Partial Hepatectomy In Rats Research Articles

Effects of Organic Apricot on Liver Regeneration After Partial Hepatectomy in Rats

BackgroundThe present study investigated the effects of (sun-dried organic apricot/SDOA) supplementation in chow on liver regeneration after partial hepatectomy/(PH) in rats. MethodIn this study, 28 female rats were randomized into four groups. On the 7th day of the study, group 1 underwent laparoscopic intervention while a PH was performed on the other three cohorts. On day 28, all rats were humanely killed. Blood and liver tissue samples were subjected to biochemical determinations, histological examinations, and measurement of tissue oxidative stress enzyme activity. ResultsSerum levels of alanine transaminase (ALT), alkaline phosphatase (ALP), and liver tissue glutathione (GSH) activities were affected by PH and/or SDOA consumption (P < .05). Moderately staining cell counts in group 4 were significantly different from the other three groups (P < .05). However, no significant differences were detected among all groups in regard to aspartate aminotransferase (AST) serum levels or liver tissue superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) or glutathione peroxidase (GSHpx) activities (P < .05). ConclusionThe 5% SDOA supplementation over a 21-day feeding period showed a beneficial effect on liver regeneration in rats, as reflects by Ki-67 finding although there was no change in ALT or ALP or in liver tissue GSH activity.

Administration of rhIL-2 upregulates HGF in the cirrhotic liver of partial hepatectomized rats

Liver regeneration is a process involving highly organized and ordered tissue growth triggered by the loss of liver tissue. This process has remained a fascinating topic in medicine. In this study, we examined the effects of recombinant human interleukin-2 (rhIL-2) in rats under cirrhotic conditions. Thirty rats were divided into three groups, and liver cirrhosis was induced by injecting diethylnitrosamine for 2 weeks and CCl4 for 8 weeks. After induction of cirrhosis, rhIL-2 was injected daily into their tail vein for three days and then hepatocyte growth factor (HGF), immunocytes in the blood, and TNF-α production were analyzed. The analysis showed that TNF-α was significantly downregulated in the liver without significant changes in nitric oxide and caspase-3/7 activities in primary cultured liver cells. In terms of the number of immunocytes, there were no significant differences between the control and the treated groups. However, HGF levels were significantly higher in the treated groups than the control (saline administration) group. Treatment of rhIL-2 significantly increased the proliferation of primary cultured liver cells. The proliferation rates of primary cultured liver cells were correlated with the upregulation of HGF by treatment with rhIL-2 in vivo and in vitro. These results suggest that rhIL-2 affects liver regeneration, which is at least related to the upregulation of HGF under cirrhotic conditions in rats.

Bone Marrow-Derived Mesenchymal Stem Cells Promote Hepatic Regeneration after Partial Hepatectomy in Rats

Objectives: Our goal was to study the ability of mesenchymal stem cells (MSCs) to stimulate liver regeneration after partial hepatectomy in rats. Methods: MSCs were isolated from bone marrow and cultured in vitro. Their characteristics were analyzed by flow cytometry. After 70% partial hepatectomy, Sprague-Dawley rats were randomly divided into three groups: a control group that was injected with saline, animals that received bone marrow-derived MSCs (BM-MSCs) by tail vein injection (the BM-MSC-TV group) and animals that received BM-MSCs by portal vein injection (the BM-MSC-PV group). The injected BM-MSCs were traced by labeling with 4′,6-diamidino-2-phenylindole, and cell proliferations were determined by immunohistochemical staining with Ki-67 and 5-bromo-2′-deoxyuridine. Results: After the third passage, the cultured BM-MSCs had a fibroblast-like morphology and expressed high levels of stem cell markers CD29 and CD90. The levels of albumin rose significantly in the BM-MSC-TV and BM-MSC-PV groups compared with the control group. The number of 4′,6-diamidino-2-phenylindole-positive liver cells in the BM-MSC-PV group was significantly higher than in the BM-MSC-TV group. The levels of Ki-67 and 5-bromo-2′-deoxyuridine were significantly higher in the BM-MSC-TV and the BM-MSC-PV groups than in the controls. Conclusion: Taken together, these results indicate that BM-MSC injections enhance liver regeneration after partial hepatectomy in rats.

