Cellular cytoskeletal changes after oxidant injury and possible healing assisted by thymoquinone, the main constituent of Nigella sativa / Mohd Suhaimi Draman

Mohd Suhaimi, Draman (2021) Cellular cytoskeletal changes after oxidant injury and possible healing assisted by thymoquinone, the main constituent of Nigella sativa / Mohd Suhaimi Draman. Masters thesis, Universiti Malaya.

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Abstract

A wide-ranging spectrum of stem cell research receives intensively growing attention since the last decade. It promises the cure for a lot of apparently incurable diseases (e.g., paralysis or spinal cord injury). One of them is in the cytoskeleton concerning cell death/cell injury. For instance, oxidative injury by hydrogen peroxide (H2O2) has been linked to triggering cell death especially programmed cell death or apoptosis in bone marrow stem cells. The cytoskeleton commonly plays a big role in protecting cells against oxidative damage and, itself is a target for oxidative stress-induced free radical damage. Thus, drugs that protect the cytoskeleton could be one of the ways to save the cells from dying. Thymoquinone compound, which has been suggested to have protective and healing function against injury caused by an oxidant agent, is one of the candidates. In the current study, rat bone marrow mesenchymal stem cells (rBMSCs) were cultured and treated with different concentrations of H2O2 (50 μM, 500 μM, and 5000 μM) to damage the cells. This treatment caused morphological changes in the cells reminiscent of necrotic and apoptotic cell death. The number of cells surviving after the treatment was counted by MTT assay. At the end of the experiment, the cells were fixed and immunocytochemistry for the F-actin, microtubules, and nucleus was each performed using Alexa Fluor-488 phalloidin, Cy3-labeled anti-β-tubulin monoclonal antibody, and 4′,6-diamidino-2-phenylindole (DAPI), respectively. Three-channel fluorescence microscopy along with phase-contrast images was obtained by using fluorescence microscope to obtain digitally merged images of the cytoskeleton and the nucleus. The cells were also treated with thymoquinone (TQ) with different concentrations (0.5 μM, 5 μM, 50 μM, and 500 μM) before and after H2O2 oxidative damage treatments (50 μM, 500 μM and 5000 μM) to observe the protective and healing effects on the cytoskeleton of the rBMSCs. H2O2 was observed to cause cell death of rBMSCs in a concentration-dependent manner. H2O2 induced morphological changes in the cytoskeleton of the cells were devastating as shown by immunocytochemistry of the F-actin and microtubules. The morphology of stress fibres of the F-actin was disrupted, and the microtubules appeared to be missing. The cell nuclei were shrunken, and this involved a big cell number. Pre-treatment with TQ appeared to partially prevent these changes in the cytoskeleton. However, higher concentrations of TQ appeared to be toxic to the cells. Therefore, the results demonstrated that these compounds at high concentrations were pro-oxidant, whereas at low dosage they acted as antioxidants. These results suggested that the active compounds which can found in Nigella sativa could help in preserving the bone marrow rBMSCs and, thus, could help in regeneration against several degenerative diseases.

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