Effect of sorafenib on liver regeneration after partial hepatoectomy in liver cirrhotic rats

AIM: To study the effect of sorafenib on the liver regeneration after partial hepatectomy (PH) in cirrhotic rats. METHODS: Thirty Wistar rats with liver cirrhosis induced successfully with diethylnitrosamine (DEN) underwent 30% PH and then were randomly divided into 2 groups (n=15). The rats in experimental group were fed with sorafenib at dose of 30 mg·kg^-1·d^-1 from the 1st day to the 10th day after PH, while those in control group were fed with vehicle by gavage. The blood and liver tissues of the rats were collected after PH and at the end of the experiment. Liver regeneration rate (LRR) and proliferating cell nuclear antigen (PCNA) expression were assessed for determining the hepatocyte proliferation. The content of alanine transaminase (ALT), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), angiogenesis related factors including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β) and micro-vessel density (MVD) were measured in both groups. RESULTS: LRRs on day 10 after PH were 45.43%±3.36% and 44.21%±2.77% in experimental group and control group, respectively (P>0.05), and the expression of PCNA in hepatic tissues of the rats was not found by the method of immunohistochemistry in both groups. Liver function index had no significant difference between the 2 groups (P>0.05). However, other than VEGF, sorafenib resulted in inhibition of VEGFR-2 and PDGFR-β expression and reduction of MVD in experiment group, and significant difference between the 2 groups was observed (P<0.01). CONCLUSION: Sorafenib does not influence live regeneration after PH in liver cirrhotic rats.     

A review of augmenter of liver regeneration

Augmenter of liver regeneration (ALR) is a novel hepatic stimulator. Owing to its peculiar characteristic of gene structure and many kinds of biological functions, it is thought to be an important hepatotrophic factor. This review summarized the advance in ALR research in recent years including characteristics of ALR gene, molecular structure, physiochemical characteristics, biological functions and roles in liver injury.     

Molecular mechanisms of liver regeneration following cold ischemia injury after liver transplantation in rat

AIM: To study the molecular mechanisms of liver regeneration following different cold ischemia (CI) times after liver transplantation in a rat model. METHODS: A model of rat orthotopic liver transplantation was established. The rats were divided into 3 groups: 1 h CI group, 8 h CI group and 16 h CI group. Survival rate in each group was recorded. Specimen were collected at predetermined intervals from 90 min, 1, 4 and 7 d post-reperfusion. The patterns of TNF-α, IL-6 and STAT3 activation were determined in liver grafts with 1 h, 8 h and 16 h CI times. Expression of cyclin D1 and hepatocyte replication with bromodeoxyuridine (BrdU) were confirmed by immunohistochemistry. The results of TNF-α and IL-6 expression in all groups were analyzed after whole liver transplantation. Statistical analysis was used to compare BrdU positively stained hepatocytes at 48 h post-reperfusion. RESULTS: Liver transplantation was successfully performed in all experimental groups. Survival rate in each group was 100% (>14 d). Compared with 1 h CI, TNF-α expressions in whole liver grafts with 8 h and 16 h CI were markedly increased at 90 min after reperfusion(P<0.05). Compared with 1 and 8 h CI, IL-6 expression in liver grafts preserved for 16 h were markedly increased at 90 min after transplantation (P<0.05). With 8 and 16 h CI, STAT3 activity was markedly increased. Cyclin D1 expression in 8 CI group was demonstrated with cytoplasmic and nuclear staining at 24 h in liver grafts. Cyclin D1 expression was mainly nuclear in 16 h CI group. Extensive hepatocyte replication was present. The numbers of hepatocytes with positively stained nuclei in 16 h CI group were more than those in 1 and 8 h CI group at 48 h after transplantation(P<0.05). CONCLUSION: Rat whole liver grafts with 16 h CI injury still initiate and complete liver regeneration and graft recovery after liver transplantation. Liver regeneration following transplantation may be through TNF-α/IL-6/STAT3/cyclin D1/DNA synthesis pathways.     

