International Journal of Biomedicine. 2019;9(4):350-355.
Originally published December 15, 2019
Background: Mitochondrial oxidative phosphorylation (OxPhos) accounts for more than 90% of the cellular ATP production, plays the role in reactive oxygen species (ROS) generation and programmed cell death. In addition, it contributes to such cellular processes as proliferation, differentiation and cell aging. Currently, several signaling systems are known to participate in regulation of OxPhos and activity of cytochrome c-oxidase (CcO), the terminal enzyme of the mitochondrial electron transport chain. However, data on mechanisms and key units involved in the signal transduction are still being supplemented.
Methods: Peritoneal fibroblasts were isolated from the omentum of Wistar rats by fragmenting the dissected tissue and disaggregating the fragments in collagenase solution (200 U/ml). The primary culture of fibroblasts was cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% antibiotic/antimycotic at 37°C, 80% RH and 5% СО2 in Biostation CT, Nikon. To obtain a culture, the fibroblasts were subcultured every 7 days. After the third passage the culture was treated with SB203580 at the concentration of 10 μM or with SB203580 in combination with bFGF at the dose of 133 pg/ml. Cells for immunofluorescent studies were fixed with 70% ethanol and stained with antibodies to CcO subunit I.
Results: When exposed to SB203580 or the combination of SB203580 and bFGF, marked changes were observed in the fibroblast culture: in both cases there was intensive collagen destruction; attached fibroblasts rounded and detached from the substrate. When exposed to SB203580, non-rounded cells started to vacuolate actively while vacuoles occupied the entire cytoplasm. Introduction of the p38 inhibitor into the culture of activated fibroblasts caused a more intensive cell detachment and collagen destruction. Moreover, the formation of large conglomerates containing several dozens of cells connected with collagen fibers was observed in the areas characterized by the highest cell density. Immunofluorescent staining made it possible to reveal a certain increase in the cell area occupied by CcO after fibroblasts exposure to SB203580 and a significant increase in the area of the enzyme distribution in the studied cells (more than 5-fold in comparison with the control group) when simultaneously adding SB203580 and bFGF. In addition, one-way ANOVA test demonstrated a statistically significant increase in the CcO fluorescent staining intensity in both cases.
Conclusion: The analysis of the results indicates that SB203580, a p38 MAPK inhibitor, influences both peritoneal fibroblast morphology and the energy status of the cells under study increasing the amount of CcO, the terminal enzyme of the mitochondrial electron transport chain, in the cells, although no cell change to active proliferation or apoptosis was observed. Fibroblast culture stimulation by bFGF significantly enhances the effect of SB203580, which implies a greater OxPhos complex expression in case of impaired p38 MAPK signaling pathway activation.
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Received October 28, 2019.
Accepted November 27, 2019.
©2019 International Medical Research and Development Corporation.