门静脉分支结扎后门静脉压力变化与肝再生的关系

目的 探讨90%门静脉分支结扎后大鼠门静脉压力变化与肝再生的关系.方法 45只雄性SD大鼠行90%门静脉分支结扎术,其中5只进行假手术作为对照.观察不同时相点门静脉压力和非结扎侧肝脏质量变化,光学显微镜下观察非结扎侧肝细胞的形态学变化,免疫组织化学方法检测未结扎侧肝细胞的增殖细胞核抗原(PCNA),TUNEL法检测未结扎侧肝细胞的凋亡情况,并进行定最分析.采用Pearson相关分析和t检验分析数据.结果 95%(38/40)的大鼠存活.结扎侧肝叶进行性萎缩,非结扎侧肝叶占全肝质量的比例随时问推移而增加,12 h内增加较缓慢,仅为10.75%;而1～5 d则增加速度明显加快,达到27.57%;7～28 d达到平台期,缓慢增加到32.37%.术前门静脉压力为(9.1±1.8)cm H_2O(1 cm H_2O=0.098 kPa);结扎后立即升高,12 h达到高峰(15.8±2.7)cm H_2O,与术前比较差异有统计学意义(t=6.847,P<0.05);1～28 d由(13.6±2.3)cm H_2O逐渐下降为(9.3±2.0)cm H_2O.术前大鼠PCNA阳性细胞计数为7%±3%,术后12 h至3 d由14%±5%上升至21%±6%,第5天达到高峰为26%±7%,与术前比较差异有统计学意义(t=9.129,P<0.05),随后逐渐恢复正常.TUNEL法检测结果显示,术前大鼠肝脏和术后各时相点大鼠未结扎侧肝脏仅见极少量凋亡细胞.大鼠门静脉压力与非结扎侧肝叶肝细胞PCNA的表达在术后1、3、5 d呈正相关(r=0.913,0.896,0.908,P<0.05),在术后14 d时相点呈负相关(r=-0.926,P<0.05).结论 大鼠90%门静脉分支结扎术后,引起未结扎侧肝细胞的活跃再生,再生后的肝脏可恢复原来的质量;肝再生以肝细胞增殖加速为主,而非肝细胞凋亡减少;门静脉压力变化在肝再生过程中可能发挥重要作用.     

Augmentative effect of cyclosporin A on rat liver regeneration: influence on hepatocyte growth factor and transforming growth factor-beta(1).

We investigated the effect of cyclosporin A (CsA) on rat liver regeneration following partial hepatectomy with reference to cytokine production. Rats were divided into two groups: those without CsA pretreatment (group 1) and those with CsA pretreatment (group 2). Animals were given olive oil vehicle or CsA (10 mg/kg) dissolved in olive oil daily by gavage from 4 to 1 days before hepatectomy. The ratio of regenerating liver weight to initial body weight in group 2 was significantly higher than that in group 1 at 72 h. Although a peak 5-bromo-2-deoxyuridine labeling index was found at 24 h after hepatectomy in both groups, the peak value in the CsA-treated animals was significantly higher than in controls. In both groups, hepatocyte growth factor concentrations in both plasma and liver tissue showed maximal values at 12 h. Liver tissue values in group 2, however, were significantly higher from 1 to 12 h compared to group 1. Transforming growth factor-beta(1) (TGF-beta(1)) concentrations showed minimal serial changes in group 1, while those in liver tissue of group 2 rats were significantly lower than in group 1. Plasma TGF-beta(1) concentrations did not differ. These results suggest that upregulation of hepatic regeneration with CsA pretreatment might be attributed in part to changes in production of these mitogenic and mitoinhibitory cytokines.     

核因子-кB在70%门静脉分支高位结扎后大鼠肝脏增殖与凋亡中的意义

目的 探讨NF-κB在70％门静脉分支高位结扎后大鼠肝脏增殖与凋亡中的意义。方法 Wistar大鼠60只，随机分成假手术对照组和门静脉结扎组。观察术后0．5、1、2、3、7和14d肝脏大体结构和血浆转氨酶的变化。运用凝胶阻滞实验(EMSA)检测术后肝脏NF-κB的活性，用TUNEL法对结扎侧肝细胞凋亡进行定量分析，光镜下观察对侧肝细胞的增殖情况，电镜下观察结扎侧肝脏的超微结构改变。结果 70％门静脉分支高位结扎后，结扎侧肝叶呈进行性萎缩变小，对侧则成比例的代偿性增生。在整个观察过程中，全肝的总重量维持恒定，肝脏功能基本保持正常。NF-κB的活性在术后明显升高，于第2天达高峰，1周后基本恢复正常。TUNEL显示结扎侧肝细胞在术后凋亡明显增加，于第2天达高峰，同时对侧肝细胞却明显增殖。电镜显示结扎侧肝脏早期出现典型的凋亡改变，晚期明显纤维化。结论 70％门静脉分支高位结扎后，NF-κB的活性明显升高，其可能在肝脏的增殖与凋亡中具有重要意义。     