Effects of Ozone Oxidative Preconditioning on Liver Regeneration after Partial Hepatectomy in Rats

ABSTRACTAim: Similar protective effect of ischemic and ozone oxidative preconditioning (OzoneOP) in hepatic ischemia–reperfusion (I/R) injury was demonstrated, providing evidences that both preconditioning settings shared similar biochemical mechanisms of protection. We investigated the effects of OzoneOP on liver regeneration after 70% partial hepatectomy (PHx) in rats. Methods: Rats were divided into three groups: PHx, I/R + PHx, and OzoneOP + I/R + PHx groups. Ozone (intraperitoneal, 1.2 mg/kg) was given to rats subjected to I/R and 70% hepatectomy daily five times before operation. At 24 hr and 48 hr after resection, samples were collected for the measurement of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Moreover, liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling index, mitotic index, and histopathological examination were evaluated. Results: OzoneOP reduced liver injury determined by liver histology and serum transaminases. There was a rise in serum TNF-α and IL-6 levels in the I/R + PHx group whereas OzoneOP significantly decreased the rise in the level of TNF-α but not IL-6 on the 24 hr and 48 hr of reperfusion. Moreover, liver regeneration in OzoneOP + PHx group, as assessed by the regenerated liver weight, mitotic, and PCNA-labeling index, was significantly improved when compared to I/R + PHx group. Conclusion: These results suggest that OzoneOP ameliorates the hepatic injury associated with I/R and has a stimulatory effect on liver cell regeneration that may make it valuable as a hepatoprotective modality.

Effects of Urtica dioica on oxidative stress, proliferation and apoptosis after partial hepatectomy in rats

The present study was performed to investigate the effect of Urtica dioica (UD) on liver regeneration after partial hepatectomy (PH) in rats. A total of 24 male Sprague Dawley rats were divided into three groups: sham-operated, PH and PH+UD; each group contains eight animals. The rats in UD-treated groups were given UD oils (2ml/kg/day) once a day orally for 7days starting 3days prior to hepatectomy operation. At day 7 after resection, liver samples were collected. The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) were estimated in liver homogenates. Moreover, histopathological examination, mitotic index (MI), proliferating cell nuclear antigen labeling, proliferation index (PI), transferase-mediated deoxyuridine triphosphate nick end-labeling assay, apoptotic index (AI) were evaluated at day 7 after hepatectomy. As a result, UD significantly increased MI and PI, significantly decreased AI and also attenuated hepatic vacuolar degeneration and sinusoidal congestion in PH rats. UD treatment significantly decreased the elevated tissue MDA level and increased the reduced SOD activity and GSH level in the tissues. These results suggest that UD pretreatment was beneficial for rat liver regeneration after partial hepatectomy.

Lipocalin-2 gene expression during liver regeneration after partial hepatectomy in rats

BackgroundLipocalin-2 (Lcn2) is related to cell proliferation. We studied Lcn2 gene expression during liver regeneration after partial hepatectomy (PH). MethodsMale Wistar rats were sacrificed before and 2, 4, 6, 12, 24, 72 h, 7 days after 70% or 40% PH. The remnant liver weight/body weight (RLW/BW) ratio, Lcn2 gene and mRNA expression in the remnant livers were measured. Hepatocytes and nonparenchymal cells were isolated from the remnant livers. Expression of Lcn2 related protein was detected by Western blot. ResultsThe RLW/BW ratio increased to nearly 90% of the original liver 72 h after PH. Lcn2 gene expression showed upward curves from 4 to 72 h after PH in both 70% and 40% PH rats and peaked at 12 h (8 times vs 0 h). Lcn2 mRNA expression showed parallel upward curves from 2 to 72 h. The peak was significantly higher in 70% PH rats (27 times vs 0 h) than in 40% PH rats (25 times vs 0 h) 12 h after PH (p < 0.05). Lcn2 related protein in the isolated liver cells was markedly enhanced 24 h after PH, more in hepatocytes than in nonparenchymal cells. ConclusionThe expressions of Lcn2 gene and mRNA, and its related protein increased markedly after PH. Lcn2 might be important in the genetic regulation of liver regeneration after PH.

