Cyclosporine augments hepatic regenerative response in rats

A number of mechanisms participate in the hepatic injury that occurs during and following liver transplantation. A normal allograft regenerative response is probably essential for a successful transplant outcome. In this study, the effect of cyclosporine, a potent immunosuppressant used routinely after liver transplantation, on the regenerative response of the liver after partial hepatectomy was investigated. Male Wistar rats were pretreated for one week with either cyclosporine or the olive oil vehicle and were subjected to either a two-thirds partial hepatectomy or a sham operation. Animals were sacrificed at various times postoperatively and the remnant livers were weighed to determine the liver weight to body weight ratio, two biochemical measures of a regenerative response (cytosolic ornithine decarboxylase activity and thymidine kinase activity), and the hepatic content of estrogen and androgen receptors, as the content of these receptors has been shown to modulate, at least in part, the subsequent hepatic regenerative response. The preoperative hepatic cytosol content of ornithine decarboxylase, thymidine kinase, and estrogen receptor was significantly greater (P<0.05) in rats pretreated with cyclosporine than in those treated with the vehicle alone. A significant increase in ornithine decarboxylase and thymidine kinase activities occurred after partial hepatectomy in both the cyclosporine-pretreated and vehicle-pretreated animals. The absolute levels for each parameter were also greater in the cyclosporine-treated animals than in the vehicle-treated controls at 24 hr after partial hepatectomy (P<0.05). The pattern of change in the hepatic cytosolic content of estrogen and androgen receptors in both groups of animals was comparable with those described previously for regenerating liver. These data suggest that cyclosporine may predispose the liver to respond to either a regenerative signal or perceived need and thereby fortuitously enhance liver graft performance after successful surgical implantation.Supported by Research Grants from the Veterans Administration and Project Grant AM 29961 and from the NIDDK 32556 and from the NIAAA AA06601.Dr. D. Kahn was supported by a grant from the Medical Research Council of South Africa.     

5-羟色胺2A受体阻断剂对大鼠肝脏再生的影响

目的观察5-羟色胺2A受体阻断剂酮色林对大鼠肝部分切除后肝再生的影响,了解5-羟色胺及其受体在肝脏再生中的作用。方法 80只雄性Wistar大鼠随机分为实验组和对照组。采用肝大部分切除术建立肝再生模型,术后16 h分别给予腹腔内注射酮色林（实验组）和生理盐水（对照组）,采用免疫组化及流式细胞技术动态观察并比较两组大鼠术后24、36、48、72 h肝脏Ki67、增殖细胞核抗原的表达情况。结果大鼠肝大部切除术后24、36 h肝脏表达Ki67、增殖细胞核抗原最为活跃,而后表达逐渐下降。实验组大鼠肝脏表达Ki67、增殖细胞核抗原较对照组显著下降（P〈0.05）。结论 5-羟色胺2A受体阻断剂酮色林显著抑制大鼠肝大部切除术后的肝脏再生,说明5-羟色胺具有一定的促进肝再生的作用,2A受体是其重要的信号传导受体之一。     

Effects of heparin on hepatic regeneration and function after partial hepatectomy in rats

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Effect of serotonin receptor 2 blockage on liver regeneration after partial hepatectomy in the rat liver.

The effect of serotonin receptor 2 blockade (5-HT(2)) on liver regeneration after 30-34% and 60-70% partial hepatectomy in the rat liver was investigated. Materials and methods: Male Wistar rats were subjected to 60-70% (group I) and 30-34% (group II) partial hepatectomy. Serotonin receptor 2 blockade was exerted by intraperitoneal administration of ketanserin at different doses and time points after partial hepatectomy. The rats of all groups were killed at different time points until 96 h after partial hepatectomy. The rate of liver regeneration was evaluated by the mitotic index in hematoxylin and eosin sections, the immunochemical detection of Ki67 and proliferating cell nuclear antigens, the rate of [(3)H]-thymidine incorporation into hepatic DNA and liver thymidine kinase enzymatic activity. Results: Liver regeneration peaked at 24 and 32 h after partial hepatectomy in 60-70% hepatectomized rats. In 30-34% hepatectomized rats liver regeneration peaked at 60 h, whereas low rates of regenerative activity were observed between 24 and 72 h after partial hepatectomy. Ketanserin administration arrested liver regeneration only when administered at 16 h after 60-70% partial hepatectomy. Ketanserin also abrogated the observed peak of regenerative activity at 60 h in 30-34% hepatectomized rats when administered at 52 h after partial hepatectomy. All indices of liver regeneration were affected by ketanserin administration. Conclusions: Serotonin receptor 2 blockade can arrest liver regeneration only when administered close to G1/S transition point, and that while serotonin may be a cofactor for DNA synthesis, it does not play a role in initiation of liver regeneration.     

