Author Affiliations: Department of Liver Surgery, First Affiliated Hospital of Nanjing Medical University; Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing 210029, China (Hao BB, Pan XX, Fan Y, Lu L, Qian XF, Wang XH, Zhang F and Rao JH)

Corresponding Author: Feng Zhang, MD, PhD, Department of Liver Surgery, First Affiliated Hospital of Nanjing Medical University; Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, #300 Guangzhou Road, Nanjing 210029, China (Tel: +86-25-83718836ext6476; Fax: +86-25-83672106; Email: zhangfeng1958@hotmail.com)

 

© 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(16)60115-7

Published online July 13, 2016.

 

 

Contributors: WXH and ZF proposed the study. HBB and PXX performed research and wrote the first draft. FY collected and analyzed the data. LL, QXF, ZF and RJH made the critical revision of the article. HBB and PXX contributed equally to this article. ZF is the guarantor.

Funding: This study was supported by grants from the Foundation of Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Basic Research Program-Youth Fund Project of Jiangsu Province (BK20140092), and the National Natural Science Foundation of China (81400650, 81470901, 81273261 and 81270583).

Ethical approval: The protocols were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Ischemia reperfusion injury (IRI) is unavoidable in liver transplantation and hepatectomy. The present study aimed to explore the possible mechanism and the effect of oleanolic acid (OA) in hepatic IRI.

 

METHODS: Mice were randomly divided into 6 groups based on different treatment. IRI model: The hepatic artery, portal vein, and bile duct to the left and median liver lobes (70% of the liver) were occluded with an atraumatic bulldog clamp for 90 minutes and then the clamp was removed for reperfusion. The mice were sacrificed 6 hours after reperfusion, and blood and liver tissues were collected. Liver injury was evaluated by biochemical and histopathologic examinations. The expressions of Sesn2, PI3K, Akt and heme oxygenase-1 (HO-1) were measured with quantitative real-time RT-PCR and Western blotting.

 

RESULTS: The serum aminotransferases level and scores of hepatic histology were increased after reperfusion. The increase was attenuated by pretreatment with OA (P<0.01). Compared with the IR group, OA pretreatment significantly up-regulated the expression of Sesn2, PI3K, Akt and HO-1 in IR livers (P<0.05). Administration of zinc protoporphyrin (ZnPP), an inhibitor of HO-1, diminished the OA effect on HO-1 and Sesn2 expressions (P<0.05) and the protective effect of OA on IRI.

 

CONCLUSIONS: Our results demonstrate that OA can attenuate hepatic IRI. The protective mechanism may be related to the OA-induced HO-1/Sesn2 signaling pathway.

 

(Hepatobiliary Pancreat Dis Int 2016;15:519-524)

 

KEY WORDS: oleanolic acid; liver injury; ischemia reperfusion; HO-1; Sesn2

Oleanolic acid (OA), which is a pentacyclic triterpene acid, protects rats from experimental hepatic injury and has been used for human liver dysfunction as an oral remedy.^{[1, 2]} OA pretreatment also protects the liver from ischemia reperfusion injury (IRI) during the acute phase, with a significant decrease in serum alanine aminotransferase (ALT) and hepatic centrilobular necrosis in mice.^[3-5] However, the underlying molecular mechanisms of OA-mediated hepatic protection are unclear.

 

Many liver operations may result in hepatic IRI, followed by liver dysfunction.^[6] Reperfusion of the hepatic blood supply causes cellular damage in the ischemic areas, which induces the formation of reactive oxygen species, release of pro-inflammatory cytokines and hepatocyte necrosis and thus, leading to severe liver dysfunction.^[7] Despite the evidence for OA protection against IR-induced hepatic injury via the PI3K/Akt pathway, other potential mechanism should be investigated.^[5] Sestrins 2 (Sesn2) is a member of the sestrins family, which are stress-inducible proteins that protect cells and tissues from oxidative stress. Increased expression of Sesn2 could attenuate hepatocyte oxidative injury.^{[8, 9]} Heme oxygenase-1(HO-1), a phase II detoxifying enzyme, is considered to decrease hepatic IRI.^[10]

 

We hypothesized that OA suppresses hepatic oxidative injury via the enhancement of the HO-1/Sesn2 pathway.

