated after partial
hepatectomy, and its downstream target CDK/cyclin D1 was lower in the old group than in the young group. This result indicated that the cell cycle mechanism of the liver progressively decreased in the old group, and thereafter, cell cycle arrest mechanisms
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 19
were increased. Autophagy, a homeostatic, catabolic degradation process, prevents the accumulation of damaged proteins or organisms. Autophagy has cytoprotective and cytocidal effects, and is induced by ER stress, and is a downstream mediator of ER stress-related cell death. Excessive autophagy might promote cell death and intense degranulation may be the turning point that commits to a damaged cell death process rather than a cell protective process.17,25 Many studies have suggested that autophagy may mediates cell death, and autophagy has been classified as one of three morphological forms of cell death together with apoptosis and necrosis.26 However, other investigations have suggested an alternative function for macroautophagy as a critical pathway for cell survival.27 In our study, after partial hepatectomy, autophagy-related LC3 and Atg5 expressions were increased with the ER stress marker CHOP in the old group at all of the time points. CHOP is a proapoptotic transcription factor, which is expressed at low levels under physiological conditions, but is strongly induced during severe ER stress.28 Therefore, in the current study, partial hepatectomy was an excessive stress for the aged liver, and
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 20
resulted in an increase in those markers. Increased autophagy might be triggered by accumulation of damaged mitochondria and other organelles, intracellular components and unfolded proteins in aged liver. In such cases, dying cells activate autophagy to degrade by own lysosomes, that is an alternative way of cell death pathway.23,29 Cell cycle arrest, autophagy, and senescence are closely linked with conserved biological processes, such that autophagy induces senescence, and conversely, senescence induces autophagy. Cell lines overexpressing p16 are clearly more sensitive to the induction of senescence and autophagy, especially under conditions of stress. 30 SMP30 has an antiapoptotic function, which prevents cells from various injuries occurring during their lifespan. In SMP30-KO mice, hepatocytes are more susceptible to apoptosis. 31 Our study showed that SMP30 expression was lower in the old group than in the young group at all of the time points after partial hepatectomy. Alterations in Ca+2 homeostasis can negatively affect mitochondrial Ca+2 signaling. Therefore, Ca+2 is a second messenger that can initiate the intrinsic apoptotic pathway, which is responsible for releasing proapoptotic proteins from the intermembrane space to the cytoplasm. It
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 21
activates the conversion of procaspase-9 to caspase-9, which then cleaves procaspase-3 and activated caspase-3, causing the release of caspase-activated DNAse.32-35 In our study, caspase-3, which is one of the major indicators of apoptosis, was significantly higher in the old group at all of the time points. This finding suggested that antiapoptotic function was decreased in old liver regeneration after hepatectomy. All of our results demonstrated that ER stress, apoptosis, and autophagy are important for regulating liver regeneration. Liver regeneration is affected by age and aging, and cell cycle-related genes are affected by this process. In conclusion, regulation of these genes is required for liver regeneration to proceed normally. Further studies are needed to address the potential differences in old liver regeneration, and the implications of these differences. It is reasonable to assume that blocking and activating specific signaling pathways and genes will more likely lead to liver regeneration stimulus. Therefore, understanding the molecular basis for aging liver regeneration will not only help define the impaired conditions, but it will also hopefully lead to new therapeutic options for old patients.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 22
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research (C) (21462091) and Scientific Research (B) (23390324), from the Japan Society for the Promotion of Science.
References
1. Fausto N, Campbell JS, Riehle KJ. Liver Regeneration. Hepatology 2006; 43: 45-53. 2. Michalopoulos GK. Liver Regeneration. J Cell Physiol 2007; 231: 286-300. 3. Suzuki A, Sakaguchi T, Inaba K, Suzuki S, Konno H. Impact of cell cycle disruption on impaired hepatic regeneration in aged livers with ischemic insult. J Surg Res 2012; 173: 267-277.
4. Sanchez-Hidalgo JM, Naranjo A, Ciria R, Ranchal I, Aguilar-Melero P. Ferrin G, et al. Impact of age on liver regeneration response to injury after partial hepatectomy in a rat model. J Surg Res 2012; 175: 1-9.
5. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young systematic environment. Nature 2005;
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 23
433: 760-764.
6. Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, et al. Ink4a/Arf expression is a biomarker of aging. J Clin Invest 2004; 114: 1299-1307.
7. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003; 425: 962-967.
8. Rayess H, Wang MB, Srivatsan ES. Cellular senescence and tumor suppressor gene p16. Int J Cancer 2012; 130: 1715-1725.