Effect of cyclosporine on liver regeneration after orthotopic reduced-size hepatic transplantation in the rat

These experiments were undertaken to study the effects of cyclosporine A (CsA) on liver regeneration after an isogeneic orthotopic reduced-size hepatic transplantation (RSHT) in rats. Male Wistar rats were treated with or without a daily injection of CsA beginning 24 hr before surgery and were subjected to a 68% partial hepatectomy. A isogeneic orthotopic reduced-size hepatic transplantation was performed in recipient rats pretreated with or without CsA. A daily injection of CsA was continued until the recipient rats were sacrificed. Animals were sacrificed at various time points (12, 24, 36, 48, and 72 hr) postoperatively. The incorporation of bromodeoxyuridine (BrdU) into the DNA of the remnant hepatocytes was evaluated by immunohistochemical staining with a monoclonal antibody against BrdU. CsA (10 mg/kg/day) significantly augmented BrdU incorporation into hepatocytes after hepatectomy. The maximum labeling index (LI) was observed at 24 hr after hepatectomy. In contrast, the maximum LI in the recipient rats not receiving CsA was seen at 36 hr after RSHT, and 10 mg/kg/day of CsA decreased the LI at 36 hr after RSHT. A lower dose of CsA (3 mg/kg/day), however, significantly increased the LI in the recipient rats (P<0.01), and it reached a peak at 24 hr after RSHT when compared to the transplant recipients not receiving CsA. The time course of the increase in the LI in the transplant recipient rats receiving 3 mg/kg/day of CsA was similar to that observed in the rats after hepatectomy. This dosage improved the delay in the reduced-size hepatic transplant LI reaching its peak. These findings suggest that after RSHT the liver graft is more sensitive to both hepatotrophic and hepatotoxic effects of CsA.This work was supported in part by Mika Fund.     

Estradiol and testosterone levels in patients undergoing partial hepatectomy

In five adult male patients undergoing a 40–60% partial hepatectomy, serum sex hormone levels before and after hepatic resection were determined. Blood was drawn immediately prior to each surgical procedure and at specified time points postoperatively. Compared to hormone levels found prior to surgery, following major hepatic resection, estradiol levels increase at 24 and 48 hr, while testosterone levels decline, being significantly reduced at 96 and 144 hr. These data demonstrate that adult males who undergo a 40–60% partial hepatectomy experience alterations in their sex hormone levels similar to those observed in male rats following a 70% hepatectomy. These changes in sex hormone levels have been associated in animals with an alteration of the sex hormone receptor status of the liver that is thought to participate in the initiation of the regenerative response. These studies suggest, but do not prove, that in man, as in the case of the rat, sex hormones may participate in the initiation of or at least modulate in part the regenerative response that occurs following a major hepatic resection.Supported by research grants from the Veterans Administration; project grant DK29961 from the National Institutes of Health, Bethesda, Maryland; by grant 87/0129144 from the Consiglio Nazionale dell Ricerche, Italy; and by grant AA04425 from NIH.     