Effect of portal branch ligation on liver regeneration in the rat

GOAL: The aim of this study was to assess liver regeneration after partial portal ligation. METHODS: 70% partial portal occlusion was obtained by ligation of the left portal vein branch. Total liver weight ratio were measured 96 hours after partial portal occlusion and in sham operated animals. The kinetics of hepatocytes division was evaluated by measuring the incorporation of 5-bromo-21-deoxyuridine into replicating cells at various time points by immunohistochemistry. RESULTS: Partial portal occlusion did not alter the total liver weight 96 hours after surgery. It resulted in atrophy of the ligated lobes and hypertrophy of the lobes with preserved portal flow. Hypertrophy was associated to an increase of the percentage of replicating hepatocytes. The replication rate was maximum at 28 hours with a peak at 12.5% and was prolonged beyond the 48th hour. CONCLUSIONS: Partial portal occlusion results in major and prolonged regeneration process in the liver lobes with preserved portal flow.     

Reciprocal balance of hepatocyte growth factor and transforming growth factor-beta 1 in renal fibrosis in mice

BACKGROUND: Transforming growth factor-beta 1 (TGF-beta 1) plays a central role in the pathogenesis of renal fibrosis. In contrast, hepatocyte growth factor (HGF) may be an important molecule for tissue repair. As TGF-beta 1 is a suppressor molecule for HGF expression, we asked whether a decrease in HGF expression would be accompanied by an increase in TGF-beta 1 and whether the progression of renal fibrosis would be modulated. METHODS: We used the ICR strain-derived glomerulonephritis (ICGN) mice as a model of chronic renal disease and examined changes in local HGF expression during the natural course of renal fibrosis. To determine the significance of intrinsic HGF noted during progression of renal fibrosis, we administered an anti-HGF antibody to mice at the early stage of renal fibrosis. RESULTS: At an early stage of renal fibrosis, the mice showed strong peritubular HGF expression, coinciding with tubular proliferation. In the late stages, the renal HGF level was markedly decreased, coinciding with a reduction in proliferative tubular areas. Renal TGF-beta 1 levels were increased in accordance with expansion of fibrotic areas. Notably, the anti-HGF antibody treatment of early-stage mice decreased the HGF level and reduced tubular areas, whereas collagen-deposited areas were expanded in parallel with increased TGF-beta 1 levels. Consequently, in HGF-neutralized mice, there was a rapid progression of renal dysfunction. CONCLUSIONS: Not only an increase in TGF-beta 1 level, but also a decrease in local HGF expression may be responsible for the manifestation of renal fibrosis, particularly tubular destruction.     

Hepatocyte growth factor supply accelerates compensatory hypertrophy caused by portal branch ligation in normal and jaundiced rats.

BACKGROUND: Hepatocyte growth factor (HGF), first identified as the most potent mitogen for hepatocytes, significantly stimulates liver regeneration after hepatectomy. In this report, we examined whether HGF is also useful in accelerating compensatory hypertrophy caused by portal branch ligation in normal and jaundiced rats. MATERIALS AND METHODS: Normal and reversible obstructive jaundiced rats underwent portal ligation of the left lateral and median branches, which supply approximately 70% of the total volume of the liver. Simultaneously, the animals were continuously treated with either recombinant human HGF (rhHGF) or vehicle alone via an intraperitoneally implanted osmotic pump. Two and four days after portal ligation, the degree of compensatory hypertrophy in unoccluded lobes was examined by measuring the wet weight ratios of the unoccluded lobes to the whole liver and the 5-bromo-2'-deoxyuridine labeling index of hepatocytes in each group. RESULTS: The HGF treatment significantly increased the wet weight ratios and the DNA synthesis in nonoccluded lobes 2 and 4 days after portal ligation in both normal and jaundiced rats. Moreover, rhHGF supply promptly decreased serum total bilirubin level in jaundiced rats. CONCLUSIONS: Continuous rhHGF administration not only accelerates compensatory hypertrophy in normal and jaundiced rats but also ameliorates hyperbilirubinemia in jaundiced rats.     