 

 

Thirty-six male C57BL/6 mice, 8-10 weeks old (Model Animal Research Center of Nanjing University, Nanjing, China) were used in this study. The mice were housed in controlled environmental conditions with a 12-hour light-dark cycle and free access to standard rodent diet and water. The experiments were conducted in accordance with the guidelines approved by the Chinese Association of Laboratory Animal Care, and the standards for animal use and care were set by the Institutional Animal Care Committee.

 

We used a well-established mouse model of partial warm hepatic IRI.^{[11, 12]} Briefly, a midline laparotomy was performed under 10% chloral hydrate (0.3 g/kg i.p.) anesthesia in mice. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were occluded with an atraumatic bulldog clamp for 90 minutes and then the clamp was removed for reperfusion. Sham-operated controls underwent the same procedure but without vascular occlusion. The peritoneal injection included OA (30 mg/kg per day for 7 days) and/or zinc protoporphyrin (ZnPP) (1.5 mg/kg, 4 hours prior to ischemia). The mice were randomly divided into 6 groups: sham, OA+sham, OA+ZnPP+sham, IR, OA+IR, and OA+ZnPP+IR. All mice were sacrificed 6 hours after reperfusion, blood and liver tissue were collected.

 

The levels of serum ALT and aspartate aminotransferase (AST) were measured using an automated chemical analyzer (Olympus Automated Chemistry Analyzer AU5400, Japan).

 

Liver specimens were fixed with 10% neutral formaldehyde and embedded in paraffin. The specimens were sectioned at 4 µm and stained with hematoxylin and eosin (HE). The sections were used in histopathologic analysis by light microscopy. The sections were scored from 0 to 4 for sinusoidal congestion, vacuolization of hepatocyte cytoplasm, and parenchymal necrosis as described by Suzuki et al.^[13]

 

Proteins (20 µg per sample) from livers were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories Co, Ltd, Shanghai, China). Polyclonal rabbit anti-mouse β-actin (Santa Cruz Biotechnology, Shanghai, China) was used as an endogenous control. Relative protein quantities were determined with a densitometer and were expressed in absorbance units.

 

Total RNA was extracted from liver tissue and cDNA was synthesized according to the manufacturer’s instructions with a reverse transcriptase kit (TaKaRa Biotechnology Co, Ltd., Japan).^[14] The measurements of each sample were performed in triplicate. Real-time PCR was carried out using an ABI Prism 7300 (Applied Biosystems, USA). The following cycling conditions were used in the real-time PCR: 40 cycles at 95 �� for 30 seconds; 60 �� for 31 seconds; and extension at 72 �� for 30 seconds. The results were analyzed using Applied Biosystems software. Data of target gene mRNA copies were expressed as the relative quantification (RQ), and RQ was calculated using the 2^-ΔΔCt method, where ΔΔCt=ΔCt (observed)-ΔCt (control) and ΔCt=Ct (gene)-Ct (β-actin). The primer sequences used for RT-PCR were as follows: PI3K, 5’-CAT CAC TTC CTC CTG CTC TAT-3’ and 5’-CAG TTG TTG GCA ATC TTC TTC-3’; Akt, 5’-GGA CAA CCG CCA TCC AGA CT-3’ and 5’-GCC AGG GAC ACC TCC ATC TC-3’; HO-1, 5’-GTC TAC GCC CCG CTC TAC TTC CCG-3’ and 5’-TAG CCT CTT CCA CCA CCC TCT GCC-3; β-actin, 5’-AAA CGA GAC GAG ATT GGC ATG GCT TTA-3’ and 5’-GGG ATG CTC GCT CCA ACG ACT GCT-3’. The β-actin was used as an endogenous control.

 

Data were expressed as the mean and standard deviation. Differences among the experimental groups were analyzed with one-way ANOVA. All differences were considered statistically significant at P<0.05.

