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目的探讨外周苯二氮受体(peripheral benzodiaz-epine receptor,PBR)在调控心肌线粒体通透性转换(mito-chondrial permeability transition,MPT)中的作用。方法分离SD大鼠心肌线粒体,电镜观察其形态证实线粒体结构完整性。将线粒体分别和不同浓度(50,100,200μmol·L-1)的PBR拮抗剂PK11195孵育,部分线粒体和100mmol·L-1PK11195孵育之前5min加入5μmol·L-1MPT抑制剂环孢菌素A(CsA组)。不给任何处理的线粒体为阴性对照(Con组),单纯给150μmol·L-1Ca2+作阳性对照(Ca2+组)。分光光度法测520nm处吸光度的改变反映MPT变化,电镜观察线粒体超微结构改变,Western blot检测线粒体细胞色素C(CytoC)释放。结果PK11195剂量依赖性诱发MPT,不同浓度组间比较差异有显著性(P<0.05,P<0.01);PK11195导致线粒体出现空泡变性、线粒体肿胀、线粒体脊膜破裂,CytoC释放明显增多(vsCon组,P<0.01);CsA能阻断PK11195的上述作用,CsA组线粒体超微结构基本完好,线粒体CytoC释放较少(vs100μmol·L-1组,P<0.05)。结论PBR参与调控大鼠心肌MPT,其拮抗剂PK11195可剂量依赖性诱导心肌MPT,导致线粒体超微结构损伤和线粒体CytoC释放。
Objective To investigate the role of peripheral benzodiazepin receptor (PBR) in the regulation of mito-chondrial permeability transition (MPT). Methods Myocardial mitochondria isolated from SD rats were observed by electron microscopy to confirm the structural integrity of mitochondria. Mitochondria were incubated with PB11 antagonist PK11195 at different concentrations (50,100 and 200μmol·L -1) respectively. Some mitochondria and 5μmol·L -1 MPT inhibitor cyclosporine A (CsA group) were added 5 min before incubation with 100 mmol·L -1 PK11195. Mitochondria without any treatment were negative control group (Con group), and 150μmol·L-1Ca2 + was used as positive control (Ca2 + group). The change of absorbance at 520 nm by spectrophotometry reflected the change of MPT, the ultrastructure of mitochondria was observed by electron microscope, and the release of mitochondrial CytoC was detected by Western blot. Results PK11195 induced MPT in a dose-dependent manner (P <0.05, P <0.01). PK11195 resulted in vacuolar degeneration, mitochondria swelling, mitochondrial membrane rupture and CytoC release (vsCon group , P <0.01). CsA could block the above effects of PK11195. The ultrastructure of mitochondria in CsA group was basically intact and the release of CytoC in mitochondria was less (vs 100μmol·L-1, P <0.05). Conclusions PBR is involved in the regulation of MPT in rat myocardium. Its antagonist PK11195 induces MPT in a dose-dependent manner, resulting in mitochondrial ultrastructure damage and mitochondrial CytoC release.