Sphingosine mediates FTY720-induced apoptosis in LLC-PK1 cells

FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 micrometer of FTY720, sphingosine accumulated in the LLC-PK1 cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 micrometer of FTY720 treatment and by 5 micrometer of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.

An immunohistochemical study on the expression of caspase-3 in the skin wound healing / 法医学杂志

OBJECTIVE@#To investigate the expression of caspase-3 during skin wound healing and explore the applicability of caspase-3 to determination of wound age.@*METHODS@#The expression of caspase-3 were performed on 33 mice skin incised wounds at different posttraumatic intervals and 3 mice as control by immunohistochemical technique.@*RESULTS@#Expression of caspase-3 was detectable in polymorphonuclear cells (PMNs) in the wound specimens aged 6 h. In the wound specimens aged from 12 h to 24 h after injury, caspase-3 was identified in a large number of infiltrating PMNs and part of mononuclear cells (MNCs). Afterwards, the MNCs and fibroblastic cells (FBCs) accounted in the most part of the caspase-3 positive cells. Morphometrically, the ratio of the number of the caspase-3 stained PMNs, MNCs and FBCs to total number of the cells in the wounds was evaluated and calculated. The ratio of the caspase-3 positive cells was low in the wound specimens aged from 0 h to 3 h (4.53 +/- 6.53)%, and maximized in the wound specimens aged 3 day (62.66 +/- 4.84)%. Thereafter, the ratio decreased and minimized in the specimens aged 14 day (21.56 +/- 4.54)%.@*CONCLUSION@#The results suggest that caspase-3 may play an important role in inducing neutrophil, macrophage and fibroblast apoptosis during skin wound healing. Furthermore, caspase-3 may be used as a marker for the wound age determination.