摘要
AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin(GFX) to stromal fibroblasts(SFs) in vitro.METHODS: SFs were treated with GFX at different concentrations(0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine(PS) externalization, and mitochondrial transmembrane potential(MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins.RESULTS: Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2(Bcl-2) and B-cell leukemiaXL(Bcl-XL), and upregulate Bcl-2 assaciated X protein(Bax), Bcl-2-associated death promoter(Bad), Bcl-2 interacting domain(Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs.CONCLUSION: The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.
AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin(GFX) to stromal fibroblasts(SFs) in vitro.METHODS: SFs were treated with GFX at different concentrations(0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine(PS) externalization, and mitochondrial transmembrane potential(MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins.RESULTS: Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2(Bcl-2) and B-cell leukemiaXL(Bcl-XL), and upregulate Bcl-2 assaciated X protein(Bax), Bcl-2-associated death promoter(Bad), Bcl-2 interacting domain(Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs.CONCLUSION: The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.
基金
Supported by the National High Technology R&D Program of China(No.2006AA02A132)