Change of liver function in hypertrophying lobe of rabbit liver after portal branch ligation.

Preoperative portal embolization (PE) is useful for the prevention of postoperative liver failure after extended hepatectomy. However, clinical evaluation of liver function in the hypertrophying lobe after PE has not been studied. Here we report functional changes in the hypertrophying lobe using a 80% portal-branch-ligation rabbit model. Liver function was evaluated by the expression of liver-specific genes detected by Northern blot analysis and plasma disappearance rate of indocyanine green (ICG). The weight of the unligated lobe after portal ligation increased about twofold on the 7th postoperative day (POD) and about threefold on the 14th POD. The mRNA levels of the liver-specific genes (albumin, aldolase B, and tyrosine aminotransferase) in the unligated lobe decreased to about 50% on the 1st POD and returned to the preoperative levels on the 7-14th POD. In contrast, the expression of histone H2B mRNA increased on the 3rd-7th POD. The plasma disappearance rate of ICG (K-ICG) in the rabbit that has only the unligated lobe did not significantly change during the first 7 days, but then improved and recovered to 80% of that in the rabbit that has whole liver on the 14th POD. These results indicate that liver function of the hypertrophying lobe after portal branch ligation does not increase during the first 7 days despite an increase in liver weight. This finding suggests that the compensatory hypertrophying liver is enlarging without functional augmentation in the early period after PE.     

Disturbed spatial learning of rats after intraventricular administration of transforming growth factor-beta 1

Patients with subarachnoid hemorrhage (SAH) who later suffer hydrocephalus show persistently high levels of transforming growth factor-beta 1 (TGF-beta 1) in the cerebrospinal fluid after the onset of SAH. Recombinant TGF-beta 1 induces hydrocephalus in mice. This study examined the spatial learning ability of rats after intraventricular administration of TGF-beta 1. Thirteen-week-old Wistar rats were treated with 0.8 or 8.0 micrograms of human recombinant TGF-beta 1 by direct injection or via osmotic pump. Three months later, their spatial learning ability was evaluated with a Morris water maze. Ventricular size, ultrastructural features, and sodium-potassium-adenosine triphosphatase (Na+, K(+)-ATPase) activity of the subarachnoid space were examined. All three TGF-beta 1-treated groups clearly exhibited impaired spatial learning ability, but they did not exhibit ventricular dilation. Histological examination revealed subarachnoid fibrosis and deactivation of Na+, K(+)-ATPase in the arachnoid cells. These findings are similar to those of our previous experiments involving injection of TGF-beta 1 in mice. The present and previous studies suggest that subarachnoid fibrosis is an important factor in the disturbance of the spatial learning ability of rats, whereas ventricular size is less important.     

Clinical significance of lymphocytes hepatocyte growth factor mRNA expression in patients after liver transplantation

Hepatocyte growth factor (HGF) plays a key role in the regulation of liver regeneration after hepatocyte damage. Changes in HGF gene expression reflect the status of the regeneration process. AIM: The aim of this study was to ascertain the clinical significance of the expression of HGF among liver transplant patients. METHODS: Expression of the mRNA of HGF among peripheral blood lymphocytes were measured prior to as well as at 1, 2, 6, and 10 days after liver transplantation in a group of 30 liver recipients. RESULTS: In first 24 hours after reperfusion, the patients with compromised graft function (group 1) showed persistently higher HGF gene expression after reperfusion compared with patients displaying well-functioning grafts (group 0; P = .0189). Between postoperative days 1 and 10, there was a rapid decrease in gene expression among group 0 compared with group 1 (P = .0155). The significant decrease observed in the both groups reached a certain plateau after 48 hours postoperatively. There was no statistical difference in aminotransaminase levels over the days after liver transplantation. The decreased mRNA HGF expression in lymphocytes preceded the decrease in aminotransferase levels. CONCLUSIONS: HGF was more sensitive to predict early graft function than prothrombin time, aspartate aminotransferase, and alanine aminotransferase levels. The determination of HGF expression level in lymphocytes after liver transplantation may yield valuable information for evaluation of early graft function.     

