Construction of Curcumin-Loaded Mesenchymal Stem Cell-Derived Exosomes and Their Mechanism in Inhibiting Pyroptosis...

Objective: Hepatic ischemia-reperfusion injury (HIRI) is a common pathological condition in liver surgery and transplantation, and cellular pyroptosis plays a key role in its pathogenesis. However, the clinical application of curcumin is limited by its poor water solubility and low bioavailability. This study aims to develop mesenchymal stem cell (MSC)-derived exosomes loaded with curcumin (Exo-Cur). It also investigates the role and mechanism of Exo-Cur in inhibiting HIRI-related cellular pyroptosis. Methods: The preparation of Exo-Cur was optimized using orthogonal experimental design. Its solubility, stability, particle size distribution, and zeta potential were then evaluated. The morphology of Exo-Cur and its uptake in hepatocytes were observed using laser scanning confocal microscopy. The effect of Exo-Cur on HIRI was assessed through hematoxylin and eosin (HE) staining, ALT and AST measurements, TUNEL assay, CCK-8 assay, and lactate dehydrogenase (LDH) assay. Inflammatory cytokine protein levels were quantified by ELISA, and their mRNA expression was assessed by qRT-PCR. Pyroptosis was assessed by Western blot, immunohistochemistry, and flow cytometry. Additionally, protein expression changes in the PI3K/Akt/mTOR signaling pathway were analyzed using Western blot. Results: Orthogonal experiments determined that the optimal preparation method for Exo-Cur involves cell density at 95%, a curcumin concentration of 30 μg/mL, and a co-cultivation time of 12 h. Characterization results showed that Exo-Cur maintained its typical cup-shaped structure as well as stable particle size and zeta potential. Additionally, its water solubility and its stability in vitro were significantly improved compared to free curcumin. Further mechanistic studies indicated that Exo-Cur could ameliorate the abnormal morphology resulting from HIRI-induced hepatocyte pyroptosis, reduce the proportion of pyroptotic cells, and significantly downregulate the expression of NLRP3 inflammasome and downstream pyroptosis-related proteins (ASC, C-Caspase-1, GSDMD-N). Pathway analysis revealed that Exo-Cur activates the PI3K/Akt/mTOR axis, a pathway inhibited by HIRI. Moreover, rapamycin, an inhibitor of this pathway, reverses Exo-Cur's anti-pyroptosis effect. Conclusions: This study develops an efficient and stable Exo-Cur delivery system, confirming its protective effect against HIRI by activating the PI3K/Akt/mTOR axis and inhibiting NLRP3-mediated cellular pyroptosis. This innovative combination of MSC-derived exosomes combined with curcumin overcomes the limitations in clinical application of curcumin, such as poor bioavailability and stability, and offers a novel nanotherapeutic strategy to treat HIRI clinically.