Author Affiliations: Department of Hepatobiliary and Transplantation Surgery, Ganmei Affiliated Hospital, Kunming Medical University, Kunming 650011, China (Zhao YP, Li L, Ma JP, Chen G and Bai JH)

Corresponding Author: Li Li, PhD, Department of Hepatobiliary and Transplantation Surgery, Ganmei Affiliated Hospital, Kunming Medical University, No. 504, Qingnian Road, Kunming 650011, China (Tel: +86- 871-63188092; Fax: +86-871-63188091; Email: ynkmlili62@hotmail.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60347-2

Published online March 10, 2015.

 

 

Acknowledgement: We thank Professor Shu-Rong Tang (Laboratory of Ganmei Affiliated Hospital, Kunming Medical University) for the technical support.

Contributors: ZYP and LL proposed the study. ZYP and MJP performed the research and wrote the first draft. CG and BJH collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZYP is the guarantor.

Funding: This study was supported by a grant from the Committee of Science and Technology of Kunming, China (09H130201).

Ethical approval: The experimental design was approved by the local government [Department of Science and Technology, Kunming, China, No. SYXK (DIAN) 2011-0006] and the Ethics Committee of Kunming Medical University, and the study was conducted according to the regulations for animal experiments (Institutional Animal Care and Use Committee of China).

Competing interest: The authors do not choose to declare any conflict of interest related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Steatotic liver grafts, although accepted, increase the risk of poor posttransplantation liver function. However, the growing demand for adequate donor organs has led to the increased use of so-called marginal grafts. Liver X receptor alpha (LXRα) is important in fatty acid metabolism and interrelated with the specific ischemia-reperfusion injury in fatty liver transplantation. This study aimed to investigate whether LXRα RNA interference (RNAi) could improve the organ function of liver transplant recipients.

 

METHODS: Fifty Sprague-Dawley rats were fed with a high-fat diet and 56% alcohol. The livers of these animals had greater than 60% macrovesicular steatosis and were used as liver donors. The experimental donors were treated with 7×10⁷ TU LXRα-RNAi-LV of a mixture injection and control donors with negative control-LV vector injection into the portal vein 72 hours before the operation. The effects of LXRα-RNAi-LV were assessed by serum aminotransferases, histology, immunostaining, and protein levels. The transcription of LXRα mRNA was assessed by reverse transcription-polymerase chain reaction.

 

RESULTS: Compared with controls, LXRα RNAi inhibited the expression of LXRα at the mRNA (0.53±0.03 vs 0.94±0.02, P<0.05) and protein levels (0.51±0.08 vs 1.09±0.12, P<0.05). LXRα RNAi also decreased the expressions of sterol regulatory element-binding protein 1c (SREBP-1c) and CD36. LXRα RNAi consequently reduced fatty acid accumulation in hepatocytes. Compared with control animals, LXRα RNAi-treated group had lower serum alanine aminotransferase, aspartate aminotransferase, interleukin-1β, and tumor necrosis factor-alpha levels and milder pathologic damages. TUNEL analysis revealed a significant reduction of apoptosis in the livers of rats treated with LXRα-RNAi-LV, and overall survival as determined by the Kaplan-Meier method was improved among rats treated with LXRα-RNAi-LV (P<0.05).

 

CONCLUSION: LXRα-RNAi-LV treatment significantly downregulated LXRα expression and improve steatotic liver graft function and recipient survival after a fatty liver transplantation in rats.

 

(Hepatobiliary Pancreat Dis Int 2015;14:386-393)

 

KEY WORDS: fatty liver; liver transplantation; LXRα; RNA interference; ischemia-reperfusion injury

Orthotopic liver transplantation (OLT) is the most effective treatment for end-stage liver disease. However, the shortage of appropriate donor organs has led to an increased acceptance of so-called marginal grafts such as steatotic livers. However, the limited quality of the graft in addition to cold storage and ischemia-reperfusion (I/R) injury may result in poor posttransplantation liver function.^[1-3] At most centers, fatty infiltration of 10%-30% is considered the upper limit for donor selection.^[4] However, donor organ shortages prompt us to explore the possibility of liver donors with high steatotic status.

