Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration

Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-1/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-1/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-1/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice. mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced expression of mfrep-1 maintained high until 72 h and then declined slowly to the basal level. Immunohistochemistry assessment confirmed the up-regulated expression of MFREP-1 protein in parenchymal cells during liver regeneration. These data suggested that MFREP-1 might play an important role in liver regeneration and be involved in the regulation of cell growth.     

Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration

Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-l/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-l/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-l/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice, mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced express     

Hepatocyte-derived fibrinogen-related protein-1 is associated with the fibrin matrix of a plasma clot

In order to study the multiple functions of fibrinogen and fibrin, we are investigating which proteins bind to the fibrin matrix of a plasma clot by using a proteomic approach. Extracts from washed plasma clots were analysed by 2-D gel electrophoresis. A relatively abundant spot was identified as hepatocyte-derived fibrinogen-related protein-1 (HFREP-1) by MALDI-TOF analysis, molecular mass (34 kDa), iso-electric point (pI 5.5) as well as by Western blot analysis. HFREP-1 in plasma almost completely bound to the fibrin matrix during clot formation. Several purified fibrinogen preparations proved to be contaminated with HFREP-1. It is concluded that HFREP-1 (also named hepassocin), a protein with liver cell growth regulatory properties, occurs in plasma and strongly associates with fibrin and possibly fibrinogen.     

G-CSF-induced evacuation of sinusoidal NK cells and the facilitation of liver regeneration in a partial hepatectomy

Since liver regeneration after partial hepatectomy (PHx) is known to improve by pretreatment with recombinant human G-CSF (rhG-CSF), we investigated the mechanism by evaluating the distribution and activity of sinusoidal NK cells. F344 rats were treated with rhG-CSF (250 microg/kg/day) for 5 days before PHx. Pretreatment with rhG-CSF improved the serum ALT levels and DNA biosynthesis of the remnant liver tissues at 20 h after PHx. Notably, the rhG-CSF pretreatment decreased the number of NK cells in the liver determined by immunohistochemistry using anti-NKR-P1A mAb before and at 20 h after PHx with no significant change in the NK activity per cell base, while also increasing the number of NK cells in the peripheral blood detected by flow cytometry. The rhG-CSF induced a pre-PHx downregulation of the IL-12p70 protein levels, while also promoting the post-PHx reduction of the protein levels of IL-12p70 and IFN-gamma. Conversely, rhG-CSF had no effect on the pre-PHx mRNA levels or the PHx-induced upregulation of mRNA levels of TNF-alpha, IL-1beta, IL-6, TGF-beta, IL-10, HGF, and c-Met determined by real-time RT-PCR. These results strongly suggest that rhG-CSF-induced facilitation of liver regeneration is achieved by immunoregulation through the intrahepatic IL-12 downregulation and evacuation of sinusoidal NK cells.     

Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats

BACKGROUND/AIMS: Cytoplasmic liver fatty acid binding protein (L-FABP) has been suggested to be associated with cellular mitotic activity but the changes in L-FABP mRNA and protein levels during liver regeneration following partial hepatectomy (PHx) are not clear. METHODS: In the present study, we determined the time course of L-FABP mRNA expression and L-FABP levels following 70% PHx using Northern and Western blot, respectively. To elucidate one of the roles for L-FABP in PHx, [3H]-palmitic acid clearance in hepatocytes isolated from 24 h post-PHx and control animals was assessed. RESULTS: L-FABP mRNA increased at 30 min, peaked at approximately 1 h (163 +/- 17%; mean +/- SE, n = 5), and returned to control levels 6 h post-PHx. L-FABP level also increased at 1 h but peaked at 24-h (219 +/- 41%; mean +/- SE, n = 5). Hepatocyte [3H]-palmitic acid clearance increased by 29% at 24-h post-PHx, suggesting an increased intracellular transport (or binding) function by L-FABP. Pre-treatment with dexamethasone statistically reduced L-FABP levels (29%) and suppressed the regenerative process (mitotic activity). CONCLUSIONS: L-FABP mRNA increased sharply in response to PHx but the increase was short lived, while L-FABP level increased at a later stage. Both L-FABP content and fatty acid uptake increased significantly during liver regeneration induced by PHx in rats. It is likely that L-FABP is one of the factors responsible for hepatic regeneration.     

