Objective To observe the expression of caspase-3 and transposition of Omi/HtrA2 in H9C2 by erythropoietin and/or Omi/HtrA2 silencing to explore the related anti-apoptotic mechanisms.Methods The cultured H9C2 cardiomyocytes were divided into control group,H/R group (anoxia 2 h and re-oxygenation 24 h), and different concentrations of EPO treatment groups.The release rate of lactate dehydrogenase (LDH)in cell supernatant was measured in each group.Expressions of cleaved caspase-3 and Omi/HtrA2 were measured by Western blot;then the transposition of Omi/HtrA2 between cytoplasm and mitochondria was observed.Specific siRNA interfering fragment was transfected into H9C2 cardiomyocytes by liposome method.Its silencing effect on Omi/HtrA2 was measured by RT-PCR and Western blot.The survival rate,release rate and expression of cleaved caspase-3 were measured.And the expression of Omi/HtrA2 was measured in cytoplasm and mitochondria in H9C2 (transposition of Omi/HtrA2 ).Results Compared with H/R group,the release of LDH and expression of cleaved caspase-3 were decreased; the transposition of Omi/HtrA2 from mitochondria to cytoplasm in H/R treatment groups was increased compared with control group,while that in EPO (20 IU/mL)group decreased.si-HtrA2 group transfected with siRNA showed a decreased release of LDH and expression of cleaved caspase-3 with all significant variances (P <0.05).Conclusion EPO exerts a cytoprotective effect by inhibiting the transposition of Omi/HtrA2 and hence the activation of caspases-3 pathway.

Objective: To investigate the effect and mechanism of helix B surface peptide (HBSP) on myocardial ischemia reperfusion injury (MIRI) in experimental mice.
Methods: The MIRI model was established by ligation of anterior descending coronary artery of the mice for 45 min and followed by corresponding treatment at 5 min before reperfusion. A total of 64 male ICR mice were randomly assigned to 4 group:①Sham group,②MIRI group, the mice received normal saline at 5 min before reperfusion,③HBSP group, MIRI mice received HBSP at 5 min before reperfusion and④HBSP+PD98059 group, MIRI mice received PD98059 (a speciifc blocker of ERK1/2) at 20 min before reperfusion and followed by HBSP at 5 min before reperfusion.n=16 in each group, all animals were treated for 2 hours. The area of myocardial infarction (MI) was detected by TTC-EB double staining method, the myocardial apoptosis rate was examined by TUNEL method, the levels of protein expression of ERK1/2 and phosphorylation of ERK1/2 were measured by Western blot analysis.
Results: Compared with MIRI group, HBSP group presented decreased MI area, decreased myocardial apoptosis rate and increased phopsphorylation level of ERK1/2, allP<0.05. Compared with HBSP group, HBSP+PD98059 group showed decreased phopsphorylation level of ERK1/2, increased myocardial apoptosis rate and increased MI area, allP<0.05.
Conclusion: HBSP may reduce the MI area via inhibiting myocardial apoptosis and therefore, protecting the experimental mice from MIRI; the mechanism might be related to the activation of ERK1/2 pathway.

Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.
Apoptosis ; drug effects ; Cell Line, Tumor ; Enzyme Activation ; drug effects ; Humans ; Ligands ; Molecular Docking Simulation ; Nuclear Receptor Subfamily 4, Group A, Member 1 ; genetics ; metabolism ; Oximes ; metabolism ; pharmacology ; Protein Conformation ; Proto-Oncogene Proteins c-akt ; metabolism ; Pyrazoles ; metabolism ; pharmacology ; Retinoid X Receptor alpha ; chemistry ; genetics ; metabolism ; Thiazoles ; metabolism ; pharmacology ; Transcription, Genetic ; drug effects ; Transcriptional Activation ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism