ABSTRACT
OBJECTIVE@#To observe the effect of heat shock factor 1 (HSF1) on heat stress-induced apoptosis in Raw264.7 macrophages.@*METHODS@#Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-HSF1 were exposed to heat stress (42.5 degrees C +/- 0.5 degrees C) for 1 h and recovered at 37 degrees C for 6, 9, 12, and 24 h respectively. Flow cytometry (FCM), Hoechst 33258 staining and DNA ladder assays were performed to assess the apoptosis.@*RESULTS@#After heat stress, FCM showed that apoptotic cells were increased significantly and reached the peak at 9 h in Raw 264.7 cells transfected with pcDNA3.1, and were characterized with classical morphologic changes including apoptotic body and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly at 6, 9, and 12 h after the heat stress. But the overexpression of HSF1 could reduce the number of apoptotic cells and inhibit DNA fragmentation.@*CONCLUSION@#HSF1 can inhibit heat stress-induced apoptosis in Raw264.7 macrophages.
Subject(s)
Animals , Mice , Rats , Apoptosis , Cells, Cultured , DNA-Binding Proteins , Pharmacology , Heat Shock Transcription Factors , Heat-Shock Response , Macrophages , Cell Biology , Transcription Factors , Pharmacology , TransfectionABSTRACT
OBJECTIVE@#To observe whether HSP70 could protect against H2O2-induced apoptosis in C2C12 myogenic cells by inhibiting Smac release from the mitochondria.@*METHODS@#HSP70 gene and full length Smac gene was transiently transfected in C2C12 myogenic cells by lipofectamine and the protein levels of HSP70 and Smac were analysed by Western blotting. Hoechst 33 258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe the DNA fragmentation. Activities of caspase-3 and caspase-9 were assayed with Western blotting. The release of Smac from the mitochondria to the cytoplasm was observed by immunofluorescence.@*RESULTS@#H2O2 ( 0.5 mmol/L ) activated caspase-3, caspase-9 8 h after the treatment and specific morphological changes of apoptosis 12 h after the treatment, and overexpression of Smac significantly promoted H2O2-induced activation of caspase-3, caspase-9 and apoptosis in C2C12 myogenic cells. HSP70 overexpression significantly inhibited H2O2-induced and Smac-promoted apoptosis, as shown by no specific DNA ladder pattern in agarose gel electrophoresis, decrease of percentage of apoptotic nuclei, and marked inactivation of caspase-3 and caspase-9. HSP70 could inhibit the release of Smac from the mitochondria to the cytoplasm 2 h after the treatment by H2O2.@*CONCLUSION@#HSP70 inhibits Smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells.