Effect of Chitosan Nanoparticle-Loaded Thymus serpyllum on Hydrogen Peroxide-Induced Bone Marrow Stromal Cell Damage

Abstract

We have determined the protective effects of Thymus serpyllum (TS) extract and nanoparticle-loaded TS on hydrogen peroxide-induced cell death of mesenchymal stromal cells (MSCs) in vitro. Gas chromatography-mass spectroscopy confirmed the spectrum of active components in the extract. Out of the three different extracts, the hexane extract showed significant free radical scavenging activity. Treatment of MSCs with H₂O₂ (hydrogen peroxide) significantly increased intracellular cell death; however, pretreatment with TS extract and nanoparticle-loaded TS (200 μg/ml) suppressed H₂O₂-induced elevation of Cyt-c and MMP13 and increased the survival rates of MSCs. H₂O₂-induced (0.1 mM) changes in cytokines were attenuated in the extract and nanoparticles by pretreatment and cotreatment at two time points (p < 0.05). H₂O₂ increased cell apoptosis. In contrast, treatment with nanoparticle-loaded TS suppressed the percentage of apoptosis considerably (p < 0.05). Therefore, TS may be considered as a potential candidate for enhancing the effectiveness of MSC transplantation in cell therapy.

Figure 1

Gas chromatography–mass spectroscopy chromatogram of Thymus serpyllum hexane extract. Peak numbers indicate the type of active component present in the extract.

Figure 2

Free radical scavenging potential of (a) hexane extract, (b) dichloromethane extract, (c) methanol extract, and (d) ascorbic acid (control). The percentage of scavenging activity has been compared with ascorbic acid used as control.

Figure 3

Expression of BCL2, cytochrome c (Cyt-c), and matrix metalloproteinase (MMP13) in control (untreated) and experimental groups (Extract-S and Nano-S represent cotreatment with hydrogen peroxide (H₂O₂–0.5 mM)).

Figure 4

Expression of BCL2, cytochrome c (Cyt-c), and matrix metalloproteinase (MMP13) in control (untreated) and experimental groups (Extract-S and Nano-S represent cotreatment with hydrogen peroxide (H₂O₂—1 mM)).

Figure 5

Expression of BCL2, cytochrome c (Cyt-c), and matrix metalloproteinase (MMP13) in control (untreated) and experimental groups (Extract-P and Nano-P represent pretreatment with hydrogen peroxide (H₂O₂–0.05 mM)).

Figure 6

Expression of BCL2, cytochrome c (Cyt-c), and matrix metalloproteinase (MMP13) in control (untreated) and experimental groups (Extract-P and Nano-P represent pretreatment with hydrogen peroxide (H₂O₂—0.1 mM)).

Figure 7

(a–d) Levels (pg/ml) of IL8, IL2, IL1beta, and VEGF in control (untreated) and experimental groups (S—cotreatment, P—pretreatment), as well nano- and extract-treated cells at two different time points, 24 hr and 72 hr.

Figure 8

(a–d) Levels (pg/ml) of IL6, IL2, TNF-alpha, MCP1, and IL3 in control (untreated) and experimental groups (S—cotreatment, P—pretreatment), as well nano- and extract-treated cells at two different time points, 24 hr and 72 hr.

Figure 9

Flow cytometric analysis of H₂O₂ control and experimental groups such as extract- and nanoparticle-loaded Thymus serpyllum treated to cells at two different time points, 24 hr and 72 hr, as cotreatment against H₂O₂ (a–e). Relative percentage of apoptosis (f) in control and experimental groups such as extract- and nanoparticle-loaded Thymus serpyllum treated to cells at two different time points, 24 hr and 72 hr, as cotreatment against H₂O₂.