Antioxidative, antiapoptotic, and proliferative effect of curcumin on liver regeneration after partial hepatectomy in rats

The aim of the present study was to assess the influence of curcumin on liver regeneration after partial hepatectomy (PH) in rats. A total of 24 male Sprague Dawley rats were divided into three groups: sham-operated (SH), PH, and PH+curcumin; each group contains eight animals. The rats in curcumin-treated groups were given curcumin (in a dose of 100mg/kg body weight) once a day orally for 7days, starting 3days prior to hepatectomy operation. At 7days after resection, liver samples were collected. The malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were estimated in liver homogenates. Moreover, histopathological examination, mitotic index (MI), proliferating cell nuclear antigen labeling, proliferation index (PI), transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick end-labeling assay, and apoptotic index (AI) were evaluated at 7days after hepatectomy. As a result, curcumin significantly increased MI and PI and significantly decreased AI in PH rats. Additionally, curcumin remarkably inhibited MDA elevation, restored impaired antioxidant SOD activity and GSH level and also attenuated hepatic vacuolar degeneration and sinusoidal congestion. These results suggested that curcumin treatment had a beneficial effect on liver regenerative capacity of the remnant liver tissue after hepatectomy, probably due to its antioxidative, antiapoptotic, and proliferative properties.

Partial hepatectomy in rats results in immediate down-regulation of p27Kip1 in residual liver tissue by transcriptional and post-translational processes

Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h.Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1.Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH.Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.

C-kit expression in pancreas after partial hepatectomy in rats

C-kit expression in pancreas after partial hepatectomy in rats

The effects of anesthetic regimen in 90% hepatectomy in rats

To evaluate the influence of the anesthetic regimen on anesthetic recovery, survival, and blood glucose levels following a 90% partial hepatectomy in rats. Thirty adult male Wistar rats were divided into two groups according to their anesthetic regimens: intraperitoneal ketamine and xylazine or inhaled isoflurane. In order to prevent hypoglycemia, glucose was administered intraperitoneally and glucose (20%) was added to the drinking water. Anesthetic recovery time was longer in the ketamine and xylazine group. The survival rate after 72 hours was lower (log rank=0.0001) in the ketamine and xylazine group (0.0%) than in the isoflurane group (26.7%). The blood glucose after six hours was lower (p=0.017) in the ketamine and xylazine group (63 ± 31.7 mg/dL) than in the isoflurane group (98 ± 21.2 mg/dL). The prolonged anesthesia recovery time associated with ketamine and xylazine decreased the survival rate and blood glucose levels after 90% hepatectomy. Isoflurane anesthesia reduced the recovery time and incidence of hypoglycemia and increased the survival rate in the early hours, providing a therapeutic window that is suitable for experimental studies.

Human Placenta Extract Could Promote Proliferating Cell Nuclear Antigen Expression during Liver Regeneration Induced by Partial Hepatectomy in Rats

Human placenta extract (hPE) has therapeutic potential against certain diseases such as burn injury, liver cirrhosis and chronic wound through stimulating tissue repair processes. However, the effects of hPE on liver regeneration in animals are unknown. This study investigated the effect of hPE on the expression of proliferating cell nuclear antigen (PCNA) during liver regeneration induced by partial hepatectomy (PH) in rats. The activities of AST, ALT and ALP increased during a few days after PH. A high level of ALP was particularly seen at day 3 in the control group. All the levels of experimental groups were normalized by day 5 after PH. On immunohistochemistry, the expression of PCNA increased at the early days, showed a peak at day 3 after PH. The PCNA staining was more obvious in the experimental group over the whole period. By western blotting, PCNA seemed to be more strongly expressed in the hPE injected group in the early stage and fell to almost undetectable levels at day 7. On immunocytochemical observations, the number of PCNA-gold particles in the nuclei at day 1 of the hPE treated groups was more than those of the untreated groups. The results suggest that hPE could accelerate liver regeneration induced by PH involving the expression of PCNA in rats.