Mechanisms of augmenter of liver regeneration in promoting damaged hepatocyte proliferation

AIM: To investigate the mechanisms of augmenter of liver regeneration (ALR) in promoting damaged hepatocyte proliferation.METHODS: The effects of Kupffer cell condition medium (KCCM+) stimulated by ALR on damaged hepatocyte proliferation were studied by MTT. The localization of ALR binding to Kupffer cell membrane and in intact rat liver was studied by immunohistochemistry. The IL-6 expression in Kupffer cells stimulated with ALR was observed by immunohistochemistry. RESULTS: The proliferation of damaged hepatocytes stimulated with KCCM+ was increased significantly. ALR immunostaining particles in plasm of hepatocyte were found in intact liver. The rough immunostaining particles of ALR were seen on the surface of Kupffer cell membrane. Immunostaining particles of IL-6 in Kupffer cells induced by ALR increased. CONCLUSION: ALR promotes proliferation of damaged hepatocytes indirectly by stimulating Kupffer cells.     

Intrathymic inoculation of liver specific antigen protects hepatocytes from apoptosis after liver allotransplantation

AIM: To study the effects of intrathymic inoculation of liver specific antigen (LSA) on hepatocyte apoptosis after liver allotransplantation. METHODS: Orthotopic liver transplantation was used in this study. Group Ⅰ: syngenic control (Wistar-to-Wistar); Group Ⅱ: acute rejection (SD-to-Wistar); Group Ⅲ: thymus inoculation of SD rat LSA day 7 before transplantation. The observation of general situation and survival time, hepatocyte apoptosis and LAT expression in liver transplants were used to analyze immune state of animals in different groups. RESULTS: The general situation of group Ⅰ was very well after transplantation. Recipients of groupⅡ lost body weight progressively and all died within day 9 to day 13 post transplantation. As for group Ⅲ, the general situation of recipients was remarkably better than that in group Ⅱ. The positive cells of apoptosis in group Ⅲ detected by TUNEL were not significantly different from that in group Ⅰ, but was significantly lower than that in group Ⅱ. LAT was detected at any time in group Ⅱ with peak expression at day 5 and day 7 post transplantation. In contrast, LAT was not detected in any other groups. CONCLUSION: Intrathymic inoculation of LSA protects hepatocytes from apoptosis after liver allotransplantation.     

Changes of mitochondrial peripheral-type benzodiazepine receptor during rat live regeneration

AIM: To investigate the expression profile of peripheral-type benzodiazepine receptor (PBR) involved in mitochondrial permeability transition (PT) regulation, and to observe the binding dynamic of the mitochondrial PBR with specificity ligand during rat live regeneration. METHODS: Liver regeneration model was produced by 70% partial hepatectomy (PH) performed in male SD rats. The animals of sham groups underwent the same surgical operations as PH groups did, but the liver lobes were not resected. The animals in the PH groups and corresponding sham groups were sacrificed at 3, 6, 12, 24, 48, 72, 120 and 168 hours after the operation. The livers were removed, weighted and processed for isolation of mitochondria. Semi-quantitative RT-PCR was performed to examine the expression level of PBR in 70% hepatectomized rat livers during the whole regeneration process and compared to that in the sham and normal groups. Compared with healthy rats, the kinetic parameters of PBR was evaluated by using a specific radioligand ［3H］-PK11195. RESULTS: Compared with healthy rats, the expression of PBR was unchanged. Meanwhile, the results obtained in the present experiments by scatchard analysis, Bmax of PK11195 for PBR significantly decreased, returned to normal level in 168 h after PH. Kd of PK11195 for PBR significantly decreased at 72 h and 168 h after PH of rat liver regeneration (P<0.01). CONCLUSION: The mRNA expression and evaluation of kinetic parameters of PBR may be related to the time-phase change of mitochondrial PT during rat liver regeneration after partial hepatectomy.     

Effects of changing PKC activity on proliferation and telomerase expression in human hepatocellular carcinoma cells

AIM: To investigate whether protein kinase C (PKC) is involved in the proliferation and the telomerase expression in human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (BEL-7402) were treated with exogenous phorbol-12-myristate-13-acetate (PMA, PKC activator) and staurosporine (SP, PKC inhibitor) for 48 hours. The techniques of cell culture and the telomeric repeat amplification protocol silver staining in combination with computer image scanning system in vitro were used to observe the variations of the growth and the telomerase expression. RESULTS: The proliferative potential of BEL-7402 cells was decreased by the action of PMA as well as SP, and the telomerase expression was also inhibited by PMA and SP. CONCLUSION: Our findings suggest that the proliferation of human hepatocellular carcinoma cells and the telomerase expression may be related to PKC.     

