The Protective Effects of MSC-Derived Exosomes Against Chemotherapy-Induced Parotid Gland Cytotoxicity

Abstract

Background: Fluorouracil (5-FU) is one of the most popular chemotherapeutic agents used in various cancer therapy protocols. Cell-free therapy utilizing exosomes is gaining increased popularity as a safer option due to concerns over potential tumor progression following stem cell therapy. Methods: Parotid glands of albino were treated with a single bone marrow mesenchymal stem cell (BMMSC)-derived exosomes injection (100 μg/kg/dose suspended in 0.2 mL phosphate-buffered saline [PBS]), a single 5-Fu injection (20 mg/kg), and BMMSC-derived exosomes plus 5-FU and compared to control group (daily saline injections). After 30 days, the parotid glands were examined using qualitative histological evaluation, immunohistochemical evaluation using rabbit polyclonal mouse antibody to Ki-67, caspase 3, and iNOS, as well as quantitative real-time polymerase chain reaction (RT-PCR) to evaluate gene expression of TGFβ1, TNF-α, and BCL-2. Results: Histological examination of the parotid gland revealed that BMMSC-derived exosomes restored the glands' architecture and repaired most of the distortion created by 5-FU. Immunohistochemical expression of tumor proliferation and cell death markers were restored to normal levels in the exosome-treated groups that were similar to the control group. Furthermore, BMMSC-derived exosomes reversed the effects of 5-FU on quantitative gene expression levels and showed a significant decrease in TNF-α (p < 0.001) and a significant increase in TGFβ (p < 0.0001) and BCL-2 (p < 0.05) when compared to 5-FU treatment. Conclusion: Within the limitations of the current study, BMMSC-derived exosomes have the potential to counteract the cytotoxic effects of 5-FU on the parotid glands of rats in vivo. Further studies are deemed necessary to simulate clinical scenarios.

Keywords: chemotherapy; exosomes; fluorouracil; parotid gland; stem cells.

Figure 1

Characterization of BMMSCs by inverted microscope showing spindle-shaped and confluent cells (A) and flowcytometry analysis showing positive expression of CD73, 90, and 105 and negative expression of CD34 and 45 (B). Characterization of exosomes by transmission electron microscope (C) and flow cytometry analysis showing positive expression of CD63 and 81 (D).

Figure 2

Histological analysis H&E stain showing Group I (A–C), Group II (D–F), Group III (G–I), Group IV (J–L), consisting of pure serous acini (black arrows), intercalated ducts (red arrows), striated ducts (yellow arrows), excretory duct (green arrows), and blood vessels (red circles). Original magnification 400x.

Figure 3

Immuno-stained sections of iNOS showing Group I (A), Group II (B), Group III (C), Group IV (D), Ki-67 showing Group I (E), Group II (F), Group III (G), Group IV (H), and caspase 3 showing Group I (I), Group II (J), Group III (K), Group IV (L). Original magnification 400×.

Figure 4

RT-PCR analysis of TGFβ, BCL-2 and TNF-α genes in the experimental groups. Levels of significance were ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, and ⁣^∗∗∗∗p < 0.0001.

Figure 5

Scatter plot showing the correlation and variability between each sample of the RT-PCR within the individual groups. The groups that have been marked by different lowercase letters (a, b or c) showed significant differences at p < 0.05 based on Tukey HSD test.