THE ROLE OF EXOSOMES DERIVED FROM HUMAN WHARTON'S JELLY MESENCHYMAL STEM CELLS INDUCED BY BIOACTIVE COMPOUNDS FROM INDONESIAN PLANTS IN REDUCING ACE2 EXPRESSION IN VERO AND CACO-2 CELL LINES

• Main Author: Gracia Feronytha, Alfina
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/80483
• Institution: Institut Teknologi Bandung

Excessive expression and dysregulation of Angiotensin Converting Enzyme-2 (ACE2) expression trigger various diseases and health complications such as hypertension, heart failure, and atherosclerosis. Since the spread of the SARS-CoV- 2 pandemic, there has been a significant focus on developing treatments that target the regulation of ACE2 expression. One recommended treatment development is cell-free therapy. This therapy is carried out by utilizing products released by cells, such as secretomes and exosomes. The cell environment is known to influence the composition and function of secretomes and exosomes. In this study, the potential of bioactive compounds curcumin (Curcumin longa L.) and quinine (Cinchona ledgeriana) was utilized to modify the environment of human Wharton's Jelly Mesenchymal Stem Cells (hWJ-MSC). Therefore, this research aims to modulate the content of exosomes and secretomes produced by hWJ-MSC to enhance the functional aspects of exosomes and secretomes by reducing ACE2 expression in Vero and Caco-2 cells. The hWJ-MSC obtained from the primary culture was morphologically characterized. These cells were divided into 4 groups, namely cells treated with bioactive compounds (curcumin or quinine), DMSO (as a solvent), and only with culture media. Secretomes and exosomes derived from hWJMSC were isolated from all treatment groups for further analysis. Exosomes isolated from hWJ-MSC were characterized with specific markers CD63, Transmission Electron Microscopy (TEM), and Nanoparticle Tracking Analysis (NTA). Internalization of exosomes by Vero and Caco-2 cells was observed at 1, 2.5, 5, 12, and 24 hours. ACE2 expression in Vero and Caco-2 cells treated with exosomes and secretomes from hWJ-MSC was observed at the 24th hour using immunocytochemistry (ICC) and Western blotting. The results of this study indicate that hWJ-MSC (Wharton's Jelly-derived Mesenchymal Stem Cells) have differentiation abilities, and the analysis of surface markers CD44, CD73, CD90, CD105 using a flow cytometer shows percentages of 100%, 96.4%, 99.9%, 97.7% respectively. Characterization of exosomes derived from hWJ-MSC based on the specific marker CD63 reveals concentrations for DMSO at 242.81 pg/mL, Control at 438.69 pg/mL, Quinine at 347.80 pg/mL, and Curcumin at 313.97 pg/mL. Analysis using TEM (Transmission Electron Microscopy) indicates that these extracellular vesicles meet the criteria for exosomes with a diameter of 30-150 nm. NTA (Nanoparticle Tracking Analysis) results show the exosome concentration in the hWJ-MSC treatment groups as follows: control 5.6 x 107 (particles/mL), curcumin 4.5 x 108 (particles/mL), and quinine 3.7 x 108 (particles/mL). Exosomes in each treatment group can be internalized at 1 hour, 2.5 hours, 5 hours, 12 hours, and are highly optimal at 24 hours. Immunocytochemistry (ICC) results on Vero and Caco-2 cells show that the addition of derivative hWJ-MSC exosomes treated with curcumin (Kur-Exo) and quinine (Kuin-Exo) more effectively inhibits ACE2 expression compared to derivative hWJ-MSC exosomes without treatment (Kont- Exo). The addition of derivative hWJ-MSC secretome treated with curcumin (Kur- CM), quinine (Kuin-CM), and without treatment (Kont-CM) does not show a decrease in ACE2 expression in Vero and Caco-2 cells. Western blotting results indicate that Caco-2 cells significantly lower ACE2 expression compared to Vero cells. In Vero cells, the addition of exosomes or secretome derivatives of hWJ-MSC treated with quinine (p ? 0.001) and curcumin (p ? 0.001) significantly higher than the control (p ? 0.05), while in Caco-2 cells, quinine (p ? 0.0001) and curcumin (p ? 0.0001) significantly more effective than the control (p ? 0.01) in inhibiting ACE2 expression. Furthermore, the addition of exosomes to Vero and Caco-2 cells significantly higher in inhibiting ACE2 in the quinine (p ? 0.01), curcumin (p ? 0.05), and control (p ? 0.05) groups, while the addition of exosomes or secretome in DMSO treatment has no effect on inhibiting ACE2 expression. Based on the study results, it can be concluded that the addition of bioactive compounds quinine and curcumin has a significant impact on the hWJ-MSC environment, enabling the modulation of exosome and secretome compositions produced by hWJ-MSC. Furthermore, exosomes and secretomes derived from hWJ-MSC treated with curcumin and quinine show a significant decrease in ACE2 expression in Vero and Caco-2 cells. This study represents a potential therapeutic implication of bioactive compounds, particularly in reducing ACE2 expression through exosomes, providing promising opportunities for further exploration in the medical research field.