Effect of mutant β-catenin gene on the proliferation of hepatocytes

AIM:The β-catenin is a key molecule in the Wnt signal pathway, which plays a critical role in normal development and tumorigenesis. However, the mechanisms of the β-catenin on the cell growth control are still not completely defined. The aim of this study was to test the hypothesis that the mutant β-catenin may regulate the hepatocyte proliferation. METHODS: The immortalized murine hepatocyte cell line, AML12, was used for this study. A plasmid that contain mutant β-catenin S33Y was transfected into the AML12 cells and a stable cell line AML12S33Y was established. The cell growth property of this cell line and the parental cell were compared by flow cytometry analysis and direct cell count. The cells were also tested for the ability to form soft agar colonies, and the ability to form tumors in the severe immune deficient mice (SCID). RESULTS:1. The mutant β-catenin containing cell line AML12S33Y has higher proliferating index compared with the parental AML12 cells (P<0.01), suggesting that mutant β-catenin promotes cell growth. 2. The mutant β-catenin cells formed small colonies in soft agar after 4 weeks of culture, but did not generate tumor in SCID mice. CONCLUSION:The mutant β-catenin promotes liver cell growth.     

Tissue irradiation injury and past-transplantation distributing diversity of Sca-1 positive cells from murine fetal liver

AIM: To study the relationship between tissue irradiation injury and past-transplantation distributing diversity of Sca-1 positive cells from murine fetal liver and their offspring cells in multi-organs after syngeneic sex mismatch hematopoietic remodeling.METHODS: The Sca-1 positive cells from the livers of C57BL/ 6j male mouse fetus aged 14.5 day were identified and separated by quick PCR and magnetic cell sorting (MACS) techniques. In order to reconstruct hematopoiesis of the adult female mice, which were lethally irradiated with 8G of ［Co⁶⁰］, the 2×10⁴ of Sca-1+ cells were transplanted through caudal vein into each of them. After 6 months, these recipient mice were sacrificed, and their kidneys, livers, lungs, stomachs, and small intestines were taken out, fixed and slices were made. Fluorescence in situ hybridization was performed and observed by fluorescent microscope. Images were captured and analyzed through appropriative cool CCD and software. RESULTS: After 2×10⁴ of Sca-1+ cells were transfused, the hematopoietic function in the lethally irradiative female mice was completely restored. The cells containing Y chromosome were observed 6 months latter in multi-organs, including kidney, liver, lung, stomach, and small intestine. The frequency of the cells containing Y chromosome respectively was kidney (1.65±0.18)%, liver (0.90±0.10)%, lung (1.90±0.60)%, stomach (6.10±0.50)%, and small intestine (7.61±2.30)%, presented the trend of small intestine>stomach>lung>kidneys>liver. Combined informational analysis showed that the presenting frequency of the cells containing Y chromosome was consistent with the irradiative sensitivity of the organ. CONCLUSION: These findings suggest that the capability of differentiation of Sca-1 positive cells from murine fetal liver was potentially connected to the extent of damage in these organs when transferred in vivo.     

Effect of VEGF on morphology and function of rat ovarian tissues after autologous transplantation

AIM: To evaluate the effect of vascular endothelial growth factor (VEGF) on the ovarian tissues during transplantation. METHODS: Twenty regularly cycling rats were randomly divided into 2 groups: the rats in group A received fresh autologous ovarian transplantation without VEGF administration (n=10); the rats in group B received fresh autologous ovarian transplantation with VEGF administration (n=10). Four weeks after transplantation, the ovarian tissues and sera of the rats were collected for histological examination and determination of the hormone levels. RESULTS: The serum concentration of estradiol in group B was significantly higher than that in group A (P<0.05). Four weeks after transplantation, we found that the ovarian tissues were significantly developed, and the average size of the ovaries in group B was significantly larger than that in group A (P<0.05). CONCLUSION: The ovarian tissues with VEGF administration develop better than those without VEGF administration. VEGF can increase the graft vascularity and the percentage of surviving tissues.     

Effects of sulphated heparin on proliferation and apoptosis of human hepatocellular carcinoma cell line (HepG2)

AIM:To explore the effect and the mechanism of sulphated heparin on the proliferation and the apoptosis of human hepatocellular carcinoma cells.METHODS:The human hepatocellular carcinoma cell line (HepG-2) was used to identify the expression ofrasgene protein and to study the effect of sulphated heparin on proliferation and the apoptosisin vitro.RESULTS:The sulphated heparin downregulated the ras protein expression and inhibited the cell growth in HepG2 cells. In the presence of sulphated heparin, the apoptosis rate of HepG2 increased.CONCLUSION:The data suggest that the effects of sulphated heparin on the proliferation and the apoptosis of the human hepatocellular carcinoma cell are correlated with the signaling transduction mediated byrasgene protein.     

Expression of Hedgehog signaling molecules in rat livers after partial hepatectomy

AIM: To detect the expression of Hedgehog signaling molecules in rat livers after partial hepatectomy. METHODS: The model of rat partial hepatectomy was established by resecting the middle and left lobes of the liver. Eighteen male Sprague-Dawley rats were divided into 3 groups: sham operation group (group A), partial hepatectomy group 1 (group B) and partial hepatectomy group 2 (group C). Hepatic tissues were collected 24 h after the operation in group A and group B, and 48 h after the operation in group C. The expression of Ki-67,Sonic Hedgehog(Shh),Indian Hedgehog(Ihh) and Glioblastoma-2(Gli-2) in the hepatic tissues were detected by immunohistochemical staining and Western blotting. RESULTS: The edema and spotty necrosis in the hepatic tissues were observed in group B and group C by HE staining. The cells of different dividing stages were found in the hepatic tissues of group C. Compared with group A, the expression of Ki-67, Shh, Ihh and Gli-2 in group B (P<0.01) and group C (P<0.01) was significantly elevated, and the expression levels in group C were higher than those in group B (P<0.01). CONCLUSION: Hedgehog signaling in rat livers may be activated after partial hepatectomy and stimulate liver regeneration.     

Effects of β-ME and RA on expression of GFAP in mesenchymal cells derived from mouse fetal liver

AIM: To investigate the effects of β-mercaptoethanol (β-ME) and all-trans rentinal acid (RA) on glial fibrillary acidic protein (GFAP) expression in mesenchymal cells derived from mouse fetal liver in vitro. METHODS: Cells suspension from 14.5-days-old mouse fetal liver were cultured in DMEM/HEPES/F12 supplemented with 20％ FCS and mesenchymal cells were acquired after discarding nonadherent cells. The 5th passage cells were induced by β-ME and RA. The characteristics of treated cells were assayed by immunocytochemistry staining at 5 hours and 5 days after induction. β-actin as an internal control, GFAP gene expression of mesenchyal cells was detected with semi-quantitative RT-PCR. RESULTS: After being inducted by β-ME and RA, 80％ approximately of the cells exhibited typical neural morphology and about 85％ expressed GFAP phenotype. Semi-quantitative RT-PCR showed that mRNA expression of GFAP increased in treated cells versus untreated cells (P<0.01). CONCLUSION: GFAP expression in mesenchymal cells derived from mouse fetal liver in vitro increases after being treated with β-ME and RA.     

Effects of testosterone on endothelial function and intimal proliferation after balloon injury in male rabbit abdominal aorta

AIM: To investigate the effects of testosterone on endothelial function and intimal proliferation after balloon injury in male rabbit abdominal aorta. METHODS: 24 male New Zealand white rabbits were divided randomly into three groups: control group (n=8, sham castration), hypotestosteronemia group (n=8,castration) and testosterone replacement group (n=8,castration +testosterone undecanoate intramuscular injection,14mg/kg). Abdominal aorta was injured with 3 mm PTCA balloon after testosterone undecanoate had been injected for three days. Two weeks later, blood samples were obtained for detection of plasma testosterone, lipids, metabolic product of nitric oxide (NO₂^-／NO₃^-), superoxide dismutase(SOD) and malondialdehyde (MDA),and all the abdominal aorta were excised to be analyzed by computer. RESULTS: The intimal area of hypotestosteronemia group were significantly larger than that of other two groups(P＜0.01). plasma NO₂^-／NO₃^- and SOD levels were significantly decreased, while the total cholesterol(TC),triglycerides(TG), low density lipoprotein(LDL) and plasma MDA were significantly increased. No difference was observed between control group and testosterone replacement group in all parameters. CONCLUSION: Testosterone, at physiological level,had the effects of inhibiting the intimal proliferation and of protecting the endothelial function after balloon injury in male rabbit abdominal aorta.     