 

Liver X receptor alpha (LXRα) is a member of the LXR nuclear receptor superfamily, and it is activated by oxysterols and intermediates from the cholesterol biosynthetic pathway. LXRα also plays an important role in fatty acid metabolism by activating the sterol regulatory element-binding protein 1c (SREBP-1c) gene.^[5] It has been reported that LXRα expression is highly upregulated and strongly and positively correlated with that of SREBP-1c in patients with non-alcoholic fatty liver disease.^[6] Nakamuta et al^[7] found that despite an increased uptake of free fatty acids and intracellular accumulation of fatty acids and triglycerides, the expression of SREBP-1c, a key positive regulator of fatty acid synthesis, remained upregulated. Moreover, LXRα has a close relationship with fatty acid translocase (CD36), which is involved in the uptake of fatty acids. Cheng et al^[8] found that downregulated expression of LXRα in L02 steatotic hepatocytes in vitro decreased the triglyceride content of the hepatocytes and promoted the recovery of hepatocyte steatosis. The reason why the donation of a fatty liver is a major risk factor for primary graft non-function in a posttransplantation liver has not yet been elucidated. The potential reasons include several interacting mechanisms:^[9] (i) liver steatosis is clearly inverse to the blood flow in the hepatic sinuses; (ii) the liver sinusoidal endothelial cells are damaged by cytokines and oxyradicals that are activated by lipid accumulation during reperfusion; and (iii) the mitochondria of a fatty liver produce inadequate amounts of ATP during warm ischemia and cold storage. Therefore, when the sensitivity of I/R injury was alleviated, graft function may be improved by reducing the impact of future I/R injury and decreasing lipid droplet accumulation in the cytoplasm of hepatocytes.

 

We chose to establish a rat model of fatty liver transplantation to mimic the clinical situation of a "marginal graft".^[10-15] Then, mRNA downregulation of LXRα by RNA interference (RNAi) was conducted to decrease free fatty acid and intracellular fatty acid accumulation. This study aimed to evaluate the potential effect of treatment with LXRα RNAi on posttransplantation graft function in a rat model of steatotic liver transplantation.

 

 

Male Sprague-Dawley rats (weighing 90-150 g) were purchased from the Animal Research Center of Kunming Medical University (Kunming, China). The experimental design was approved by the local government [Department of Science and Technology, Kunming, China, No. SYXK (DIAN) 2011-0006] and the Ethics Committee of Kunming Medical University, and the study was conducted according to the regulations for animal experiments (Institutional Animal Care and Use Committee of China). The donor livers were obtained from rats that had been fed a high-fat diet (20% lard and 2% cholesterol) for 1 month and infused intragastrically with 56% alcohol at a 10 mL/kg dose once daily to ensure that the model was as similar to a human fatty liver donor as possible. This induced fatty liver was greater than 60% steatosis after 4 weeks upon histopathologic examination (Fig. 1).

 

Fifty Sprague-Dawley fatty liver rats served as liver donors in the experiment. Weight- and age-matched isogeneic Sprague-Dawley rats were used as the OLT recipients. The rats were randomly divided into two groups (each n=25). In experimental group, the donor animals were administered a mixture of Nr1h3 (target gene of LXRα) and GV115 (vector number) RNAi-LV (7×10⁷ TU/rat in 1.0 mL), as well as polybrene and enhanced infection solution (Shanghai Genechem Co. Ltd., China) through a branch of the superior mesenteric vein into the portal vein for hepatocyte transfection 72 hours before transplantation. In control group, the donor rats received a negative control-LV injection in the same manner. We planned to sacrifice the animals postoperative 2 and 24 hours for the cytokine levels and 3 days for a histologic evaluation, as this was a top endpoint of the posttransplantation examination. Five recipients from each group were sacrificed at specific time points to collect serum and liver tissues. The remaining recipients were analyzed for 7-day survival rate.

