Ursolic acid enhances mouse liver regeneration after partial hepatectomy

Abstract

Context: Ursolic acid is a pentacyclic triterpenoid which has hepatoprotective and antihepatotoxic activities.

Objective: This study investigated whether ursolic acid is able to stimulate liver regeneration in partially hepatectomized mice.

Materials and methods: Ursolic acid or the vehicle solution was orally administered to the experimental, sham-operated and vehicle-treated group mice for 7 days, positive control animal (mice) was treated with recombinant human hepatocyte growth factor (rhHGF), and then the 70% liver partial hepatectomy was performed. The liver mass recovery rate was estimated by measuring the ratios of mice liver weight to body weight. The liver cells undergoing DNA synthesis were identified by immunohistochemistry analysis using monoclonal anti-BrdU antibodies. The expression levels of cyclin D1, cyclin E and C/EBP proteins (C/EBPα and C/EBPβ) were detected by the Western blotting technique.

Results: Our results showed administration of ursolic acid significantly increased the ratio of the liver to body weight and BrdU labeling index at 36 and 48 h after partial hepatectomy, and the potency of UA is similar to rhHGF treated positive control mice. In addition, ursolic acid treatment significantly increased cyclin D1, cyclin E and C/EBPβ protein expression levels at 36 h after liver PHx compared with the vehicle-treated control mice.

Discussion and conclusion: All these results suggest that ursolic acid stimulates liver proliferation after partial hepatectomy, and this effect may be associated with the stimulation of C/EBPβ expression.

Keywords::

• Liver partial hepatectomy
• mice
• C/EBP

Introduction

Liver has the unique ability to regenerate itself after major tissue loss. The remaining liver can proliferate and fully restores its original size (Michalopoulos & DeFrances, 1997; Court et al., 2002; Markiewski et al., 2006). Hence, if the liver fails completely, a surgical resection (partial hepatectomy) or a liver transplant offers a chance to cure serious liver diseases. However, liver resection involves the risk of liver failure, and the survival of patients depends on the ability of the remaining hepatocytes to regenerate (Clavien et al., 2007; Clavien, 2008). Thus, developing an agent that can stimulate hepatic regeneration has tremendous therapeutic relevance. Experimental liver partial hepatectomy, which is applied by excising about 70% of the liver in mice or rats, is widely used as a model of liver regeneration and surgical stress (Michalopoulos & DeFrances, 1997; Andiran et al., 2000; Furuta et al., 2000).

Ursolic acid (UA), a naturally occurring pentacyclic triterpenoid compound, is the major component of some traditional medicinal herbs (Price et al., 1987). It has broad bioactive use including antifungal, antiinflammatory, antiulceric, antihyperlipidemic, antihyperglycemic, and antitumorgenic activity (Yan et al., 2010; Liu, 1995; Gayathri et al., 2009). Moreover, multiple lines of evidence indicate that UA has hepatoprotective and antihepatotoxic activities (Ma et al., 1986; Lin et al., 1988; Liu et al., 1994; Saraswat et al., 2000; Ovesna et al., 2004). However, the potential role of UA in liver regeneration has not been studied. Hepatocyte growth factor (HGF) is the most potent mitogen for mature hepatocytes and seems to act as a hepatotropic factor (Matsumoto & Nakamura, 1992). Ishiki et al. (1992, 1995) and others have shown that injection of recombinant human hepatocyte growth factor (rhHGF) after 70% hepatectomy produced dose-dependent increases in hepatic DNA synthesis and proliferation (Kaibori et al., 1997). In the present study, we evaluated the effect of the exogenous administration of UA on the stimulation of liver regeneration in vivo in 70% partially hepatectomized mice by measuring the liver mass recovery, Brdu uptake, cell cycle proteins and C/EBP proteins (C/EBPα and C/EBPβ) expression.

Materials and methods

Chemicals

Ursolic acid (UA, 3β-hydroxy-12-ursen-28-ic acid, formula: C₃₀H₄₈O₃, molecular weight: 456.70) (Figure 1) was obtained from Sigma Aldrich (St. Louis, MO, USA). BrdU was from Invitrogen (Carlsbad, CA, USA). rhHGF was from R&D Systems (Minneapolis, MN, USA). All other chemicals used were of analytical grade and purchased from commercial suppliers.

Figure 1. . Structure of UR solic acid.

Animals and drug administration

C57BL/6 male mice, 10–12 weeks of age, were provided by the Experimental Animal Center of Yanbian University Medical College (Yanji, Jilin, China). The mice were kept at controlled temperature 22 ± 2°C and humidity under a 12 h light-dark cycle and fed standard laboratory chow and water. All experiments were performed in accordance with the National Research Council’s guidelines. The experimental procedures were approved by the Ethical Committee for the Experimental Use of Animals at Yanbian University. The mice were randomly divided into four groups. UA (Sigma Chemical Co., St Louis, MO, USA, 80 mg/kg/day) was orally administered to the experimental group animals daily for 7 days before surgery and continually until sacrifice. The dose of the UA was selected on the basis of the literature data (Aparecida Resende et al., 2006). The sham-operated group animals were submitted to UA treatment same as experimental group animals without liver PHx. The vehicle-treated group animals were submitted to the treatment of 0.5% sodium carboxymethyl cellulose (CMC-Na) solution without or instead of UA, and subjected to PHx. The positive control mice were administered with rhHGF (1.0 μg/kg/day, i.p.) once daily before surgery and continually until sacrifice.

