Abstract
BackgroundCold atmospheric plasma (CAP) has recently been identified as a novel therapeutic strategy for supporting processes of wound healing. Since CAP is additionally known to kill malignant cells, our study intends to determine the influence of CAP on crucial molecules involved in the molecular mechanism of apoptosis in osteoblast-like cells. MethodsHuman osteoblast-like cells were CAP-treated for 30 and 60 s. CAP effects on critical factors related to apoptosis were studied at transcriptional and protein level using real time-PCR, immunofluorescence staining and western blot. Phalloidin / DAPI staining was used for analyzing the cell morphology. In addition, apoptotic outcomes of CAP were displayed using flow cytometry analysis. For studying intracellular signaling pathways, MAP kinase MEK 1/2 and PI3K were blocked. Finally, the effects of CAP on caspase-3 activity were examined using a caspase-3 assay. ResultsCAP treatment resulted in a significant downregulation of p53 and apoptotic protease activating factor (APAF)-1, caspase (CASP)9, CASP3, BCL2 Antagonist/Killer (BAK)1, and B-Cell Lymphoma (BCL)2 mRNA expression at 1 d. An inhibitory effect of CAP on apoptotic genes was also shown under inflammatory and apoptotic conditions. Nuclear translocation of p53 was determined in CAP treated cells at the early and late stage, after 15 min, 30 min, and 1 h. p53 and APAF-1 protein levels were reduced at 1 d, visualized by immunofluorescence and western blot, respectively. Moreover, a morphological cytoskeleton modification was observed after CAP treatment at 1 d. Further, both CAP-treated and untreated (control) cells remained equally vital as detected by flow cytometry analysis. Interestingly, CAP-associated downregulation of CASP9 and CASP3 mRNA gene expression was also visible after blocking MAP kinase and PI3K. Finally, CAP led to a decrease in CASP3 activity in osteoblast-like cells under normal and apoptotic conditions. ConclusionsOur in vitro-study demonstrated, that CAP decreases apoptosis related molecules in osteoblast-like cells, underlining a beneficial effect on hard-tissue cells.
Highlights
Cold atmospheric plasma (CAP) has recently been identified as a novel therapeutic strategy for supporting processes of wound healing
The extrinsic pathway can be initiated by molecules such as Tumor Necrosis Factor (TNF)α, which modulate an apoptotic cascade, which leads to the activation of CASP3
Effect of CAP on gene expression of markers of apoptosis Since CAP has been described to have a stimulating effect on multiple cells, we examined a possible anti-apoptotic effect of CAP on the gene regulation in osteoblast-like cells
Summary
Cold atmospheric plasma (CAP) has recently been identified as a novel therapeutic strategy for supporting processes of wound healing. Chronic wounds have a high proportion of inflammatory, damaged or dead cells, which are usually cleared by macrophages [4]. Another important process involved in damaged cells removal is apoptosis known as programmed cell death. The extrinsic pathway can be initiated by molecules such as Tumor Necrosis Factor (TNF)α, which modulate an apoptotic cascade, which leads to the activation of CASP3. The intrinsic pathway can be triggered by cell stress, which leads to the activation of reactive oxygen species (ROS) or the release of p53 caused by DNA damage. The apoptotic cascade is further modulated by anti-apoptotic genes, such as BCL2 Antagonist/Killer (BAK), or antiapoptotic genes, such as B-Cell Lymphoma (BCL)2 [10]
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