 

RNAi was used to silence LXRα (Nr1h3, NM-013627.2) in the fatty liver cells. The rat LXRα-RNAi target sequence was 5'-GGA GTG TGT CTT ATC AGA A-3'. The oligonucleotide sequence for the sense strand was 5'-CCG GGA GGA GTG TGT CTT ATC AHA ACT CGA GTT CTG ATA AGA CAC ACT CCT CTT TTT G-3', where that for the antisense strand was 5'-AAT TCA AAA AGA GGA GTA TAT CTT ATC AGA ACT CGA GTT CTG ATA AGA CAC ACT CCT C-3'. The negative control RNAi sequence (5'-TTC TCC GAA CGT GTC ACG T-3') was a randomly scrambled sequence not found in human or rat genome databases. They contained two endonuclease sites: AgeI and EcoRI. The lentiviral vector particle was hU6-MCS-CMV-EGFP (GV115). The RNAi was synthesized by Shanghai Genechem Co. Ltd. LXRα silencing was performed by transfecting RNAi into the hepatocytes through the portal vein.

 

The rat hepatocyte cell line BRL, which was obtained from China Cell Culture Center (Shanghai, China), was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37?�� with 5% CO₂. BRL cells were seeded at a density of 1.5×10⁷ cells/well in six-well plates and transfected with LXRα-RNAi-LV, which also included a blank control and an negative control. The multiplicity of infection (MOI) ratios (20 and 40) were tested. After 72 hours of incubation, BRL cells were washed to remove any free lentiviral particles, green fluorescent protein (GFP) expression was observed using a fluorescence microscope, and cells with >80% transfection efficiency were considered for gene interference. The silencing efficiency of LXRα was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).

 

The rats were anesthetized using isoflurane inhalation with 40% oxygen, and the OLTs were performed using a "two-cuff" technique developed by Kamada and Calne^[16] with a few modifications. The hepatic artery was not reconstructed. After systemic heparinization, the donor liver was flushed in situ with 10 mL of saline solution at 4?��, followed by 10 mL of cold (4?��) University of Wisconsin (UW) solution. An 8-mm long stent prepared from a polyethylene tube was inserted into the common bile duct and secured with sutures. Then, the liver was removed and stored in the UW solution at 4?��. The cuffs for the vascular connections were attached to the portal vein and the intrahepatic vena cava. All donor grafts were immediately transplanted orthotopically into the recipients, and the portal clamping time for all transplantation procedures conducted in this study was less than 20 minutes. The average preservation time was 46±4.7 minutes.

 

At specific time points posttransplantation, groups of rats were sacrificed, and the liver allografts were removed after drawing blood from the abdominal aorta. A portion of the liver tissues was frozen in liquid nitrogen for Western blotting, real-time RT-PCR, and lipid measurements, and other tissues were harvested for histological examination. The sera were centrifuged immediately, and the supernatant was stored at -80?�� for enzyme-linked immunosorbent assay (ELISA) and at 4?�� for biochemical testing.

 

Liver tissue was fixed in 4% formalin and stained with hematoxylin and eosin (HE) when the tissues were cut into sections from the paraffin-embedded blocks. All slides were assessed by one of the investigators blinded to the corresponding group allocation. The previously published criteria by Suzuki et al^[17] were modified to evaluate the histologic severity of the I/R injury in the OLT model. In this classification of sinusoidal congestion, hepatocyte necrosis and ballooning degeneration were graded from 0 to 4. The absence of necrosis, congestion, or centrilobular ballooning was given a score of 0, whereas severe congestion and ballooning degeneration, as well as lobular necrosis of more than 60% of the liver, was scored as 4.

 

Serum alanine aminotransferase, aspartate aminotransferase, triglyceride, and total cholesterol were detected in duplicate using an automated biochemistry analyzer (OLYMPUS, Japan) kits according to the manufacturer's protocols.

