| BACKGROUNDRegeneration of the liver is an important pathophysiological process after partial hepatectomy and living donor liver transplantation. It is extremely important that appropriate surgical approaches and postoperative treatment for the recovery of residual liver function, preventing small-for-size syndrome (SFSS), reducing complications and improving the liver regenerative capacity. However, it has been widespread during the liver surgery and postoperative therapy, both of which can cause disorder in bile acid metabolism. The most common reason is external biliary drainage (such as T-tube drainage) that can lead to the loss of bile acids and the reduction of bile acid pool size, and long-term parenteral nutrition which can result in the reduction of bile secretion and the disorder of bile acid enterohepatic circulation. All of these are able to cause the lack of intestinal bile acids and the decrease of total circulation of bile acid. Besides the roles of bile acids in dietary lipid absorption and cholesterol homeostasis, it has become clear that bile acids are also signaling molecules. Three major signaling mechanisms have been identified. Bile acids activate mitogen-activated protein kinase (MAPK) pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5, and activate nuclear hormone receptors such as farnesoid X receptor a (FXR, NR1H4), all of which have been found to play an important role in the regulation in cell proliferation, bile acid metabolism, lipid metabolism and energy metabolism, and liver regeneration must rely on their normal metabolism of bile acids, glucose, lipid and energy. Therefore, under the pathophysiology of the lack of bile acids in the intestine, the study of pathological process of liver regeneration has important theoretical and clinical significance for correctly understanding of the clinical application value of the external drainage.OBJECTIVE1. The bile acids overload can lead to liver cell apoptosis and necrosis. However, studies of the effect of the low-concentration bile acids on liver cell proliferation are still few. Therefore, the first study was to investigate the effect of the low-concentration cholic acid (CA) on rat primary hepatocyte cytotoxicity and DNA synthesis, and to explore the effect of it on JNK, p38-MAPK protein expression in vitro cytologic assay. 2. To define the effect of the lack of intestinal bile acids on liver regeneration in the animal model of both changing intestinal bile acids level and liver regeneration, and to observe the characteristics of bile acid metabolism, in particular the changes of serum bile acids level as well as the farnesoid X receptor (FXR) and its target genes cholesterol 7a-hydroxylase (CYP7A1) mRNA expression and the Na+-taurocholate cotransport protein (NTCP) expression in these models. Combining the course of liver regeneration with the feature of bile acids metabolism, the relationship between bile acids metabolism and liver regeneration was explored.3. To investigate the effect of intestinal bile acid on immediate-early gene c-jun and c-fos, and cyclin D1 expression during liver regeneration in the early period in rats, the mechanism of both liver regeneration and intestinal bile acids was investigated.METHODS1. Rat hepatocytes were cultured in primary culture for 48 hour, then they were incubated in medium containing 0,10,20,40,80μmol/L CA for 12 hours. The cell survival rate was determined by MTT assay. Liver cell proliferation index was counted by BrdU-labeling method. The cell cycles were detected by flow cytometry, and the relative percentage of S phase was calculated. The expression of JNK and p38-MAPK protein was detected by western blotting.2. Interference with intestinal bile acid metabolism in rats was established through feeding rats 0.2% cholic acid (cholic acid loading group),2% cholestyramine resin (lack of bile acids group) and feeding the standard diet as the control group. All rats were performed 70% partial hepatectomy (PH). Liver regeneration was compared among three groups at 0,1,2,3,7 d after PH in rats:calculation of restoration of liver mass, immunohistochemical examination of PCNA and Ki-67, serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum total bilirubin (TBIL), and total bile acids (TBA) were analyzed with an automatic multichannel analyzer. The mRNA expression of FXR and its target genes CYP7A1 was examined by Quantitative RT-PCR at 0,1,2,3,7 d after PH. The expression of NTCP protein was detected by western blotting at 1 d after PH.3. The mRNA expression of c-jun and c-fos was examined by Quantitative RT-PCR at 0, 0.5,1,3,6 h after PH. The expression of cyclin D1 protein was detected by western blotting at 6 h,12 h after PH.RESULTS 1. No significant difference in cell viability was found in 10,20μmol/L and 40μmol/L group, however,80μmol/L CA can make the liver cell survival rate drop to 66.7%, significantly lower than other groups (P< 0.01). With the increase of CA concentration, BrdU labeling index also increased. Liver cell proliferation