Chronic ethanol feeding enhances miR-21 induction during liver regeneration while inhibiting proliferation in rats

Liver regeneration is an important repair response to liver injury. Chronic ethanol consumption inhibits and delays liver regeneration in experimental animals. We studied the effects of chronic ethanol treatment on messenger RNA (mRNA) and microRNA (miRNA) expression profiles during the first 24 h after two-thirds partial hepatectomy (PHx) and found an increase in hepatic miR-21 expression in both ethanol-fed and pair-fed control rats after PHx. We demonstrate that the increase of miR-21 expression during liver regeneration is more robust in ethanol-fed rats. Peak miR-21 expression occurs at 24 h after PHx in both ethanol-fed and control rats, corresponding to the peak of hepatocyte S phase in control rats, but not in ethanol-exposed livers in which cell cycle is delayed. The induction of miR-21 24 h after PHx in control rats is not greater than the increase in expression of miR-21 due to sham surgery. However, in the ethanol-fed rat, miR-21 is induced to a greater extent by PHx than by sham surgery. To elucidate the implications of increased miR-21 expression during liver regeneration, we employed unbiased global target analysis using gene expression data compiled by our group. Our analyses suggest that miR-21 may play a greater role in regulating gene expression during regeneration in the ethanol-fed rat than in the control rat. Our analysis of potential targets of miR-21 suggests that miR-21 affects a broad range of target processes and may have a widespread regulatory role under conditions of suppressed liver regeneration in ethanol-treated animals.     

Liver proteome analysis of adaptive response in rat immediately after partial hepatectomy

The extraordinary ability of the liver to regenerate after resection continues to be an important fascination to mammalian liver researchers. However, at present, there are still several central questions regarding the process of liver regeneration that are not clear. In our study, we try to clarify how the liver is able to maintain its functions as well as to initiate liver regeneration after a significant loss of two-thirds. Here differentially expressed proteins in rat livers at 1 h after partial hepatectomy (PHx) and sham operation were analyzed using 2-DE combined with MALDI-TOF/TOF MS. After the analysis, 24 significantly changed spots (ratio> or =2, p<0.05) were identified. Those proteins are involved in important liver functions such as metabolism, detoxification, and inflammation. Based on the changes in the protein levels found in our data, we identified two aspects of remnant liver immediately after PHx, which focused on the hepatic adaptation and the inflammatory response associated with the initiation of liver regeneration after PHx. For the first time, the differential expression of pyruvate dehydrogenase complex (PDHX), paraoxonase 1 (PON1), thyroid hormone receptor beta, GAP43 (where GAP stands for growth-associated protein), and interleukin-2 (IL2), after PHx, were validated by Western blot.     

Acid-labile subunit regulation during the early stages of liver regeneration: implications for glucoregulation

The initiation of liver regeneration is regulated by endogenously produced growth factors and cytokines and is accompanied by suppression of growth hormone (GH) binding to hepatocytes. We have demonstrated some of these factors, particularly GH, which modulate acid-labile subunit (ALS) expression in vitro. Consequently, we investigated ALS hepatic mRNA and serum levels in rats for 24 h after partial hepatectomy (PHx). There was a significant suppression of ALS gene expression (approximately 50%, P < 0.005) and serum levels (approximately 30%, P < 0.02) by 12 h in PHx rats relative to controls. Relative to intact animals, hepatic mRNA and serum levels of ALS were suppressed by approximately 60% at 24 h. Similarly, hepatic GH receptor mRNA levels were significantly reduced in PHx animals. Moreover, hepatocytes isolated from PHx animals were less responsive to GH than those from controls. Overall, our results demonstrate that suppression of ALS gene expression and serum levels during liver regeneration relates to lowered hepatic GH sensitivity. Suppressed circulating ALS may alter insulin-like growth factor bioavailability and constitute a mechanism to maintain relatively normal glucoregulation after loss of liver mass.     

