RNA Interference-induced LXRα Downregulation And RHuEPO Enhance Hepatic Fuction After Transplantation Of Fatty Livers:A Study In Rat Models

Part One Establishment of a rat model of fatty liver donationObjective To establish a stable rat model of severe fatty liver graft and lay a foundation for the study of posttransplant graft function in a rat model of marginal graft liver transplantation.Methods 310 male Sprague-Dawley (SD) (60-90g) rats were purchased from the Model Animal Research Center of Kunming Medical University. The donor livers were obtained from SD rats (n=155) that had been fed for 2 months with a high-fat diet and infused intragastrically with 56% white spirits once each two days for making a model closely like the donor of human fatty liver. Experimental effects were assessed by the measurements of hepatic index, total blood fat, transaminases and histology at 2,4 and 8 week. Evaluations were done by histodiagnosis.Results The diet induced fatty involution of the donor liver with a degree of at least 50% steatosis after 8 weeks. The values of measurements of rats with high-fat diet were significant higher than the rats with normal diet (P<0.05).Conclusions The high-fat diet and alcohol would induce a rat model of fatty liver after 8 weeks successfully.Part Two Construction and functional identification of a recombinant lentiviral vector mediated RNAi targeting LXRaObjective A recombinant lentiviral vector mediated RNAi targeting LXRa gene was constructed and was used to infect the BRL cells. The knocking down efficiency on LXRa gene was analysed by RT-PCR.Methods The rat LXRa RNAi target sequences were designed. The lentiviral vector was constructed and amplified and BRL cells were transfected. The green fluorescent protein (GFP) expression which was measurement of the virus infection efficiency was evaluated by observing the frozen section of liver tissue with fluorescence microscope. Two multiplicity of infection (MOI) ratios (20 and 40) were tested. After 72 h of incubation, BRL cells were washed to remove free lentiviral particles and GFP expression was observed with fluorescence microscope and cells with>80% transfection were qualified for gene interference. The knocking down efficiency on LXRa gene were analysed by reverse transcription (RT)-PCR.Results RNA interference was used to knock down LXRa (Nrlh3, NM-013627.2) in fatty liver cells. The four constructed lentiviral vectors were: Nr1h3/GV115-RNAi-LV#1,Nr1h3/GV115-RNAi-LV#2,Nr1h3/GV115-RNAi-LV#3 and Nrlh3/GV115-RNAi-LV#4. The transfection ratio of target cells was over 80%. The real-time PCR analysis showed that LXRa gene expression was knocked down significantly over 80% compared with NC cell. The Nr1h3/GV115-RNAi-LV#4 was the set of most efficient.Conclusions 1.Successfully construct the LXRα-RNAi-LV of over 80% interference efficiency.2. The BRL cells were infected by LXRα-RNAi-LV and the gene expression was knocked down significantly.Part three Establishment of reduced-size liver transplantation with fatty liver donor in ratObjective To establish a suited and stable model of reduced-size liver transplantation for fatty liver donor in rat, reducing the difficulty in operation and make a foundation for the next studyMethods 20 donors were chosed randomly in the fatty liver transplantation. The rats in experimental group received reduced the left lateral liver transplantation and the rats in control group received orthotopic liver transplantation. The breath, heart rate, vessel filling situation, survival conditions and liver function of rats were observed after reperfusion. Histology and survival curve were assessed.Results After graft reperfusion, the respiratory rates, heart rates of rats in control group were faster than those in experimental group(P<0.05). The vessel filling of inferior vena cava in control group was poorer than that in the experimental group. Rats in experimental group recovered better and sooner and the operative time was shorter (P<0.05). The mortalities of rats in control group were higher and the causes of death included intra-abdominal hemorrhage, air embolism and hypovolemic shock. The survival time, histology and liver function postoperatively of living recipients were without significant difference.Conclusions The establishment of reduced-size liver transplantation model with fatty liver donor were successful. The recipients in experimental group were with the same values in hepatic function and death rate. The new surgical procedure was an ideal method on fatty liver transplantation research in rat which worked out on poor operation field exposure and unstable circulation.Part four RNA interference-induced LXRa downregulation and rHuEPO Enhance Hepatic Fuction After Transplantation of Fatty Livers:A study in Rat ModelsObjective To investigate whether LXRa RNA interference (RNAi) and rHuEpo can improve the organ function of rat liver transplant recipients after fatty liver transplantation.Methods 100 SD rats were fed with high-fat diet and used as donors which displayed over 50% macrovesicular steatosis in livers. Recipients received fatty liver transplantation after donor organ treatment with 7×107TU Nrlh3/GV115-RNAi-LV mixture injection 72h before operation (Group A and B) and rHuEpo (4000IU/kg) 30min before organ harvest(Group A and C) or NC-LV injection (Group D) and saline (Group E) into portal veins. Recipients also received rHuEpo (4000IU/kg) at reperfusion (Group A and C). Experimental effects were assessed by the measurements of fluorescence microscope, transaminases, blood fat, bilirubin, histology, apoptosis rate, cytokins, immunohistologic staining, protein levels(LXRa, SREBP-1c and CD36), and reverse transcriptase-polymerase chain reaction of LXRa messenger RNA at defined time points.Results 1.There were abundant green fluorescence of GFP in fatty liver donors after 72h and focused on hepatic lobular regions and central vein regions. That is to say the lentiviral vectors tranfected successfully and effectively into the hepatocytes.2. The combined treatment group displayed more obvious reduction in serum ALT, TBIL, IL-1β and TNF-a values than single treatment groups and control groups postoperatively (P<0.05). TG and TC levels were not different between the groups at any time point. The TG content in the liver tissue was lower in the animals of group A, B than in the control animals at all time points.3. After OLT, histological findings revealed that the hepatic lobules were expanded by inflammatory infiltration in all 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 liver cells in the centrilobular areas. An evaluation using the Suzuki pathological score revealed least pathologic damage in the combined treated rats compared with the other groups (P<0.05).4. TUNEL staining:the degree of apoptosis in the graft tissue of combined treated group was lowest after 24h to 72h postoperatively compared with other groups (P< 0.05).5. NF-κBp65 staining:the absorbances of combined treated group were lowest at 4h time point but higher than group B at 24h point and then declined. The values of rHuEPo treated group were highest at 24h point.6. Data demonstrated that the lentivirus-LXRa RNAi vectors inhibited the expression of LXRa gene at mRNA and protein levels, in addition to levels of SREBP-1c and CD36 which reduced the fatty acid accumulation in hepatocytes, as compared with the effects of the control (P< 0.05).7. The overall survival as determined by Kaplan-Meier analysis was improved among rats treated with LXRα-RNAi-LV and rHuEPo.Conclusions LXRα-RNAi-LV treatment significantly downregulated LXRa expression and LXRa-RNAi-LV and rHuEpo combined treatments improved steatotic graft function and recipient survival after a fatty liver transplantation in rats.