9. Kim WY, Sharpless NE. The regulation of INK4/ARF in cancer and aging. Cell 2006; 127: 265-275.
10. Gil J, Peters G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 2006; 7: 667-677.
11. Bernales S, McDonald KL, Walter P. Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLos Biol 2006; 4: e423.
12. Lin JH, Walter P, Yen TS. Endoplasmic reticulum stress in disease pathogenesis. Annu
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 24
Rev Pathol 2008; 3: 399-425.
13. Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y, Jungreis R, et al. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic retilculum. Genes Dev 2004; 18: 3066-3077.
14. Rutkowski DT, Arnold SM, Miller CN, Wu J, Li J, Gunnison KM, et al. Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and
proteins. PLos Biol 2006; 4: e374.
15. Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, et al. Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 2006; 26: 9220-9231.
16. Cuervo AM, Bergamini E, Brunk UT, Droge W, Ffrench M, Terman A. Autophagy and aging: the importance of maintaining “clean” cells. Autophagy 2005; 1: 131-140.
17. Zhang J, Morris MW Jr, Dorsett-Martin WA, Drake LC, Anderson CD. Autophagy is involved in endoplasmic reticulum stress-induced cell death of rat hepatocytes. J Surg Res 2013; 183: 929-935.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 25
18. Zarnegar R, DeFrances MC, Kost DP, Lindroos P, Michalopoulos GK. Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy. Biochem Biophys Res Commun 1991; 177: 559-565.
19. Nejak-Bowen K, Orr A, Bowen WC Jr, Michalopoulos GK. Conditional genetic elimination of hepatocyte growth factor in mice compromises liver regeneration after partial hepatectomy. PLoS One 2013; 8: e59836.
20. Patijn GA, Lieber A, Schowalter DB, Schwall R, Kay MA. Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice. Hepatology 1998; 28: 707-716.
21. Weber A, Boger R, Vick B, Urbanik T, Haybaeck J, Zoller S, et al. Hepatocyte-specific deletion of the antiapoptotic protein myeloid cell leukemia-1 triggers proliferation and hepatocarcinogenesis in mice. Hepatology 2010; 51: 1226-1236.
22. Xiong Y, Fang JH, Yun JP, Yang J, Zhang Y, Jia WH, et al. Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology 2010; 51: 836-845.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 26
23. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007; 8: 741-752.
24. Sharpless NE. Ink4a/ Arf links senescence and aging. Exp Gerontol 2004; 39: 1751-1759. 25. Passegue E, Wagner EF. JunB suppresses cell proliferation by transcriptional activation of p16 (INK4a) expression. EMBO J 2000; 19: 2969-2979.
26. Ito M, Nakagawa H, Okada T, Miyazaki S, Matsuo S. ER-stress caused by accumulated intracistinal granules activates autophagy through a different signal pathway from unfolded protein response in exocrine pancreas cells of rats exposed to fluoride. Arch Toxicol 2009; 83: 151-159.
27. Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 2008; 9: 1004-1010.
28. Bhogal RH, Weston CJ, Curbishley SM, Adams DH, Afford SC. A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress. Autophagy 2012; 8: 545-558.
29. Levine B, Yuan J. Autophagy in cell death: an innocent convict?. J Clin Invest 2005; 115:
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 27
2679-2688.
30. Capparelli C, Chiavarina B, Whitaker-Menezes D, Pestell TG, Pestell RG, Hulit J, et al. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neoangiogenesis. Cell cycle 2012; 11: 3599-3610.
31. Galehdar Z, Swan P, Fuerth B, Callaghan SM, Park DS, Cregan SP. Neuronal apoptosis induced by endoplasmic reticulum stress is regulated by ATF4-CHOP-mediated induction of the Bcl-2 homology 3-only member PUMA. J Neurosci 2010; 30: 16938-16948.
32. Chandra D, Bratton SB, Person MD, Tian Y, Martin AG, Ayres M, et al. Intracellular nucleodites act as critical prosurvival factors by binding to cytochrome C and inhibiting
apoptosome. Cell 2006; 125: 1333-1346.
33. Saelens X, Festjens N, Vande Walle L, van Gurp M, van Loo G, Vandenabeele P. Toxic proteins released from mitochondria in cell death. Oncogene 2004; 23: 2861-2874.
34. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 2003; 4: 552-565.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 28
35. Enari M, Sakahira H, Yokohama H, Okawa K, Iwamatsu A, Nagata S. A caspase activated DNAse when released from mitochondria. Nature 1998; 391: 43-50.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 29
Figure Legends Figure 1. A.
