Effect And Mechanism Of RhALR On Liver Regeneration After Partial Hepatectomy And Partial Orthotopic Liver Transplantation In Rats

Objective: To establish the animal model of partial hepatectomy and partial orthotopic liver transplantation in rats, and analysis the variability of liver regeneration in different groups. After the rat model of 40% partial hepatectomy and 60% partial orthotopic liver transplantation have been established, recombinant human augmenter of liver regeneration (rhALR) was administered intraperitoneally. The serum levels of alanine aminotransferase(ALT) and albumin(ALB), proliferating cell nuclear antigen labeling index (PCNA LI) in liver sections, hepatocyte proliferation index (PI) and apoptotis index (AI) was detected in order to evaluate the effects of rhALR on hepatic regeneration. Further more, the mRNA levels of interleukin-6 (IL-6) and the cell-cycle mediators, Cyclin D1, natural killer cells (NK cells) activity in hepatic tissues was detected so as to explore the mechanism of rhALR in hepatic regeneration.Methods: 1. Establishment of the rat model: 40% partial hepatectomy rat model was established by resecting left lobe and double caudal lobe. 60% partial orthotopic liver transplantation model was established by removing full liver and implanted into a 60% partial liver with the modified two-cuffed vascular anastomoses technique of Kamada. 2. Wistar rats model of partial hepatectomy were divided into two groups randomly, in group A, 40ug/kg rhALR was administered intraperitoneally twice a day after operation, and in group B, same vehicle of sterile water for injection was administrated as control. Rats model of 60% partial orthotopic liver transplantation were divided into two groups randomly, in group C, 40ug/kg rhALR was administered intraperitoneally twice a day after liver transplantation, and in group D, same vehicle of sterile water for injection was administrated as control. 3. Experimental items and methods: 5 rats were sacrificed randomly on the day of 1,2,3,5 and 7 respectively after operation in every group. The serum levels of ALT and ALB was detected with automatic biochemistry analyzer, the expression of PCNA was detected in liver sections with immunohistochemistry assay, hepatocyte PI and AI was evaluated with flow cytometry, the expression of interleukin-6 (IL-6) mRNA and Cyclin D1 mRNA in hepatic tissue was detected with real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique, and natural killer cells (NK cells) activity in hepatic tissue was detected by methyl thiazolyl tetrazolium (MTT) reduction assay. 4. The data were expressed as mean±SD. Statistical analysis of the results was performed by one-way analysis of variance(ANOVA) and unpaired LSD t-test. P values of less than 0.05 were considered statistically significant.Results: 1. Stable rat model of partial hepatectomy and partial orthotopicd liver transplantation had been established successfully. There was no significantly difference between A and B group in the duration of operation and loss of blood, and there was no significantly difference too between C and D group in the duration of operation, cold-ischemia time, anhepatic phase time and loss of blood (P > 0.05) . 2. Serum ALT level of group A was significantly lower than that of group B on the 1^th post operation day (POD), ALT level of group C was significantly lower than that of group D on the 1^th and 2^th POD and there was no significantly difference for the rest. Comparing serum ALT between the two control groups, ALT level of group D was significantly higher than that of group B on the 1^th and 2^th POD (P < 0.05) . 3. Serum ALB level of group A was significantly higher than that of group B on the 7^th POD, ALB level of group C was significantly higher than that of group D on the 5^th and 7^th POD, and there was no significantly difference for the rest. Comparing serum ALB between the two control groups, ALB level of group D was significantly lower than that of group B on each POD (P< 0.05) . 4. The peak of hepatocyte PCNA LI in every group was all on the 2th POD, the level of PCNA LI of group A was significantly higher than that of group B on the lth,2th,3th and 5^th POD, and the same results appeared in group C and D. Comparing PCNA LI between the two control groups, PCNA LI of group D was significantly lower than that of group B on the 1th POD (P < 0.05) . 5. The peak of hepatocyte AI in every group was all on the 2th POD, the level of AI of group A was significantly lower than that of group B on the 2th POD, and the same results appeared in group C and D. Comparing AI between the two control groups, the hepatocyte AI of group D was significantly higher than that of group B on the every POD except for 7^th day (P < 0.05) .6. The level of PI of group A was significantly higher than that of group B on the 2th and 3th POD, the level of PI of group C was higher than that of group D on the 2th POD, and there was no significantly different for the rest. Compare PI between the two control groups, the hepatocyte PI of group D was lower than that of group B on the 1th POD, but higher on the 3th day (P < 0.05) , there was no significantly different for the rest. 7. The expression peak of IL-6 mRNA (inversely proportional to value of ACT) in hepatic tissue in every group was all on the 1th POD, there was no significantly difference in the expression of IL-6 mRNA between A and B groups on every POD, so did it between C and D groups. Comparing the expression between the two control groups, the expression of group D was significantly lower than that of group B on the 1th POD (P < 0.05) . 8. The expression of Cyclin D1 mRNA of group A was significantly higher than that of group B on the 1th POD, so did it between C and D groups. Comparing it between the two control groups, the expression of Cyclin D1 mRNA of group D was significantly lower than that of group B on the 1th POD, and higher on the 3th POD, there was no significantly difference for the rest. 9. The level of NK cells activity of group A was significantly lower than that of group B on the 1th POD, and the level of group C was significantly lower than that of group D on the lth,2th POD, Comparing it between the two control groups, the level of NK cells activity of group D was significantly higher than that of group B on the lth,2th and 3th POD (P<0.05) .Conclusions: 1. The Wistar rat model of hepatectomy and partial orthotopic liver transplantation was stable and reliable, and ensured the comparability between comparison groups. 2. Compared to group B of following hepatectomy, Hepatocyte regeneration in group D of following partial orthotopic liver transplantation was relative delayed. 3. rhALR can promote the regeneration of hepatocyte following partial hepatectomy and partial orthotopic liver transplantation, decrease the level of serum ALT, and preserve hepatic function. 4. The delayed regeneration of hepatocyte following partial orthotopic liver transplantation was probably concerned with lower expression level of IL-6mRNA and Cyclin D1mRNA in hepatic tissue compared to that following partial hepatectomy. 5. The mechanism of rhALR in promoting hepatocyte regeneration could probably be to up-regulate the expression level of Cyclin D1mRNA in hepatic tissue but not IL-6mRNA. 6. The mechanism of rhALR in promoting hepatocyte regeneration may probably be relevant to lower activity of NK cells in hepatic tissue after partial hepatectomy and partial orthotopic liver transplantation.