Gastric estrogen increases pituitary estrogen receptor α and prolactin mRNAs during the different pathological conditions of the liver

Mammalian liver is an estrogen-responsive tissue mediated by hepatic estrogen receptors. Although Ueyama et al. (Endocrinology 143:3162-3170, 2002) have reported the presence of aromatase and active production of gastric 17β-estradiol in parietal cells, there are a few studies on gastric 17β-estradiol exploring the relationship between gastro-hepato function and the gastro-pituitary-gonadal axis. The alteration of gastric 17β-estradiol flow into the systemic circulation by portal vein ligation (PVL) or partial hepatectomy (PH), and the effect of gastric 17β-estradiol on the pituitary function was investigated. In the PVL rats, arterial 17β-estradiol increased 9.5 times that of controls on day 3, and gradually decreased near to control levels in the portal vein by 4 weeks, which was still 5 times higher than those in the arteries of the control rats. In the PH rats, arterial 17β-estradiol increased 2 times that of controls on day 3, and gradually decreased to the control levels. Regeneration and growth of the liver remnants were observed about 2 weeks after PH. In the PVL and PH animals, pituitary ERα and prolactin mRNAs levels increased, positively correlating with an increase of arterial 17β-estradiol levels. Both reduced LHβ mRNA. It is apparent that hepatic dysfunction causes changes in gastric 17β-estradiol levels in the systemic circulation; and that elevated gastric 17β-estradiol affects pituitary function(s). This data suggest that gastric 17β-estradiol has a pivotal role in the regulation of the gastro-hepato-pituitary axis.

Sildenafil Accelerates Liver Regeneration after Partial Hepatectomy in Rats

ObjectiveThe regeneration process causes the liver to achieve an adequate volume and function after major hepatectomy or living donor liver transplantation. Sildenafil, a selective phosphodiesterase-5 inhibitor used for erectile dysfunction, impacts the liver by enhancing the effects of nitric oxide. The aim of this study was to investigate the influence of sildenafil on liver regeneration in rats after partial hepatectomy. MethodsSixty young female Wistar Albino rats were randomly divided into three equal groups before 70% hepatectomy. Thereafter, we administered intraperitoneal saline to the control group (G1); 10 μg/kg sildenafil to the low-dose group (G2) and 100 μg/kg to the high-dose sildenafil group (G3). Half of the rats per group were sacrificed on the first and the other half on the fifth postoperative day after partial hepatectomy. Regeneration was assessed using three methods: (1) the formula described by Kwon et al formula, (2) the average number of mitotic figures in 10 microscopic fields, and (3) the average of Ki-67–positive nuclei in 1000 cells using immunohistochemistry. ResultsAlthough, the hepatic regeneration and mitosis rates were similar in all three groups, Ki-67 levels were significantly higher in both G2 and G3 than the control group on the first postoperative day. Hepatic regeneration was significantly greater in G2 and G3 than the control group as was the mitosis rate in the G2 group versus the two groups. By the 5th postoperative day Ki-67 levels were similar in the three groups. ConclusionSildenafil treatment accelerated hepatic regeneration after partial hepatectomy in rats.