 

The liver tissue was prepared with 10% homogenate in ice water, and the supernatant was used to detect triglyceride after centrifugation. The triglyceride content of the liver tissue was measured using a commercial kit (Furui Biological Engineering Co. Ltd., Beijing, China).

 

The two cytokines were measured using a rat IL-1β/TNF-α Platinum ELISA Kit (eBioscience, Bender MedSystems GmbH, Vienna, Austria) for a sandwich ELISA analysis with streptavidin-HRP according to the manufacturer's procedural instructions (n=5 for each time point). Aliquots of the spiked serum samples, which were stored at -80?��, were thawed at room temperature and diluted for the test. The absorbance of the samples was read by a spectrophotometer using a 450-nm primary wavelength with a plate reader that was analyzed with Multiskan Ascent (Thermo Fisher Scientific Inc, MA, USA).

 

The liver embedded in paraffin was cut into 6-µm slices and mounted on a glass slide. Apoptotic hepatocytes nuclei were identified using an ApopTag Peroxidase in situ apoptosis detection kit and the TUNEL assay (Intergen Co, New York, NY, USA) according to the manufacturer's instructions. Brown-stained nuclei were considered positive hepatocytes and were counted in 1000 hepatocytes (200-fold magnification), and the apoptosis index (%) was defined. Examination and grading of all tissue samples were performed by the same investigator who had been blinded to the study group.

 

The frozen samples were homogenized in a lysis buffer, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Sigma, Israel). Rat-specific rabbit polyclonal antibodies against LXRα, SREBP-1c, and CD36 were supplied by Proteintech Group Inc (Chicago, IL, USA). A densitometry analysis for protein levels was performed by a Bio-Gel Imaging system equipped with the Genius synaptic gene tool software.

 

Silencing of LXRα was detected by real-time RT-PCR. The total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA was obtained using an RT procedure with M-MLV reverse transcriptase (Promega, Madison, WI, USA). The target genes were analyzed by real-time RT-PCR with the use of primers (forward, 5'-GTA CAA CCC TGG GAG TGA GA-3'; reverse, 5'-GGT TGA TGG AGA CAT AGG CA-3') and GAPDH as an internal control (forward, 5'-TTC AAC GGC ACA GTC AAG G-3'; reverse, 5'-CTC AGC ACC AGC ATC ACC-3'). The PCR products were quantified using SYBR Green I (TAKARA, Dalian, China) and an iCycler (TAKARA) to measure the amount of double-stranded DNA in each well during each cycle and obtain a critical threshold value.

 

Data were expressed as mean±standard deviation (SD). Differences between the groups were estimated using analysis of variance followed by Fisher's exact test. SPSS version 13.0 (SPSS Inc, USA) was used for statistical analysis. A P value <0.05 was considered statistically significant.

 

 

The BRL cells infected with LXRα-RNAi-LV were photographed using a fluorescence microscope after 3 days. The transfection ratio of the target cells exceeding 80% at an optimal MOI of 20. The real-time RT-PCR analysis illustrated that LXRα expression was silenced by more than 80% compared with that of the control group (0.17±0.01 vs 1.00±0.04; P<0.05, Fig. 2).

 

After OLT, histological examination revealed that the hepatic lobules were expanded by inflammatory infiltration in the two groups. Thrombi were observed in the interstitial spaces and central veins. The LXRα-RNAi-LV-transfected rats exhibited notable amelioration of fatty infiltration, especially in the hepatocytes in the centrilobular areas. Significantly increased necrosis was observed in the control group compared with that in the RNAi-treated group 3 days after OLT. Pleomorphic nuclei pyknosis of the hepatocytes was observed in both groups. An evaluation using the Suzuki pathological score revealed severe pathologic damage in the control rats (score 9.67±1.51), compared with the mild damage observed in the RNAi-treated group (score 3.33±0.82, P<0.05).

 

The blood biochemical results after OLT are shown in Fig. 3. All liver enzyme levels were increased 24 hours later after liver transplantation. The enzyme levels were significantly lower in animals treated with LXRα-RNAi-LV than those in the control group (P<0.05). We did not find the differences of triglyceride and total cholesterol levels between the two groups at any time point (data not shown).

