Tauroursodeoxycholic acid and 4-phenyl butyric acid alleviate endoplasmic reticulum stress and improve prognosis of donation after cardiac death liver transplantation in rats

Hao Lu, Ling Lu, Zhen-Chao Xu, Yun-Jie Lu, Bo Zhao, Lin Zhuang, Bao-Bing Hao and Feng Zhang

Nanjing, China

Tauroursodeoxycholic acid and 4-phenyl butyric acid alleviate endoplasmic reticulum stress and improve prognosis of donation after cardiac death liver transplantation in rats

Hao Lu, Ling Lu, Zhen-Chao Xu, Yun-Jie Lu, Bo Zhao, Lin Zhuang, Bao-Bing Hao and Feng Zhang

Nanjing, China

BACKGROUND:Inevitable warm ischemia time before organ procurement aggravates posttransplantation ischemiareperfusion injury. Endoplasmic reticulum (ER) stress is involved in ischemia-reperfusion injury, but its role in donation after cardiac death (DCD) liver transplantation is not clear and the effect of ER stress inhibitors, tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (PBA), on the prognosis of recipient of DCD liver transplantation remains unclear.

METHODS:Male Sprague-Dawley rats (8-10 weeks) were randomly divided into control group: liver grafts without warm ischemia were implanted; DCD group: warm ischemia time of the liver grafts was 60 minutes; TUDCA and PBA groups: based on the DCD group, donors were intraperitoneally injected with TUDCA or PBA 30 minutes before the organ procurements. Serum aminotransferase levels, oxidative stress activation and expression of ER stress signal molecules were evaluated. Pathological examinations were performed. The survivals of the recipients in each group were compared for 14 days.

RESULTS:Compared with the control group, DCD rats had significantly higher levels of serum aminotransferase at 6 hours, 1 day and 3 days after operation (P＜0.01, 0.01 and 0.05, respectively) and oxidative indices (P＜0.01 for both malondialdehyde and 8-hydroxy deoxyguanosine), more severe liver damage (P＜0.01) and up-regulated ER stress signal expressions (P＜0.01 for GRP78, phos-eIF2α1, CHOP, ATF-4, ATF-6, PERK, XBP-1 and pro-caspase-12). All recipients died within 3 days after liver transplantation. Administration of TUDCA or PBA significantly decreased aminotransferase levels (P＜0.05), increased superoxide dismutase activities (P＜0.01), alleviated liver damage (P＜0.01), down-regulated ER stress signal expressions (P＜0.01) and improved postoperative survivals (P＜0.01).

CONCLUSIONS:ER stress was involved with DCD liver transplantation in rats. Preoperative intraperitoneally injection of TUDCA or PBA protected ER stress and improved prognosis.

(Hepatobiliary Pancreat Dis Int 2014;13:586-593)

donation after cardiac death;

liver transplantation;

ischemia-reperfusion injury;

endoplasmic reticulum stress

Introduction

As the only effective therapy for end-stage liver diseases, liver transplantation (LTx) did bring hopes to the patients. However, growing shortage of liver graft led to a large number of on-list patients delisted or died.[1]To enlarge the graft pool and fill the gap between supply and demand, donation after cardiac death (DCD) was re-introduced and re-attracted increasing attention from transplantation surgeons.[1,2]

Contrasting to donation after brain death and livingdonation, DCD liver grafts underwent a prolonged warm ischemia time (WIT) comprising the time before cardiac death because of poor cardiac output and organ perfusion and "no-touch" time,[3,4]which resulted in more postoperative complications, e.g. ischemic cholangiopathy,[5]hepatic artery thrombosis and stenosis,[6,7]and primary non-function[8-11]at early stage and a higher rate of graft or patient loss 3 years after operation,[12-15]while severe ischemia-reperfusion injury (IRI) was the main cause of the bad prognosis of DCD LTx.[16-18]