Direct inhibitory effect of estrogen on LH-stimulated androgen synthesis by ovarian cells cultured in defined medium

The direct inhibitory effect of estrogen on ovarian androgen synthesis was investigated. When primary cultures of rat ovarian theca-interstitial cells were grown in defined medium with LH there was a marked increase in androgen synthesis of which 98% was androsterone (control = 11 ± 2 ng; LH = 1219±217 ng/ml/10⁶ cells). Diethylstilbestrol (DES), estrone (E₁), estradiol (E₂), and estriol (E₃) inhibited LH-stimulated androsterone synthesis by 81%, 81%, 81%, and 47%, respectively. The ED₅₀'s of the estrogens were: DES = 4.2±2.l × 10^?9M; E₁ = E₂ = 9.5±2.4 × 10^?8 M; and E₃ = 3.8±2.6 × 10^?7 M. The estrogen effect was very rapid ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ = 10 min) and long-lasting. Metabolic studies revealed that estrogen inhibited androsterone, androstenedione, 5α-androstane-3α, 17β-diol, and testosterone accumulation by 80%, dehydroepiandrosterone and 17α-hydroxypregnenolone by 40%, 17α-hydroxyprogesterone by 30%, while pregnenolone and progesterone were unchanged. These results prove, for the first time, that estrogen can directly inhibit LH-stimulated androgen production in ovarian theca-interstitial cells and suggest that mechanism involves, at least in part, a rapid selective inhibition of the 17α-hydroxylase/C_17–20 desmolase activities.     

Quantitation of estrogen and androgen receptors in hepatocellular carcinoma and adjacent normal human liver

Sex hormones have been shown to influence the development and course of several liver diseases. The worldwide predominance of hepatocellular carcinoma (HCC) in males has led to the suggestion that this disease might be hormone-responsive. Therefore, the hepatic estrogen (ER) and androgen receptor (AR) status of liver specimens from such patients was investigated. Samples were obtained from three female and six male patients undergoing liver resection; in each case, a small sample of both the tumor and adjacent normal tissue was collected. All patients had primary hepatocellular carcinoma without cirrhosis. In most cases, the tumor and the normal specimen had an equivalent content of cytosolic ER; however, three of the tumor samples (one female and two male) displayed considerably elevated cytosolic ER levels as compared to that of the normal tissue. In every sample, the tumor contained less nuclear ER than did the normal liver. When AR was measured, tumors of three patients (one female and two male) demonstrated a twofold elevation in cytosolic AR as compared to adjacent normal tissue. In the two male patients, an approximately twofold greater nuclear AR was found. Two other samples from male patients showed a modest elevation of cytosolic AR in the tumors. The patients whose tumors showed elevations in ER were not the same patients as those in whom the AR was elevated. Thus, these studies indicate that certain, but not all, specimens of HCC demonstrate either elevated ER or AR and suggest that a determination of receptor content might be useful prior to initiation of certain antihormone therapies.Presented at the Proceedings of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.     

Circadian rhythm of hepatic cytosolic and nuclear estrogen and androgen receptors

Mammalian liver is a sex steroid-responsive tissue. The effects of these hormones presumably are mediated by hepatic estrogen receptors (ER) and androgen receptors (AR). Serum levels of sex hormones display circadian rhythms. Further, estrogens and androgens are commonly administered; administration of these agents is associated frequently with liver disease. Therefore, we investigated whether the cytosolic and nuclear sex steroid receptors also display a similar circadian rhythm, and whether variations occurred in the distribution of receptors between cytosolic and nuclear compartments. Animals were killed every 4 h from midnight till the following midnight; cytosolic and nuclear levels of both ER and AR were measured. Cytosolic ER reached a maximum level at 4 AM, and a minimum at 8 PM and midnight of both days. Nuclear ER was highest at 8 AM and lowest at 4 PM and 8 PM, a pattern which parallels variations in serum estradiol levels. Cytosolic AR was highest at 8 PM and lowest at midnight and 4 AM. Nuclear AR was highest at 4 AM and lowest at 4 PM and 8 PM. The highest level of nuclear AR does not correspond to the maximum serum testosterone level, which occurred at 4 PM. The total hepatic content of both ER and AR was not constant over the 24-h period, but varied considerably with time of day. These studies suggest that both ER and AR show a distinct circadian rhythm in subcellular compartmentalization, and that total hepatic content of ER and AR varies significantly during a 24-h period.     

