Partial hepatectomy and mediators of inflammation decrease the expression of liver alpha 2-HS glycoprotein gene in rats

Liver mRNA levels of two acute phase reactant (APR) proteins, alpha 2-HS glycoprotein (a major negative APR) and alpha 1-acid glycoprotein (a major positive APR) were measured in male rats at different times after the administration of turpentine, of tumor necrosis factor, or following partial hepatectomy. In every case, a marked decrease in mRNA levels of alpha 2-HS glycoprotein was observed which reached a maximum at 24 h. A concomitant increase of alpha 1-acid glycoprotein mRNA levels was observed under the same conditions. These results indicate that the decreased levels of alpha 2-HS glycoprotein induced by the acute-phase response following inflammatory mediators and partial hepatectomy are due to a down-regulation of the gene expression of this protein in rat liver.     

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.     

Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%-hepatectomy

Mast cells (MC) can undergo significant changes in number and phenotype; these alterations result in the differential expression of growth factors and cytokines. Kit ligand (KL; stem cell factor) is produced by mesenchymal cells, and in the liver by biliary epithelial cells. Recent studies suggest that KL, and its receptor c-kit, may be involved in liver regeneration after loss of liver mass. However, KL is also the major growth, differentiating, chemotactic, and activating factor for MC. The aim of our study was to elucidate the dynamics and phenotype of hepatic MC and KL/c-kit expression during liver regeneration after partial (70%) hepatectomy in the rat. Regenerating livers were harvested after 1, 3, 7, and 14 days, respectively (n = 6 each day). MC were stained for naphthol-AS/D-chloroacetate esterase and counted as MC per bile ductule. MC phenotype was assessed by rat MC protease (RMCP)-1 and -2 immunofluorescence staining, in order to distinguish RMCP-1 positive connective tissue MC (CTMC) from RMCP-2 positive mucosa MC (MMC). mRNA expression of RMCP, c-kit, and the differentially spliced variants of KL was quantified by RT-PCR. MC counts per bile ductule decreased in regenerating rat liver tissue at day 3, compared with native livers, and became normal thereafter. Hepatic MC were predominantly of a CTMC phenotype expressing RMCP-1, as previously published; after hepatectomy, between 76 and 99% of all MC double-expressed RMCP-1 and -2, compatible with an MMC phenotype. The ratio of the two alternatively spliced mRNAs for KL (KL-1 : KL-2), and c-kit mRNA expression did not differ significantly between regenerating livers and the livers of sham operated animals. These results suggest that hepatic mast cells are restored during liver regeneration after partial hepatectomy in the rat. Restored MC express an MMC phenotype, suggesting migration from outside into the regenerating liver. Alternative splicing of KL is affected by the surgical procedure in general, and, together with its receptor c-kit, doesn't seem to be involved in liver regeneration after partial hepatectomy in the rat. Further functional studies, and studies in regenerating human livers might offer the possibility of elucidating the role of the hepatic mast cell, and its different protease phenotypes during liver regeneration after surgical loss of liver mass.     

Microtubule depolymerization inhibits the regulation of alpha 1-acid glycoprotein mRNA by hepatocyte stimulating factor

Hepatocyte stimulating factor (HSF, a polypeptide cytokine) is a major regulatory hormone responsible for hepatic acute-phase reactant (APR) induction following acute systemic injury. The mechanisms by which HSF regulates APR synthesis in the liver are unknown. Microtubules are involved in a number of polypeptide hormone-mediated events which can be modified, either positively or negatively, by microtubule depolymerizing agents. In this study we have used colchicine (a microtubule depolymerizing drug) to assess whether or not HSF-mediated changes in rat hepatic alpha 1-acid glycoprotein (AGP) or albumin mRNA levels require an intact microtubule cytoskeletal system. Cultured rat hepatocytes were pretreated for 30 min with either colchicine (10(-6) M), or the inactive isomer lumicolchicine (10(-6) M), or fresh medium. Following pretreatment, purified murine macrophage HSF (10 units/ml) was added and the cells were incubated for an additional 12 h. Colchicine, but not lumicolchicine, significantly inhibited the HSF-dependent regulation of mRNA for the positive APR, AGP, but had no effect on the mRNA levels of albumin, a negative APR. Furthermore, removal of colchicine from previously inhibited cultures allowed HSF to restimulate AGP mRNA expression. These data suggest that microtubules may play a regulatory role in controlling the expression of the genes for positive acute-phase proteins and may explain the temporal differences found in vivo between positive and negative APR expression.     

