Oliveira André G, Andrade Viviane A, Guimarães Erika S, Florentino Rodrigo M, Sousa Pedro A, Marques Pedro E, Melo Flávia M, Ortega Miguel J, Menezes Gustavo B, Leite M Fatima
Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Liver Int. 2015 Apr;35(4):1162-71. doi: 10.1111/liv.12587. Epub 2014 Jun 2.
BACKGROUND & AIMS: Liver regeneration is a multistage process that unfolds gradually, with different mediators acting at different stages of regeneration. Calcium (Ca(2+) ) signalling is essential for liver regeneration. In hepatocytes, Ca(2+) signalling results from the activation of inositol 1,4,5-trisphosphate receptors (InsP3 R) of which two of the three known isoforms are expressed (InsP3 R-I and InsP3 R-II). Here, we investigated the role of the InsP3 R-I-dependent Ca(2+) signals in hepatic proliferation during liver regeneration.
Partial hepatectomy (HX) in combination with knockdown of InsP3 R-I (AdsiRNA-I) was used to evaluate the role of InsP3 R-I on liver regeneration and hepatocyte proliferation, as assessed by liver to body mass ratio, PCNA expression, immunoblots and measurements of intracellular Ca(2+) signalling.
AdsiRNA-I efficiently infected the liver as demonstrated by the expression of β-galactosidase throughout the liver lobules. Moreover, this construct selectively and efficiently reduced the expression of InsP3 R-I, as evaluated by immunoblots. Expression of AdsiRNA-I in liver decreased peak Ca(2+) amplitude induced by vasopressin in isolated hepatocytes 2 days after HX. Reduced InsP3 R-I expression prior to HX also delayed liver regeneration, as measured by liver to body weight ratio, and reduced hepatocyte proliferation, as evaluated by PCNA staining, at the same time point. At later stages of regeneration, control hepatocytes showed a decreased expression of InsP3 R, as well as reduced InsP3 R-mediated Ca(2+) signalling, events that did not affect liver growth.
Together, these results show that InsP3 R-I-dependent Ca(2+) signalling is an early triggering pathway required for liver regeneration.
肝再生是一个逐步展开的多阶段过程，不同的介质在再生的不同阶段发挥作用。钙（Ca(2+)）信号传导对肝再生至关重要。在肝细胞中，Ca(2+)信号传导源于肌醇1,4,5-三磷酸受体（InsP3R）的激活，已知的三种亚型中有两种表达（InsP3R-I和InsP3R-II）。在此，我们研究了InsP3R-I依赖性Ca(2+)信号在肝再生过程中肝脏增殖中的作用。
采用部分肝切除术（HX）联合InsP3R-I敲低（AdsiRNA-I）来评估InsP3R-I对肝再生和肝细胞增殖的作用，通过肝体质量比、PCNA表达、免疫印迹以及细胞内Ca(2+)信号测量进行评估。
如β-半乳糖苷酶在整个肝小叶中的表达所示，AdsiRNA-I有效地感染了肝脏。此外，通过免疫印迹评估，该构建体选择性且有效地降低了InsP3R-I的表达。HX后2天，肝脏中AdsiRNA-I的表达降低了离体肝细胞中血管加压素诱导的Ca(2+)峰值幅度。HX前InsP3R-I表达的降低也延迟了肝再生，通过肝体重比测量，同时通过PCNA染色评估降低了肝细胞增殖。在再生后期，对照肝细胞显示InsP3R表达降低，以及InsP3R介导的Ca(2+)信号传导减少，这些事件不影响肝脏生长。
总之，这些结果表明InsP3R-I依赖性Ca(2+)信号传导是肝再生所需的早期触发途径。
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