Exosomes derived from TGF-β1-pretreated mesenchymal stem cells alleviate biliary ischemia–reperfusion injury through Jagged1/Notch1/SOX9 pathway

This study aimed to evaluate the efficacy of exosomes (EXO) derived from TGF-β1-pretreated mesenchymal stem cells (MSCs) on biliary ischemia reperfusion injury (IRI) and further reveal the possible mechanisms.Bone marrow-derived MSCs were treated with exogenous TGF-β1, Jagged1/Notch1/SOX9 pathway inhibitor LY450139, or their combination. Then, EXO were isolated from the culture supernatants and further characterized. After establishing IRI model of biliary epithelial cells (EpiCs), EXO derived from differently-treated MSCs were applied to detect their protective effects on EpiCs, and LY450139 was applied in EpiCs to detect the possible mechanisms after treatment with MSCs-EXO. EXO derived from differently-treated MSCs were further injected into the hepatic artery immediately after establishment of intrahepatic biliary IRI for animal studies.Pretreatment with TGF-β1 significantly enhanced MSCs-EXO production and elevated the levels of massive miRNAs associated with anti-apoptosis and tissue repair, which were evidently decreased after TGF-β1 plus LY450139 cotreatment. Notable improvement was observed in EpiCs after MSCs-EXO treatment, evidenced by reduced cellular apoptosis, increased cellular proliferation and declined oxidative stress, which were more evident in EpiCs that were treated with EXO derived from TGF-β1-pretreated MSCs. However, application of EXO derived from TGF-β1 plus LY450139-cotreated MSCs reversely enhanced cellular apoptosis, decreased cellular proliferation and anti-oxidants production. Interestingly, LY450139 application in EpiCs after treatment with MSCs-EXO also reversed the declined cellular apoptosis and enhanced oxidative stress induced by TGF-β1 pretreatment. In animal studies, administration of EXO derived from TGF-β1-pretreated MSCs more effectively attenuated biliary IRI through reducing oxidative stress, apoptosis, inflammation and enhancing the expression levels of TGF-β1 and Jagged1/Notch1/SOX9 pathway-related markers, which were reversed after administration of EXO derived from TGF-β1 plus LY450139-cotreated MSCs.Our results provided a vital insight that TGF-β1 pretreatment endowed MSCs-EXO with stronger protective effects to improve biliary IRI via Jagged1/Notch1/SOX9 pathway.