Liver partial hepatectomy

Experimental liver partial hepatectomy was used as a model of liver regeneration and surgical stress (Michalopoulos & DeFrances, 1997; Andiran et al., 2000; Furuta et al., 2000). Under anesthesia, median and left lateral lobes of the liver (about 70%, two-thirds) were surgically removed and regeneration was allowed to proceed for 24, 36 and 48 h. The liver mass recovery rate was calculated by measuring the ratios of mice liver weight to body weight at different times. Liver samples were also collected, frozen in liquid nitrogen for further analyses.

BrdU incorporation index

Mice were injected with 50 mg/kg BrdU intraperitoneally. After 2 h, mice were anesthetized and the livers were removed. Liver specimens were made into paraffin-embedded tissue sections (5 μm thick), and the liver cells undergoing DNA synthesis were immunohistochemically identified using monoclonal anti-BrdU antibodies on formalin-fixed and paraffin-embedded liver tissues according to manufacturer’s instructions and counting the number of labeled nuclei per 1000 nuclei in randomly selected fields under a light microscope.

Protein isolation and Western blotting

Nuclear extracts were isolated from livers as described previously (Wang et al., 2001; Jin et al., 2009). Nuclear extracts (50 to 100 µg) were loaded on the gradient (4–20%, Bio-rad) polyacrylamide gels, and transferred onto membranes, and probed with antibodies. To verify protein loading, each filter was re-probed with TATA binding protein (TBP) and/or stained with Coomassie blue. Antibodies to cyclin D1, cyclin E, C/EBPα and C/EBPβ were purchased from Santa Cruz Biotechnology. Monoclonal anti-TBP antibody was from Abacam.

Statistical analysis

All data are expressed as means ± SD. Unpaired Student’s t-test was used in all the experiments. P values <0.05 were considered statistically significant.

Results

UA enhances liver mass recovery rate after PHx

After UA treatment, the ratios of the liver to body weight were significantly increased at 36 and 48 h after PHx compared with those of the vehicle-treated control group, the potency is similar to the rhHGF treated group, while the application of UA for 7 days did not change the liver to body weight in the sham-operated mice (Figure 2). In addition, UA did not induce liver overgrowth after 14 days of PHx. Moreover, after careful examination of liver histopathology, we have not observed any liver damage on control normal mice liver after 14 days of treatment by UA (data not shown). These results suggest that UA accelerates liver regeneration to its original size, and this stimulatory effect does not induce overgrown liver or liver damage.

Figure 2.  Stimulatory effects of UA on liver mass recovery after 70% PHx. liver to body weight ratio was examined in the livers at pre PHx, and at 0, 24, 36, 48, 72, 168 and 336 h after PHx. Data are expressed as means ± SD. n = 5 in each group. *P < 0.05 versus sham group; ^#P < 0.05 versus vehicle-treated control group. UA, ursolic acid.

UA stimulates hepatocyte DNA synthesis after PHx

To observe if liver regeneration by UA is due to the stimulation of the hepatocyte DNA synthesis, we measured the mitotic index of nuclear thymidine analog BrdU incorporation into hepatocyte DNA, which is a useful indicator of cell proliferation and cell kinetics (Iatropoulos & Williams, 1996; Nolte et al., 2005). As shown in Figure 3, after UA treatment, the BrdU labeling indexes increased significantly compared with the vehicle-treated control animals, the potency is similar to the rhHGF treated positive control animals at 36 and 48 h after PHx, while no BrdU positive cell were observed in the sham-operated control animals. Overall, these results indicate that UA markedly stimulates DNA synthesis in hepatocytes and thereafter accelerates liver regeneration after PHx in vivo.

Figure 3.  Hepatocyte proliferation enhancement by UA following 70% PHx (A) Representative sections of BrdU labeling of hepatocytes nuclei in the sham, control, UA and HGF treated mice at 36 h following PHx. (Magnification, × 200). (B) Summary of BrdU labeling index. Values are means ± SD (n = 5). *P < 0.05; **P < 0.01 versus sham group; ^#P < 0.05, ^{# #}P < 0.01 versus vehicle-treated control group.