Endoplasmic reticulum (ER) plays an essential role in protein synthetizing, folding and trafficking. These functions might be impaired by IRI.[19]Mal-folded protein accumulation might trigger unfolded protein reaction (UPR) and subsequent dissociation of glucose-regulated protein 78 (GRP78) could activate following signals:[20]activating transcription factor (ATF)-6, inositol-requiring enzyme-1α (IRE1α) and protein kinase like ER kinase (PERK), leading to down-regulated protein production and enhanced protein folding. Sustained insult might aggravate ER stress and further up-regulate expression of CCAAT/enhancer binding protein homologous protein (CHOP) and induce apoptosis of involved cells.[21,22]

According to Anderson et al,[23]up-regulated expressions of GRP78, ATF-4, IRE1α and CHOP after steatotic LTx might be reversed by administration of tauroursodeoxycholic acid (TUDCA), leading to improved liver function after operation. Meanwhile, another small molecular chaperone 4-phenyl butyric acid (PBA) could alleviate liver damage and hepatocytes death involving ATF-6 activating and X box protein (XBP)-1 splicing.[24]Both TUDCA and PBA inhibited ER stress and improved prognosis in rat partial hepatectomy/ischemia-reperfusion models.[25]However, therapeutic effects of TUDCA and PBA on DCD LTx and underlying mechanisms remain unclear.

In the present study, rat DCD LTx models were established to elucidate the role of ER stress and evaluate therapeutic effects of TUDCA and PBA on DCD LTx.

Methods

Animals

Male Sprague-Dawley rats (250±20 g, 8-10 weeks) were purchased from the Vital River Laboratories (Beijing, China). The rats were maintained on standard laboratory diet and waterad libitumin the clean environment with a 12/12 hours light/dark cycle.

Experimental design

Rats were divided into 4 groups. In the control group, livers were flushed one minute after systemic heparinization without warm ischemia. Then, the liver grafts were preserved in cold UW solution (4 ℃) for 6 hours. In the DCD group, cardiac death was induced by incising the diaphragm after heparinization. The donor was then put on a water bath (37 ℃) pad and the WIT was 60 minutes. Then, the liver grafts were flushed with and preserved in cold UW solution (4 ℃) for 6 hours. In the TUDCA group, the donor was administered with TUDCA (100 mg/kg body weight, i.p.) 30 minutes before operation. Liver procurement and preservation of this group was as same as that of the DCD group. In the PBA group, the donor was administered with PBA (100 mg/kg body weight, i.p.) 30 minutes before operation. Liver procurement and preservation of this group was as same as that of the DCD group.

The protocol of DCD LTx was similar to that previously reported.[26-29]The doses of TUDCA and PBA were chosen according to previous studies.[19,23,25,30]Our preliminary studies using different doses of TUDCA or PBA (25, 50, 100, 200 and 400 mg/kg body weight, for both) indicated that the doses of TUDCA or PBA chosen in this study were most effective.

For each group, 15 operations were performed. Five recipients were executed for liver tissue sampling 6 hours after the operations while the rest rats were observed for 14 days.

The experimental protocol was approved by the Medical Ethical Committee of Nanjing Medical University.

Surgical procedures

Rat orthotopic LTxs (OLTs) were performed according to the reports.[31-33]Briefly, an upper abdominal cross incision was performed under general anesthesia with isoflurane. The ligands of the hepatic and splenic vein, the hepatic artery, the right renal and adrenal vein, the left inferior phrenic vein, and the esophageal venous plexus were ligated and transected. The inferior hepatic vena cava, the portal vein and the common bile duct were skeletonized. The common bile duct was transected after inserting a 24-G stent. Then the donor was systemically anti-coagulated with 250 U of heparin.

The liver was flushed with and preserved in precooling UW solution (4 ℃). After preservation, the liver graft was implanted into the recipient. The removal of the native liver was similar to graft procurement but a mid-line incision was used and the splenic vein, the right renal and adrenal vein were reserved. Penicillin (100 000 units) and meloxicam (1 mg/kg body weight) were injected subcutaneously 30 minutes before and every 12 hours after operation for 3 days. All rats were fed regularly 2 hours after operation.