Single dose of anti-transforming growth factor-beta1 monoclonal antibody enhances liver regeneration after partial hepatectomy in biliary-obstructed rats

BACKGROUND: Transforming growth factor (TGF) beta is a potent inhibitor of hepatocyte DNA synthesis and liver regeneration. TGF-beta(1) expression progressively increases in obstructive jaundice. We investigated the effect of TGF-beta(1) blockage on liver regeneration in rats induced with obstructive jaundice. MATERIALS AND METHODS: Male Wistar-albino rats were divided into three groups: sham, control, and study groups. In the study and control groups, the common bile duct was ligated and divided, and 7 days later a partial hepatectomy was performed. In the study group, anti-TGF-beta(1) monoclonal antibody (10-microg single dose) was administered immediately after the 70% hepatectomy. In the control group, those rats in which obstructive jaundice was induced received normal saline after the 70% hepatectomy, and nonjaundiced rats received anti-TGF-beta(1) monoclonal antibody after the 70% hepatectomy. Rats were sacrificed after 48 or 72 h. Relative liver weight, AST, ALT, total and conjugated bilirubin, and TGF-beta(1) levels were measured. The mitotic index and proliferating cell nuclear antigen (PCNA) labeling index were evaluated as histopathologic parameters. RESULTS: At 72 h, the TGF-beta(1) level in the study group was similar to that in the sham group, whereas TGF-beta(1) in the study group was significantly lower than that of the jaundiced control group at 48 or 72 h (P < 0.001). The relative liver weight, mitotic index, and PCNA labeling index were significantly higher in the study group than in the jaundiced control group at 48 and 72 h (P < 0.001). The AST, ALT, and TGF-beta(1) levels were significantly higher in the jaundiced control group compared to the study group after 48 and 72 h, whereas these values were significantly lower in the nonjaundiced control group (P < 0.001). CONCLUSIONS: In obstructive jaundiced rats, TGF-beta(1) blockage with anti-TGF-beta(1) monoclonal antibody after liver resection improved liver regeneration both morphologically and functionally.     

The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor-beta1

AIMS: Smad7 is an inhibitory Smad that regulates transforming growth factor-beta (TGF-beta) signaling. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of the fibrogenic effects of TGF-beta1. SMAD binding sites have been identified in both TGF-beta and CTGF promoters. The effect of CTGF on Smad7 expression and its role in the regulation of Smad7 induced by TGF-beta1 in renal tubular cells is unknown. METHODS: Human model of proximal tubular cells (HK-2 cells) was used and confirmed using a diabetic rat model. RT-PCR was performed to measure Smad7, TGF-beta1 and Smad2 and ELISA was performed to measure active TGF-beta1. CTGF or TGF-beta1 was silenced in HK-2 cells using siRNA methodology. RESULTS: TGF-beta1 induced Smad7 in a time-dependent manner, peaking at 30 min (P<0.0005) but sustained up to 24 hrs (p<0.005). Conversely, CTGF reduced Smad7, which was maximal at 24 hrs (p<0.05). This was supported by our in vivo data demonstrating that CTGF protein significantly increased while Smad7 mRNA level was reduced in a diabetic rat model. The basal expression level of Smad7 decreased in TGF-beta1 silenced cells compared to cells transfected with non-specific siRNA (p<0.0005). The basal expression level of Smad7 increased in CTGF silenced cells (p<0.05), which was increased by TGF-beta1 (p<0.005). Both mRNA and protein levels of TGF-beta1 decreased in CTGF silenced cells (p<0.05 and p<0.005 respectively) accompanied by reduction in Smad2 mRNA level in CTGF silenced cells. CONCLUSIONS: Smad7 is induced rapidly by TGF-beta1 limiting the response to TGF-beta1. CTGF likely plays a key role in promoting TGF-beta1 activity by decreasing the availability of Smad7 and increasing Smad2.     

Expression of epidermal growth factor, basic fibroblast growth factor and insulin growth factor-1 and relation to myocyte regeneration of obstructed ureters in rats

