Changes in liver-to-body weight ratio duirng rat liver regeneration
In this study, rat body weight (g) and regenerating liver weigh (g) at each timepoint were weighed, and the ratio of liver weigh to body weight was defined as liver coefficient (Lc) . According to the calculation results, the liver coefficients at 0, 6, 12, 24, 72, 120, and 168h after PH in rats were 1.35%, 1.58%, 1.86%, 1.86%, 3.69%, 4.08% and 4.61%, respectively (Figure 1A), and the time-dependent increase in liver coefficients demonstrated the sucessesful liver regeneration after PH.To testify whether the livers reliably regenerated after PH at the molecular level, we chose PCNA (an auxiliary protein of DNA polymerase delta that accumulates in the late G1 and early S-phase and whose level correlates with cellular prolifeative activity) to perform a western-bloting assay. As shown in Figure 1B, compared to the control (0 h), PCNA levels started to increase at 6 hours and peaked at 24 hours after PH, followed by a gradual reduction, indicating an enhanced cell proliferation during 12-24 hours, which is consistent with previous reports by other researchers.
Comparison of structural changes in normal and regenerating livers in rats
HE staining indicated the normal hepatic architecture with typical hepatic lobule and hepatic sinusoid, radial liver cell cord and uniform distribution of the cells in the control samples. By contrast, pathological changes were seen in the 2/3 hepatectomized livers: 6 h to 24 h post PH, there was more and more serious liver necrosis whose feature was that a number of hepatocytes showed a marked increase in nuclear size, accompanied with vesicular bodies and prominent nucleoli caryocinesia; 72 h to 120 h, the degeneration of hepatic cells and destruction of hepatic architecture were alleviated, but there were still many hepatocytes with enlarged nuclei indicative of cell division. Until 168 h after PH, the histological structure of regenerating liver closely resembled the normal liver tissue (Figure 2).
Differentially expressed proteins in regenerating livers after 2/3 PH in rats
We collected regenerating liver samples from 6h through 168h post 2/3 PH, and analyzed protein expression profiling at six different timepoints after surgery.To save space, we only showed the 2-DE maps of SO group and PH group at 24 hour because this is the time point when DNA and protein synthesis are most active (Figure 3). Among 2546 and 2554 differentially expressed proteins identified respectively in sham-operated groups and 2/3 hepatecyomized groups, 220 showed statistically significant differences (P < 0.05) in levels between PH group and SO group. These proteins were also called LR-related proteins whose volume changes in sham-operated rats and 2/3 hepatectomized rats were detailedly shown in Additional file 1: Table S1 of the Supporting Information. They were categorized into six groups based on expression changes: group 1, 125 proteins were up-regulated after 2/3 PH; group 2, 26 newly induced proteins were detected only in PH group but not in SO group; group 3, 5 proteins were down-regulated only in SO sample; group 4, 28 proteins were down-regulated after 2/3 PH; group 5, 34 proteins were below detection limit in PH group (detected only in SO sample but not in 2/3 PH sample); and group 6, 2 proteins were up-regulated at early phase but down-regulated at late phase during LR. In a general sense, proteins in group 1, 2 and 3 (totally 156) were viewed as up-regulated proteins during LR, and ones in group 4 and 5 (totally 62) as down-regulated proteins, and the remaining two in group 6 as up/down-regulated proteins.
Protein functional categorization
The 220 LR-related proteins identified in this study were divided into eight groups according to their biological functions (shown in Additional file 1: Table S1): (1) carbohydrate, lipid, protein and energy metabolism, involving 63 proteins; (2) amino acid and nucleic acid metabolism, involving 32 proteins; (3) biotransformation, involving 9 proteins; (4) cell proliferation-ralated protein, involving 13 proteins; (5) cell differentiation and development-related proteins, involving 55 proteins; (6) signal transmission, involving 23 proteins; (7) inflammatory factors and related proteins, involving 12 proteins; and (8) others, involving 13 proteins which were mainly up-regulated in LR and were hard to categorize into special biological activity.
