Study On The Mechanism Of Hepatic Ischemia-reperfusion Injury By Exosomes Derived From Dendritic Cells

Objective: Liver ischemia-reperfusion injury(IRI)is a common injury during liver resection and liver transplantation,and it is also an important cause of slow recovery and dysfunction of liver function after surgery.In addition,the immune response induced by it often causes distant organs damage,which affects the recovery of function of various organs after surgery,and even results in the death of the patient.Studies have shown that dendritic cells(DCs)play an important role in liver IRI.Exosomes are small double-membrane vesicles secreted by various cells such as dendritic cells,macrophages,and T cells under physiological and pathological conditions.However,the mechanism of action of dendritic cell-derived exosomes(DEXs)in liver IRI remains unclear.In this study,we examined the secretion of DEXs after ischemia-reperfusion,and performed relevant functional experiments to explore the mechanism of DEXs in regulating liver IRI in mice.Methods: Primary hepatocytes were isolated and used to mimic the liver ischemia-reperfusion microenvironment.The extracted bone marrow-derived dendritic cells(BMDC)were subjected to different stimulation to extract exosomes.Exosomes secreted under different conditions were injected into mice by the tail vein before liver IR and the therapeutic effect was evaluated.Serum aminotransferases(aspartate aminotransferase(AST)and alanine aminotransferase(ALT)),inflammatory cytokines,and histological changes were measured by enzyme-linked immunosorbent assay(ELISA)and pathological analysis,respectively.DEXs were separated by gradient ultracentrifugation and identified by transmission electron microscopy(TEM)and nano-trace analysis(NTA)and Western blot(WB).We then co-cultured BMDCs/DEXs with na?ve T cells to induce differentiation of na?ve T cells into Treg/Th17 cells for flow cytometry(FCM),ELISA and confocal imaging analysis.Finally,the HSP70 inhibitor cmHSP70.1,PI3 K inhibitor BKM120 and mTOR inhibitor rapamycin were used to study the signaling mechanism of the DEXs-derived HSP70 affecting the differentiation and function of Th0 cells.Results: The number of regulatory T cells(Treg)in the H/R-BMDCs group was significantly higher than that of the other groups,whereas the expression of helper T cells 17(Th17)was lower than that of the other groups,but this effect was significantly suppressed after the release of DEXs was suppressed.In addition,confocal imaging showed that the uptake of H/R-DEX by naive T cells was greater than either the control or the negative group,and this increase was closely related to the differentiation of Treg and Th17.In addition,administration of H/R-DEXs improved liver function in mice after IR.Finally,inhibition of HSP70,PI3 K,and mTOR abolished the effect of DEX on naive T cells.Conclusion: Bone marrow-derived dendritic cells regulate the balance between Treg and Th17 cells through exosomes,thereby alleviating hepatic ischemia-reperfusion injury.In addition,DEXs relieve liver ischemia-reperfusion injury by transporting HSP70 to naive T cells and stimulating the PI3K/mTOR axis to regulate the balance between Treg and Th17 cells.