Investigation of Cytotoxicity Apoptotic and Inflammatory Responses of Biosynthesized Zinc Oxide Nanoparticles from Ocimum sanctum Linn in Human Skin Keratinocyte (Hacat) and Human Lung Epithelial (A549) Cells

Abstract

Pristine and engineered metal nanoparticles are widely applied in various fields of industry, and as consequences, they are useful as well as harmful to human health and environment. This study aimed at synthesizing the green zinc oxide nanoparticles (ZnNPs) using the leaf extract of Ocimum sanctum Linn and assessing its toxicity on human skin epidermal (HaCaT) and human lung epithelial (A549) cells. The synthesized green ZnNPs (gZnNPs) were characterized by using dynamic light scattering (DLS) and a high-resolution transmission electron microscope. The average size of gZnNPs obtained was 62 nm with a spherical shape. The effects of gZnNPs on the viability of HaCaT and A549 cells were investigated using tetrazolium salt (MTT) for 24 h. We have seen more reduction of cell viability of A549 cells in comparison to HaCaT cells. The induction of intracellular reactive oxygen species (ROS) was measured using DCFDA assay and showed a slightly high intensity of green fluorescence in A549 than HaCaT cells. The different oxidative stress biomarkers such as ROS generation and lipid peroxide were increased, and GSH was decreased in a dose-dependent manner. The apoptotic and necrotic effect of gZnNPs in both cells was carried out using Annexin-V-FITC and propidium iodide staining. More apoptotic and necrotic cells were found at a higher concentration of gZnNPs exposure. Also, we determined the effect of gZnNPs at the molecular level by evaluating the apoptotic and inflammatory markers, in which gZnNPs downregulated Bcl2 and upregulated Bax, caspase-3, and TNF-α in HaCaT and A549 cells. Ultimately, gZnNPs exerted toxicity and apoptosis in HaCaT and A549 cells.

Figure 1

Schematic diagram of green synthesis of zinc oxide nanoparticles (gZnNPs) using zinc acetate salt and Ocimum sanctum leaf extract. (a) TEM image of gZnNPs. (b) Size distribution of gZnNPs (%).

Figure 2

Cytotoxicity of gZnNPs on HaCaT and A549 cells for 24 h, as measured by MTT assay. Each value represents the mean ± SE of three experiments. n = 3, ∗p < 0.05, ∗∗p < 0.01 vs. control.

Figure 3

(a) Photomicrograph of green fluorescence (DCF intensity) in HaCaT and A549 cells after exposure to gZnNPs for 24 h. (b) Intracellular intensity of ROS production in HaCaT cells (c). Intracellular intensity of ROS production in A549 cells. Each value represents the mean ± SE of three experiments. ∗p < 0.05 and ∗∗p < 0.01 vs. control.

Figure 4

Oxidative stress biomarkers after exposure to gZnNPs on HaCaT and A549 cells for 24 h. (a) LPO in HaCaT. (b) LPO in A549 cells (c). GSH in HaCaT cells. (d) GSH in A549 cells. Each value represents the mean ± SE of three experiments. ∗p < 0.05 and ∗∗p < 0.01 vs. control.

Figure 5

Scatter diagram after treatments of gZnNPs to HaCaT and A549 cells for 24 h. (a) HaCaT cells at 0 μg/ml. (b) HaCaT cells at 25 μg/ml. (c) HaCaT cells at 35 μg/ml. (d) A549 cells at 0 μg/ml. (e) A549 cells at 25 μg/ml. (f) A549 cells at 35 μg/ml. (g) Percentage of early and late apoptotic cells after treatment of ZnNPs (0, 25, and 35 μg/ml) for 24 h. (h) Caspase-3 activity after treatment of ZnNPs (0, 6, 25, and 35 μg/ml) for 24 h. Data represents the mean ± SE of three experiments. ∗p < 0.05 and ∗∗p < 0.01 vs. control.

Figure 6

ZnNPs induce apoptosis and inflammatory response of HaCaT and A549 cells. Western blot analysis was carried out to assess apoptosis and inflammatory response-related protein expression in HaCaT and A549 cells. β-Actin was used as an internal reference. ZnNPs significantly increased the expression of Bax, caspase-3, and TNF-α and decreased the expression of Bcl-2 at a concentration 35 μg/ml (a, b). Data represents the mean ± SE of three experiments. ∗p < 0.05 and ∗∗p < 0.01 vs. control.

Figure 7

ZnNPs induce apoptosis and inflammatory response of HaCaT and A549 cells. mRNA expression analysis was carried out to assess apoptosis and inflammatory response-related gene expression in HaCaT cells (a) and A549 cells (b). β-Actin was used as an internal reference. ZnNPs significantly increased the gene expression of Bax, caspase-3, and TNF-α and decreased the expression of Bcl-2 in a concentration-dependent manner (a, b). Data represents the mean ± SE of three experiments. ∗p < 0.05 and ∗∗p < 0.01 vs. control.