Effect of L-arginine supplement on liver regeneration after partial hepatectomy in rats

BackgroundNitric oxide (NO) has been reported to be a key mediator in hepatocyte proliferation during liver regeneration. NO is the oxidative metabolite of L-arginine, and is produced by a family of enzymes, collective termed nitric oxide synthase (NOS). Thus, administration of L-arginine might enhance liver regeneration after a hepatectomy. Another amino acid, L-glutamine, which plays an important role in catabolic states and is a crucial factor in various cellular and organ functions, is widely known to enhance liver regeneration experimentally. Thus, the present study was undertaken to evaluate the effects of an L-arginine supplement on liver regeneration, and to compared this with supplementation with L-glutamine and L-alanine (the latter as a negative control), using a rat partial hepatectomy model.MethodsBefore and after a 70% hepatectomy, rats received one of three amino acid solutions (L-arginine, L-glutamine, or L-alanine). The effects on liver regeneration of the administered solutions were examined by assessment of restituted liver mass, staining for proliferating cell nuclear antigen (PCNA), and total RNA and DNA content 24 and 72 hours after the operation.ResultsAt 72 hours after the hepatectomy, the restituted liver mass, the PCNA labeling index and the DNA quantity were all significantly higher in the L-arginine and L-glutamine groups than in the control. There were no significant differences in those parameters between the L-arginine and L-glutamine groups, nor were any significant differences found between the L-alanine group and the control.ConclusionOral supplements of L-arginine and L-glutamine enhanced liver regeneration after hepatectomy in rats, suggesting that an oral arginine supplement can clinically improve recovery after a major liver resection.

Serum LBP levels reflect the impaired synthetic capacity of the remnant liver after partial hepatectomy in rats

Lipopolysaccharide (LPS) binding protein (LBP) is up-regulated in inflammation and infection. Its use as a biomarker of severe infection and sepsis is currently discussed controversially. Here a novel LBP-ELISA was established to assess serum LBP-levels after various degree of in a rat partial hepatectomy (PH) model. The LBP enzyme-linked immunosorbent assay (ELISA) was designed based on the binding between LPS and LBP. LPS was employed as capture molecule. An anti-LBP antibody was used as detection antibody. Serum LBP levels were measured in serum obtained 24h after 30% PH, 70% PH and 90% PH in rats using newly established LBP ELISA method. Expression of hepatic LBP mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The detection range of the ELISA was from 0.1μg/ml to 60μg/ml. The correlation between ELISA and western blotting was strong (r=0.885, p<0.0001). Hepatic LBP mRNA expression was upregulated after PH. Of note, elevations of serum LBP protein levels were positively correlated to the remnant liver mass (R=0.821, p<0.0001), serum albumin levels (R=0.532, p<0.05) and total protein levels(R=0.813, p<0.0001). In conclusion, we established an economic and rapid ELISA assay for rat serum LBP quantification. Our results speak against using LBP as diagnostic marker of the acute phase response after liver resection as its elevation is related to the size and thereby the synthetic capacity of the small remnant liver.

Effects of human vascular endothelial growth factor 165 on liver regeneration in hepatocirrhosis after partial hepatectomy in mice

Objective To study the effects of exogenous vascular endothelial growth factor 165 (VEGF165) on liver regeneration and the recovery of liver function in hepatic cirrhosis after partial hepatectomy in rats.Methods The lentivirus vector containing human VEGF165 gene was constructed by using gene recombinant technology.CCl4-induced cirrhotic models in 150 rats were established.The 126 cirrhotic rats after hemihepatectomy were randomly divided into three groups:group A (VEGF165 group),group B (empty virus group) and group C (control group).The expression of VEGF165 mRNA and protein in liver tissue,liver regeneration rate,the positive rate of proliferating cell nuclear antigen ( PCNA),alanine transaminase (ALT) and aspartate aminotransferase (AST) contents in the blood of portal vein were detected before and 12 h,24 h,72 h,7 days,14 day,and 28 days after operation.Results The lentivirus vector construction was proved to be correct and could make the rats liver expressing VEGF165 mRNA and protein stably. The success rate of cirrhotic models in rats was 96.18％. The liver regeneration rate (34.98 ±6.22) ％,(62.19 ± 1.81 )％,(83.54 ±4.50) ％ and (92.23 ±4.41 ) ％ during 72 h to 28 days after operation and the positive rate of PCNA ( 18.05 ± 0.92 ) ％,(42.89 ± 5.52 ) ％,( 35.56 ±3.86) ％,( 26.37 ± 1.35 ) ％ and ( 19.48 ± 2.70) ％ during 24 h to 28 days after operation in VEGF165 group were obviously higher than those in the control group ( P ＜0.01 ).The serum concentrations of ALT (258.14± 12.94),(215.67 ±22.12),(175.92 ±8.78) and (133.26 ± 13.92) U/L and AST (553.34±14.10),(481.37 ±14.74),(425.95 ±5.04),(388.29 ± 15.99) U/L during 72 h to 28 days after operation in VEGF165 group were evidently lower than those in the control group ( P ＜ 0.01 ).Conclusion The exogenous VEGF165 has significantly positive effect on liver regeneration and the recovery of liver function after partial hepatectomy in rats with hepatic cirrhosis. Key words: Liver cirrhosis; Partial hepatectomy; Liver regeneration; Vascular endothelial growth factor 165 ; Lentiviral vector