Western blotting

Livers were flushed with precooling saline 6 hours after operation and liver tissue samples were collected. All these samples were subjected to Western blotting. Antibodies against ATF-4, ATF-6, phos-eIF2α1, pro-caspase-12, XBP-1 and CHOP were all obtained from Abcam Inc. (Cambridge, USA). GRP78 was from Epitomics (Burlingame, USA) and PERK from Cell Signaling Technology Inc. (Danvers, USA). Gel-Pro Analyzer software (Rockville, USA) was used for quantitative measurements.

Biochemical tests

Blood samples were collected via the caudal vein from the rest recipients under general anesthesia 6 hours after operation and on postoperative days 1, 3, 7 and 14 for all recipients survived and diluted for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) measurements.

Oxidative stress determination

Six hours after transplantation, the levels of plasma malondialdehyde (MDA), 8-hydroxy deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) were determined to evaluate lipid peroxidation, DNA injury and anti-oxidation capacity, respectively.

Histology

After tissue collection for Western blotting 6 hours after operation, the residual livers were fixed in 10% formalin, embedded in paraffin, and cut into 4 μm sections. Hematoxylin and eosin staining was performed and sections were evaluated with light microscopy. Injury degree was scored according to Ishak et al.[34]

Survival analysis

For each group, ten OLTs were performed for survival analysis. The whole observation period was 14 days. The information about the weight of donors and recipients, anhepatic phase time, and operation time was collected. Warm and cold ischemia time was not calculated in the operation time. Survival time was recorded and survival rate was analyzed.

Statistical analysis

Statistical analysis was performed using SPSS 18.0 software. For normal distributed continuous variables, data were presented as mean±standard deviation (SD); and for skewed distributed variables and missing data, medians (25th-75th interquartile) were presented. After performing homogeneity test of variances, Bonferroni or Dunnett's T3 test was used for the comparison of means among these groups, while rank sum was tested using the Mann-WhitneyUtest. In addition, The Kaplan-Meier method and log-rank test were performed for survival analysis. ThePvalue was calculated by Bonferroni's correction method. APvalue of ＜0.05 was considered statistically significant.

Results

Surgical information

As shown in Table 1, there is no difference in donors weight, recipients weight, operation time or anhepatic phase time in each group (P＞0.05 for all). Donor weight, recipient weight, operation time and anhepatic phase time ranged from 240 to 260 g, 250 to 270 g, 115 to 135 minutes and 13 to 19 minutes for all operations, respectively.

Intra- and postoperation observation

Liver grafts in the control group were well reperfused after re-construction of blood vessels and bile production could be observed immediately (within one minute). But in the DCD group, color of re-perfused liver was deep and unevenly distributed. Bile production could not be observed until the operations were finished. However, re-perfused liver color and bile production were improved in the TUDCA group or PBA group. Benefited from the analgesic, recipients in the control group showed normal activities and could seek food and water. Rats in the DCD group were characterized by delayed awakening, poor vitality, no spontaneous foodtaking, coagulation malfunction suggested by bloody ascites and progressive ataxia, and were finally dead. These symptoms were alleviated in the TUDCA group and PBA group.

Table 1.Surgical information analysis

Serum aminotransferase levels

Recipients' serum ALT and AST levels of the control, TUDCA and PBA groups declined sharply, but stayed high in the DCD group (Fig. 1). At 6 hours, 1 day and 3 days' time points, ALT and AST levels of the TUDCA and PBA groups improved significantly in comparison with the DCD group. On postoperative days 7 and 14, serum aminotransferase levels in the TUDCA and PBA groups showed no differences from the control group except ALT in the PBA group on postoperative day 7 [238 (215-244) vs 368 (309-407),P＜0.01].

Fig. 1.Postoperative levels of serum aminotransferase. The levels of ALT and AST are shown as median for each group. *:P＜0.05, compared with the control group; **:P＜0.01, compared with the control group; #:P＜0.05, compared with the DCD group; ##:P＜0.01, compared with the DCD group.