Distribution of mRNAS of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of alpha-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5-6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2-3-fold). There were no alpha-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Differential expression of apoptosis-associated genes post-hepatectomy in cirrhotic vs. normal rats

Liver regeneration after partial hepatectomy or liver injury is controlled by a wide variety of growth factors that are proven activators or inhibitors of hepatocyte proliferation. Liver regeneration post-hepatectomy has been proven to be decreased and delayed in cirrhotic vs. normal liver. Apoptosis seems to play an important role in cellular proliferation and in liver regeneration. Therefore, this study has analyzed the expression of apoptosis-associated genes following 2/3 hepatectomy in cirrhotic vs. normal rats. Cirrhosis was induced by a weekly intragastric administration of CCl4 for 16 weeks followed by hepatectomy and histological examination of the resected liver. Rats were sacrificed at 6 h, 12 h, 24 h, or 72 h after liver resection. The expression of proapoptotic (Bad, Bak, Bax) and antiapoptotic (Bcl-2, Bcl-XL) genes was analyzed by quantitative RT-PCR. We have observed an early increase in antiapoptotic mRNA levels and a delayed increase in proapoptotic mRNA levels in normal liver following hepatectomy. Before resection, proapoptotic mRNA levels were significantly higher in cirrhotic vs. normal liver. After hepatectomy, apoptotic mRNA levels were decreased and delayed as compared with that observed following hepatectomy in normal liver. These results indicate that apoptosis takes place in liver during CCl4-induced cirrhosis and could participate in the impaired regenerative response observed in cirrhotic liver.     

柴芩承气汤对重症急性胰腺炎并发肝损伤大鼠的治疗作用及其机制

目的：研究柴芩承气汤（CQCQD）对重症急性胰腺炎（SAP）并发肝损伤大鼠的治疗作用及其机制。方法：72只SD大鼠随机分为3组（n=24）：假手术（sham）组,重症急性胰腺炎模型（SAP）组和柴芩承气汤治疗（CQCQD）组。去氧胆酸钠胰胆管逆行注射建立SAP大鼠模型,CQCQD组给予柴芩承气汤治疗,于造模后1 h、5 h、10 h观察各组不同时间点的胰腺、肝脏组织病理学变化,检测血清中淀粉酶（AMS）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）活性、白介素-6（IL-6）水平及胰腺、肝脏组织单核细胞趋化蛋白-1（MCP-1）和IL-6 mRNA的表达情况。结果：与sham组比较,SAP组血清AMS、ALT、AST活性及IL-6水平明显升高,胰腺、肝脏组织MCP-1及IL-6 mRNA表达升高（P<0.05）;与SAP组比较,CQCQD组血清AMS、ALT、AST活性及IL-6水平明显降低;胰腺和肝脏组织病理损伤减轻,胰腺、肝脏组织MCP-1及IL-6 mRNA表达明显减弱（P<0.05）。结论：MCP-1参与了SAP并发肝损伤的进展;柴芩承气汤能显著抑制胰腺、肝脏组织MCP-1的表达,减轻SAP时胰腺、肝脏组织病理损伤,对SAP并发肝损伤起到治疗作用。     

Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs.