Survival in the young group was 100% after surgery and old group survival was
60%.
B.
Liver weight/body weight (LW/BW) ratio in both group after partial hepatectomy.
LW/BW ratio was significantly low at 48h and 72h in old group.
Figure 2. A. LDH were significantly elevated at 24h and 72h in old group.
B. Serum level of ALT was significantly elevated at 48h and 72h in old group. C. AST was markedly elevated at 48h and 72h in old group. D. Total bilirubin level of old group was increased markedly at 72h.
Figure 3. A.
Level of HGF was lower in old group at all-time points but significantly
decreased at 24h.
B.
Met was significantly lower at 48h in old group.
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 30
C.
CyclinA2 was significantly decreased at 24h, 48h and 72h in old group.
D.
CyclinD1 was found significant at 48h.
Figure 4. A.
PCNA was significantly well stained in the young group compared with old
group at 24h, 48h and 72.
B.
PCNA positive nuclei in young group at 72h (×200).
C.
PCNA positive nuclei in old group at 72h (×200).
D.
Western blot analysis for LC3-I and LC3-II expression at 24h, 48h and 72h.
Figure 5. A. SMP30 was significantly higher at 24h, at 48h and 72h in young group. B. p16 expression level were significantly higher in old group at 48h and 72h. C. Caspase3 was higher in old group and significantly at 48h and 72h.
Figure 6. A.
LC3 protein expression in old group at 48h after hepatectomy (×200).
B.
LC3 protein expression in young group at 48h after hepatectomy (×200).
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 31
C.
Atg5 protein expression in old group at 72h after hepatectomy (×200).
D.
Atg5 protein expression in young group at 72h after hepatectomy (×200)
E.
Caspase3 protein expression in old group at 72h after hepatectomy (×200)
F. Caspase3 protein expression in young group at 72h after hepatectomy (×200)
This article is protected by copyright. All rights reserved.
Accepted Article
Enkhbold Ch, et al 32
Figure 1
Young Old
A
Survival after hepatectomy
B
LW/ BW Ratio
% 100 Young (n=20)
*
**
48h
72h
4.0
p=0.04
50
3.0 Old (n=20) 2.0 0
0 0
2
4
6 Days
8
10
This article is protected by copyright. All rights reserved.
24h
Accepted Article
Enkhbold Ch, et al 33
Figure 2
Young
LDH (U/L)
A 25000
*
20000
**
B **
Old
ALT (U/L) 7000 6000
**
**
48h
72h
5000
15000
4000 3000
10000
2000 5000 0
1000
24h
48h
AST (U/L)
C
0
72h
Total Bilirubin (U/L)
D
14000
24h
8
12000
**
10000
**
6
*
8000 4
6000 4000
2
2000 0
24h
48h
72h
This article is protected by copyright. All rights reserved.
0
24h
48h
72h
Accepted Article
Enkhbold Ch, et al 34
Figure 3
Young
HGF B
**
1.2 1.0 0.8 0.6 0.4 0.2 0
24h
48h
Relative mRNA expression
5
** **
4
**
3 2 1 0
24h
48h
72h
This article is protected by copyright. All rights reserved.
*
1.2 1.0 0.8 0.6 0.4 0.2
24h
48h
72h
Cyclin D1
D
6
1.4
0
72h
Cyclin A2
C
1.6 Relative mRNA expression
Relative mRNA expression
1.4
2.5 Relative mRNA expression
A
Old
Met
*
2.0 1.5 1.0 0.5 0
24h
48h
72h
Accepted Article
Enkhbold Ch, et al 35
Figure 4
Young Old
PCNA
A 100
Labeling index
90
**
**
B **
80 70 60 50 40 30
200µm
C
D LC3-I 15kDa
LC3-II GAPDH 24h 48h 72h 24h 48h 72h
Young 200µm
This article is protected by copyright. All rights reserved.
Old
Accepted Article
Enkhbold Ch, et al 36
Figure 5
Young
SMP30 B
Relative mRNA expression
14 12
*
**
10
**
8 6 4 2 0
24h
48h
72h
Caspase3 C
**
Relative mRNA expression
3.5 3.0
*
2.5 2.0 1.5 1.0 0.5 0
24h
48h
72h
This article is protected by copyright. All rights reserved.
3.0 Relative mRNA expression
A
Old
p16 **
**
48h
72h
2.5 2.0 1.5 1.0 0.5 0
24h
Accepted Article
Enkhbold Ch, et al 37
Figure 6
A
B
200µm
D
C
200µm
E
200µm
This article is protected by copyright. All rights reserved.
200µm
F
200µm
200µm
