Cytoskeletal Morphological Changes of Mesenchymal Stem Cells after Oxidant Damage and its Prevention by Thymoquinone

Abstract

The functional integrity of the cytoskeleton of mesenchymal stem cells (MSCs) is essential for its differentiation into multiple cell lineages including adipocytes, chondrocytes, and osteoblasts. Abnormalities in the cytoskeletal proteins such as actin and microtubule can cause disrupted cell signalling and irregular movements of organelles leading to cell death. This study investigated cytoskeletal and nuclear morphological changes of the MSC due to oxidative damage by hydrogen peroxide (H2O2) and the possible prevention of these changes by the antioxidant thymoquinone (TQ). Bone marrow MSCs from Sprague Dawley rats were cultured and treated with different concentrations of H2O2 with or without TQ to observe the potential protective activity. Triple-label fluorescence immunocytochemistry was performed post-treatment to observe the nucleus, actin and microtubules using 4’,6-diamidino-2-phenylindole (DAPI), Alexa Fluor 488-labelled phalloidin and Cy3-labelled anti-tubulin antibody, respectively. The normal stem cell cytoskeleton demonstrated intact actin and microtubule structures along with normal appearance of the nucleus. However, oxidative damage by H2O2 caused a severe disruption of the cytoskeletal morphology of the actin and microtubule along with apoptosis and necrosis of the nucleus. Interestingly, both immunocytochemical and Fluorescence-Activated Cell Sorting (FACS) results showed that these morphological changes were prevented by TQ at low concentrations while higher concentrations of TQ were harmful. This study suggested that TQ could save MSCs from oxidative-induced cell death.