Increased rOGG1 expression in regenerating rat liver tissue without a corresponding increase in incision activity

Rapidly proliferating tissue with synthesis of a large number of cellular macromolecules including DNA, may require enhanced DNA repair capacity in order to avoid fixation of promutagenic DNA lesions to mutations. This hypothesis was addressed by assessing the incision activity and the mRNA level of the DNA repair protein rat 8-oxodeoxyguanosine glycosylase (rOGG1) as well as the level of the oxidative stress biomarker 8-oxodeoxyguanosine (8-oxodG) in rat liver tissue before and after partial hepatectomy. A five-fold increase in rOGG1 expression was found at 24h after PHx relative to the control levels. At 48h the rOGG1 mRNA levels were reduced to three-times the control values. The corresponding incision activities of rOGG1 in the crude tissue extract as measured by the incision assay were slightly increased both at 24 and 48h after partial hepatectomy although the changes failed to be statistically significant (P=0.07 and 0.06, respectively). The levels of 8-oxodG were unaltered at 24h but increased to 1.8 times the control values at 48h after partial hepatectomy. The study showed that rapid proliferating liver tissue in vivo had an increased expression of the DNA repair protein rOGG1, without significantly increased incision activity on a 8-oxodG-containing substrate and with unchanged levels of 8-oxodG/10(6) dGuo after 24h of regeneration. At 48h the rOGG1 expression was decreased, and the levels of 8-oxodG/10(6) dGuo increased but still significant changes in the incision activity could not be detected. Thus, we can conclude that the rOGG1 expression is temporarily up-regulated by the proliferating events elicited by partial hepatectomy.     

Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration

Liver regeneration in response to various forms of liver injury is a complex process, which ultimately results in restoration of the original liver mass and function. Because the underlying mechanisms that initiate this response are still incompletely defined, this study was aimed to identify novel factors. Liver genes that were up-regulated 6 h after 70% hepatectomy (PHx) in the rat were selected by cDNA subtractive hybridization. Besides known genes associated with cell proliferation, several novel genes were isolated. The novel gene that was most up-regulated was further studied. Its mRNA showed a liver-specific expression and encoded a protein comprising 367 amino acids. The mouse and human cDNA analogues were also isolated and appeared to be highly homologous. The human gene analogue was located at an apolipoprotein gene cluster on chromosome 11q23. The protein encoded by this gene had appreciable homology with apolipoproteins A-I and A-IV. Maximal expression of the gene in the rat liver and its gene product in rat plasma was observed 6 h after PHx. The protein was present in plasma fractions containing high density lipoprotein particles. Therefore, we have identified a novel apolipoprotein, designated apolipoprotein A-V, that is associated with an early phase of liver regeneration.     

Liver regeneration after partial hepatectomy in rat is more impaired in a steatotic liver induced by dietary fructose compared to dietary fat

Hepatic steatosis (HS) has a negative effect on liver regeneration, but different pathophysiologies of HS may lead to different outcomes. Male Sprague-Dawley rats were fed a high fructose (66% fructose; H-fruc), high fat (54% fat; H-fat), or control chow diet for 4 weeks. Based on hepatic triglyceride content and oil red O staining, HS developed in the H-fruc group, but was less severe compared to the H-fat group. Hepatic mRNA expression levels of fatty acid synthase and fructokinase were increased and those of carnitine palmitoyltransferase-1 and peroxisome proliferator-activated receptor-α were decreased in the H-fruc group compared to the H-fat group. Liver regeneration after 70% partial hepatectomy (PHx) was evaluated by measuring the increase in postoperative liver mass and PCNA-positive hepatocytes, and was impaired in the H-fruc group compared to the H-fat and control groups on days 3 and 7. Serum levels of tumor necrosis factor-α, interleukin-6 and hepatocyte growth factor did not change significantly after PHx. In contrast, serum TGF-β1 levels were slightly but significantly lower in the control group on day 1 and in the H-fat group on day 3 compared to the level in each group on day 0, and then gradually increased. However, the serum TGF-β1 level did not change after PHx in the H-fruc group. These results indicate that impairment of liver regeneration after PHx in HS is related to the cause, rather than the degree, of steatosis. This difference may result from altered metabolic gene expression profiles and potential dysregulation of TGF-β1 expression.     

