Vitamin C Promoted Liver Regeneration Following Partial Hepatectomy-induced Hepatic Injury in Senescence Marker Protein-30-deficient Mice

비타민 C 투여는 간 부분절제술에 의한 간 재생을 촉진 시킴

Abstract

The capacity for liver regeneration involves a variety of nutritional factors. Vitamin C has multiple metabolic and antioxidant functions. In this study, we investigated the role of vitamin C in liver regeneration following hepatectomy in senescence marker protein (SMP)-30 knockout (KO) mice. Partial hepatectomy was performed by resecting the median and left lateral lobes of mice. Vitamin C accelerated liver recovery in SMP30 KO mice treated with vitamin C (KV). The livers of the KV mice exhibited lower levels of aspartate aminotransferase and lower injury than those of the KO mice. Increased type II transforming growth factor-β receptor (TGF-βRII)-mediated regeneration signaling was accompanied by HGF and cMet in the KV but not the KO mice. Consistent with this, the expression of cell cycle regulatory proteins, including cyclin D1 and proliferating cell nuclear antigen (PCNA), increased rapidly in the KV mice. Enhanced activation of ERK and GSK-3β proteins and a significantly increased number of binuclear hepatocytes were also detected in the livers of the KV mice. Moreover, the KV mice synthesized the highest levels of albumin. These data suggest that treating SMP30 knockout mice with vitamin C resulted in earlier recovery and liver regeneration by activation of the regeneration system.

비타민 C는 신진대사에 연관되어 있으며 특히 항산화 기능을 가지고 있다. 본 연구에서는 생체에서 비타민 C를 합성할 수 없는 SMP 30 녹아웃 마우스에 간 절제술을 시행하여 간 재생에서 비타민 C의 역할을 관찰하였다. 간 절제술은 마우스 중간엽 및 좌엽을 제거한 부분절제술을 수행하였다. 마우스는 간 절제술 후 비타민 C를 투여한 군(KV)와 비타민 C를 투여하지 않는 군(KO)로 나누어서 비타민 C의 효과를 관찰하였다. 결과 비타민 C를 투여한 KV 마우스의 간 회복이 투여하지 않는 KO 마우스에 비해 촉진되었다. KV 마우스의 혈액에서 관찰된 간소상 지표인 아스파르타산 아미노전달효소 및 간 손상 정도가 KO 마우스에 비해 낮게 관찰되었다. KV 마우스에서는 HGF와 c-Met에 의해서 TGF-베타 수용체 신호전달계가 활성화되고 세포주기 조절인자인 cyclin D1과 PCNA의 발현이 빠르게 증가되었다. 반면 KO 마우스에서는 활성화 되지 않았다. 또한 ERK와 GSK-3β 단백질의 활성화가 관찰되었으며 세포분열 간세포들의 유의적인 증가가 관찰되었다. 그리고 KV 마우스에서는 혈중 알부민의 농도가 높은 것으로 확인되었다. 따라서 본 실험결과는 SMP 30 결핍 마우스에서 비타민 C 투여는 간 재생시스템의 활성화와 이에 따른 빠른 회복을 초래한다.

Introduction

The liver has a remarkable capacity to regenerate. Although hepatocytes rarely divide under normal conditions, they proliferate rapidly in response to the loss of liver mass caused by surgical removal, chemical injury, or viral infection [11, 21, 36]. Liver regeneration is a well-orchestrated process in which complex signaling pathways coordinate the progression of hepatocytes through three distinct stages: withdrawal from quiescence (“priming phase”), cell cycle entry and progression, and the cessation of cell division with a return to quiescence [11]. The cell cycle entry and progression phase occurs in two steps. After the priming phase, hepatocytes enter the G1 phase of cell cycle and activate immediate early genes (IEGs) such as c-fos and c-jun. Activation of IEGs results in the expression of cell cycle regulatory genes including G1 cyclin and cyclin D [4, 9, 29]. The induction of cyclin D1 is the most reliable marker of cell cycle progression (G1 phase) in hepatocytes, as demonstrated both in vivo and in vitro [1]. In the second step, hepatocytes leave G1 and enter S phase, which is accompanied by the phosphorylation of retinoblastoma protein (pRb) and the upregulated expression of a number of genes including cyclin E, cyclin A, and DNA polymerase [9, 10, 32]. In addition, the expression of the nuclear protein proliferating cell nuclear antigen (PCNA) is maximally elevated in the S phase of proliferating cells, which has been used as a marker of liver regeneration after partial hepatectomy (PHx, the removal of approximately 70% of the liver) in rats [17].

