Journal of Pharmacopuncture (Feb 2001)
The Antiapoptic Effects of Hominis Placenta Extract
Abstract
Purpose. Free radicals are implicated in the pathophysiology of aging, ischemic injury and neurodegenerative disorders. To deform]no whether Hominis Placenta extract prevents H2O2-induced apoptosis, we have performed morphological and biochemical analyses for the detection of apoptotic phenomena in the pineal tumor cell line PGT-beta We have also peformed cytochemical and immunocytochemical analyses for the detection of changes in nitric oxide synthase (NOS) activity and estimated the expression . of apoptotic genes using reverse transcription-polymerase chain reaction (RT-PCR) Methods. PGT-beta cells were pretreated with Hominis Placenta extracts (0,10-2/ug/ml) for 2 hours and then exposed to H2O2 (0, 50uM) for 3 hours. Appearance of apoptotic characteristics were monitored using 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining assay, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and flow cytometric analysis. NOS activity was measured by NADPH-diaphorase cytochemistry. Expression of inducible NOS (iNOS) and nuclear factor kappa B (NF k B) was assessed via immunocytochemistry. The expression of apoptotic genes was examined by RT-PCR. Results. After 3 flours of exposure to H2O2, it was shown that PGT-beta cells treated with H2O2(50 uM) exhibit classical apoptotic features and increases in NOS activity and caspase-3 expression. Treatment with Hominis Placenta extract resulted in a reduced occurrence of apoptotic features. DAPI staining, TUNEL and flow cytometric assays revealed decreases in the occurrence of nuclear fragmentation and in the sub-Gl fraction in the PGT-beta cells treated with Hominis Placenta extract. Cells treated with Hominis Placenta extract also showed lower activity of NADPH-diaphorase and immunoreactivities of both iNOS and NF k B than those of H2O2-treated cells which were not treated with Hominis Placenta extract. By RT-PCR, it was shown that the level of caspase-3 mRNA was derreased In the cells treated with Hominis Placenta . extract. Conclusions. This study shows that Hominis Placenta extract prevents H2O2-induced apoptosis in PGT-beta cells; inhibitions of iNOS and caspnse-3 are possible mechanisms of the protection against apoptosis.
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