 

 

As shown in Fig.1A and B, ALT and AST levels were increased significantly in the IR group compared to the sham controls (P<0.05). These increases were significantly attenuated in the OA+IR group (P<0.01). These data were consistent with HE scores of histologic liver IR damage (Fig. 1C and D). OA-pretreated IR mice showed minimal sinusoidal congestion and vacuolization without edema or necrosis. In contrast, the IR group displayed moderate-to-severe edema and extensive hepatocyte necrosis after 6 hours of reperfusion.

 

OA pretreatment significantly increased the Sesn2 expressions in IR livers in our mice in both mRNA and protein levels compared with those in the IR group (P<0.05, Fig. 2).

 

RT-PCR showed that OA pretreatment significantly increased mRNA expressions of PI3K and HO-1 in the IR mice compared with those in IR group (P<0.05, Fig. 3A). Fig. 3B shows that the expressions of P-Akt, the activated Akt, and HO-1 were increased significantly in the OA+IR group compared with those in the IR animals (P<0.05).

 

ZnPP, an HO-1 inhibitor, significantly reversed OA-mediated Sesn2 elevation to almost the same level as the IR group in both mRNA and protein expressions. These data may indicate that OA-induced Sesn2 is partially mediated by HO-1 (Fig. 4).

 

Serum ALT and AST levels were significantly increased in the OA+ZnPP group compared with the OA group (P<0.05) (Fig. 5A and B). These serum aminotransferases changes were in line with liver pathology (Fig. 5C and D). Indeed, the OA+ZnPP group showed marked liver sinusoidal congestion and vacuolization, severe edema, and extensive hepatocellular necrosis. ZnPP significantly aggravated liver IRI in IR mice pretreated with OA, accompanied with decreased Sesn2 expression.

 

 

The present study demonstrated that OA pretreatment alleviated hepatic IRI, as evidenced by significant down-regulation in both serum ALT and AST levels and histologic improvement. These results were associated with an obvious increase in PI3K/Akt, HO-1 and Sesn2 protein expression, indicating that PI3K/Akt or HO-1 may play an important role in increased expression of Sesn2-induced by OA in hepatic IRI.

 

It is reported that OA has potent antioxidant activity and may protect cells and tissues from oxidative stress.^[15] The antioxidant effects of OA is mediated through activation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and HO-1 in chronic cyclosporine nephropathy.^[16] OA pretreatment protects liver from IRI partially through the PI3K/Akt pathway.^[5] The novelty of the present study is that OA also attenuates hepatic IRI via the HO-1/Sesn2 signal pathway.

 

Sestrins, which include Sesn1, Sesn2, and Sesn3 in mammals, are highly conserved proteins encoded by genes whose expression is up-regulated in cells exposed to DNA damage, oxidative stress, and hypoxia. Sesn2 was shown to mediate the antioxidant activities in different regulating activities. Sesn2-mediated oxidative stress suppression could attenuate the detrimental consequences of oxidative stress and ensure cell viability and function.^[8] In many other cell types, Sesn2 is induced by Nrf2, c-Jun N-terminal kinase (JNK), activator protein 1 (AP-1), hypoxia inducible factor-1 (HIF-1), and p53;^{[17, 18]} however, there were not any results presented in hepatic IRI. In this study, expression of Sesn2 was significantly increased in ischemic liver tissue. In addition, OA pre-treatment further induced Sesn2 expression in ischemic liver tissue. Thus, Sesn2-mediated antioxidant activities may play a key role during OA protection against hepatic IRI, which was further studied in this study.

