Study On The Molecular Mechanism Of EGF Abated Apoptosis In HaCat Cells Induced By TNF-α Through The Activated PPARβ

PART ⅠStudy on the transcription activity of PPARp in the apoptotic HaCat cells induced by TNF-αOBJECTIVE: To explore the change of transcription activity of PPARp in the apoptotic HaCat cells induced by TNF-a.METHODS: HaCat cells were exposed to different concentration TNF-α for indicated durations. Apoptotic morphological changes and percentage of apoptotic nuclei were assayed with Hoechst 33258 staining. Activities of caspase-9,8,3 were analyzed with Caspase Colorimetric Assay Kit. HaCat cells viability were evaluated by MTT method. The expression of PPARβ in HaCat cells treated with TNF-α was observed by Western-blot and RT-PCR. Electrophoretic mobility shift assays demonstrated a impermanency increase in PPARβ binding activity with DNA. Forthermore, luciferase assay system were employed to analyze PPARβ transcription activity.RESULTS: The apoptosis of HaCat cells treated with TNF-α (10ng/mL) were increased by Hoechst 33258 stained, and fluorescent microscopy showed apoptotic cells with condensed chromatin in 12 hours and 24 hours. Percentage of apoptotic nuclei were raised in HaCat cells exposed to TNF-α (10ng/mL) for indicated durations. After exposure to different concentration of TNF-α (5, 10,10ng/mL) for 24 hours, the HaCat cell showed a lot of apoptotic cells ,and Percentage of apoptotic nuclei respectively were (12.20±3.28)%, (32.12±3.81)%, (56.66±4.51) %. The activation of Caspase-3,8,9 were enhanced in HaCat cells treated with TNF-α (10, 20ng/mL) for indicated durations. The effect of TNF-α with 10ng/mL and 20ng/mL on HaCat cells viability were degraded by...