糖原合成酶激酶3β介导心房钠尿肽对H9c2心肌细胞线粒体保护作用的研究

Protective mechanism of ANP on mitochondria of H9c2 myocardial cells via GSK-3β

摘要:

目的  探讨心房钠尿肽（ANP）对H9c2心肌细胞缺血再灌注损伤的保护作用机制。方法  采用四甲基罗丹明乙酯（TMRE）荧光染料和激光共聚焦显微镜成像技术测定H9c2心肌细胞TMRE荧光强度的变化，即线粒体膜通透性转换孔（mPTP）开放程度。利用双氧水H2O2诱导H9c2心肌细胞线粒体mPTP的开放，预处理不同浓度的ANP后，观察ANP对mPTP开放的影响；同时利用Western blot检测H9c2心肌细胞的糖原合成酶激酶3β（GSK-3β Ser9）磷酸化程度，即失活程度。利用激活型GSK-3β质粒转染的H9c2心肌细胞（GSK-3β-S9A-HA），来观察ANP对GSK-3β-S9A-HA的线粒体mPTP开放程度。结果  与对照组（600μmol/L H2O2）比较，0.01、0.10、1.00和10.00 nmol/L ANP明显抑制H2O2对TMRE荧光强度的衰减效应（P <0.05），其中1.00 nmol/L的ANP效应最强。Western blot检测结果显示，0.01、0.10、1.00和10.00 nmol/L ANP明显增强GSK-3β Ser9的磷酸化（P <0.05），即抑制GSK-3β的活性。利用GSK-3β-S9A-HA后发现，ANP（1.0 nmol/L）不能抑制mPTP开放（与H9c2比较P <0.05）。结论  ANP通过调节GSK-3β活性来抑制H9c2心肌细胞mPTP的开放，从而保护H9c2心肌细胞的缺血再灌注损伤。

Abstract:

Objective To investigate protective mechanism of atrial natriuretic peptide (ANP) on H9c2 myo-cardial cell mitochondria via glycogen synthase kinase 3β (GSK-3β). Methods The change of mitochondrial membrane potential was determined using fluorescent dye tetramethylrhodamine ester (TMRE) and laser confocal microscopy imaging technique. The open degree of mitochondrial permeability transition pore (mPTP) was reflected as TMRE fluorescence intensity and the GSK-3β Ser9 phosphorylation (deactivation degree) was detected by Western blot. Then the protective effect of ANP on H9c2 cells transfected with activated GSK-3β plasmid (GSK-3β-S9A-HA) was observed. Results Compared to the control group (600 μmol/L H2O2), 0.01, 0.10, 1.00 and 10.00 nmol/L ANP significantly inhibited attenuation effect of TMRE fluorescence intensity induced by H2O2 (P < 0.05); of which 1.00 nmol/L ANP had the strongest effect. It suggested that ANP may modulate the mPTP opening. Western blot result showed that 0.01, 0.10, 1.00, 10.00 and 100.00 nmol/L ANP significantly increased the level of phosphorylation of GSK-3β (P < 0.05); which indicated that ANP could inhibit the activity of GSK-3β; however, 1.00 nmol/L ANP was not able to inhibit mPTP opening in cells of GSK-3β-S9A-HA. Conclusions ANP prevents the mPTP opening by inactivating GSK-3β so as to protect H9c2 myocardial cells during ischemia-reperfusion injury.