OBJECTIVE: To investigate the roles of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and insulin growth factor-1 (IGF-1) in the myocyte regeneration of obstructed ureters. MATERIAL AND METHODS: The expression of EGF, bFGF, IGF-1 and proliferation cell nuclear antigen (PCNA) was studied immunohistochemically in 54 female Sprague-Dawley rats. RESULTS: Tissue damage to the smooth muscle layer in the obstructed ureters was aggravated during the period of obstruction. The expression of EGF, bFGF and IGF-1 in myocytes was detected using the method of concurrent immunohistochemical staining. The expression of EGF, bFGF and IGF-1 in the smooth muscle layer was found from Day 14 after ligation. The expression of EGF, bFGF and IGF-1 increased to a peak on Day 21 and then declined. The expression of PCNA in the smooth muscle layer was also found from Day 14 after ligation and increased to a peak on Day 21. The expressions of EGF, bFGF and IGF-1 were significantly correlated with the expression of PCNA in the smooth muscle layer (r=0.7982, 0.6264 and 0.5840, respectively; p-values all <0.002). Co-expression of EGF, bFGF, IGF-1 and PCNA was determined using the method of double immunofluorescence staining. Co-expression of PCNA was observed in 34% of EGF-positive myocytes, 53.6% of bFGF-positive myocytes and 41.1% of IGF-1-positive myocytes at Day 21 post-ligation. CONCLUSIONS: Expression of EGF, bFGF and IGF-1 may contribute to myocyte regeneration of damaged ureters in rats with obstructive uropathy.     

Role of upstream stimulatory factors in regulation of renal transforming growth factor-beta1

We previously identified an E-box to be implicated in high-glucose-induced transforming growth factor-beta1 (TGF-beta1) gene stimulation in murine mesangial cells. In the present study, we evaluated the role of upstream stimulatory factors (USFs) in mediating glucose-induced stimulation of TGF-beta1. Mesangial cells cultured in glucose concentrations exceeding 2.7 mmol/l D-glucose exhibited increased levels of USF1 and USF2 protein by Western analysis and electrophoretic mobility shift assay (EMSA). An E-box element from the murine TGF-beta1 promoter revealed USF1 and USF2 binding by EMSA. Chromatin immunoprecipitation assay revealed in vivo binding of USF1 to a glucose-responsive region of the TGF-beta1 promoter. Transient cotransfection studies of 293 cells with USF1 led to a twofold increase in TGF-beta1 promoter activity and a 46% increase in secreted TGF-beta1 protein levels. Wild-type and USF2 knockout mice exhibited a 2.5-fold stimulation of renal TGF-beta1 expression upon fasting and refeeding with a carbohydrate-rich diet, whereas USF1 knockout mice exhibited only a minimal increase of renal TGF-beta1 upon refeeding. USF1 mRNA levels were increased in mouse kidneys with carbohydrate refeeding, and USF1 protein was increased in diabetic rat kidneys compared with controls. We conclude that USF1 is stimulated by modest increases in glucose concentration in murine mesangial cells, bind to the murine TGF-beta1 promoter, contribute to carbohydrate-induced renal TGF-beta1 expression, and may play a role in diabetes-related gene regulation in the kidney.     

Deletion variant of hepatocyte growth factor prolongs allograft survival after liver transplantation in rats.

BACKGROUND: Despite continued progress in the development of immunosuppressive agents, allograft rejection remains an important cause of morbidity and mortality after liver transplantation. We examined the effect of the deletion variant of hepatocyte growth factor (dHGF) on allograft rejection after liver transplantation. METHODS: Male Dark Agouti rats (RT1a) were selected as donors and male Lewis rats (RT1l) as recipients for a rejection model. The recipients were divided into 2 groups after orthotopic liver transplantation (OLTx): in the dHGF group dHGF was given intravenously twice a day (1 mg/kg/day) after OLTx, whereas in the control group vehicle buffer was given intravenously daily twice after OLTx. The survival period, serum chemistry studies, and histopathologic findings were then compared between the 2 groups. RESULTS: The mean survival period after OLTx in the dHGF group was significantly longer than that in the control group (21.4 +/- 1.3 days vs 11.8 +/- 0.4 days, P < .001). On the 10th posttransplant day the serum albumin level significantly improved in the dHGF group (P < .01), and the serum total bilirubin and aspartate aminotransferase levels were significantly lower in the dHGF group (P < .01 and P < .05, respectively). On the 10th posttransplant day a histologic examination revealed no apparent difference in the severity of rejection between the 2 groups. The number of proliferating cell nuclear antigen-positive hepatocytes in the dHGF group significantly increased (P < .01), whereas the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive hepatocytes were significantly reduced in the dHGF group (P < .01) in comparison with those in the control group. CONCLUSION: dHGF has an antiapoptotic property as well as a proliferative and protective effect on hepatocytes under allograft rejection. dHGF might serve as a novel agent for reducing the harmful effects of hepatic allograft rejection in rats.