Multiple Doses of Overexpression of Cellular Prion Protein inAdipose-derived Mesenchymal Stem Cells Safely Provided AdditionalProtection to the Lungs Against Bleomycin-induced Damage in Rodents

Introduction: We tested whether early administration of multiple doses of adipose-derived mesenchymal stem cells (ADMSCs) overexpressing cellular prion protein (PrPC-OVE) could preserve lung parenchyma and function in a rodent model of bleomycin-induced pulmonary fibrosis (PF). Methods: Cell culture, immunohistochemistry, immunofluorescence, and western blot analyses were conducted. Animals were categorized into sham-control (Group 1), PF (Group 2), PF + ADMSCs (one dose; Group 3), PF + ADMSCs (three doses; Group 4), PF + PrPC-OVE in ADMSCs (one dose; Group 5), and PF + PrPC-OVE in ADMSCs (three doses; Group 6). Results: In vitro, bleomycin suppressed L2 cell proliferation and increased apoptosis and epithelialmesenchymal transition (EMT) markers, which were significantly reversed by ADMSCs and further enhanced by PrPC-OVE in ADMSCs. TGF-β/Smads signaling-mediated EMT was identified as a crucial mechanism in cellular fibrosis and PF, which was significantly suppressed by silencing TGF- β in L2 cells and PrPC-OVE in ADMSCs co-cultures and lung tissue. By days 28 and 42 after PF induction, O₂ saturation (%) was highest in Group 1, lowest in Group 2, and notably higher in Group 6 than in Groups 3-5. Right-ventricular (RV) systolic blood pressure showed an inverse trend. RV and left lung weights-to-tibial length ratios were significantly greater in Group 2 at both time points. By day 42, Group 2 exhibited the highest lung injury, fibrosis, and protein levels of fibrotic, EMT, inflammatory, and oxidative stress markers, whereas these parameters were lowest in Group 1 and significantly higher in Group 4 than in Group 6. Discussion: This study, which investigated the therapeutic role of PrPC-OVE in ADMSCs in protecting lung parenchyma against bleomycin-induced damage, provides several important preclinical insights. First, the in vitro study demonstrated that bleomycin activated TGF-β/Smads signaling, inducing EMT upregulation in lung epithelial cells (L2 cell line), which plays a crucial role in the initiation and propagation of PF. Second, both in vitro and in vivo studies showed that PrPC-OVE played a fundamental role in attenuating bleomycin-induced PF, mainly through regulation of TGF-β/Smads signaling. Third, ADMSCs were less effective than PrPC-OVE in ADMSCs, and repeated doses of ADMSCs were less effective than repeated doses of PrPC-OVE in ADMSCs in protecting lung function and parenchyma against bleomycin-induced damage. Conclusion: Repeated PrPC-OVE-ADMSCs effectively preserved lung function and parenchyma by suppressing TGF-β/Smads signaling in bleomycin-induced PF.