 

OA has been shown to mediate anti-inflammatory effects and the antioxidant activities in different ways. OA protects against hepatic injury by inducing antioxidant enzymes, such as HO-1,^[19] and reduces lipopolysaccharide (LPS)-induced inflammation by inhibiting hyperpermeability and leukocyte adhesion and migration.^[20] A recent report^[5] showed that OA pretreatment exerts hepatoprotective effects in a rat model of hepatic IR, likely involving the PI3K, P-Akt, and P-GSK-3β pathways. To detect a potential signaling pathway, PI3K/Akt and HO-1 were detected. In our study, OA pretreatment significantly increased expression of PI3K/Akt and HO-1 increased in ischemic liver tissue. Because of antioxidant activities of HO-1, ZnPP was used to inhibit HO-1 in hepatic IRI. We found that expression of OA-induced Sesn2 significantly inhibited when pretreated with ZnPP. These results support that OA-induced Sesn2 attenuates hepatic IRI partially via HO-1 regulation.

 

One main feature of hepatic IR is inflammation-induced hepatopathology.^{[21, 22]} In the acute phase, neutrophils, triggered by pro-inflammatory cytokines, are released in the ischemia stage, and are further activated to infiltrate into liver tissue. Hepatic enzymes and reactive oxygen species are then released to aggravate the associated liver tissue injury.^{[23, 24]} OA exerts antioxidant and protective effects against hepatic injury by inducing antioxidant enzymes, such as HO-1 and Sesn2, and exerts anti-inflammatory effects and protective effects against hepatic injury by multiple signaling pathways, including AMPK-mTORC1-regulating pathway and p53.^[8] Our results confirmed that OA pretreatment considerably reduces the hepatic IRI by up-regulation of HO1/Sesn2, which may control oxidative stress injury in ischemic liver. In addition, anti-inflammatory effects of OA will be further researched in the future.

 

There are some limitations in our study. For example, the HO-1 inhibitor, ZnPP, only partially decreased Sesn2 expression, indicating that some other mechanisms were involved in OA-induced Sesn2 expression in hepatic IRI. Moreover, OA treatment promoted the increase of both PI3K/Akt and HO-1 levels in ischemic liver, but the relationship between PI3K/Akt and HO-1 was not investigated. These limitations will be resolved in future research.

 

In conclusion, our data suggest that OA could partially attenuate hepatic IRI via the HO-1-mediated Sesn2 signaling pathway, which may supply some beneficial information for future clinical therapeutic strategies to attenuate IR-related hepatic injury.

 

 

1 Han DW, Ma XH, Zhao YC, Yin L, Ji CX. Studies on the preventive action of oleanolic acid on experimental cirrhosis. J Tradit Chin Med 1982;2:83-90. PMID: 6765707

2 Liu J. Oleanolic acid and ursolic acid: research perspectives. J Ethnopharmacol 2005;100:92-94. PMID: 15994040

3 Liu J, Liu Y, Klaassen CD. The effect of Chinese hepatoprotective medicines on experimental liver injury in mice. J Ethnopharmacol 1994;42:183-191. PMID: 7934088

4 Liu J, Liu Y, Parkinson A, Klaassen CD. Effect of oleanolic acid on hepatic toxicant-activating and detoxifying systems in mice. J Pharmacol Exp Ther 1995;275:768-774. PMID: 7473165

5 Gui B, Hua F, Chen J, Xu Z, Sun H, Qian Y. Protective effects of pretreatment with oleanolic acid in rats in the acute phase of hepatic ischemia-reperfusion injury: role of the PI3K/Akt pathway. Mediators Inflamm 2014;2014:451826. PMID: 24829521

6 Liu A, Jin H, Dirsch O, Deng M, Huang H, Bröcker-Preuss M, et al. Release of danger signals during ischemic storage of the liver: a potential marker of organ damage? Mediators Inflamm 2010;2010:436145. PMID: 21197406

7 Kloek JJ, Maréchal X, Roelofsen J, Houtkooper RH, van Kuilenburg AB, Kulik W, et al. Cholestasis is associated with hepatic microvascular dysfunction and aberrant energy metabolism before and during ischemia-reperfusion. Antioxid Redox Signal 2012;17:1109-1123. PMID: 22482833

8 Lee JH, Budanov AV, Karin M. Sestrins orchestrate cellular metabolism to attenuate aging. Cell Metab 2013;18:792-801. PMID: 24055102

