Preoperative portal vein embolization (PVE) has become a standard procedure before extended hepatectomy for hepatoma (1), metastatic colorectal cancer (2), and biliary cancer (3). After PVE, the nonembolized lobe enlarges and ensures the safety of a major hepatectomy. Although the mechanism of hepatic regeneration after PVE is similar to that after a hepatectomy, the details are still unknown.
Clinical PVE is simulated by an experimental model, which is called portal branch ligation (PBL); this procedure consists of ligating the branch of the portal vein that feeds approximately 70% of the liver volume. Our previous study has shown that estrogen promotes hepatic regeneration after PBL (4). In male rats, the serum estrogen levels were rapidly increased after PBL. Furthermore, chronic inhibition of the estrogen receptor by antagonists revealed a significantly lower regeneration rate and less activation of liver regeneration-related genes in the nonligated lobe as compared with vehicle treatment. These results indicated that estrogen plays an important role in the process of liver regeneration after PBL. However, in female rats, for which estrogen may play a more important role in regulating homeostasis than in male rats, the role of estrogen in the process of liver regeneration after PBL has not yet been determined.
Recently, platelet-derived serotonin has emerged as a novel important promoter of liver regeneration (5). In a mouse model of liver regeneration, thrombocytopenia or impaired platelet activity resulted in a failure to initiate cellular proliferation in the liver. Antagonists of serotonin receptors or depletion of tryptophan hydroxylase 1 (TH-1; the rate-limiting enzyme for the synthesis of peripheral serotonin) also inhibited liver regeneration. The main peripheral source of serotonin is the intestinal tract (6) upstream of the liver with respect to mesenteric blood flow. Therefore, the intestinal tract may play an important role in the regulation of liver regeneration. Moreover, a number of articles have described the modulation of serotonin function by estrogen in the brain (7, 8) and other organs (9, 10). Therefore, it could be hypothesized that estrogen may promote liver regeneration through the activation of the serotonin system in which the intestinal tract may play some regulatory role.
Therefore, the aim of this study was to determine whether estrogen plays any role in the process of liver regeneration after PBL using female ovariectomized rats with or without estrogen pellet implantation. The effect of estrogen on the activation of the serotonin system during liver regeneration was also investigated.
MATERIALS AND METHODS
An estrogen pellet (0.5 mg) was purchased from Innovative Research of America (Sarasota, Fla). All other chemicals, including ketanserin, a serotonin receptor antagonist, were purchased from Sigma (St. Louis, Mo).
Measurement of serum estradiol levels
Serum estradiol levels were measured using commercially available enzyme-linked immunosorbent assay kits (Cayman Chemical Company, Ann Arbor, Mich). The assay was performed as recommended by the manufacturer's instruction.
Animal and surgical procedure (ovariectomy and PBL)
Female Wister rats (280-320 g) were purchased from SLC (Tokyo, Japan). The animals were kept in a temperature- and humidity-controlled environment in a 12-h light-dark cycle and allowed free access to water and food at all times. All rat experiments were approved by the university committee on animal research and received humane care in accordance with National Institutes of Health publication 86-23, the "Guide for the Care and Use of Laboratory Animals." All rats were ovariectomized 2 weeks before PBL. For PBL, the rats were anesthetized with an injection of pentobarbital sodium (50 mg·kg−1, i.p.), after which the abdomen was opened by a subcostal incision. The branch of the portal vein that feeds the left lateral and median lobes (equivalent to 70% of the liver) was carefully dissected without producing any injury to the hepatic artery or the bile duct. A 7-0 poly (hexafluoropropylene-vinylidene fluoride) suture (PRONOVA; Ethicon, Cincinnati, Ohio) was placed around the portal branch. A suture knot was made without ligation, and both ends of the suture were passed out from the peritoneal cavity through both sides of the flank. Subsequently, the peritoneal cavity was closed layer to layer by continuous suture. Under light anesthesia, the portal branch can be ligated by pulling both ends of the suture after 1 week, when the effects of laparotomy disappeared. This procedure was described in our previous study as "non-stress PBL model," in which the effects of laparotomy were minimized and more accurately reproduce clinical PVE (11, 12). On days 2 and 7 after PBL or a sham operation (only laparotomy), the animals were killed, and sampling was performed. Upon killing, the ligation of the portal vein was verified. If it was incomplete, the animal was omitted from the study.
