The amount of dye present in the supernatant is inversely proportional to the membrane potential of the cells. Caspase-3 and -9 activities were measured using colorimetric assays based on the identity of specific amino acid sequences by these caspases. The amount of caspase activity present in the sample is proportional to the intensity of yellow color produced upon cleavage. Then, absorbance was measured at nm. After incubation for 15 minutes, the sample absorbance was again recorded against blank DTNB. Data are shown in millimolar. To investigate the molecular mechanisms of apoptosis in PaTu cells, the expression levels of 15 apoptosis-related genes were examined by quantitative real-time reverse transcription PCR Table 1.
The pellets were washed three times with sterile phosphate-buffered saline for RNA isolation. Cycle number C t of each reaction was calculated from the amplification curve to determine the relative gene expression using the comparative cycle threshold method. All C t values calculated from the target genes were calibrated using that from the untreated control cells. All independent experiments were repeated four times. It was shown that the mitochondrial activity of PaTu cells decreased with increasing concentrations of DZ after 24 hours of exposure in comparison with untreated samples control group.
Figure 1 The inhibitory effects of cell viability on treatment with various concentrations of DZ in the PaTu cell line compared to untreated sample Con. In case of MgO NPs, by increasing the concentration, the viability of the cells increased, but in the Se NPs, by increasing the concentration, the viability of the cells decreased when compared with untreated samples control group. Accordingly, in the rest of the experiments, we evaluated the effect of and 0.
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In combination of MgO and Se NPs, no significant improvement in the release of C-peptide was observed as compared with DZ, the level of C-peptide was still lower than the control group. It was measured as percentage of control Figure 4B.
The MMP levels of groups that were treated with MgO and Se NPs and their combination demonstrated a considerable increase in comparison with DZ, but it did not show a significant difference when compared with control. As seen, most of the untreated control cells were characterized by a bright green nucleus with uniform intensity and slight uptake of EB, whereas the cells treated with DZ were orange red in appearance and showed obvious nuclear disintegration after 24 hours of treatment.
Fluorescence microscopic images clearly revealed morphological alterations of the DZ-treated cells compared to the untreated control cells. Notes: A Viable cells appear uniformly green; early apoptotic cells show characteristic loss of membrane integrity and chromatin condensation, stain bright green to yellow; late apoptotic cells incorporate EB and therefore stain yellow orange and show condensed and often fragmented nuclei; necrotic cells are orange red in appearance.
B AO is a vital dye and stains both live and dead cells, but EB stains only cells that have lost membrane integrity. So, an increase in the percentage of EB-stained cells indicates an increase in cell damage apoptosis and necrosis. It was shown that cell incubation with DZ resulted in the reduction of cell viability and similar increase of apoptosis and necrosis. In contrast, NPs-treated MgO and Se NPs and their combination groups displayed lower intensity of EB compared to DZ group 44, 45, and 46 vs 58 , revealing a decrease in the numbers of apoptotic and necrotic cells.
Interestingly, there were no significant differences in intensity of AO green viable cells in comparison with control group 56, 55, and 54 vs To investigate the molecular mechanism of apoptosis in PaTu cell line, the expression levels of 15 apoptosis-related genes were examined Table 2. The expression levels of these 15 genes were analyzed by real-time PCR to evaluate the effect of DZ and NPs on the expression of intrinsic and extrinsic pathway proteins 7 and 8 genes, respectively.
The analysis of data showed that the changes in the gene expression induced by DZ were significantly different in the PaTu cells compared to changes induced by NPs. The expression changes of these genes were significantly different in MgO NPs and combination groups when compared with DZ group. For example, these factors lead to the development of hyperglycemic and hemostatic disorders related to glucose metabolism and can be regarded as precursors to diabetes. They could penetrate into a different place and have protective effects against oxidative stress-induced cell death through different mechanisms.
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Our results demonstrated that using MgO and Se NPs could improve the release of insulin, proinsulin, and C-peptide all of which were inhibited after exposure to DZ , one of the main functions of the PaTu cells. In this area of research, previous studies have shown that metal oxide NPs, including CeO 2 , Y 2 O 3 , ZnO, and MgO, were capable of enhancing cell survival and function under conditions of oxidative stress; 33 — 37 the reason for increasing insulin by metal oxide NPs was justified by their antioxidant property.
In addition, DZ can induce mitochondrial damage, such as the loss of MMP, and lead to a decrease of mitochondrial activity in human PaTu cells, which suggests that mitochondrial damage might play a role in DZ-induced cell death. Mitochondrial potential loss triggers the caspase cascade leading to increased apoptosis. Therefore, the caspase enzyme activity, as one of the mechanisms for justifying the apoptosis, 39 was assessed in this study by measuring caspase-3 and -9 activities.
On the basis of our results, it was hypothesized that using MgO NPs with the potential to prevent caspase enzyme activity could enable PaTu cells survive the cytotoxicity of DZ exposure. OP-induced cell death specially by DZ has been described generally in different cell types, including ovarian follicular cells, cardiac muscle cells, NTera2-D1 cells, peripheral blood lymphocytes, and skeletal muscles.
Moreover, to investigate in greater detail the factors that are possibly responsible for apoptosis, the expression levels of 15 apoptosis-related genes were analyzed by real-time PCR. Depending on the trigger of the cell death program, the basic mechanism of apoptosis has been divided into intrinsic mitochondrial and extrinsic death receptor pathways. While inhibition of Bclmediated intrinsic pathway results in the activation of caspases 9 and 3, death receptor-mediated extrinsic pathway involves caspases 10 and 8 in inducing apoptosis.
Notably, both pathways converge at caspase-3, leading to activation of other proteases.