Mechanism underlying NO-induced apoptosis in human gingival fibroblasts
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Nitric oxide (NO) acts as an intracellular messenger at thephysiological level but can be cytotoxic at highconcentrations. The cells within periodontal tissues, such asgingival and periodontal fibroblasts, contain nitric oxidesyntheses and produce high concentrations of NO whenexposed to bacterial lipopolysaccharides and cytokines. However, the cellular mechanisms underlying NO-inducedcytotoxicity in periodontal tissues are unclear at present. Inour current study, we examined the NO-induced cytotoxicmechanisms in human gingival fibroblasts (HGF). Cellviability and the levels of reactive oxygen species (ROS)were determined using a MTT assay and a fluorescentspectrometer, respectively. The morphological changes inthe cells were examined by Diff-Quick staining. Expressionof the Bcl-2 family and Fas was determined by RT-PCR orwestern blotting. The activity of caspase-3, -8 and -9 wasassessed using a spectrophotometer. Sodium nitroprusside(SNP), a NO donor, decreased the cell viability of the HGFcells in a dose- and time-dependent manner. SNP enhancedthe production of ROS, which was ameliorated by NAC, afree radical scavenger. ODQ, a soluble guanylate cyclaseinhibitor, did not block the SNP-induced decrease in cellviability. SNP also caused apoptotic morphological changes,including cell shrinkage, chromatin condensation, and DNAfragmentation. The expression of Bax, a member of the proapoptoticBcl-2 family, was upregulated in the SNP-treatedHGF cells, whereas the expression of Bcl-2, a member of theanti-apoptotic Bcl-2 family, was downregulated. SNP augmented the release of cytochrome c from themitochondria into the cytosol and enhanced the activity ofcaspase-8, -9, and -3. SNP also upregulated Fas, acomponent of the death receptor assembly. These resultssuggest that NO induces apoptosis in human gingivalfibroblast via ROS and the Bcl-2 family through bothmitochondrial- and death receptor-mediated pathways. Our data also indicate that the cyclic GMP pathway is notinvolved in NO-induced apoptosis.
Abstract
Nitric oxide (NO) acts as an intracellular messenger at thephysiological level but can be cytotoxic at highconcentrations. The cells within periodontal tissues, such asgingival and periodontal fibroblasts, contain nitric oxidesyntheses and produce high concentrations of NO whenexposed to bacterial lipopolysaccharides and cytokines. However, the cellular mechanisms underlying NO-inducedcytotoxicity in periodontal tissues are unclear at present. Inour current study, we examined the NO-induced cytotoxicmechanisms in human gingival fibroblasts (HGF). Cellviability and the levels of reactive oxygen species (ROS)were determined using a MTT assay and a fluorescentspectrometer, respectively. The morphological changes inthe cells were examined by Diff-Quick staining. Expressionof the Bcl-2 family and Fas was determined by RT-PCR orwestern blotting. The activity of caspase-3, -8 and -9 wasassessed using a spectrophotometer. Sodium nitroprusside(SNP), a NO donor, decreased the cell viability of the HGFcells in a dose- and time-dependent manner. SNP enhancedthe production of ROS, which was ameliorated by NAC, afree radical scavenger. ODQ, a soluble guanylate cyclaseinhibitor, did not block the SNP-induced decrease in cellviability. SNP also caused apoptotic morphological changes,including cell shrinkage, chromatin condensation, and DNAfragmentation. The expression of Bax, a member of the proapoptoticBcl-2 family, was upregulated in the SNP-treatedHGF cells, whereas the expression of Bcl-2, a member of theanti-apoptotic Bcl-2 family, was downregulated. SNP augmented the release of cytochrome c from themitochondria into the cytosol and enhanced the activity ofcaspase-8, -9, and -3. SNP also upregulated Fas, acomponent of the death receptor assembly. These resultssuggest that NO induces apoptosis in human gingivalfibroblast via ROS and the Bcl-2 family through bothmitochondrial- and death receptor-mediated pathways. Our data also indicate that the cyclic GMP pathway is notinvolved in NO-induced apoptosis.
참고문헌 (25)
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