Determination of tissue mRNA expression by comparative quantitative real-time reverse-transcriptase-polymerase chain reaction
The mRNA levels of IL-6, TNF-α, hepatocyte growth factor (HGF), c-fos, c-jun, c-myc, and serotonin receptors H2A (5-HT2A) and H2B (5-HT2B) in the liver were determined by comparative quantitative real-time polymerase chain reaction (PCR) using the Mx3000P real-time PCR system (Stratagene, La, Jolla, Calif). The mRNA levels of TH-1, a rate-limiting enzyme in serotonin biosynthesis, was also determined in the small intestine. Total RNA was isolated from the liver and small intestine using the Qiagen RNeasy mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. cDNA was generated from the total RNA samples using a SuperScript III reverse-trascriptase reagent (Invitrogen, Carlsbag, Calif). Each reaction was performed in a 20-μL reaction mixture containing cDNA, 2× PCR Master Mix (Applied Biosystems, Foster City, Calif), and each probe and primer set. TaqMan gene expression assays (Applied Biosystems) for IL-6, TNF-α, HGF, c-fos, c-jun, c-myc, 5-HT2A, 5-H2B, TH-1, and 18S rRNA (endogenous control) were purchased as a probe and primer set (IL-6, Rn00561420_m1; TNF-α, Rn01525860_g1; HGF, Rn00566673_m1; c-fos, Rn00582193_m1; c-jun, Rn00572991_s1; c-myc, Rn00561507_m1; 5-HT2A, Rn00568473_m1; 5-HT2B, Rn00568450_m1; TH-1, Rn01476862_m1; and 18S rRNA, Hs99999901_s1). The reaction mixture was denatured for one cycle of 2 min at 50°C, 10 min at 95°C, and incubated for 40 cycles (denaturing for 15 s at 95°C and annealing and extending for 1 min at 60°C). All samples were tested in duplicate, and average values were used for quantification. Analysis was performed using MxPro software version 2.00 (Stratagene) according to the manufacturer's instructions. The comparative cycle threshold method (ΔΔCT) was used for quantification of gene expression. The average of the sham group was set to one, and other data were adjusted to that baseline.
Measurement of the hepatic blood flow
The procedure for the measurement of hepatic blood flow was previously described by Yokoyama et al. (13). Briefly, the rats were anesthetized with an injection of pentobarbital sodium (50 mg·kg−1, i.p.). The right carotid artery was cannulated using a PE-50 catheter, and it was advanced to the left ventricle. The left femoral artery was also cannulated with a PE-50 catheter for the measurement of MAP, heart rate, and blood sampling during the fluorescent microsphere injection study. The reference blood sample was withdrawn from the femoral artery for 60 s at a rate of 1.0 mL·min−1. Ten seconds after the beginning of blood withdrawal approximately 75,000 fluorescent microspheres (15 ± 3 μm in diameter) were injected into the left ventricle at a rate of 20 μL·s−1 for 20 s. The spleen, stomach, small intestine, large intestine, and liver were harvested carefully. After digesting the tissues with 5 to 7 mL of 2.3 M ethanolic KOH with 0.5% Tween-80, the microspheres were recovered by sedimentation as described previously (13). Finally, 3 mL of ethoxyethyl acetate was added to the pellet to dissolve the fluorescent microspheres. All blood and tissue samples were centrifuged at 2,000 × g for 20 min, and the supernatant was measured on the same day using a CytoFluor (Applied Biosystems).
Flow to each organ was calculated by the following equation:
where Qorg is the blood flow rate of the sample, FLorg is the fluorescence reading of the sampled organ, FLref is the fluorescence reading of the reference blood sample, and R is the withdrawal rate of the reference blood flow sample. The hepatic arterial flow rate was calculated from samples of the liver. Portal venous inflow was calculated from the sum of arterial blood flow to the stomach, small intestine, large intestine, and spleen as described by Vorobioff et al. (14). The cardiac output (CO), cardiac index (CI), and total peripheral resistance (TPR) were calculated by the following equation:
where FLinj is the fluorescence reading of the injected suspension.
There were 5 to 12 animals in each group. The results were presented as mean ± SE. One-way ANOVA, followed by the Student-Newman-Keuls test for multiple comparisons, was used to determine the significant differences among the experimental groups. When criteria for parametric testing were violated, the appropriate nonparametric (Mann-Whitney U test) test was used. Student t test was used to compare two groups. A P value less than 0.05 was considered to indicate a significant difference.
Serum estradiol levels
The serum estradiol levels in the ovariectomized rats with an estrogen pellet implantation (E group) were significantly higher than those without an estrogen pellet (non-E group; Fig. 1). The serum estradiol levels in the non-E group are compatible with those in male or estrus female rats. In contrast, the levels of serum estradiol in the E group are compatible with those in proestrus female rats (4, 15).