Hepatic estrogen and androgen receptors and binding proteins in streptozotocin-diabetic male Wistar rats

Summary We have previously shown that there are decreases in the sex differences seen in certain hepatic drug and steroid metabolising enzymes in rats with early (4 day) streptozotocin-induced diabetes [31]. We postulated that hepatic sex hormone receptors or binding proteins might be involved in modulation of the sex differences noted in metabolism. In the present study, we measured the binding kinetics of the hepatic cytosolic estrogen receptor and androgen receptor, along with the high capacity-low affinity estrogen binding protein. At 4 or 10 days post-streptozotocin (60 mg/kg intravenously), there was no change in the maximum binding capacity of the estrogen receptor, nor in the hormone affinity of any of the three proteins. However, the binding capacity of the androgen receptor and estrogen binding protein in the diabetic animals was decreased to less than half of control levels. This effect could not be reversed by hormone replacement with any of the following regimens: protamine zinc insulin, 10 U/kg subcutaneously once a day; Toronto insulin, 15 U/kg subcutaneously twice a day; testosterone enanthate, 1 mg/kg s.c. once a day; triiodothyronine, 30 g/kg s.c. daily; ovine growth hormone: 0.02 U/h s.c., 30 g s.c. 7 times daily, 30 g i.v. 4 times daily; or various combinations of these hormones. Stress, such as 4 intravenous injections of saline per day, was noted to decrease the binding capacity of the estrogen binding protein. Therefore, we measured the basal serum corticosterone levels, which were not significantly different from control values in untreated or insulin-treated diabetic rats. In addition, the hepatic cytosolic glucocorticoid receptor capacity was not significantly changed in the diabetic animals. This provides evidence that at 4 days post-streptozotocin, the diabetic state is not so stressful as to result in major alterations in these two parameters.In summary, because insulin is known to restore the sex differences in hepatic drug and steroid metabolism to control levels but does not restore the capacity of the cytosolic androgen receptor or estrogen-binding protein, we conclude that they are not of primary importance in regulation of the metabolic enzymes.     

Changes in circulating cytokine levels and lymphocyte subsets in healthy liver donors after partial hepatectomy

Aim: Studies of animal models have determined that liver regeneration after partial hepatectomy is mediated by a various cytokines. The aim of the present study was to evaluate the levels of these cytokines and subsets of circulating lymphocytes in healthy humans after partial hepatectomy. Methods: Four individuals underwent partial hepatectomy for living-related donor liver transplantation. We also evaluated for comparison, three patients with myoma uteri who underwent hysterectomy. Blood samples were obtained before surgery and on postoperative days (PD) 1, 3, and 7. Serum levels of hepatocyte growth factor, interleukin (IL)-6, -10, and plasma levels of transforming growth factor beta were measured. Results: Increased circulating levels of hepatocyte growth factor and transforming growth factor beta were observedafter hepatectomy. The levels of IL-6 and IL-10 peaked on PD 1. Circulating white blood cell counts increased remarkably, whereas lymphocyte count decreased particularly on PD 1 and 3. CD4/CD8 and T-helper cell (Th)1/Th2 ratios were still decreased on PD 7. The percentage of natural killer cells was increased on PD 1. Partial hepatectomy in healthy humans leads not only to decreased lymphocyte counts, but also to remarkable changes in lymphocyte subsets. Conclusion: These findings suggest that immune suppression after partial hepatectomy involves decreases in CD4(+) helper T cells, particularly Th1 cells.     

