Department of Hepatobiliary Surgery II, State Key Laboratory of Organ Failure Research, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China;Department of General Surgery, The Second Hospital of Shenzhen Baoan People's Hospital Group, Shenzhen, China.
Department of Hepatobiliary Surgery II, State Key Laboratory of Organ Failure Research, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China.
Turk J Gastroenterol. 2020 Feb;31(2):167-179. doi: 10.5152/tjg.2020.18507.
BACKGROUND/AIMS: Autophagy plays a positive role in the prevention of liver damage after hepatic ischemia-reperfusion injury (HIRI); however, the molecular mechanism is still a mystery. Understanding the molecular events behind this injury may have important implications for devising proper strategies for managing liver injury. This study investigated the effects of Frizzled-2 expression on autophagy as well as Ca2+ concentration and apoptosis in BRL-3A cells.
BRL-3A cells exposed to the hypoxia/reoxygenation (H/R) condition were used as an in vitro HIRI hepatic cell model. The transfection of Frizzled-2 small interfering RNA (siRNA) or expression vector was performed to silence or overexpress Frizzled-2 in BRL-3A cells. The intracellular Ca2+ concentration was monitored by the fluorescence of Ca+. Western blot was used to detect autophagy-related proteins and apoptotic marker Caspase-3. The cellular autophagosome was observed by a transmission electron microscope.
Beclin-1 and Atg7 expressions were considerably induced by H/R treatment, and this induction was attenuated by Frizzled-2 siRNA in BRL-3A cells. The LC3B-II/I ratio was inhibited by H/R treatment, although it was considerably induced by Frizzled-2 siRNA. The overexpression of Frizzled-2 induced intracellular Ca2+ concentration and expressed autophagy-related proteins and Caspase-3 except for the suppression of LC3B-II/I ratio in BRL-3A cells in the normoxia condition.
The overexpression of Frizzled-2 mimicked H/R treatment and suppressed autophagy activity, whereas Frizzled-2 siRNA induced cellular autophagy and attenuated the H/R-induced hepatic injury in BRL-3A cells. These developments suggest that Frizzled-2 siRNA protects hepatic BRL-3A cells from the injury of H/R via autophagy modulation.
背景/目的：自噬在肝缺血再灌注损伤（HIRI）后预防肝损伤中发挥积极作用；然而，其分子机制仍然是个谜。了解这种损伤背后的分子事件可能对制定适当的肝损伤管理策略具有重要意义。本研究探讨了卷曲蛋白 2（Frizzled-2）表达对 BRL-3A 细胞自噬以及 Ca2+浓度和细胞凋亡的影响。
将暴露于低氧/复氧（H/R）条件的 BRL-3A 细胞用作体外 HIRI 肝细胞模型。通过转染 Frizzled-2 小干扰 RNA（siRNA）或表达载体沉默或过表达 BRL-3A 细胞中的 Frizzled-2。通过 Ca2+荧光监测细胞内 Ca2+浓度。Western blot 用于检测自噬相关蛋白和凋亡标志物 Caspase-3。透射电镜观察细胞自噬体。
H/R 处理可显著诱导 Beclin-1 和 Atg7 的表达，而 Frizzled-2 siRNA 可减弱 BRL-3A 细胞中的这种诱导作用。LC3B-II/I 比值受 H/R 处理抑制，但 Frizzled-2 siRNA 可显著诱导该比值。在常氧条件下，Frizzled-2 的过表达可诱导细胞内 Ca2+浓度以及自噬相关蛋白和 Caspase-3 的表达，而 LC3B-II/I 比值受到抑制，Frizzled-2 过表达在 BRL-3A 细胞中模拟了 H/R 处理并抑制了自噬活性，而 Frizzled-2 siRNA 则诱导了细胞自噬，并减轻了 BRL-3A 细胞中 H/R 诱导的肝损伤。这些发现表明，Frizzled-2 siRNA 通过调节自噬来保护肝 BRL-3A 细胞免受 H/R 损伤。