Cell cycle progression is driven by growth factors including hepatocyte growth factor (HGF), epidermal growth factor (EGF), and transforming growth factor-α (TGF-α). Hepatocyte growth factor (HGF) has been known to play major roles in embryonic organ development, wound healing, and adult organ regeneration [24]. Specifically, previous studies suggested that HGF is a potent mitogen in mature hepatocytes, and acts as a hepatotropic factor during liver regeneration. These growth factors activate various mitogenic signaling cascades, including the mitogen-activated protein kinase (MEK)/extracellular signal regulated kinase 1/2 (ERK1/2) and phosphatidylinositol3-kinase (PI3K)/Akt pathways [6, 23, 36]. ERK1/2, which is a member of the mitogen-activated protein kinase (MAPK) family, phosphorylates several downstream substrates involved in cell growth and cell cycle control [19, 35]. GSK3β is downstream of PI3K/Akt kinase, and is a ubiquitously expressed multifunctional serine/threonine protein kinase. It was originally identified as a key regulator of insulin-dependent glycogen synthesis, and has also been shown to support cell proliferation and liver regeneration [8, 14, 31, 38].

Senescence marker protein (SMP) 30 was first identified in 1992; it is mainly expressed in hepatocytes and the proximal tubular cells of kidneys, and its expression decreases with age [12]. Its expression is maintained at a high level throughout the tissue maturation process, and then decreases in an androgen-independent manner during the senescent stages [13]. Interestingly, SMP30 knockout (KO) mice developed symptoms of scurvy and aging when fed a vitamin C-free diet [26]. Vitamin C (ascorbic acid) is a nutrient that is required for a variety of biological functions. Humans, primates, and other animals such as guinea pigs depend on their diet as a source of vitamin C to prevent scurvy (the disease of vitamin C deficiency) and to maintain general health. Vitamin C can also function as a source of the signaling molecule hydrogen peroxide and as a Michael donor to form covalent adducts with endogenous electrophiles in plants [37]. There is considerable evidence reporting roles of vitamin C in inhibiting the proliferation, migration, and constriction of fibroblasts and lens epithelial cells [5]. Vitamin C is also required for some step(s) of G1 and G2, prior to the onset of DNA replication and the G2 mitosis transition, respectively [2, 20, 27].

In this study, we investigated the effect of vitamin C on hepatectomy-induced liver regeneration in SMP 30 KO mice.

 

Materials and Methods

Animal experiments

SMP30 KO mice were kindly provided by Dr. Akihito Ishigami, Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan) [16]. The SMP30 KO and WT mice were housed in a room at 22±3℃, relative humidity of 50±10%, and a 12 hr light-dark cycle, and were given food and water ad libitum. Mice were intraperitoneally injected with 1 ml/kg of body weight CCl4 in olive oil vehicle. One day after CCl4 injection, 70% PHx was performed by resecting the median and left lateral lobes of the liver in 8-week-old SMP30 KO mice and WT mice (C57BL/6 background) according to the method described by Higgins and Andersen [36]. Mice were divided into three groups: group 1 contained WT mice (WT), group 2 was vitamin C-treated SMP30 knockout (KV) mice, and group 3 was control untreated SMP30 knockout (KO) mice. Five mice from each group were sacrificed at 0, 24, 48, 72, and 120 hr after PHx. Liver remnants were then removed, weighed, and either snap-frozen in liquid nitrogen or processed for histology. The WT and KO groups were fed a vitamin C-free diet, whereas the KV group mice were given 5 ml of vitamin C water containing 1.5 g/l of vitamin C. Animal procedures were performed in accordance with the National Institutes of Health (NIH, Bethesda, USA) guidelines for the care and use of laboratory animals and approved by the Kyungpook National University Institutional Animal Care for the care and use of laboratory animals [Approval No. : KNU 2011-11].

Liver regeneration ratio

The regeneration ratio (R) was calculated from the wet weight of the resected livers at PHx and the remnant livers at sacrifice. The regeneration ratio was calculating as the following: R (%) = 100×0.7× (W2/W1) [39], where W1 is the weight of the liver resected during PHx, and W2 is the weight of the regenerating liver at sacrifice.