Amelioration of hepatic ischemia/reperfusion injury in the remnant liver after partial hepatectomy in rats

BACKGROUND AND AIMS: Reactive oxygen species have been implicated in the development of hepatic ischemia/reperfusion (I/R) injury. I/R injury remains an important problem in massive hepatectomy and organ transplantation. The aim of this study was to examine the effect of edaravone, a newly synthesized free radical scavenger, on I/R injury in the remnant liver after partial hepatectomy in rats. METHODS: Partial (70%) hepatic ischemia was induced in rats by occluding the hepatic artery, portal vein, and bile duct to left and median lobes of liver. Total hepatic ischemia (Pringle maneuver) was induced by occluding the hepatoduodenal ligament. Edaravone was intravenously administered to rats just before reperfusion and partial (70%) hepatectomy was performed just after reperfusion. RESULTS: Edaravone significantly reduced the increases in the levels of serum alanine aminotransferase and aspartate aminotransferase in rats with liver injury induced by 90-min of partial ischemia followed by 120-min of reperfusion. Histopathological analysis showed that edaravone prevented inflammatory changes in the livers with I/R injury. Edaravone also decreased the levels of myeloperoxidase activity, which is an index of neutrophil infiltration, and interleukin-6 mRNA, which is a proinflammatory cytokine. Additionally, edaravone improved the survival rate in partial hepatectomy rats with I/R injury induced by the Pringle maneuver. CONCLUSIONS: Edaravone administration prior to reperfusion protected the liver against I/R injury. Edaravone also improved the function of the remnant liver with I/R injury after partial hepatectomy. Therefore, edaravone may have applicability for major hepatectomy and liver transplantation in the clinical setting.     

颅内动脉瘤患者瘤壁雌激素和雄激素受体的表达及其作用

目的探讨颅内动脉瘤壁雌激素和雄激素受体表达水平的意义。方法前瞻性纳入2007年11月至2016年6月在四川大学华西医院接受开颅手术夹闭颅内动脉瘤的患者32例,获得颅内动脉瘤壁19个,颞浅动脉分支26个,共45个合格标本。采用免疫组化法检测颞浅动脉分支和颅内动脉瘤壁平滑肌层及内皮细胞的雌激素受体-α、β及雄激素受体表达水平。以Image Pro Plus6.0软件分析检测阳性细胞表达水平积分光密度值。统计学分析采用χ2检验,成组Mann-Whitney秩和检验。结果动脉瘤壁的雌激素受体-α、β表达中位数(M)与四分位距(P25,P75)分别为3 049(2 112,5 554)和4 364(2 314,5 667),低于颞浅动脉分支的6 544(3 507,10 103)和6 972(5 694,10 024)。动脉瘤壁雄激素受体表达水平3 299(1 375,4 895),高于颞浅动脉分支的1 130(794,1 922),两组比较差异均有统计学意义(均P0.05)。结论脑血管壁雌激素受体-α、β表达水平的降低,雄激素受体表达水平的升高,可能促进了颅内动脉瘤的进展,但具体机制尚需进一步研究。     

Effects of estrogen and androgen deprivation on the progression of non-alcoholic steatohepatitis (NASH) in male Sprague–Dawley rats

Aim:  We studied the mechanisms of estrogen/androgen involvement in the induction of NASH by treating Sprague–Dawley (SD) rats fed with a normal or high fat (HF) diet by depriving them of endogenous estrogens/androgens.
Methods:  Male adult SD rats ( n  = 10/group) on normal or HF diets were treated for 75 days either with tamoxifen (Tam) or flutamide (Flu) or Tam + Flu in order to induce NASH. We analyzed histopathologically the liver samples from the treated groups for NASH, checked the serum biochemical and lipid profile markers and finally analyzed the signal pathways underlying the molecular mechanisms for the induction process of NASH.
Results:  Deprivation of endogenous estrogens and/or androgens (Tam or Flu or Tam + Flu) without the HF diet did not induce NASH. Tam or Tam + Flu induced NASH, compared to milder lesions without fibrosis in HF diet and Flu-treated liver. Serum alanine aminotransferase or lipid profile markers further proved the Tam, Flu or Tam + Flu effects on the induction of NASH in conjunction with a HF diet. Tam treatment predominantly downregulated the ERα and FAS and upregulated UCP2 and TNF-α.
Conclusions:  Deprivation of endogenous estrogen/androgens in conjunction with a HF diet may induce NASH where the downregulated ERα and FAS, and upregulated UCP2 and TNF-α could be involved in their molecular pathomechanism pathways. These results could suggest the potential negative roles of estrogenic/androgenic depriving compounds in the induction of NASH, along with obesity.     

Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation.

BACKGROUND: Expression of neurotrophins (NTs) and their receptors is increased during hepatic regeneration, but their role is not well understood. METHODS: NTs and their receptors were investigated by RT-PCR and immunostaining in regenerating livers after two-thirds hepatectomy (PH) and in hepatocytes and hepatic stellate cells (HSCs) isolated from regenerating livers in mice. Induction of apoptosis after treatment with NGF and the expression of alpha-smooth muscle actin (SMA), interleukin 6 (IL-6) and hepatocyte growth factor (HGF) were also investigated in regenerating HSCs. RESULTS: Nerve growth factor (NGF) and p75 NT receptor (p75NTR) mRNA were elevated after PH, while other NTs and NT receptors showed no remarkable change. NGF was detected in regenerating hepatocytes, but not in normal hepatocytes. Regenerating HSCs expressed increased p75NTR and SMA in vivo and showed an activated phenotype and the high expression of HGF and IL-6 in vitro. Enhanced cell death was seen in HSCs, both from normal and regenerating liver, after treatment with NGF. CONCLUSIONS: Although activated HSCs may produce the factors that regulate liver regeneration, the de novo NGF production by regenerating hepatocytes may induce the death of activated HSCs via p75NTR, leading to termination of hepatic regeneration.     

Effect of in situ hypothermic perfusion on intrahepatic pO2 and reactive oxygen species formation after partial hepatectomy under total hepatic vascular exclusion in pigs

Abstract: Aim: This study examined attenuation of ischemia and reperfusion (I/R) induced liver injury during liver resections by hypothermic perfusion of the liver under total hepatic vascular exclusion (THVE). Method: Reactive oxygen species (ROS) formation, microcirculatory integrity and endothelial cell damage were investigated. Left hemihepatectomy (LHX) was performed without in situ perfusion (control‐LHX, n = 5) or with concomitant in situ perfusion with hypothermic (4 °C) Ringer‐glucose (cold‐LHX, n = 5) or normothermic (38 °C) Ringer‐glucose (warm‐LHX, n = 5). Glutathione (GSH) and malondialdehyde (MDA) concentrations, tissue pO₂ levels and hyaluronic acid (HA) uptake capacity were determined. Results: After cold, warm and control‐LHX, 24 h survival was 5/5, 0/5 and 3/5, respectively. GSH levels were best preserved after cold‐LHX during reperfusion. MDA levels increased in all groups without significant differences between the groups during reperfusion. Tissue pO₂ levels increased after cold‐LHX whereas after warm‐LHX and control‐LHX, pO₂ levels decreased during reperfusion. HA uptake capacity remained normal after cold‐LHX. After warm‐LHX and control‐LHX, HA uptake capacity decreased after 6 h of reperfusion but recovered after 24 h of reperfusion in the control‐LHX group. Conclusion: Moderate hypothermic perfusion protects the liver from I/R injury during LHX under THVE. This protective effect depended on maintenance of liver microcirculation rather than a reduction in ROS formation.     

生长激素对鼠部分肝切除术后肝再生影响

目的　探讨生长激素对 70 %肝切除后肝再生的影响。方法　 60只SD大鼠随机分为对照组及生长激素组 ,按Higgins方法行 70 %肝切除术 ,术后给药并分批于术后 6、2 4、48、72、96h处死 ,作如下比较 :①残肝肝重 ;②增殖细胞核抗原 (PCNA)标记指数 ;③图像定量分析法测量PCNA阳性产物面积及灰度值。结果　与对照组比较 ,生长激素组残肝肝重、PCNA标记指数、PCNA阳性产物面积在术后均显著增高 (P <0 .0 5 ) ,而灰度值则显著降低 (P <0 .0 5 )。结论　生长激素具有强烈促进肝细胞增殖和刺激肝再生的作用