9 Ke B, Shen XD, Ji H, Kamo N, Gao F, Freitas MC, et al. HO-1-STAT3 axis in mouse liver ischemia/reperfusion injury: regulation of TLR4 innate responses through PI3K/PTEN signaling. J Hepatol 2012;56:359-366. PMID: 19502171

10 Luo YH, Li ZD, Liu LX, Dong GH. Pretreatment with erythropoietin reduces hepatic ischemia-reperfusion injury. Hepatobiliary Pancreat Dis Int 2009;8:294-299. PMID: 20460768

11 Rao J, Zhang C, Wang P, Lu L, Zhang F. All-trans retinoic acid alleviates hepatic ischemia/reperfusion injury by enhancing manganese superoxide dismutase in rats. Biol Pharm Bull 2010;33:869-875. PMID: 17241879

12 Pietrangelo A, Dierssen U, Valli L, Garuti C, Rump A, Corradini E, et al. STAT3 is required for IL-6-gp130-dependent activation of hepcidin in vivo. Gastroenterology 2007;132:294-300. PMID: 7685932

13 Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulating effects of FK506 and cyclosporine. Transplantation 1993;55:1265-1272. PMID: 23750267

14 Rao J, Qin J, Qian X, Lu L, Wang P, Wu Z, et al. Lipopolysaccharide preconditioning protects hepatocytes from ischemia/reperfusion injury (IRI) through inhibiting ATF4-CHOP pathway in mice. PLoS One 2013;8:e65568. PMID: 21756853

15 Wang X, Liu R, Zhang W, Zhang X, Liao N, Wang Z, et al. Oleanolic acid improves hepatic insulin resistance via antioxidant, hypolipidemic and anti-inflammatory effects. Mol Cell Endocrinol 2013;376:70-80. PMID: 23791844

16 Hong YA, Lim JH, Kim MY, Kim EN, Koh ES, Shin SJ, et al. Delayed treatment with oleanolic acid attenuates tubulointerstitial fibrosis in chronic cyclosporine nephropathy through Nrf2/HO-1 signaling. J Transl Med 2014;12:50. PMID: 24559268

17 Shin BY, Jin SH, Cho IJ, Ki SH. Nrf2-ARE pathway regulates induction of Sestrin-2 expression. Free Radic Biol Med 2012;53: 834-841. PMID: 22749810

18 Zhang XY, Wu XQ, Deng R, Sun T, Feng GK, Zhu XF. Upregulation of sestrin 2 expression via JNK pathway activation contributes to autophagy induction in cancer cells. Cell Signal 2013;25:150-158. PMID: 22982090

19 Reisman SA, Aleksunes LM, Klaassen CD. Oleanolic acid activates Nrf2 and protects from acetaminophen hepatotoxicity via Nrf2-dependent and Nrf2-independent processes. Biochem Pharmacol 2009;77:1273-1282. PMID: 19283895

20 Lee W, Yang EJ, Ku SK, Song KS, Bae JS. Anti-inflammatory effects of oleanolic acid on LPS-induced inflammation in vitro and in vivo. Inflammation 2013;36:94-102. PMID: 22875543

21 Liaskou E, Wilson DV, Oo YH. Innate immune cells in liver inflammation. Mediators Inflamm 2012;2012:949157. PMID: 22933833

22 van Golen RF, van Gulik TM, Heger M. The sterile immune response during hepatic ischemia/reperfusion. Cytokine Growth Factor Rev 2012;23:69-84. PMID: 22609105

23 Vollmar B, Richter S, Menger MD. Liver ischemia/reperfusion induces an increase of microvascular leukocyte flux, but not heterogeneity of leukocyte trafficking. Liver 1997;17:93-98. PMID: 9138279

24 Walsh KB, Toledo AH, Rivera-Chavez FA, Lopez-Neblina F, Toledo-Pereyra LH. Inflammatory mediators of liver ischemia-reperfusion injury. Exp Clin Transplant 2009;7:78-93. PMID: 19715511