Proliferative and nutritional dependent regulation of glyceraldehyde-3-phosphate dehydrogenase expression in the rat liver

Glyceraldehyde-3-phosphate dehydrogenase is a multifunctional protein possessing numerous cytoplasmic and nuclear functions associated with cellular proliferation. Despite the emerging role of glyceraldehyde-3-phosphate dehydrogenase in regulating the proliferative process, there is a paucity of data regarding its expression and intracellular distribution in non-malignant proliferating hepatocytes. Thus the aim of the present study was to document the intracellular distribution of glyceraldehyde-3-phosphate dehydrogenase protein in proliferating hepatocytes derived from regenerating rat livers, and glyceraldehyde-3-phosphate dehydrogenase gene expression in fasted and re-fed rats following partial hepatectomy (PHx). Glyceraldehyde-3-phosphate dehydrogenase mRNA and protein expression were documented by Northern and Western blot analyses, respectively, at various times following 70% PHx in adult Sprague-Dawley rats. At 24 h post-surgery, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was significantly increased in both PHx and sham operated rats (P < 0.001), respectively. Despite the increase in glyceraldehyde-3-phosphate dehydrogenase mRNA expression in both groups, only PHx rats had a significant increase in the nuclear fraction of glyceraldehyde-3-phosphate dehydrogenase protein (threefold increase compared to sham and baseline levels, P < 0.01), cytoplasmic levels of glyceraldehyde-3-phosphate dehydrogenase protein remained unaltered in both groups. In terms of the effects of feeding and fasting on rats there were no significant differences in glyceraldehyde-3-phosphate dehydrogenase mRNA levels, whether fasted or refed, in rats that had undergone PHx, 8 h earlier. On the other hand, glyceraldehyde-3-phosphate dehydrogenase mRNA levels were significantly increased in refed compared to fasted sham operated rats 8 h following surgery. Serum insulin concentrations were higher in the refed PHx and sham groups compared to their fasted counterparts. The results of this study indicate that although glyceraldehyde-3-phosphate dehydrogenase mRNA are altered to the same extent in PHx and sham-operated rats following surgery, increases in the nuclear fraction of glyceraldehyde-3-phosphate dehydrogenase protein only occur in PHx rats. The results also indicate that glyceraldehyde-3-phosphate dehydrogenase expression is affected by the nutritional status of animals undergoing abdominal sham surgery.     

短暂前脑缺血小鼠海马脑红蛋白表达的动态变化

研究了小鼠短暂前脑缺血再灌注后不同时相点脑红蛋白表达的动态变化及其意义。用夹闭双侧颈总动脉的方法建立C57BL/6小鼠缺血再灌注动物模型;采用RT-PCR及Western blotting方法检测各组小鼠海马组织中脑红蛋白在转录和翻译水平表达的动态变化。结果显示,脑红蛋白在mRNA水平表达的动态变化为:与假手术对照组(100±0.00)比较,再灌注后6h(132.59±28.26,P<0.05)开始升高;24h(157.36±13.85,P<0.001)达高峰;48h(146.55±23.17,P<0.01)开始下降;72h(118.42±34.23,P>0.05)基本恢复至正常水平。脑红蛋白在蛋白水平表达的动态变化为:与假手术对照组(100±0.00)比较,再灌注后6h(111.46±23.54,P>0.05)轻微升高,24h(141.25±32.12,P<0.01)达高峰,48h(138.02±19.68,P<0.05)开始下降,72h(119.29±35.18,P>0.05)基本恢复至正常水平。结果提示,脑缺血再灌注各时相点的脑红蛋白mRNA及蛋白表达水平均增加,可能是机体的应激反应,但持续时间较短(48h以内)。     

The nucleolar protein NML regulates hepatic ATP levels during liver regeneration after partial hepatectomy