Oxidative stress

MDA and 8-OHdG of the control group were 11.41± 0.62 nmol/mL and 2.89±0.58 ng/mL, respectively, which were elevated substantially in the DCD group (40.28 ± 1.48 nmol/mL and 19.79±1.66 ng/mL, respectively,P＜0.01). TUDCA and PBA significantly decreased the plasma levels of MDA and 8-OHdG (20.75±1.80 nmol/ mL, 9.55±1.48 ng/mL and 23.80±1.93 nmol/mL, 11.92±1.17 ng/mL, respectively,P＜0.01). Activity of SOD was abated in the DCD group but boosted in the TUDCA and PBA groups (Fig. 2).

Expressions of ER stress signals

As shown in Fig. 3, expressions of GRP78, phoseIF2α1, CHOP, ATF-4, ATF-6, PERK, XBP-1 and procaspase-12 were all up-regulated in the DCD group compared with the control group, intraperitoneal administration of TUDCA or PBA before organ procurement significantly attenuated their expressions.

Pathological examinations

Six hours after LTx, livers in the control group suggested an almost normal lobular architecture with widened sinusoidal intervals, mild lymphocyte infiltration in portal areas, and few vacuolization, karyolysis and necrosis (Fig. 4A). In the DCD group, lobular architecture was destroyed. Severe confluent necrosis was observed (Fig. 4B). However, liver damages were significantly alleviated in the TUDCA or PBA group (Fig. 4C and D) and the damage scores were also significantly decreased (Fig. 4E).

Survival curve

Survival rates of the four groups on postoperative day 14 were 90%, 0%, 70% and 60%, respectively (Fig. 5). Log-rank test suggested that the survival was significantly lower in the DCD group, whereas no differences were found among the other three groups (Table 2).

Fig. 2.Induction of oxidative stress 6 hours after operation. The levels of MDA, 8-OHdG and SOD are shown as mean±SD (n=15 for each group). **:P＜0.01, compared with the control group; ##:P＜0.01, compared with the DCD group.

Fig. 3.Protein expressions of ER stress signals. Western blotting (A) and densitometry analysis for GRP78 (B), phos-eIF2α1 (C), CHOP (D), ATF-4 (E), ATF-6 (F), PERK (G), XBP-1 (H) and procaspase-12 (I). **:P＜0.01, compared with the control group; ##:P＜0.01, compared with the DCD group.

Fig. 4.Pathological examinations 6 hours after operation. Representative photographs of pathological changes in the control (A), DCD (B), TUDCA (C) and PBA (D) groups and damage scores (E). *:P＜0.05 and **:P＜0.01, compared with the control group; ##:P＜0.01, compared with the DCD group.

Fig. 5.Survival curve of the recipients in different groups. Being censored indicated the survival until postoperative day 14 (n=10 for each group).

Table 2.Log-rank test for survival analysis

Discussion

Postoperative morbidity and mortality were higher after DCD LTx.[7,35-37]In the present study, although the donors were systemic heparinized before induction of cardiac death, livers were variegated after flushing with precooling UW solution, suggesting abundant blood thrombi formation, leading to the absence of organ preservation solution and deprivation of nutrient supply during cold storage and inflammation activation and non-perfusion after reconstructions of blood vessels. All these pathological processes resulted from severe IRI after DCD LTx.

Fernández et al[38]reported that liver IRI causes significant serum aminotransferases elevation, histological damage, oxidation/anti-oxidation imbalance, and glutathione consumption. Accordingly, recipients after DCD LTx (DCD group) were characterized by elevated serum aminotransferase levels, severer oxidative injury evidenced by excessive lipid peroxidation (MDA), DNA damage (8-OHdG) and impaired anti-oxidation capacity (SOD), and typical pathological changes including destroyed lobular architecture, severe confluent necrosis, nucleolysis and inflammatory infiltration. All of these led to the whole mortality in the DCD group within 3 days after operation. Autopsy showed mal-reperfusion and cholestasis, which were attenuated in the TUDCA and PBA groups. A small amount of food was in the stomach and the rats were emaciated in the DCD group.

