Mitochonrial Related Pathway Involved In Apoptosis In RAW264.7 Cells Induced By Uranium Dust

Objective: To explore the mechanism of mitochondrial-related pathway of apoptosis induced by uranium dust(UD) in RAW 264.7 cell, to clarify the mechanism of uranium dust induced lung lesion.Methods: The experiment was divided into two groups, the control(0 μg/ml) and the uranium dust groups(25, 50, 100, 200, 400 μg/ml). The survival rate and time-efficiency of rat macrophage RAW 264.7 were determined by MTT, the morphology and rate of apoptosis were tested by Hoechst33258 fluorescent labeling and Annexin-V FITC/PI dual-dyeing,respectively. Rhodamine123 fluorescent probe was used to detect the mitochondrial membrane potential altering in RAW264.7 cell. The expression of apoptotic proteins including intracellular Cyt-c, mitochondrial Cyt-c as well as cytosol Cyt-c, Cleaved-Caspase-3, Cleaved-Caspase-9 and Cleaved-PARP, Bcl-2, Bax was tested with Western Blot. The expression of Bcl-2 and Bax m RNA was examined by Q-PCR.Results: The outcomes of MTT test showed a drop of survival rate in a time-dose effective relationship. The apoptosis rate increased in correlating with the UD concentration for 24 hour after contamination indicated by both Hoechst 33258 fluorescent observation and Annexin-V FITC/PI dual-dyeing flow-cytometry. A decrease of membrane potential correlating with the rise of UD concentration was observed by spectrophotofluorimetry after Rh123 dyeing. The result of Western Blot showed the protein expression of Bcl-2 and Bax were in accordance with gene expression. The expression of intracellular Cyt-c and intracytoplasm Cyt-c was rising, but mitochondrial Cyt-c expression dropped. The expression of Cleaved-Caspase-3, Cleaved-Caspase-9 and Cleaved-PARP were increasing, which inferred that the precursors of them were activated. Along with the increasing of UD concentration, the expression decreasing of Bcl-2 gene and and icreasing of Bax gene was discerned by Q-PCR.Conclusions: 1. The apoptosis of rat macrophage RAW 264.7 cell could be induced by UD. 2. The Caspase-dependent mitochondrion pathway might be the mechanism of UD induced apoptosis of the RAW 264.7 cells 3. The Bcl-2 family protein might make a vital role in the UDinduced apoptosis of RAW 264.7cells.