mTOR signaling in liver regeneration: Rapamycin combined with growth factor treatment.
mTOR signaling in liver regeneration: Rapamycin combined with growth factor treatment.
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DOI：
10.5500/wjt.v3.i3.36
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发表时间：
2013-09-24
期刊：
影响因子：
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通讯作者：
de Jonge, Jeroen
中科院分区：
文献类型：
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作者：
Fouraschen, Suomi Mg;de Ruiter, Petra E;de Jonge, Jeroen
目的：在手术切除模型中研究哺乳动物雷帕霉素靶蛋白（mTOR）抑制对肝再生和自噬的影响。对C57 BL/6小鼠进行70%的部分肝切除术（PH），并每24小时用mTOR抑制剂雷帕霉素（rapamycin Omg/kg/天）和类固醇地塞米松（2.0mg/kg/天）的磷酸盐缓冲盐水（PBS）溶液中或与单独的PBS作为媒介物对照。在免疫抑制剂组中，在PH前4小时和PH后24小时，用PBS中的人重组白细胞介素6（IL-6; 500 μ g/kg/天）和肝细胞生长因子（HGF; 100 μ g/kg/天）的组合皮下处理部分组。PH后2、3、5 d处死动物，取肝组织和血液进行分析。5-溴-2 '-脱氧尿苷（BrdU）的免疫组织化学染色用于定量肝细胞增殖。Western blotting检测肝微管相关蛋白1轻链3（LC 3）-II蛋白表达作为自噬的标志。在伊拉斯谟大学医学中心的临床化学核心设施中，通过实时逆转录聚合酶链反应检测了增殖、炎症和血管生成相关基因的肝脏基因表达水平，并分析了血清胆红素和转氨酶水平。mTOR抑制显著抑制再生，表现为肝细胞增殖减少（2% vs 12%，P < 0.01）;第5天0.8% vs 1.4%，P = 0.02）和肝脏重量重建（第3天为初始总肝重的63% vs 76%，P = 0.04），此外，血清转氨酶水平升高（第2天天冬氨酸转氨酶641 U/L vs 185 U/L，P = 0.02）。自噬标志物LC 3-II的表达在正常肝再生期间减少，在mTOR抑制后增加（在第2天增加46%，P = 0.04）。肝脏基因表达显示炎症相关反应增加[肿瘤坏死因子（TNF）-α在第2天上调3.2倍，P = 0.03; IL-1 Ra在第2天上调6.0倍，在第5天上调42.3倍，P < 0.01]，细胞周期进展和血管生成相关因子表达减少（第2天HGF降低40%;第2天和第5天血管内皮生长因子受体2降低50%;第2天血管生成素1降低60%，所有P ≤ 0.01）。用再生刺激细胞因子IL-6和生长因子HGF治疗可以克服对肝脏重量的抑制作用（第3天为初始总肝脏重量的75%，与单独免疫抑制相比P = 0.02，与对照相比P = 0.90），并部分逆转雷帕霉素引起的基因表达变化（第2天TNF-α和IL-1 Ra水平恢复至对照水平）。然而，肝细胞增殖，血清损伤标志物或自噬无显着变化被发现。结论：mTOR抑制严重损害肝再生和增加自噬后PH。这些影响部分逆转刺激IL-6和HGF途径。
AIM: To investigate the effects of mammalian target of rapamycin (mTOR) inhibition on liver regeneration and autophagy in a surgical resection model.METHODS: C57BL/6 mice were subjected to a 70% partial hepatectomy (PH) and treated intraperitoneally every 24 h with a combination of the mTOR inhibitor rapamycin (2.5 mg/kg per day) and the steroid dexamethasone (2.0 mg/kg per day) in phosphate buffered saline (PBS) or with PBS alone as vehicle control. In the immunosuppressant group, part of the group was treated subcutaneously 4 h prior to and 24 h after PH with a combination of human recombinant interleukin 6 (IL-6; 500 mug/kg per day) and hepatocyte growth factor (HGF; 100 mug/kg per day) in PBS. Animals were sacrificed 2, 3 or 5 d after PH and liver tissue and blood were collected for further analysis. Immunohistochemical staining for 5-Bromo-2'-deoxyuridine (BrdU) was used to quantify hepatocyte proliferation. Western blotting was used to detect hepatic microtubule-associated protein 1 light chain 3 (LC3)-II protein expression as a marker for autophagy. Hepatic gene expression levels of proliferation-, inflammation- and angiogenesis-related genes were examined by real-time reverse transcription-polymerase chain reaction and serum bilirubin and transaminase levels were analyzed at the clinical chemical core facility of the Erasmus MC-University Medical Center.RESULTS: mTOR inhibition significantly suppressed regeneration, shown by decreased hepatocyte proliferation (2% vs 12% BrdU positive hepatocyte nuclei at day 2, P < 0.01; 0.8% vs 1.4% at day 5, P = 0.02) and liver weight reconstitution (63% vs 76% of initial total liver weight at day 3, P = 0.04), and furthermore increased serum transaminase levels (aspartate aminotransferase 641 U/L vs 185 U/L at day 2, P = 0.02). Expression of the autophagy marker LC3-II, which was reduced during normal liver regeneration, increased after mTOR inhibition (46% increase at day 2, P = 0.04). Hepatic gene expression showed an increased inflammation-related response [tumor necrosis factor (TNF)-alpha 3.2-fold upregulation at day 2, P = 0.03; IL-1Ra 6.0-fold upregulation at day 2 and 42.3-fold upregulation at day 5, P < 0.01] and a reduced expression of cell cycle progression and angiogenesis-related factors (HGF 40% reduction at day 2; vascular endothelial growth factor receptor 2 50% reduction at days 2 and 5; angiopoietin 1 60% reduction at day 2, all P ≤ 0.01). Treatment with the regeneration stimulating cytokine IL-6 and growth factor HGF could overcome the inhibitory effect on liver weight (75% of initial total liver weight at day 3, P = 0.02 vs immunosuppression alone and P = 0.90 vs controls) and partially reversed gene expression changes caused by rapamycin (TNF-alpha and IL-1Ra levels at day 2 were restored to control levels). However, no significant changes in hepatocyte proliferation, serum injury markers or autophagy were found.CONCLUSION: mTOR inhibition severely impairs liver regeneration and increases autophagy after PH. These effects are partly reversed by stimulation of the IL-6 and HGF pathways.