We previously identified a novel protein complex, eNoSC, which senses intracellular energy status and epigenetically regulates the rDNA locus by changing the ratio between the numbers of active and silent gene clusters. eNoSC contains a novel nucleolar protein, Nucleomethylin (NML), which has a methyltransferase-like domain and binds to Lys9-dimethylated histone H3 at the rDNA locus, along with the NAD⁺-dependent deacetylase SIRT1 and the histone methyltransferase SUV39H. The aim of this study was to determine the role of NML in liver after partial hepatectomy (PHx). We assessed liver regeneration and lipid metabolism after PHx in wild-type (WT) and NML transgenic (NML-TG) mice. Survival rates of NML-TG mice were reduced after PHx. We found that hepatic triglyceride content in NML-TG mice remained elevated 48 h after PHx, but not delayed liver regeneration. Moreover, hepatic ATP levels in NML-TG mice were higher than that in WT 48 h after PHx. These observations suggest that NML may regulate consumption of hepatic triglyceride in liver regeneration after PHx due to storage of excess ATP. The delayed consumption of hepatic triglyceride may be the cause of reduced survival rate in NML-TG mice.     

大鼠2/3肝切除72 h后再生肝脏质膜比较蛋白质组学研究

大鼠2/3肝切除模型为研究肝细胞增殖和生理性血管生成提供了一个很好的活体内模型.为了揭示肝再生过程中与肝细胞增殖终止相关及与血管生成启动相关的质膜蛋白质,本研究对大鼠肝2/3部分切除72 h后的肝脏质膜进行了研究：利用两步蔗糖密度梯度离心法对切除组和假手术组的肝脏质膜进行纯化;然后通过双向电泳和质谱技术对肝切除样品进行了比较分析并对几个关键蛋白程序性凋亡相关蛋白-6和丝蛋白-A进行了免疫印迹验证.相对于假手术对照组(Sham组),21种蛋白质在切除后72 h的肝脏中上调,15种蛋白质下调.所鉴定的差异表达蛋白参与了血管生成、细胞分裂增殖和凋亡、细胞分化调控、肝脏组织重新构建、代谢及应急反应.本研究为肝脏再生及其血管生成的研究提供了理论依据.     

Keap1/Nrf2/ARE通路相关基因在热诱导的氧化应激小鼠肝脏中的表达

为了研究热应激对小鼠肝脏抗氧化功能及Keap1 (kelch-like ECH-associated protein- 1)/Nrf2（NF-E2-related factor 2）/ARE (antioxidant response element)通路相关基因表达的影响,选用30只8周龄雄性小鼠随机分成6组,每 d连续42 ℃热处理2 h,分别在热处理0 d(对照组)、1 d、2 d、4 d、8 d和12 d时观察肝脏组织形态学和免疫组织化学分析,另取一部分肝脏组织保存于-80 ℃用于后续荧光定量PCR实验,检测肝脏抗氧化指标及Keap1/Nrf2/ARE通路相关基因的表达．结果显示：小鼠的体表温度和直肠温度在热处理后都极显著高于热处理前．组织形态学观察发现,热处理导致小鼠肝脏组织充血和肝细胞水肿．小鼠肝脏氧化应激指标 MDA (malondialdehyde)含量在热处理第2 d较对照组显著升高,GSH (glutathione)含量、GSH-PX (glutathione peroxidase)活力和总SOD (superoxide dismutase)活力在第4 d和12 d都有升高．免疫组织化学发现,与对照组和第12 d组相比,Nrf2蛋白在第1 d,2 d,4 d,8 d表达明显,其中Nrf2蛋白在第4 d表达最为显著. 荧光定量RT PCR结果表明,与对照组比较Keap1基因的表达量从热处理第1 d开始显著降低,Nrf2基因的表达量在第4 d和12 d显著升高,HO-1 (Heme oxygenase-1)基因的表达量在第1 d显著升高,NQO1 (Quinone oxidoreductase)和GCLC (Glutamate cysteine ligase catalytic)基因的表达量在第1 d和4 d显著升高．上述结果表明,热应激引起了小鼠肝脏氧化损伤, Keap1/Nrf2/ARE通路可能参与了肝脏自身缓解热应激的过程.