Effects Of Akt/TSC1-TSC2/mTOR Signaling Pathway On Liver Regeneration After Partial Hepatectomy In Rats

Background and Objective:Despite its large metabolic load, liver is a largely quiescent organ in terms of cell proliferation, with only 0.0012% to 0.01% of hepatocytes undergoing mitosis and proliferation at any given time. This low cell turnover in healthy liver tissue can be altered by toxic liver injury or surgical resection, which results in sudden, massive hepatocyte proliferation, producing recovery of functional liver mass within 2 weeks after the loss of up to two thirds of the liver. In recent years a number of studies on the mechanism of liver growth and development have examined by 70% partial hepatectomy(PH) model or chemical injury model. Furthermore, significant advancements have been achived by application of transgenic mice or knock out mice model. Liver regeneration is controlled by multiple signaling pathways induced by a variety of growth factors, hormones and cytokines. Most studies have examined hepatocyte proliferation as the major mechanism for liver regeneration, and have focused mainly on the cell cycle-regulatory system. In the case of resection, this results in hypertrophy of the remnant liver rather than restoration of the resected lobes. This phenomenon is actually compensatory hyperplasia but not true regeneration. Cell proliferation ceases once liver mass reaches a species- and age-specific fraction of total body mass. It is well known that both cell growth (increase in cell size) and cell proliferation (increase in cell number) contribute to the formation of multicellular organisms. However,the role of cell growth in liver regeneration is not thoroughly understood. Cell growth and cell proliferation are two distinctive but relative concepts which play an important role in organ growth, tissue formation, and tumorigenesis etc. One single cell does not divide into two daughter cells until the cell grows up to a certain size. So cell proliferation bases on cell growth. What has become clear in recent years is that Akt/TSC1-TSC2/mTOR signaling pathway, which is a conservative signal pathway from Drosophila melanogaster to human, can regulate protein synthesis, cell size, cell growth, cell number, cell proliferation, and tumor development, etc. However, it's still unknown how Akt/TSC1-TSC2/mTOR signaling pathway acts in the course of liver regeneration after partial hepatectomy. We speculate that this signaling pathway may play a key role in liver regeneration. It would be significant for better understanding liver regeneration to systematically investigate the changes and effects of this signaling pathway in hepatocytes.The aim of the present study is to study Akt/TSC1-TSC2/mTOR signaling pathway in hepatocytes in liver regeneration and to prove that this pathway plays a key role in liver regeneration.Methods:The left and medium lobes of normal rat's liver were ligatured and resected by the classics methods of partial hepatectomy established by Higgins & Aderson. The rate of resected liver was counted. Rat hepatocytes were separated on five time points after partial hepatectomy in normal rat, namely (0h,2h,6h,24h,72h).and the method to separate and culture hepatocytes was established using collagenase circumfusion in situ as well as in vitro and Percoll centrifugation methods. Cells'activity was measured by trypan blue dye exclusion. Hepatocytes were counted and the concentration was adjusted the concentration to (1.0-1.5)×106/ml,then cultured in 37℃,5%CO2.After 2 hours pre-culture, the separated hepatocytes were divided into three groups:control group (CO), Rapamycin group (RA), and Triciribine group (TR). Extracts of the hepatocytes from remnant liver were assayed for the phosphor-TSC2 and phosphor-mTOR through western blotting. The level of protein synthesis in regenerative hepatocytes was assayed by 3H-leucine incorporation. The cross-sectional area of hepatocytes was assayed by scanning electron microscope(SEM)and computer-assisted image analysis system(BEIHANG CM2000B). Cell proliferation was accessed through a flow cytometer.Results:1. The total amount of hepatocytes in a rat was about 1.21×108, and every gram of tissue can yields 9.5×106 hepatocytes. The average activity of hepatocytes was 96%.The forms of separated cells were agreed well with those of classic hepatocytes. The freshly separated hepatocytes were single, round, similar in term of size, and high refraction. 4h later they gradually became stretched. 24h later most of them adherenced to the culture dish and became flat. 48h later they turned to the classic epithelioid shapes. The experiments were repeated by multiple trials and reproducible results were observed.2. In group CO and group RA the protein phosphor-TSC2 expression began to increase at 2h and peaked at 6h(P<0.01) after hepatectomy, then decreased gradually, But the protein phosphor-TSC2 expression was greatly suppressed in group TR. In group CO the protein phosphor-mTOR expression was significantly enhanced at 2h and at 6h with a peak at 6h after hepatectomy, then decreased gradually, but the protein phosphor-mTOR expression was suppressed in group RA and group TR.3. Our experiments show 3H-leucine CPM of regenerating hepatocytes cultured in group CO was higer than that in group RA or group TR after partial hepatectomy; Significant difference was observed at time points of 0h, 2h, 6h and 24h after PH(P<0.05),and very significant difference on the time point of 72h(P<0.01).4. Compared with group RA and group TR, the cross-sectional area of hepatocytes increased significantly in group CO after partial hepatectomy at 2h and 6h(P<0.05). At time point of 24h, there were no significant difference between group CO and RA while cross-sectional area in group CO was still larger than in group TR. Most hepatocytes in group CO were in division states while many single cells were found in group RA and TR by scanning electron microscopy. In group CO, the cross-sectional area of hepatocytes was gradually increased from 2h to 24h, clearly trend was observed from 2h to 6h. Group RA had similar change with group CO.5. Cell cycle study by flow cytometry: Compared with two suppressant groups(TR and RA), the percentage of cells in G0/G1 phase gradually decreased while that in S and G2/M phase gradually increased after partial hepatectomy, the peak wasobserved at 24h, with a obvious decrease at 72h. Furthermore, the significant difference (P<0.01) between group RA and TR was only found at 24h after partial hepatectomy.Conclusion:1. The methods of separation, cultivation and evaluation of rat liver hepatocytes are economical, convenient and reliable.2. The expression of phosphor-TSC2 increased slightly at 2h,with a peak at 6h after partial hepatectomy. It can obviously influence the expression of phosphor-mTOR.Our data suggested that Akt/TSC1-TSC2/mTOR signaling pathway is rapidly activated in the residual hepatocytes at early stage of liver regeneration after partial hepatectomy. 3. Akt/TSC1-TSC2/mTOR signaling pathway activated at early stage of liver regeneration after partial hepatectomy can strongly promote protein synthesis and cell growth in hepatocytes. Our experiment show that 3H-leucine CPM and cross-sectional area of hepatocytes in group CO increased greatly with time points. We think that the role of this signaling pathway contribute to body recovery after partial hepatectomy, on the other hand it also settle a substance basis for hepatocyte proliferation4. Hepatocytes were clearly in proliferative state after partial hepatectomy. The percentage of cells in G0/G1 phase gradually decreased while that in S and G2/M phase gradually increased in all points after partial hepatectomy; the peak was at 24h, with an clear decrease at 72h. Furthermore, the significant difference (P<0.01) between group RA and TR was only found at 24h after partial hepatectomy. This proliferative state is tightly related to the activation of Akt/TSC1-TSC2/mTOR signaling pathway early stage of liver regeneration after partial hepatectomy. Our results strongly suggest that this signaling pathway plays a pivotal pathophysiological role in hepatocyte proliferation in liver regenerative responseFurther study on Akt/TSC1-TSC2/mTOR signaling pathway and the relationship between cell growth and cell proliferation will greatly enrich the theory of liver regeneration...