Biochemical measurements

All serum samples were collected and stored at -70℃ prior to analysis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using standard enzymatic procedures (Hitachi, Tokyo, Japan). In addition, serum vitamin C levels were measured using a high performance liquid chromatography (HPLC)-electrochemical detection method [18]. Briefly, 100 μl of serum collected from the blood of mice by centrifugation at 3,000× g for 15 min was mixed with 450 μl of 3% metaphosphoric acid. After centrifugation at 10,000× g for 10 min, the supernatant was collected and the serum vitamin C levels were measured by HPLC using an Atlantis dC18 5 μm column (4.6×150 mm, Nihon Water, Tokyo, Japan). The mobile phase consisted of 50 mM phosphate buffer (pH 2.8), 0.2 g/l EDTA, and 2% methanol at flow rate of 1.3 ml/min. Electrical signals were recorded using an electrochemical detector with a glassy carbon electrode at +0.6 V.

Liver histopathology and immunohistochemistry

Liver tissues were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Sections 4-μm in thickness were cut, deparaffinized in xylene and toluene, and then rehydrated in a graded alcohol series. The sections were stained with hematoxylin and eosin (H&E) for histological examination. Immunohistochemistry was performed using primary antibodies against PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Slides were incubated in 3% hydrogen peroxide in methanol for 30 min, and then microwaved at 750 W for 10 min in 0.01M citric buffer for antigen retrieval. The antigen-antibody complexes were visualized using an avidin-biotin-peroxidase complex solution with a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA, USA) with 3,3-diaminobenzidine (Zymed Laboratories, San Francisco, CA, USA).

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was isolated from frozen mouse livers using TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA was used as the template for reverse transcribing RNA into cDNA using AccuPower RT/PCR PreMix (Bioneer Corporation, Daejeon, Korea). The following primers were used: TGF-β RII, 5‘-TTCGCCGAGGTCTACAAG-3’ and 5‘-CTCTTGAGGT CCCTGTGAA-3’; HGF, 5‘-CTGGGGCTACACTGGATTG-3’ and 5‘-GATGCTTCAAACACACTGGC-3’; cMet, 5‘-TCACTCTTGGGAATCTGCCTGA- 3’ and 5‘-GCAACAGAGAAGGATATGG-AGC-3’; GSK-3β, 5‘-ATGGTCTGCAGGCTGTGTGT- 3’ and 5‘-TGTGCCTTGATTTGAGGGAA-3’; and GAPDH, 5‘-ACTCACGGCAAATTCAACGG-3’ and 5‘-ACCAGTGGATGCAGGGATGA-3’. The PCR products were visualized using 1.5% agarose gel electrophoresis.

Immunoblotting

Snap-frozen liver tissues were homogenized in radioimmunoprecipitation assay buffer containing 1 mM Na3VO4, 50 mM NaF, and protease inhibitor cocktail tablets (Roche, Basel, Swiss). The lysates were centrifuged at 3,000 rpm for 10 min at 4℃ to remove solid tissues and debris. Subsequently, the supernatants were centrifuged at 14,000 rpm for 20 min at 4℃ to obtain soluble cytosolic proteins. The protein concentrations were then determined using the Bradford method. Protein samples were separated by 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For immunoblotting, proteins were electrotransferred onto PVDF membranes (Schleicher & Schuell, Dassel, Germany) and blocked with 3% bovine serum albumin in Tris-buffered saline (TBS). The following antibodies were used for immunoblotting: anti-ERK (dilution 1:200), anti-p-ERK (1:1,000), anti-PCNA (1:500), anti-cyclin D1 (1:800, all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GAPDH (1:1,000, Cell Signaling Technology, Danvers, MA, USA). The primary antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary IgG. Specific binding was then detected using the Super Signal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA) and exposure to medical X-ray film (Kodak, Tokyo, Japan). The band intensities were quantified using Image J software (NIH).

Statistical analysis

All data are expressed as means ± standard errors (SEMs). The statistical significance between experimental groups was determined using Student’s t-test or one-way analysis of variance (ANOVA). A p-value of <0.05 was considered to indicate statistical significance. All statistical analyses were performed using the SPSS 19 statistical software program.

 

Results

Vitamin C increased the liver regeneration ratio

To examine the effects of vitamin C on restoration of liver mass, the weight of the livers was measured at each time point after PHx. The liver regeneration ratios were calculated as described previously. As shown in Fig. 1, the liver regeneration ratio was increased gradually in the KV group compared with KO, suggesting that treatment with vitamin C stimulated liver regeneration.

Fig. 1.The ratio of liver regeneration. R (%) = 100×0.7× (W2/W1), where W1 is the weight of the liver resected during the operation, and W2 is the weight of the regenerating liver. Significantly increased liver regeneration was observed in the KV group. Each value is presented as the mean ± SEM. *p<0.05, 0 vs. 120 hr of WT; #p<0.05, 0 vs. 120 hr of KV.