Sixty three of the identified proteins were involved in carbohydrate, lipid, protein and energy metabolism. Of these proteins, 43 proteins were upregulated including 9 carbohydrate metabolism-related (mainly involved in catabolic process, i.e., G6PD), 13 lipid metabolism-related (mainly lipid degradation proteins, i.e., ACADVL, HADH), 17 protein metabolism-related, and 4 energy metabolism-related proteins (i.e., COX5A, UQCRC1, ATP6V1B2, ATP5B); 20 proteins were downregulated including 4 carbohydrate metabolism-related, 8 lipid metabolism-related, 7 protein-metabolism and 1 energy metabolism-related proteins. Thirty-two proteins were found to be functionally related to amino acid and nucleic acid metabolism, 18 proteins of which were upregulated in PH rats at early phase after operation, such as GOT1, ARG1; the remaing 14 were decreased in expression levels in 2/3 hepatectomized rats versus sham-operated rats, such as GLUL, RNASEH1.
Thirteen proteins, increased primarily in the middle phase of rat LR, were functionally related to cell proliferation. These proteins were involved in various events occurring in the cell cycle, such as chromatid separation (i.e., TOP2a), G1/S transition (i.e., PSMC4), and the regulation of cell cycle progression (i.e., CDC42).
Fifty-five proteins were functionally associated with cell differentiation and development, such as extracellular matrix organization-involved components (i.e., VWA1), cytoskeleton organization-related factors (i.e., NUP35) and so on. Out of these proteins, 43 were increased in hepatectomized rats, and the other 12 were decreased.
Twelve proteins were identified as inflammatory factors and the functionaly related proteins. Eight of them were upregulated after PH in rats, and other four were down-regulated. These proteins mainly play roles in antigen processing and presentation (i.e., PSMA6), macrophage chemotaxis (i.e., EDN2), and histocompatibility antigens (i.e., RT1-B) as well.
Twenty-three proteins were identified to be responsible for signal transduction, the majority of which (involving 18 proteins) were up-regulated during the regeneration process. They participate in various signal pathways, such as Ras signaling pathway (i.e., RASA2), cAMP pathway (i.e., CAP1), epidermal growth factor receptor signaling pathway (i.e., PDGFRB), GABA signaling pathway (i.e., GABRB2), Wnt receptor signaling pathway (i.e., AXIN2), Rho signal transduction (i.e., ARHGDIA), and insulin receptor signaling pathway (i.e., AKT2). These signaling pathways modulate multiple biological processes as mentioned above, such as cellular metabolism, cell proliferation, cell differentiation and inflammatory response etc.
Pathways regulating rat liver regeneration after 2/3 PH
All the differentially expressed proteins at each time point after PH, that’s 125 proteins at 6 h, 143 proteins at 12 h, 121 proteins at 24 h, 124 proteins at 72 h, 119 proteins at 120 h, and 131 proteins at 168 h, were respectively subjected to pathway analysis using Ingenuity Pathway Analysis 9.0 software. For audience’s convenience, we only presented the networks constructed by differentially expressed proteins at the representative time points during LR (e.g., 6 h within early phase, 72 h within middle phase and 168 h within terminal phase), as displayed in Figure 4. The networks from six recovery timepoints was attached in the Additional file 2: Figure S1.
As Additional file 2: Figure S1 indicated, the differentially expressed proteins of rat regenerating livers from each timepoint after 2/3 PH were connected to each other in one way or another to construct a network distinct from one another. Despite the great difference from each other, the common point between these networks was that 19 of 125, 24 of 143, 14 of 121, 19 of 124, 18 of 119, 14 of 131 proteins at six time points were all clustered into YWHAE protein-mediated pathway. Obviously, YWHAE protein was placed at the center of all the networks and modulated the diverse biological activities, such as cell division, apoptosis, cell differentiation, cell development and so on. According to pathway analysis, at the late phase of LR, besides YWHAE protein, CDC42 is considered as another important factor involved in the events occuring the termination of LR.
Validation by Western blot analysis
To testify the reliability of identification of differentially expressed proteins, six differential proteins in our experiments, including β-Actin, G6PD, AKT2, CDC42, YWHAE and APC, were picked out for Western blotting using available specific antibodies. Figure 5A showed the Western blot results of the six proteins. Figure 5B showed the gray values of Western blot bands obtained with BandScan 5.0 software. The results showed that G6PD was down-regulated in the PH group compared to the sham operation group, while AKT2, CDC42, YWHAE and APC were obviously up-regulated in the PH grou, which were generally consistent with the results of 2-DE experiments.