UA activates cell cycle regulatory proteins and CCAAT/enhancer binding protein beta expression after PHx

To investigate the possible mechanism of UA-induced liver regeneration, we first examined the expression of cell cycle proteins in liver. We found that the protein levels of cyclin D1 and cyclin E were significantly increased in livers of UA-treated mice at 36 and 48 h after PHx, compared with the sham-operated and vehicle-treated control mice (Figure 4A). Since the activation of C/EBPβ has been reported to contribute to an increase in cyclin E expression level (Cho et al., 2009), and both C/EBPα and C/EBPβ and plays a critical role in the regulation of liver biology and liver regeneration (Cressman et al., 1996; Greenbaum et al., 1998; Ankoma-Sey, 1999; Wang & Timchenko, 2005), we measured the hepatic expression of C/EBPα and C/EBPβ. Our results showed that after the UA treatment, both C/EBPα and C/EBPβ expressions did not change before PHx (data not shown). However, after PHx, UA significantly increased the expression of both LAP and LIP, which are the isoforms of C/EBPβ, compared with the vehicle-treated animals (Figure 4A). No significant differences were observed in the expression level of C/EBPα in experiment group animal compared with the vehicle-treated control mice after PHx. Therefore, these results suggest that the UA-induced enhancement in the liver proliferation after PHx may be associated with the stimulation of C/EBPβ expression and the consequent activation of the cyclin E expression (Figure 4).

Figure 4.  Western blotting for cell cycle proteins and C/EBPβ expression. (A) Expression of cyclin D1 and cyclin E proteins were increased at 36 and 48 h after PHx in UA treated group. (B) Expression of C/EBPβ proteins was increased at 36 h after PHx in UA treated group animals. Changes in the protein levels relative to control were assessed by scanning densitometry of the immunoblots. Data represent mean ± SD (n = 3). *P < 0.05 versus sham group; ^#P < 0.05 versus vehicle-treated control group.

Discussion

Normal liver is well known for its capacity to regenerate itself after partial hepatectomy (Francavilla et al., 1990; Court et al., 2002). Liver resection is often the best option in primary and secondary hepatic malignancies patients (Tsim et al., 2010). Partial hepatectomy induces a rapid regenerative process in the remnant liver tissue, and the liver returns to its original size and the liver regeneration is terminated within 2 weeks (Michalopoulos & DeFrances, 1997; Andiran et al., 2000; Furuta et al., 2000). However, the major liver resection, both in patients with liver tumors and for organ donation, involves the risk of liver failure (Clavien et al., 2007; Clavien, 2008). Therefore, it is very important to investigate and find an effective therapeutic agent to improve the liver regeneration after partial hepatectomy without side-effects. In the present study, we discovered that UA can improve liver regeneration after PHx through increasing the liver mass recovery and accelerating liver proliferation.

Previous studies demonstrated that UA has pro-apoptotic effect on hepatoma cell line (Lauthier et al., 2000; Achiwa et al., 2005; Harmand et al., 2005). However, on normal livers, we have not observed any pro-apoptotic or toxic effect after UA treatment. Moreover, the application of UA produced a more rapid increase in the BrdU labeling indexes at 36 and 48 h after PHx, suggesting that UA can more rapidly stimulate DNA synthesis and thereafter accelerate liver regeneration after PHx.

Liver proliferation is controlled by a complex cooperation of several signal transductions pathways (Chamuleau & Bosman, 1988; Michalopoulos & DeFrances, 1997). Two members of C/EBP family, C/EBPα and C/EBPβ, are expressed in the liver and play a critical role in the regulation of liver biology. It has been also shown that the C/EBPα is involved in the inhibition of cell proliferation (Wang & Timchenko, 2005); while C/EBPβ, is a vital component of the hepatic regenerative response (Cressman et al., 1996; Greenbaum et al., 1998; Ankoma-Sey, 1999). Liver regeneration normally begins with a priming phase, which is followed by a spurt of regeneration during which most of the hepatocytes enter the cell cycle (Michalopoulos, 2007). Cyclin D1 and cyclin E are important cell cycle proteins which are induced after PHx, and are involved in the promotion of liver proliferation (Schwabe et al., 2003; Kato et al., 2005). Activation of C/EBPβ enhances cyclin E expression (Nakano et al., 2001). We found that UA increase the C/EBPβ and cell cycle proteins cyclin D1 and cyclin E expression after PHx, suggesting that the activation of C/EBPβ expression by UA after PHx may induce the cyclin E expression and contribute to the stimulation of mice liver regeneration. We do not know yet mechanisms by which UA regulates C/EBPβ in livers after PHx. However, from previous observations (Zhang et al., 2010), it is possibly through regulation of the ERK pathway, which can also be activated by HGF (Li et al., 2011). Further studies are required to elucidate how UA regulates C/EBPβ expression.

Taken together, our findings indicate that UA may play a role in accelerating liver proliferation, recovering liver function, and protecting the integrity of hepatocytes against liver damage. And we postulate that this function may be through an activation of C/EBPβ expression. Hence it merits further development for exploiting it as a therapeutic agent.

Conclusion

In conclusion, ursolic acid accelerates liver mass recovery, stimulates hepatocyte proliferation after partial hepatectomy, and this effect may be associated with the stimulation of C/EBPβ expression.

Acknowledgement

This study was supported by the Yanbian University Foundation of Jilin Province, China, No. 200009.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper