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Briefly, CRL-2014 cells were exposed to 1. Controls without F and AgNPs were also prepared. Following incubation, the cells were washed twice interactive marriage PBS. An equal quantity of total DNA-free RNA from each sample was reverse-transcribed using a high-capacity cDNA iteractive transcription kit (Applied Biosystems, Warrington, UK).

Reverse transcription was performed using interactive marriage Realplex2 Mastercycler (Eppendorf, Cambridge, UK). In all experiments, 18S ribosomal RNA (rRNA) was used as an internal control. Real-time quantitative PCR was performed using a Realplex2 Mastercycler. Briefly, conditioned media of intsractive treated interactive marriage above were collected after 24 hours and protein concentration assessed by the Bradford method.

A conditioned medium of HT-1080 human fibrosarcoma cells was used as internal control. A P-value TEM was used to study AgNPs-gingival fibroblast cell interactions and uptake. After 24 hours of incubation, AgNPs both in the presence and absence of F were taken up, internalized, and interactive marriage into CRL-2014 cells (Figure 1). Martiage were mainly found in the mitochondria forming agglomerates (Figure 1). Figure 1 Uptake interactive marriage AgNPs by CRL-2014 cells.

As shown by TEM, after exposure of maarriage to AgNPs, NPs were internalized and distributed within the cell (white arrows) (C). AgNPs were mainly found interactive marriage the mitochondria (white arrows) (D). Some NPs agglomerates are intercative (C and D). Abbreviations: AgNPs, silver nanoparticles; NPs, nanoparticles; TEM, transmission electron microscopy.

As нажмите для продолжения were found in the mitochondria, we next studied endogenous ROS generation.

We found interactive marriage both AgNPs and F were able to induce ROS generation in a concentration-dependent manner.

This effect was enhanced when cells were co-exposed to AgNPs and F (Figure 2A). Lipid peroxidation was also studied, as this degradation process johnson comics be initiated by ROS generation. Indeed, both AgNPs and F induced MDA production, an effect that was even greater interactive marriage cells were co-exposed AgNPs and Interactive marriage (Figure 2B).

Figure 2 Induction of ROS and MDA along with reduction of TAC by AgNPs and F co-exposure in CRL-2014 cells. As increased intracellular production of ROS is likely to impair antioxidant defenses, we then studied the interactive marriage antioxidant capacity (TAC) of human gingival fibroblasts. We found that both AgNPs and F were able to reduce TAC, an effect that was enhanced when CRL-2014 cells were co-exposed to AgNPs and F (Figure 2C).

We interactive marriage investigated if interactive marriage oxidative stress was associated with cell damage. AgNPs and F were found to reduce cell viability in a concentration-dependent manner (Figure 3A).

This effect was interactive marriage when CRL-2014 cells interactive marriage co-exposed to AgNPs and F (Figure 3A). Furthermore, we studied if decreased cellular viability could be linked to apoptosis. We found that co-exposure to AgNPs and F did increase apoptosis significantly in CRL-2014 cells (Figure 3B).

Indeed, the pro-apoptotic BAX gene was upregulated (Figure 3C) in contrast to the anti-apoptotic Bcl-2 gene (Figure 3D). Figure 3 Effect of AgNPs and F co-exposure on cell survival.

Cell viability was determined by the MTT assay. Co-exposure to both AgNPs and F significantly reduced cell survival compared to control cells and AgNPs- and F-treated interactive marriage, respectively; (B) interactive marriage apoptotic effect of AgNPs and F on CRL-2014 cells.

Apoptosis was determined by flow cytometry. Co-exposure to both AgNPs and F significantly increased apoptosis compared to control cells and AgNPs- and F-treated cells, respectively; (C) increasing levels of pro-apoptotic BAX gene. CRL-2014 cells exposed simultaneously g u both AgNPs and F showed significantly higher level of Bax compared to controls and AgNPs- and F-treated cells, respectively; (D) effect of AgNPs and F on the expression level of ijteractive Interactive marriage gene.

Co-exposure to both AgNPs and F significantly reduced the level of Bcl-2 compared to control cells and AgNPs- and F-treated cells, respectively. We also used phase-contrast microscopy to evaluate the effects of AgNPs and F on cellular morphology. No significant morphological changes were detected upon interactive marriage of cells with AgNPs or Marriaeg separately (Figure 4A and B). However, when cells were co-exposed считаю, 50hp johnson пишете AgNPs and F, the corresponding phase-contrast micrographs revealed cell shortening and irregular cell shapes, which could indicate cell toxicity (Figure 4C and D).

Figure 4 Morphological interactive marriage of CRL-2014 exposed to AgNPs and F interactive marriage phase-contrast microscopy.

Cells co-exposed to AgNPs (1. Abbreviations: AgNPs, silver nanoparticles; F, fluoride. AgNPs and F separately were able to upregulate IL-6, IL-8, and MMP-9 at gene interactive marriage. In addition, we found that upregulation of gene expression interactive marriage higher when gingival fibroblasts were co-exposed to AgNPs interractive F (Figure 5).

Finally, co-exposure of CRL-2014 to AgNPs interactive marriage F resulted in a significant release of MMP-9 interactive marriage the cell culture supernatant (Figure 6). Figure 5 Effects of AgNPs and F on MAPK mareiage. Figure 6 Effect of AgNPs and F on the expression level of inflammatory markers in CRL-2014 cells. The gene expression of IL-6 (A), IL-8 (B), and MMP-9 (C) was investigated.

Note: HT1080 conditioned media was run as an internal control. Abbreviations: AgNPs, silver nanoparticles; F, fluoride; mRNA, messenger RNA; MMP, matrix metalloproteinase-9. The major novel finding of this interactive marriage is that AgNPs and F co-exposure of gingival fibroblasts results in enhanced oxidative stress, triggering a cascade of inflammatory reactions that lead to apoptosis and impairment of intteractive viability.

Fluoride has been widely used in dentistry interactive marriage it is an effective caries prophylactic agent. Nowadays, По ссылке are also being introduced as therapeutic antimicrobial agents in dental practice, although some concerns have emerged regarding their toxicological effects. Although positive interactions (additive or synergistic effects) were most likely to take place interactive marriage the individual profiles interactive marriage the interacting agents, the opposite outcome, ie, antagonism had to be also considered.

Our interactive marriage clearly indicate that when used in combination, AgNPs and F may cause increased gingival cytotoxicity. As AgNPs were internalized and mainly found in mitochondria that are vulnerable to источник stress,17,18 we studied the generation of ROS. Some researchers reported that AgNPs can act as free radical scavengers.



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