 

The triglyceride content in the liver tissue is shown in Table. Triglyceride levels were lower in the treated animals than in the control animals at 2-h and 24-h time points.

 

Serum IL-1β and TNF-α levels were lower in rats pretreated with LXRα-RNAi-LV than in the control animals 24 hours later after liver transplantation (IL-1β: 1342.46±141.02 vs 3243.50±294.47 pg/mL, P<0.05; TNF-α: 879.00±97.43 vs 2242.88±402.80 pg/mL, P<0.05; Fig. 4).

 

Two hours after reperfusion, the degree of apoptosis was 19% in the LXRα-RNAi group and 28% in the control group; after 24 hours, this difference was significant (26.4% vs 68.3%, P=0.012). This trend continued until 72 hours postoperatively, at which point the values were 29.5% and 33.5%, respectively (Fig. 5).

 

Twenty-four hours after OLT, the LXRα mRNA expression was significantly reduced in the LXRα-RNAi treated rats compared with that in the control group (0.53±0.03 vs 0.94±0.02, P<0.05; Fig. 6).

 

LXRα RNAi significantly reduced LXRα protein levels in the rat liver allografts 24 hours after liver transplantation (normalized protein level: 0.51±0.08 vs 1.09±0.12; F=77.53; P<0.05). The other lipid metabolism related proteins, CD36 and SREBP-1c, were downregulated in the LXRα-RNAi-treated group compared with the control group (CD36: 0.28±0.05 vs 0.69±0.09; F=75.26; P<0.05; SREBP-1c: 0.20±0.04 vs 0.67±0.13; F=62.20; P<0.05; Fig. 7).

 

The 7-day survival rate was significantly increased in the LXRα-RNAi-LV-treated rats compared with that of the control group (75.0% vs 30.0%; P=0.045; n=10; Fig. 8).

 

 

Given the severe shortage of liver donors, marginal grafts are considered to have optimal utility for patients as a unique solution.^[18] The present study demonstrated that treatment of donor livers with LXRα-RNAi-LV application attenuated I/R injury and improved survival after steatotic liver transplantation in rats. It was reported that the different features of an I/R injury in a steatotic liver graft, as compared to that of a normal liver graft, were closely related to primary graft non-function. Necrosis, instead of apoptosis, is the predominant form of cell death in fatty rats which decreases the capacity of liver tissue regeneration.^{[19, 20]} The mechanisms remain unclear, the following aspects may play a role.^[9] (i) The degree of steatosis in hepatocytes is inversely proportional to the hepatic sinusoidal blood flow. Intracellular lipids increase cell volume, liver sinusoidal obstruction, and vascular resistance, which sensitize the liver tissue to anoxia and I/R injury. (ii) The reduced production and significant loss of adenosine triphosphate in the mitochondria during cold preservation and reperfusion make a fatty liver graft vulnerable to I/R injury. (iii) Sinusoidal endothelial cell injury and blood cell adhesion to the endothelial cells are markedly increased by fatty liver transplantation, and free radicals and cytokines from activated Kupffer cells and neutrophils are increased. Triglycerides and free fatty acids from the damaged cells activate phospholipases, resulting in lipid peroxidation, which produces oxygen radicals and accelerates injury to cellular functions.

 

To reduce the accumulation of lipids in the hepatocytes of a fatty liver donor in a short period of time, we targeted LXRα, a gene involved in fatty acid metabolism, with lentiviral-based RNAi to protect the graft from I/R injury. The present study clearly demonstrated that the lentiviral-based RNAi effectively silence LXRα expression in vitro, and thus, it can be tested in vivo.