Effect of vitamin C on biochemical changes during liver regeneration

Because serum ALT levels reflect the severity of liver damage, ALT levels are commonly used as a specific indicator of liver necrosis [25]. The KV and KO groups exhibited higher ALT levels than did the WT (Fig. 2A). There were no significant differences in ALT levels between the KV and KO groups. By contrast, AST levels in the KO group were significantly higher than those in the KV and WT groups were (Fig. 2B). Next, we measured the serum vitamin C levels. As shown in Fig. 2C, decreased serum vitamin C levels were observed in not only the WT group, but also in the KV group after liver damage. However, normal serum vitamin C levels were restored in the WT group 120 hr after PHx. The serum vitamin C levels decreased at the initial stages of liver regeneration, but then increased by 120 hr when complete liver regeneration was achieved in the KV and WT groups. In contrast, serum vitamin C levels were undetectable throughout the study in KO mice (Fig. 2C). These data suggest that vitamin C could be used to promote liver regeneration after liver damage.

Fig. 2.Serum measurements of ALT, AST, and vitamin C. There were no significant changes in ALT (A) and AST (B) in the KO group. However, the levels were lower in the KV group than those in the KO group were. (C) The serum levels of vitamin C decreased during liver regeneration. Each value is presented as the mean ± SEM. *p<0.05, KV vs. KO.

Effect of vitamin C on the protein levels of growth factors

HGF/SF-Met and TGF-β/TGF-βRII (type II transforming growth factor-β receptor) mediate hepatocyte proliferation after PHx by activating several signaling pathways [22, 33]. The levels of these growth factors increase during the early stages of liver regeneration, and decrease after complete liver regeneration. We examined the protein levels of these growth factors using immunoblotting. Fig. 3 reveals that the expression of TGF-β and TGF-βRII increased gradually up to 48 hr after PHx, decreased at 72 hr, and was barely detectable at 120 hr in WT and KV groups. In contrast, increased levels of both proteins were detected 120 hr after PHx in the KO group (Fig. 3).

Fig. 3.TGF-β, TGF-β-receptor II, and growth factor expression in the regenerating liver. (A) The growth factor levels were increased in the early stages of liver regeneration, and decreased in complete liver regeneration. However, the expression was lower in the KO group than in other groups. Expression was upregulated in all groups from 24-72 hr, and then downregulated at 120 hr. The expression patterns were similar in the KV and WT groups. (B) The relative band densities were normalized to GAPDH. Each value is presented as the mean ± SEM. *p<0.05, KV vs. KO.

Effect of vitamin C on the levels of proliferationrelated signaling proteins

Several studies have provided evidence for the essential role of the ERK pathway in proliferating hepatocytes [41]. These kinases phosphorylate a number of substrates and mediate the induction of several genes including c-fos and cyclin D1; this subsequently leads to cell cycle progression and growth in hepatocytes [35]. To investigate the effects of vitamin C on ERK activation, the phosphorylation of ERK was measured by immunoblotting. Vitamin C treatment led to the early activation of ERK in the KV and WT groups at 24 hr; in contrast, activation was only detected at 48 hr in the KO group (Fig. 4). Next, we examined the protein levels of cyclin D1 and PCNA. Cyclin D1 was first detected 24 hr after PHx in the WT and KV groups. However, it was only detected at 48 hr in KO mice (Fig. 5). The levels of PCNA protein increased during liver regeneration from 24-72 hr in all groups. However, levels were significantly higher in the KV group than those in the KO group were at 48 hr (Fig. 6).

Fig. 4.The activation of ERK during liver regeneration. (A) Immunoblotting for ERK and p-ERK. (B) The relative band densities were normalized to GAPDH. Each value is presented as the mean ± SEM. *p<0.05, KV vs. KO.

Fig. 5.Alteration in cyclin D1 protein levels during liver regeneration. (A) Immunoblotting for cyclin D1. (B) The relative band densities were normalized to GAPDH. Each value is presented as the mean ± SEM. **p<0.05, KV vs. KO.

Fig. 6.Alteration in PCNA protein levels during liver regeneration. (A) Immunohistochemistry for PCNA (original magnification ×200). PCNA-positive hepatocytes were counted in five fields (×400) of each sample. (B) Immunoblotting for PCNA. (C) The relative band densities were normalized to GAPDH. Each value is presented as the mean ± SEM. *p<0.05, KV vs. KO.

