Ursolic Acid Improves Liver Transplantation and Inhibits Apoptosis in Miniature Pigs Using Donation After Cardiac Death

Liver transplantation is the only effective treatment for end-stage liver disease, which brings therapeutic hopes to patients with end-stage liver diseases [1, 2]_ENREF_1_ENREF_1. However, insufficient donor restricts the development of liver transplantation. Donation after cardiac death (DCD) enriches the source of donor to some extent [3, 4]. However, the organs experience a process of ischemia and hypoxia before cardiac arrest [5, 6]_ENREF_5_ENREF_1. Therefore, organ injury will aggravate and complications will increase after surgery. Severe ischemia / reperfusion injury (IRI) is a leading cause of poor prognosis of DCD liver transplantation [7]_ENREF_2. Therefore, a lot of methods were tried to improve the function of grafted DCD [8]. Current studies have indicated that endoplasmic reticulum stress (ERS) plays an important role in ischemia injury after DCD liver transplantation [9]_ENREF_1, especially the protein kinase-like ER kinase (PERK) pathway [10]_ENREF_1.

External factors stimulate the accumulation of unfolded and incorrectly folded proteins in the endoplasmic reticulum, which will lead to ERS [11]_ENREF_3. The unfolded protein response (UPR) was stimulated in ERS. UPR initiates to decrease new protein synthesis, correct protein folding and modification by molecular chaperone, and promote the protein degradation through ubiquitin-proteasome system to rescue endoplasmic reticulum [12]_ENREF_1. However, once the stimulus is too strong or persistent or surpasses the endurance capacity of ER, it will activate the ERS-related apoptosis [13, 14]_ENREF_12_ENREF_1.

The UPR was achieved by three transmembrane molecules–PERK, inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6). There was study implicating that PERK-eIF2α-ATF4-CHOP pathway was obviously activated in the rat liver transplantation model [10]_ENREF_1. Moreover, this signaling pathway regulates the ischemia reperfusion injury after DCD liver transplantation through apoptosis [10]_ENREF_1. Meanwhile, endoplasmic reticulum stress is also involved in diseases such as non-alcoholic fatty hepatitis, myocardial injury in myocardial infarction [15, 16]_ENREF_15_ENREF_1.

Ursolic acid (UA) belongs to pentacyclic triterpenes and widely exists in natural plants. The pharmacological activities of UA include anti-oxidation, anti-infection, anti-inflammation, anti-tumor, liver protection, etc [15, 16]_ENREF_16_ENREF_1. Previously, UA was reported to down-regulate PERK-eIF2α -ATF4-CHOP pathway to reduce apoptosis [15]_ENREF_1. In this study, we aimed to investigate the effects of UA on the tissue damage after DCD liver transplantation and mechanisms involved.

24 healthy adult Bama miniature pigs (12 months, weight 30-40 kg, half male and half female) were provided by Shenyang Agricultural University (China). As a reference, miniature pigs in both genders were applied in the transplantation experiments [17]. In our study, the pigs were randomly divided into two groups: control group and experimental group (12 in each group). Each group had 6 donors and 6 recipients and pigs in each pair weighed similarly. The pigs in both groups received 6 DCD orthotopic liver transplantations. DCD model was established using the method of asphyxia as previously described [18], that is, stopping oxygen supplement after anesthesia and muscle relaxation. The donors were anesthetized by pump controlled intravenous injection of propofol and inhalation of isoflurane, and we need to do tracheal intubation. During this period, the blood pressure was monitored by cephalic artery catheterization. Abdomen was opened and hepatic portal vein and infrarenal aorta abdominalis were isolated. Pancuronium was applied to relax the muscle. After that, the respirator was withdrawn. The donor pigs went into cardiac arrest. The time period from respirator withdrawing to circulatory arrest was about 19.25±3.4 min, comparable between two groups. The standard of circulatory arrest is heart stoppage or arterial blood pressure lower than 25 mmHg and pulse pressure lower than 20 mmHg. The cold perfusion was completed by using 4°C UW solution, respectively 1000ml through portal vein and aorta abdominalis. Orthotopic liver transplantation was conducted and blood flow was initiated after the portal vein and suprahepatic vena cava were anastomosed. In the two groups, anhepatic phase was about 41.7±2.8 min. Blood loss was estimated to be 300-500 ml. After surgery, all the transplanted livers and recipients survived for at least 24 hours. 24 h after transplantation, the pigs were euthanized. In control group, the pigs received DCD orthotopic liver transplantation without intervention. In experimental group, 4 hours before the surgery, recipient pigs received 120 mg/kg UA by intraperitoneal injection. Selection of the dose of UA was referred to the literature [19]. The liver tissues and venous blood were collected at 0 h(donor liver), and 1 h, 3 h, 6 h, 12 h and 24 h after when the blood circulation of transplanted liver was established (anastomosis of suprahepatic vena cava and hepatic portal vein were completed and blood flow was opened).