was significantly higher in 20μmol/L,40μmol/L CA group (27.81±3.13,30.21±5.29 vs 18.35±4.88, P< 0.05, P< 0.01). BrdU labeling index in 80μmol/L CA group was significantly decreased (23.53±6.01), there was no significant difference with 0μmol/L CA(P> 0.05) The relative percentage of S phase cell was significantly higher in 20μmol/L,40μmol/L group than 0μmol/L group (40.49±7.23,47.81±9.14 vs 35.69±5.64, P< 0.05, P< 0.01), but it was significantly decreased in 80μmol/L group (30.56±4.99, P< 0.01). In addition, liver cell DNA synthesis showed a time-dependent in 40μmol/L group, increased more significantly between 6～12 hour (P< 0.01). The relative expression of JNK protein was significantly higher in 20μmol/L and 40μmol/L group than in 0μmol/L group (P< 0.05, P< 0.01), whilst it was significantly decreased in 80μmol/L group(P< 0.01), in which, however, p38-MAPK protein expression increased significantly (P< 0.01); there was no difference in p38-MAPK protein expression among the others.2. On the day 1 after PH, there was no significant difference in the levels of serum ALT and AST among three groups, and serum TBA and TBIL in the lack of bile acids group was significantly lower than the other two groups (P< 0.01, P< 0.05). The rate of liver regeneration was significantly lower on the day 3,7 after PH in the lack of bile acids group than the other groups (P< 0.05). At day 1, the labeling indices of PCNA and Ki-67 in the lack of bile acids group(22.21±2.31%,17.25±6.50%)was lower than the cholic acid loading group (44.4±4.92%,30.83±3.91%) and control group (38.74±6.42%,27.04±7.22%) and the peaking of labeling indices was delayed. On the day 1, CYP7A1 mRNA expression was as follows:the cholic acid loading group< the normal group< the lack of bile acids group (P< 0.05), but FXR mRNA expression was just the opposite. After PH, the mRNA expression of FXR was significantly lower than the other groups, however, CYP7A1 mRNA had a trend towards increase after PH and was higher than the other groups. The protein expression of NTCP was highest in the lack of bile acids group, followed by the control group and the cholic acid loading group (P<0.05).3. At 1 hour after PH, the c-jun expression was significantly higher in the cholic acid loading group than the control group (P< 0.01), and it was significantly lower in the lack of bile acids group than that in control group (P< 0.01). At 3,6 hour, the c-jun mRNA remained at a high level in the cholic acid loading group, significantly higher than the other two groups, and it was decreased obviously in the others. In terms of c-fos expression, it was significantly different at only 3 hour after PH, and was as followed: the cholic acid loading group> the control group> in the lack of bile acids group. At 6 hour after PH, the protein expression of cyclin D1 was as follows:the cholic acid loading group> the normal group> the lack of bile acids group (P< 0.05). At 12 hour after PH, the protein expression of cyclin D1 was no significant difference, but significantly lower than the cholic acid loading group (P< 0.05)CONCLUSION1. There was no significant difference in survival rate below the concentration of 40μmol/L CA; however, the relative percentage of S phase and JNK protein expression were significantly increased by 40μmol/L CA.80μmol/L CA significantly decreased survival rate, relative percentage of S phase, and JNK expression but significantly increased p38-MAPK protein expression. Therefore, low-concentrations CA could promote liver cell DNA synthesis, which might be caused by an increase of the JNK protein expression.2. Compared with the standard feed,0.2% cholic acid feed could effectively reduce the expression of CYP7A1 mRNA, and 2% colestyramine feed could validly enhance the expression of CYP7A1 mRNA in liver tissue without substantial liver toxicity, which indirectly reflected that these doses of cholic acid and colestyramine were able to change the intestinal bile acids content effectively.3. The lack of intestinal bile results in delayed liver regeneration of normal rat liver after hepatectomy accompanied by decreased serum total bile acids levels, decreased expression of FXR mRNA and increased expression of CYP7A1 mRNA. The mechanism might be a fact that the lack of bile acids in the body made the expression of FXR reduced, and then made FXR to lost the inhibited effect on CYP7A1 expression, or reactive regulation in the absence of bile acids in the body during liver regeneration. We proposed that liver regeneration was required in order to generate an appropriate state of bile acid pool and the associated expression level of bile acid receptor FXR.4. Transcriptional activation of immediate-early genes c-fos,c-jun and cyclin D1 have been considered to play an important role in regulating cell cycle. The lack of intestinal bile acids could significantly decrease the expression of c-jun and c-fos mRNA after partial hepatectomy, and could reduce the expression of cyclin D1 protein.
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