Effect of vitamin C on hepatic necrosis

Livers were harvested, and then stained with H&E. Widespread centrilobular necrosis and recruited inflammatory cells were observed when sections were analyzed microscopically (Fig. 7). Severe necrosis was observed immediately following CCl4 treatment, but then decreased during liver regeneration. In the KO group, necrosis was detected across a larger area than in the WT and KV groups. Gross findings revealed that the proliferation rate of inflammatory cells was faster at 48 and 72 hr than at other times. At the end of regeneration, the hepatic cord was rearranged and inflammatory cells had disappeared. Although inflammatory cells were not detected in significant numbers at 72 hr in the WT and KV groups, they were still observed until 120 hr in the KO group. These data suggest that liver regeneration occurred more slowly in vitamin C-deficient mice; these mice were also more susceptible to CCl4–induced injury than mice supplemented with vitamin C.

Fig. 7.Histological features during liver regeneration. (A) Histological image of a liver after H&E staining. CCl4-treated groups exhibited widespread centrilobular necrosis. Original magnification ×100; (B) ×200.

 

Discussion

The major finding of this study is that vitamin C promotes liver regeneration in vitamin C-deficient mice by upregulating growth factors and genes related to cell proliferation. We also identified the involvement of vitamin C in the hepatic regenerative response on the first day after PHx. Vitamin C-treated KO mice showed higher liver regeneration ratio than did water-treated KO mice. This enhanced regeneration was associated with increased DNA synthesis and cell cycle progression stimulated by the activation of TGF-β, TGF-βRII, HGF, c-met, and cyclin D1 in response to PHx.

TGF-β is member of a superfamily of growth and differentiation factors that interact with an equally diverse group of cell surface receptors [3]. TGF-β can regulate cell proliferation during both embryogenesis and liver regeneration [7, 15]. Consistent with this, increased TGF-β mRNA expression was detected in hepatocytes after PHx and during liver regeneration [33]. In addition, the expression of TGF-β RII was restored to normal levels in the later stages of regeneration [34]. In the current study, the expression of TGF-β was increased during the early stages of liver regeneration, and then decreased when liver regeneration was complete in the WT and KV groups. However, levels in the KO group were lower than in either KV or WT mice. In addition, the levels of TGF-βRII were decreased to a lesser extent in KO than in the WT and KV groups.

We investigated the mechanism by which vitamin C regulates cell proliferation in the liver by assessing key signaling pathways that play roles in cell cycle progression. ERK and Akt both transmit mitogenic signals via the Ras pathway, and they are activated by phosphorylation in the regenerating liver in response to growth factors [6]. In this study, ERK was phosphorylated more rapidly and robustly in the KV group than in the KO group, suggesting that vitamin C could modulate hepatocyte replication by interacting with ERK signaling pathways. Interestingly, HGF and c-met levels were upregulated early after PHx in the KV group, but remained low in the KO group. In addition, p-ERK was activated 24 h after PHx in the KV group. The upregulation of growth factors might activate specific signaling cascades, and thus partially contribute to enhancing proliferative signals in the KV group. The signaling cascades activated by ERK have been implicated in controlling a diverse range of cell cycle proteins. Activated ERK translocates to the nucleus and induces the expression of, or activates, transcription factors including c-fos and c-jun, which in turn increase the transcription of genes involved in cell cycle progression such as cyclin D1 [28, 30, 40]. In the present study, the depletion of vitamin C altered the expression of cell cycle regulators following PHx, which was accompanied by rapid and robust ERK phosphorylation. These results provide a molecular link between increased levels of p-ERK and enhanced cell cycle progression in hepatocytes from the KV group.

Compared with the KO group, the remaining livers of the KV group showed a significant increase in weight 48 hr after PHx, which was due to increased cell proliferation. Because G0-M phase cells can be recognized by PCNA immunohistochemical staining in the liver, we performed immunoblotting and immunohistochemistry with PCNA antibodies to detect proliferating cells; the expression levels of PCNA correlated with the pattern of cell proliferation [35]. The KV group exhibited the highest expression of PCNA, whereas the KO group showed relatively reduced expression. This is consistent with the levels of cyclin D1, which is required for the G1/S transition; levels were also increased significantly in the KV group following PHx. Therefore, liver recovery was faster in the KV group than in KO as a consequence of the increased hepatocyte proliferation.

H&E staining revealed that the presence of inflammatory cells was sustained in the KO group compared with KV. This suggests that vitamin C depletion delayed liver regeneration. In addition, the vitamin C-treated group showed higher albumin synthesis than did the other groups.

To summarize, our data demonstrate that vitamin C plays an important role in liver regeneration, at least in part by regulating the signaling pathways involved in hepatocyte proliferation. The present study is the first report to demonstrate that vitamin C promotes liver regeneration. Moreover, these results suggest that vitamin C might be useful to accelerate liver regeneration after liver resection and transplantation.