TUDCA and PBA significantly attenuated serum aminotransferase levels at 6 hours, 1 day and 3 days. Compared with the control group, only ALT level in the PBA group was increased, showing statistical significance at 7 days. Lipid peroxidation and DNA damage were milder in TUDCA or PBA treated rats; the oxidation states were well balanced with antioxidation evidenced by raised SOD capacities. Also, the pathological examinations revealed that the severe histological changes were attenuated in both TUDCA and PBA groups and the damage scores were significantly reduced.

In rodents ischemia-reperfusion/partial hepatectomy and steatotic LTx models, induction of ER stress was definite, which was caused by IRI and oxidative stress.[23-25]These findings gave clues to the potential role of ER stress in DCD LTx. In this study, protein expression profile confirmed the involvement of ER stress in graft damage after DCD LTx. GRP78 was dissociated after induction of UPR and activated the following downstream signal pathways: PERK-eIF2α1-ATF-4-CHOP, IRE1α-XBP-1 and ATF-6-CHOP. These responses resulted in decreased synthesis, enhanced degradation and accelerated folding of proteins, promoting the recovery of ER functions. But sustained UPR, or ER stress, caused further activations of CHOP and pro-caspase-12 inducing cell apoptosis. According to our preliminary results and previous studies,[19,23,25,30]TUDCA and PBA significantly inhibited of ER stress, accompanied with milder liver injury, suggesting the essential role of ER stress and therapeutic effects of TUDCA and PBA in DCD LTx.

In addition, both TUDCA and PBA improved the quality of life after DCD LTx. Majority of recipients in both groups showed activities in seeking food and water after complete revival of anesthesia; while in the DCD group, rats manifested delayed awakening, poor vitality, no spontaneous food-taking, coagulation malfunction, progressive ataxia, and finally death within 3 days. Cumulative survival rates in the TUDCA and PBA groups showed no differences compared with the control group but significantly improved, compared with the DCD group.

ER stress participated in the underlying mechanisms of graft injury and impaired prognosis after DCD LTx; either TUDCA or PBA inhibited ER stress, alleviated the liver damage, improved the prognosis and prolonged the survival after DCD LTx. Our findings demonstrated that TUDCA or PBA inhibited ER stress, prevented postoperative complications and improved prognosis after DCD LTx.

Contributors:LH and LL contributed equally to this article. LL, ZB and ZF proposed the study. LH and LL designed the experimental program and wrote the first draft. LH, XZC, ZL and HBB performed the operations. LH and LYJ collected and analyzed the data. All authors contributed to further drafts. ZF is the guarantor.

Funding:This study was supported by a grant from the National Natural Science Foundation of China (81273262).

Ethical approval:The experimental protocol was approved by the Medical Ethical 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.

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Received March 18, 2014

Accepted after revision May 20, 2014

Author Affiliations: Translation Medicine Research Center, Affiliated Jiangning Hospital; Department of Liver Surgery, First Affiliated Hospital, Nanjing Medical University; Key Laboratory of Living Donor Liver Transplantation, National Health and Family Planning Commission of China, Nanjing 210029, China (Lu H, Lu L, Xu ZC, Lu YJ, Zhuang L, Hao BB and Zhang F); School of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China (Zhao B)

Feng Zhang, MD, PhD, Translation Medicine Research Center, Affiliated Jiangning Hospital; Department of Liver Surgery, First Affiliated Hospital, Nanjing Medical University; Key Laboratory of Living Donor Liver Transplantation, National Health and Family Planning Commission of China, Nanjing 210029, China (Tel: +86-25-68136476; Fax: +86-25-86660751; Email: zhangfeng1958@hotmail.com)

? 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(14)60269-1

Published online June 23, 2014.