The effect of estrogen on the volume change of the liver after PBL
We examined the effects of estrogen on the volume change of the liver after PBL. An estrogen pellet (E group) or a vehicle pellet (non-E group) was implanted while performing the ovariectomy. The pellet releases estrogen continuously and maintains high serum estrogen levels in ovariectomized rats, whereas serum estrogen was almost undetectable in the ovariectomized with vehicle pellets (data not shown). Two weeks later, the rats were subjected to PBL. The weights of the regenerating lobes (nonligated lobe = right and caudate lobes) and atrophying lobes (ligated lobe = left and middle lobes) were measured on days 1, 2, and 7 after PBL, re spectively. The weight of each lobe was expressed as a proportion of the body weight (Fig. 2). Although there was no change in the total body weight between the E and non-E groups, the weight of the nonligated lobe per body weight on days 1, 2, and 7 after PBL was significantly greater in the E group as compared with the non-E group (Fig. 2A). There was no difference in the volume of the ligated lobe among these groups (Fig. 2B).
Real-time reverse-transcriptase-PCR for hepatic regeneration-related factors after PBL
We next examined the activation of liver regeneration-associated factors in the nonligated lobe for both the E and non-E groups. The expression of hepatic regeneration-associated factors in the nonligated lobe after PBL was detected by real-time reverse-transcriptase (RT)-PCR, and it was compared with the sham operation (ovariectomy without PBL). The factors examined were IL-6 and TNF-α as triggering factors for liver regeneration (16); HGF as a representative growth factor for liver regeneration (17); and c-fos, c-jun, and c-myc as immediate early genes (18). All of these factors, except for c-jun, were significantly up-regulated on day 1 after PBL as compared with sham operation controls (Fig. 3). These changes were further enhanced by estrogen administration (in the E group). These results indicated that administration of estrogen promotes liver regeneration and is accompanied with an up-regulation of liver regeneration-related genes in the liver.
The expression of serotonin receptors in the liver
In a previous study, the up-regulation of serotonergic receptor subtype 5-H2A and 2B receptors was observed at 2 days after hepatectomy (5). These data suggest that the type 2A and 2B receptors contribute to promote liver regeneration Therefore, we next tested whether an administration of estrogen alters the gene expression of serotonin receptors 5-HT2A and 5-HT2B in the liver by real-time RT-PCR. Although the expression of 5-HT2B did not show any change after PBL, the expression of 5-HT2A was increased after PBL, and the level of increase was significantly higher in the E group as compared with the non-E group (Fig. 4). We further found that the gene expression of the major serotonin synthetic enzyme TH-1 was significantly up-regulated in the small intestine in the E group (Fig. 5). These results implied that under the condition of PBL, estrogen not only enhances the activation of the serotonin system in the liver but also increases the production of serotonin in the small intestine, which is a major source of serotonin and upstream of the liver in terms of the portal venous flow.
Measurement of hepatic blood flow
We hypothesized that the higher regeneration rate and enhanced expression of liver regeneration-associated factors in the E group are due to the higher blood flow rate to the liver. To test this hypothesis, we measured the organ blood flow rate using fluorescent microsphere technique for the rats on day 7 after PBL. Systemic hemodynamics also was monitored during the experiments (Table 1). There was no significant difference between the E group and non-E group with respect to systemic hemodynamic parameters such as CO, CI, MAP, and total peripheral resistance. Interestingly, total intestinal flow, portal venous flow, and hepatic arterial flow were all significantly higher in the E group compared with the non-E group (Fig. 6).
The effect of serotonin receptor blockade on liver regeneration
Finally, we tested the effect of serotonin receptor antagonists (ketanserin) on liver regeneration in PBL rats with estrogen treatment. Because we observed an up-regulation of 5-HT2A receptor gene expression in the E group after PBL, we used ketanserin (a subtype-specific antagonist for 5-HT2A) in the antagonist study. The dose of ketanserin (6 mg kg−1, i.p., on days 0, 3, and 6 after PBL) was determined according to the previously published study (5).
As expected, the enhanced regeneration rate of the nonligated lobe in the E group was significantly attenuated by using serotonin receptor antagonists (Fig. 7). Therefore, we concluded that the effect of estrogen on liver regeneration was at least partially mediated by an activation of the serotonin system in the liver after PBL.