The 0-h time point referred to the time immediately after laparotomy in donors. The 1-h time point after opening of blood flow was just before the abdomen was closed (we successively sewed up the infrahepatic vena cava, hepatic artery, and common bile duct). The liver tissue was also obtained at 3-h, 6-h, 12-h and 24-h after transplantation under anesthesia through the incision which was partially closed. All experimental procedures were approved by the Animal Care and Use Committee of the First Affiliated Hospital of China Medical University.

Immunostaining of histological sections was performed using monoclonal antibodies against Bax (1: 200, Abcam, USA), Caspase 3 (1: 200, Abcam, USA) and Bcl-2 (1: 200, Abcam, USA). Endogenous peroxidase activity was blocked with 3% (vol/vol) H₂O₂ for 5 minutes. After that, tissues were incubated with primary antibody overnight at 4 °C followed by a 30-minute incubation with secondary antibody (Dako, Carpinteria, CA) and visualization with DAB chromagen for 3 minutes.

Protein was abstracted from liver tissue for western blotting as previously described [20]_ENREF_1. The antibodies, including anti-PERK, anti-p-PERK, anti-eIF2α, anti-p-eIF2α, anti-ATF4 and anti-CHOP (Santa Cruz, USA) were incubated overnight at 4°C. The ECL reagent kit was applied to assist the staining. The blots were scanned by ChemiDocTM XRS (Bio-Rad, USA). The grey density was analyzed by Image J 7.0 software.

Serum alanine aminotransferase (ALT) and liver malondialdehyde (MDA) were detected according to the assay kits (Beyotime, Ningbo, China).

Apoptosis was detected using Tunel assay according to the instruction of the assay kit (Beyotime, Ningbo, China).

Data were presented as means ± standard deviations. One-way analysis of variance with post-hoc Bonferroni test for multiple comparisons was performed. Differences were considered significant at p < 0.05.

HE staining was applied to detect the morphological changes of the transplanted liver tissue. As shown in Fig. 1, there were no morphological difference between control group and UA-treated group at the 0-h time point. However, cell edema, liver sinus congestion, local necrosis, unclear cell boundary, infiltration of periportal lymphocyte gradually appeared in control group, especially at 24-h after transplantation. Although these pathological changes were also observed in experimental group, UA obviously mitigated those abnormalities.

ALT level was also detected in our study. As shown in Fig. 2, serum ALT level was significantly decreased in experimental group at 3 h, 6 h, 12 h and 24 h time points compared with control group.

Serum MDA level was also compared between two groups. As shown in Fig. 3, significant difference was observed between the two groups at 12 h and 24 h after transplantation.

We compared the apoptosis between control and experimental groups. As shown in Fig. 4, apoptotic cells appeared in control group at the 3-h and aggravated at 6-h time point. In UA treated group, although apoptosis was still found, the level was decreased obviously compared with control group.

The apoptosis-regulators were also compared (Fig. 5). Compared with control, Caspase 3 and Bax were significantly decreased, whereas Bcl-2 was increased 6 h after transplantation in experimental group.