 

The protective effects of the lentiviral treatment were reflected by reductions in serum liver enzyme levels after 24 hours. In addition, evaluation of the triglyceride content and HE staining revealed an obvious amelioration of fatty infiltration of the liver tissue and reduced necrosis of the hepatocytes, particularly around the central veins, in the RNAi transfection group. These findings were only limited in histology, and the hyperlipidemia was not improved. It was proposed that the reduction of fat accumulation decreased the obstruction of the hepatic sinusoid and improved the function of mitochondrial oxidative phosphorylation. Consequently, injury to sinusoidal endothelial cells was reduced, and ATP synthesis was increased. The improved cellular situation would diminish free radical formation, which was associated with severe graft injury, poor graft function, and decreased survival.^[21] However, the local action of RNAi did not improve the general hyperlipidemia.

 

In this study, the level of steatosis in the hepatocytes of the liver graft was significantly and quickly diminished upon transfecting hepatocytes with lentivirus-mediated LXRα-RNAi. The data demonstrated that LXRα-RNAi effectively inhibited LXRα expression in the liver tissue at both the mRNA and protein levels in vivo. The protein levels of SREBP-1c in the rat liver allografts were downregulated synchronously. It has been reported that SREBP-1c is positively regulated by the transcription factor LXRα, which forms a heterodimer with retinoid X receptor α.^[22] In addition, fatty acid synthesis in hepatocytes is positively regulated by the transcriptional factor SREBP-1c.^[23] The upregulation of SREBP-1c contributes to the increased de novo synthesis of fatty acids, even in hepatocytes with fatty acid accumulation.^[24] Thus, it could be demonstrated that LXRα-RNAi influenced the level of steatosis in the hepatocytes by inhibiting the expression of the transcriptional factor SREBP-1c. In addition, the protein expression of CD36 was decreased in the LXRα-RNAi-LV group. It was reported that the hepatic uptake of fatty acid is facilitated by cell surface receptors, including CD36. Overexpression of CD36 results in increased fatty acid and lipoprotein influx and utilization in tissues.^[25] Moreover, CD36 has been found to be transcriptionally regulated by several nuclear receptors including LXR. A recent report^[26] illustrated that LXR might also promote lipogenesis by activating the expression of CD36. Another study confirmed that intact expression, activation, or both of CD36 are required for the steatotic effect of LXR agonists.^[27] Therefore, effective silencing of LXRα expression also inhibits the expression of CD36 to reduce intracellular fat accumulation.

 

TUNEL staining and an evaluation of HE-stained tissue revealed that the liver had significantly milder apoptosis and necrosis in the LXRα-RNAi-LV-treated animals. The TUNEL test might not be specific for detecting apoptosis, as it also detects necrotic injury.^[28] However, the effect of the LXRα-RNAi-LV was identical because both cell death mechanisms play important roles in fatty liver I/R injury. Published findings in murine non-alcoholic steatohepatitis models demonstrated that overexpressed CD36 may activate death receptors and induce apoptosis.^[29-31] Moreover, it has been shown that CD36/FAT acts pro-apoptotically.^[32] Thus it may be suggested that LXRα-RNAi-LV treatment reduces the number of hepatocytes undergoing apoptosis as well as the necrosis by reducing the expression of fatty acid transport proteins including CD36/FAT.

 

I/R injury-associated tissue damage is well reflected by IL-1β and TNF-α levels. The levels were significantly reduced in our study after the LXRα-RNAi-LV treatment. It is known that the two cytokines display the greatest activity in I/R injury, which can lead to microcirculatory disturbance in the liver through interactions between neutrophils and endothelial cells.^[33] The results demonstrated the effectiveness of the treatment for the reduction of ischemic tissue damage.

 

Compared with those received transplanted fatty livers treated with negative control-LV, rats received transplanted fatty livers treated with LXRα-RNAi-LV exhibited improved initial graft function. Most importantly, the utilization of this construct significantly prolonged the survival time of allogeneic rat liver transplant recipients.

 

In conclusion, the application of LXRα-RNAi-LV in an adipohepatic marginal donor organ is valuable for the improvement of organ function and survival after OLT of fatty livers in rats. This effect may be induced by the downregulation of LXRα, CD36 and SREBP-1c expressions and alleviation of I/R injury.

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