Portal vein embolization is widely used before a major hepatectomy to induce hypertrophy of the future liver remnant and to reduce the risks of a major hepatectomy (3, 19). Several reports have analyzed the factors that affect the outcome of PVE in human clinical studies (20, 21). A clinical study analyzing 84 consecutive PVE patients revealed that the extent of hypertrophy in the nonembolized lobe was significantly greater in female patients (22). Moreover, our recent study analyzing 88 PVE patients demonstrated a gender dimorphism in the outcome of PVE (23). These results indicated that females are better positioned for a good outcome. However, most of the female patients involved in these studies were postmenopausal, and whether estrogen, a representative female sex hormone, is actually necessary to promote liver regeneration in these patients is still unknown. However, it is almost impossible to test this hypothesis in human subjects. The pathophysiology of PVE has been extensively studied using the rat PBL model (24). Therefore, in this study, we used female ovariectomized rats with or without estrogen supplementation to determine the exact role of estrogen in the process of liver regeneration after PVE.
Several previous studies elucidated the mechanisms of estrogen in promoting liver regeneration (4, 25). Serum concentrations of estrogen were elevated after major hepatectomy both in animals (26) and in humans (27). Nuclear estrogen binding 48 h after 70% partial hepatectomy was elevated, although no alteration in affinity of the receptor for estrogen has been observed (25). In our previous study using male rats, the administration of estrogen receptor antagonists after PBL significantly attenuated the liver regeneration rate (4). This was associated with attenuated expression of liver regeneration-related genes, indicating that estrogen plays an important role in promoting liver regeneration. In addition to estrogen, a recent study has clearly shown that serotonin, another multipotent hormone that regulates homeostasis, is involved in the process of liver regeneration (5).
The mRNA expression of serotonin receptor subtype 5-HT2A increases after partial hepatectomy, and antagonists of these receptors inhibited liver regeneration. Because estrogen is known to modulate serotonin action in the brain (7, 28) and the intestine (10), we further hypothesized that the promotion of liver regeneration by estrogen is partly mediated by serotonin activation. As expected, administration of estrogen to the ovariectomized female rats significantly improved the liver regeneration rate as compared with nonestrogen administration. Furthermore, these effects were correlated with increased serotonin receptor (5-H2A) expression in the liver. In the peripheral system, serotonin is mainly produced and stored in the intestine (29). We also found that the rate-limiting enzyme of serotonin synthesis (TH-1) (30) was significantly up-regulated in the intestine in the estrogen-treated group. Although we did not measure the circulating serotonin levels in the blood, these results indicated that estrogen administration leads to enhanced liver regeneration after PBL at least partly through an activated serotonin system in the liver.
Our results clearly showed increased serotonin receptor expression in the liver after PBL, especially with estrogen supplementation. It was associated with a greater expression of liver regeneration-related genes. However, these are only indirect observations, and we have never determined whether the serotonin system is really activated in the regenerating liver. Moreover, the levels of serotonin in the blood (mostly stored in platelets) (31) or the amount of serotonin storage in the gastrointestinal tract (the major source of serotonin in the body) (29) have not been evaluated. These are issues that should be addressed in future studies. It should be noted, however, that our in vivo study using serotonin receptor antagonists (ketanserin) in estrogen-treated rats clearly inhibited estrogen-mediated hepatoproliferative effect on the nonligated lobe. This result implied an important role of the estrogen-serotonin interaction in the process of liver regeneration after PBL.
To the best of our knowledge, this is the first report that showed a hepatoproliferative effect of estrogen in female animals without ovaries. In the clinical setting, most of the women who need to undergo PVE before hepatectomy are postmenopausal because these procedures are used for hepatoma (1), metastatic colorectal cancer (2), and biliary cancer (3) (most of these patients are older than 50 years). The results of our study indicated that estrogen supplementation in postmenopausal women could promote liver regeneration after PVE. However, further evidence is needed to clarify the importance of estrogen therapy in postmenopausal women who are going to undergo PVE.
In summary, this study demonstrated that estrogen plays an important role in the process of liver regeneration after PBL in female rats. Enhanced expression of the serotonin receptor (5-HT2A) in the regenerating liver was also observed after administering estrogen to ovariectomized female rats. Moreover, a specific serotonin receptor antagonist (ketanserin) alleviated estrogen-mediated hepatoproliferative effect. These results indicate that estrogen promotes hepatic regeneration at least in part through activation of the serotonin system.
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Liver; regeneration; portal vein embolization; ovariectomy; estrogen pellet; serotonin receptor; sex difference; hepatic regeneration; portal branch ligation; ovariectomized rat