Effects of rhBMP-2/collagen composite on the remodeling of rat interparietal suture after rapid expansion

AIM: The purpose of this study was to investigate the effects of rhBMP-2/collagen composite on bone regeneration during expansion of the interparietal suture in the rats. METHODS: 32 10-week old SD rats were divided into groups consisting of 8 rats each. They were comprised of normal control group, expansion control group and the treatment group, the two treatment groups were covered with atelo-typeⅠcollagen and rhBMP-2/collagen composite on the suture before subjected to the expansion force. The bone regeneration in the interparietal suture was estimated by histological method, the osteocalcin content was measured by radioimmonoassay and the calcium content was determined by atomic absorption spectrophotometer. RESULTS: The bone regeneration was more active in the suture after giving an expanding force than in the suture without any intervention. Even bone bridge was formed in the rhBMP-2/collagen composite group. Both the osteocalcin content and calcium content were much higher in the rhBMP-2/collagen composite group than in other three groups (P<0.01). CONCLUSION: The study shows that rhBMP-2/collagen composite promotes the bone regeneration in the expanded suture. It's suggested that rhBMP-2/collagen composite may be of therapeutic benefit in inhibiting relapse and shorting the retention period through acceleration of bone regeneration in the midpalatal suture.     

Effects of RNA interference targeting CDC25agene on proliferation of human liver cancer HepG2 cells

AIM: To investigate the effect of silencing cell division cycle 25a (CDC25a) gene on the proliferation of human hepatoma HepG2 cells. METHODS: CDC25agene in human hepatoma HepG2 cells was silenced by RNA interference. Real-time PCR was applied to detect the expression of CDC25a, cyclin E and CDK2 at mRNA levels in the HepG2 cells. Western blotting was applied to detect the expression of CDC25a at protein level. In addition, MTT assay, Giemsa staining and flow cytometry were used to measure the proliferation of human hepatoma HepG2 cells. RESULTS: The expression of CDC25a at mRNA and protein levels in RNA silence group was lower than those in negative control group and normal control group (P＜0.05). The mRNA expression of cyclin E and CDK2 in silence group was lower than that in negative control group and normal control group (P＜0.05). The cell proliferation in silence group was lower than that in negative control group and normal control group (P＜0.05). The results of flow cytometry revealed that the cells in silence group were blocked in G1 phase. CONCLUSION: Infection of LV-CDC25a-RNAi recombinant to the HepG2 cells effectively inhibits the CDC25agene expression and the proliferation of human hepatoma cells, and arrests the cells in G1 phase, suggesting that CDC25agene may be a key target for the treatment of liver cancer.     

Relationship of Ki-67 expression,microvessel density and therapeutic effect of sorafenib on patients with tumor recurrence after liver transplantation

AIM: To study the therapeutic effect of sorafenib on patients with tumor recurrence after liver transplantation and its relation with the expression of Ki-67 and microvessel density(MVD). METHODS: Thirty patients with tumor recurrence after liver transplantation were treated with sorafenib. The therapeutic effect of sorafenib was observed. The expression of Ki-67 and CD34 in the tissue samples of liver cancer were examined by immunohistochemistry. The MVD was also calculated according to the expression of CD34. RESULTS: In 30 patients treated with sorafenib, none of them achieved complete response (CR), 13 achieved partial response (PR), 8 had stable disease (SD), and 9 had progressive disease (PD). The therapeutic effect of sorafenib was associated significantly with Ki-67 expression .The mean counts of MVD were 356.45±156.13 in PR patients, and 99.39±49.88 in PD patients. A significant difference of MVD between PR and PD patients was observed. CONCLUSION: There is a better therapeutic effect of sorafenib in treating tumor recurrence after liver transplantation in the patients with Ki-67-positive expression and high MVD.     

Effects of uremic serum on the proliferation and trans-differentiation of human renal tubular epithelial cells

AIM: To explore the effects of uremic serum on proliferation and trans-differentiation of human renal tubular epithelial cells. METHODS: Human renal tubular epithelial cell line (HK-2) was cultured in RPMI-1640 medium. The proliferation effects of uremic serum at different concentrations were evaluated by methylene blue assay (MTT method) and flow cytometry. The positive cells percentage of α-smooth muscle actin(α-SMA)in different concentration uremic serum medium was also measured by flow cytometry in vitro. RESULTS: Absorbance 490 (A₄₉₀) was increased in 5%-20% uremic serum groups compared with that in normal controls with the use of MTT. Cells in G₁ phase were decreased, but proliferation index (PI) was increased in 10%-20% uremic serum groups compared with that in normal controls with the use of flow cytometry. No significant difference of cell proliferation index was found among uremic serum groups. The positive percentage of α-SMA cells was increased significantly in uremic serum groups compared with that in normal controls, and increased in parallel with the increasing of uremic serum concentration. CONCLUSION:These data suggest that AGEs could induce a high expression of MIP-1αm RNA and protein in cultured HUVECs in a dose-dependent and time-dependent manner.