Influences of cold atmospheric plasma on apoptosis related molecules in osteoblast-like cells in vitro

Abstract

Abstract Background Cold 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. Methods Human 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. Results CAP 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. Conclusions Our in vitro-study demonstrated, that CAP decreases apoptosis related molecules in osteoblast-like cells, underlining a beneficial effect on hard-tissue cells.

Keywords

• Cold atmospheric plasma
• Apoptosis
• Inflammation
• Bone remodelling
• Osteoblast like cells