Hepatocyte growth factor (HGF) is a potent growth factor for various epithelial cells including mature hepatocytes and renal tubular cells. When 70% of the rat liver was excised, HGF mRNA in the intact lung markedly increased at 6 h later, then decrease to normal levels at 24 h. A similar marked increase of HGF mRNA was found in the lung of rats with hepatitis induced by CCl4. Moreover HGF mRNA in the intact lung also increased to about a 5 times higher level than the normal, within 12 h after unilateral nephrectomy. Isolated alveolar macrophages significantly expressed HGF mRNA, yet the amount remained unchanged after injury of the liver. The marked increase of HGF mRNA in lungs of partially hepatectomized rats remained even after removal of alveolar macrophages. In situ hybridization showed a marked increase of HGF mRNA signal found in endothelial cells in the lung after partial hepatectomy. We postulate that endothelial cells in the lung recognize damage of distal organs through a mediator and that lung-derived HGF may contribute to tissue repair or regeneration of injured organs, through endocrine-related mechanisms.     

Hepatotropin mRNA expression in human foetal liver development and in liver regeneration

A 569 bp probe against the β-chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.     

Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes

Proto-oncogene fos mRNA levels are rapidly and transiently elevated 12-fold in regenerating liver 10-60 min following partial hepatectomy. This response, and the induction of fos protein synthesis, has been simulated qualitatively and quantitatively in long term primary cultures of quiescent adult rat hepatocytes where proliferative transitions can be initiated directly in serum-free medium by known hepatocyte mitogens like epidermal growth factor. Expression of a second proto-oncogene, c-rasH, in proliferatively activated hepatocyte cultures between 6 and 24 h also simulates the delayed hepatic response that occurs in vivo following partial hepatectomy. These results suggest that sequential proto-oncogene expression during liver regeneration is caused directly by hepatocellular interactions with specific mitogens. In addition, a role for monovalent cations in the regulation of hepatocyte gene expression is implicated from findings that Na+ deprivation inhibits induction of fos expression in cultured hepatocytes by epidermal growth factor under chemically defined conditions.     

Notch-Hif-1-alpha信号通路参与调控大鼠肝再生过程的研究

目的：探讨使用酌分泌酶抑制剂Fli-06 特异性阻断Notch 信号通路后,大鼠肝部分切除术后肝再生的情况并初步阐明 Notch-Hif-1-alpha信号通路调控肝再生的可能机制。方法：取SD 大鼠分为对照（生理盐水注射组,n=24）和抑制剂组（酌分泌酶抑制剂 注射组,n=24）。给予药物处理后,两组分别施行大鼠肝部分切除术,术后0 d,1 d,3 d,5 d,每个时间点分别留取对照组（n=6）及抑 制剂组（n=6）再生的肝组织,并检测相应肝重体重比,免疫组化法检测再生肝PCNA（增殖细胞核抗原,Proliferation Cell Nuclear Antigen）表达,RT-PCR 检测再生肝组织中的Notch1、Hes1、VEGF mRNA的变化,Western-Blot 法检测NICD（Notch 胞内段,Notch Intracellular Domain）、Hif-1-alpha（低氧诱导因子-1-alpha,Hypoxia Inducible Factor-1琢)蛋白在肝再生过程的变化情况。结果：1、肝部分切除 术后3 d和5 d,抑制剂组肝重体重比明显低于对照组差异有统计学意义（P<0.05）;2、免疫组织化学染色结果提示：抑制剂组再生 肝PCNA阳性细胞率在术后1 d,3 d,5 d 均明显低于对照组（P<0.05）;3、Western blot 结果表明：NICD 和Hif-1-alpha蛋白水平明显低 于对照组（P<0.05）;同时RT-PCR 结果提示：抑制剂组Hes1 的mRNA表达量术后1 d,3 d明显低于对照组,差异具有统计学意义 （P<0.05）。同时,抑制剂组VEGF mRNA 水平在术后3 d,5 d明显低于对照组,差异具有统计学意义（P<0.05）。结论：在大鼠肝部分 切除术后肝再生过程中,使用酌分泌酶抑制剂Fli-06 抑制Notch 信号通路后,大鼠的肝再生能力明显降低,Notch-Hif-1alpha信号通 路可能参与调控了大鼠肝部分切除术后肝再生过程。