We also detected the PERK-CHOP signaling pathway by western blotting. As shown in Fig. 6, UA treatment deceased PERK, p-PERK, p-eIF2α, eIF2α, ATF4 and CHOP expression 1 h, 3 h and 6 h after transplantation, whereas significant difference was observed at 3 h and 6 h regarding p-PERK, PERK, eIF2α, ATF4 and CHOP. These results suggested that UA treatment decreased PERK-CHOP signaling pathway.

DCD is the main source of donor organs in the field of organ transplantation. Because of the special characteristics in the process of DCD liver transplantation, the time of ischemia and hypoxia is prolonged, and the ischemia reperfusion injury is severe [5, 6]_ENREF_6_ENREF_1. It is of particular importance to find the mechanism of the liver injury during the process of ischemia and reperfusion and to search relevant interventions to reduce those injuries. In our study, we reported that UA treatment could attenuate DCD liver transplantation-related liver injury. Moreover, serum ALT level and liver tissue MDA level decreased in UA treated group. In our study, we only detected the 24-h pathological changes after transplantation. Although the long-term effect of UA was not evaluated, our study provided positive implication for future study of drug preconditioning in facilitating liver transplantation, especially for the DCD liver transplantation.

Accumulating studies have implicated that liver cell apoptosis is an important form of cell death in both ischemia period or during reperfusion after liver transplantation_ENREF_1. In this study, we disclosed that apoptosis was the major mechanism contributing to the liver injury after transplantation [21-23]. Most importantly, UA pretreatment could alleviate the apoptosis [24]. There are a lot of studies confirmed the role of ERS in ischemia reperfusion injury after liver transplantation [25, 26]_ENREF_22_ENREF_1. Ischemia reperfusion injury leads to a significant increase of liver enzymes, pathological changes and the imbalance of oxidation of glutathione and pathological consumption [27]_ENREF_1. In our study, we evidenced that apoptosis appeared 3-h and aggravated 6-h after transplantation. In addition, we also detected the expressions of apoptosis-related regulators. Caspase-3 is an apoptosis effector and the high level of caspase-3 indicates the increased apoptosis [24, 28]_ENREF_1. Although we also observed obvious apoptosis in UA treated group, this drug could increase the resistance of the transplanted livers. In our study, we also found significant expression changes of Bax and Bcl-2. UA treatment obviously decreased Bax and increased Bcl-2 expression. In this study, we verified that UA treatment in the experimental group decreased the expressions of various molecules of endoplasmic reticulum stress, including PERK, eIF2α, ATF4 and CHOP within 6 hours after transplantation. Although Bax and Bcl-2 were also altered in UA treated group, it did not exclude the endoplasmic reticulum stress involved in the apoptosis.

UA is a type of compound with the structure of pentacyclic triterpenes. The pharmacological activities have been extensively studied, including antioxidant, antitumor, anti-aging, liver protection etc [29, 30]._ENREF_26_ENREF_1. Because antioxidant has been verified in the protection of organ ischemia reperfusion [19]_ENREF_1, we selected UA in this study and demonstrated that UA likely functioned through decreasing ER stress–related apoptosis. Nevertheless, the reason why UA down-regulates PERK-CHOP pathway is still open. In addition, UA possesses anti-inflammation effects, as well as promotes inflammation and causes DNA injury [31]. Although we did not evaluate the potential toxic effect of UA, it deserves more investigations.

We reported a novel approach which can ameliorate the DCD liver transplantation related injury. UA likely decreases liver tissue injury through decreasing PERK-CHOP signaling pathway mediated apoptosis. These data implicate that UA might have clinical efficacy to improve DCD liver transplantation.

UA (ursolic acid); DCD (donation after cardiac death); MDA (malondialdehyde); PERK ( protein kinase-like ER kinase); ALT (aminotransferase); IRI (ischemia/reperfusion injury); ERS (endoplasmic reticulum stress); UPR (unfolded protein response); IRE1α (inositol-requiring enzyme 1α); ATF6 (activating transcription factor 6);

This study is financed by Ministry of health industry fund (201302009, 201002004).

None.