缺氧再给氧对肝细胞膜纤维形肌动蛋白微丝损伤的分子机制

Molecular mechanism of hypoxia-reoxygenation injury to hepatocyte membrane F-actin microfilaments

背景:建立人肝细胞体外缺氧再给氧损伤模型,模拟移植器官缺血再灌注损伤,从细胞分子水平探讨缺血再灌注所致纤维形肌动蛋白微丝损伤的机制,目前尚无相关研究报道。目的:分析缺氧再给氧对肝细胞膜纤维形肌动蛋白微丝损伤的分子机制。方法:建立体外大鼠肝细胞缺氧再给氧模型。肝细胞随机分为正常对照组、缺氧再给氧组。缺氧再给氧组又分为缺氧再给氧2 h、缺氧再给氧4 h、缺氧再给氧6 h组(分别为细胞缺氧3 h后再给氧2,4,6 h)。光镜观察细胞形态,电镜观察超微结构的改变,共聚焦激光显微镜观察纤维形肌动蛋白微丝含量变化,Real-time PCR检测HSP27、丝切蛋白基因的转录水平,Western blot检测HSP27、丝切蛋白蛋白的表达水平。结果与结论:光镜下缺氧再给氧各组梭形细胞显著增多,且脱落细胞明显增多;透射电镜下缺氧再给氧组与对照组相比内质网数量明显减少,线粒体密度深,糖原消失;共聚焦激光显微镜可见缺氧再给氧组纤维形肌动蛋白纤维荧光紊乱,形态明显改变,荧光染色明显减弱,平均荧光强度缺氧再给氧各组明显低于对照组(P<0.05);Real-time PCR和Western blot检测H/R各组HSP27、丝切蛋白基因转录和蛋白表达水平显著低于正常对照组(P<0.05)。表明缺氧再给氧可能是通过抑制肝细胞内HSP27、丝切蛋白的蛋白表达和基因转录,从而影响纤维形肌动蛋白微丝的正确装配以及减弱纤维形肌动蛋白的正常循环,进而改变纤维形肌动蛋白微丝骨架。

BACKGROUND： Human hepatocyte models of hypoxia-reoxygenation injury are established to simulate the ischemia/reperfusion injury of transplanted organ. There have been no research reports addressing the molecular mechanism underlying hypoxia-reoxygenation injury to hepatocyte membrane F-actin microfilaments. OBJECTIVE： To analyze the molecular mechanism of hypoxia-reoxygenation injury to hepatocyte membrane F-actin microfilaments.METHODS： Rat hepatocyte models of hypoxia-reoxygenation in vitro were established and then randomly divided into control and hypoxia-reoxygenation groups. The hypoxia-reoxygenation group was also subdivided into three subgroups： hypoxia-reoxygenation-2, 4 and 6 hours（after 3 hours of hypoxia, hepatocytes were given oxygen for 2, 4 and 6 hours respectively）. Cell morphology was observed by light microscope, ultrastructuralchanges by transmission electron microscope and the change in F-actin microfilament content by confocal laser microscopy. HSP27 and cofilin gene and protein levels were determined by real-time polymerase chain reaction and western blot assay respectively. RESULTS AND CONCLUSION： Light microscope observations showed that spindle cells and exfoliated cells significantly increased in hypoxia-reoxygenation group. Transmission electron microscope observations showed that in the hypoxia-reoxygenation group, the amount of endoplasmic reticulum significantly decreased, mitochondrial density increased and glycogen disappeared compared with the control group. Confocal laser microscopy observations showed that in the hypoxia-reoxygenation group, F-actin microfilament fluorescence disordered, F-actin microfilament morphology significantly changed, staining intensity significantly attenuated and mean fluorescence intensity was significantly lower than that in control group（P 〈0.05）. Real-time PCR and western blot detection results showed that HSP27, cofilin gene and protein expression levels in the hypoxia-reoxygenation group were significantly lower than in the control group（P 〈0.05）. These results demonstrate that hypoxia-reoxygenation affects the correct assembly of F-actin microfilaments and weakens the normal cycle of F-actin microfilaments through inhibiting the protein expression of HSP27 and cofilin, and gene transcription in hepatocytes, thereby changing the skeleton of F-actin microfilaments.