Proliferative effect of aqueous extract of Hyptis fructicosa on liver regeneration after partial hepatectomy in rats

To evaluate the effect of aqueous extract of Hyptis fructicosa on hepatic regeneration after partial hepatectomy in rats. Sixteen rats were divided in two groups: C (Control Group) and HF (Whose rats received aqueous extract of Hyptis fructicosa during 4 days using the dose of 100 mg/kg/day). On the consecutive day of this treatment, the animals of both groups underwent hepatectomy of about 67% of liver. Twenty four hours later, they were sacrificed, and the remaining mass of liver was removed and prepared to be studied through the PCNA immunohistochemical technique. The liver regeneration index of HF group was 53.56 ± 18.91%, while in C group was 21.12 ± 8.29% (p=0.0003). These results show that the administration of aqueous extract of Hyptis fructicosa using the dose of 100mg/kg/day increased the hepatocyte proliferation in the group HF.

Intrasplenic transplantation of bioencapsulated mesenchymal stem cells improves the recovery rates of 90% partial hepatectomized rats.

Mesenchymal stem cells (MSCs) derived from bone marrow can secrete cytokines and growth factors and can transdifferentiate into liver cells. We transplanted polymeric membrane bioencapsulated MSCs into the spleens of 90% partial hepatectomized rats. This resulted in 91.6% recovery rates. This is compared to a recovery rate of 21.4% in the 90% hepatectomized rats and 25% in the 90% hepatectomized rats receiving intrasplenic transplantation of free MSCs. After 14 days, the remnant livers in the bioencapsulated MSCs group are not significantly different in weight when compared to the sham control group. From day 1 to day 3 after surgery, in the bioencapsulated MSCs group, the plasma HGF and IL-6 were significantly higher than those in the free MSCs group and control group (P < 0.01); plasma TNF-α was significantly lower (P < 0.001). We concluded that the intrasplenic transplantation of bioencapsulated MSCs significantly increases the recovery rates of 90% hepatectomized rats. It is likely that the initial effect is from proliver regeneration factors followed later by the transdifferentiated hepatocyte-like cells. However, histopathological analysis and hepatocyte proliferation study will be needed to better understand the regenerative mechanisms of this result. This study has implications in improving the survival and recovery of patients with very severe liver failure due to hepatitis, trauma, or extensive surgical resection.

Interplay of neuropilin-1 and semaphorin 3A after partial hepatectomy in rats

To elucidate the role of neuropilin-1 (Nrp-1) and semaphorin 3A (Sema3A) in sinusoidal remodeling during liver regeneration in rats. Male Wistar/ST rats at 7 wk of age, weighing about 200 g, were used for all animal experiments. In vivo, at 24, 48, 72, 96, 144 and 192 h after two-thirds partial hepatectomy (PHx), the remnant livers were removed. Liver tissues were immunohistochemically stained for Nrp-1, Sema3A and SE-1, a liver sinusoidal endothelial cell (SEC) marker. Total RNA of the liver tissue was extracted and reversely transcribed into cDNA. The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18. In vitro, SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor. Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A. Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method. In vitro, immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs, respectively, in normal rat liver tissues. Nrp-1 expression in SECs was quantified by the percentage of immunostained area with anti-Nrp-1 antibody in relation to the area stained with SE-1. Between 24 h and 96 h following resection of liver, Nrp-1 expression in SECs was transiently increased. Compared with the baseline (5.2% ± 0.1%), Nrp-1 expression in SECs significantly increased at 24 h (17.3% ± 0.7%, P < 0.05), 48 h (39.1% ± 0.6%, P < 0.01), 72 h (46.9% ± 4.5%, P < 0.01) and 96 h (29.9% ± 3.8%, P < 0.01) after PHx, then returned to the basal level at termination of liver regeneration. Interestingly, the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx. Sema3A mRNA expression was significantly reduced by about 75% over the period 24-144 h after PHx (P < 0.05), and returned to basal levels at 192 h after PHx. In vitro, SECs isolated from rats after PHx (PHx-SECs) were observed to migrate to the lower chamber of the cell transwell system after incubation for 24 h, but not cells from normal rats (CONT-SECs), indicating that mobility of PHx-SECs increases as compared with that of CONT-SECs. Moreover, recombinant Sema3A significantly attenuated migration in PHx-SECs in primary culture (vehicle-treated 100% ± 7.9% vs Sema3A-treated 42.6% ± 5.4%, P < 0.01), but not in CONT-SECs. Compared with CONT-SECs, the apoptotic rate of PHx-SECs decreased by 78.3% (P < 0.05). There was no difference in apoptosis between CONT-SECs that were treated with vehicle and Sema3A. However, in PHx-SECs, apoptosis was induced by the presence of 5 nmol Sema3A for 24 h (vehicle-treated 21.7% ± 7.6% vs Sema3A-treated 104.3% ± 8.9%, P < 0.05). In addition, immunohistochemistry confirmed the increased expression of Nrp-1 in PHx-SECs, while it was noted to a lesser extent in CONT-SECs. The interplay of Nrp-1 and Sema3A shown in our results may lead to a better understanding of interaction between sinusoidal remodeling and SECs during liver regeneration.

Threshold dose of liver tumor promoting effect of β-naphthoflavone in rats

To determine the threshold dose of β-Naphthoflavone (BNF) that induces hepatocellular tumor promoting effects, reactive oxygen species (ROS) generation and thiobarbituric acid-reactive substance (TBARS) formation, and drug-metabolizing enzymes that protect against ROS generation, two-stage liver carcinogenesis model was used. Partial hepatectomized rats (n = 11 to 12) were fed diets containing 0, 0.03, 0.06, 0.125 or 0.25% BNF for 6 weeks after an intraperitoneal injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, glutathione S-transferase placental form (GST-P)-positive foci significantly increased in rats given 0.25% BNF. No marked changes in ROS production and TBARS contents were observed between the BNF treated and DEN alone groups. Real-time RT-PCR showed that the expression of Cyp1a1, Cyp1a2, Cyp1b1 and Nqo1 significantly increased in the groups given 0.03% BNF or more, but Ugt1a6, Akr7a3 and Gstm1 significantly increased in the groups given 0.125% BNF or more. Gpx2 and Yc2 significantly increased in the groups given 0.06% BNF or more and 0.25% BNF, respectively. Inflammation-related genes such as Ccl2, Mmp12, Serpine1 and Cox-2 significantly increased in the 0.25% BNF group. In immunohistochemistry, the number of cyclooxygenase-2 (COX-2)-positive cells increased in rats given 0.25% BNF. These results suggest that 0.25% BNF is the threshold dose for liver tumor promotion, and the fact that inflammation-related genes and COX-2 protein increased in the 0.25% BNF group strongly suggests that inflammation is involved in the liver tumor promoting effect of BNF in rats.