Human Umbilical Cord Mscs Derived Exosomes Transport USP9X To Inhibit Liver Sinusoidal Endothelial Cells Capillarization And Alleviate Liver Fibrosis

Objective:Liver fibrosis is closely related to the occurrence and development of end-stage liver diseases.But there is no reliable method for liver fibrosis at present.Liver sinusoidal endothelial cell（LSEC）capillarization can induce pathological vascular hyperplasia of liver and promote inflammation and collagen deposition of liver tissue,which is the characteristic change of early liver fibrosis and an important target for the treatment of liver fibrosis.Our previous studies have found that MSC-ex can effectively relieve liver fibrosis and restore liver function,but the exact mechanisms remain unclear.This study aims to explore the regulation of MSC-ex on the capillarization of LSEC,and to provide a new target and basis for the inhibition of liver fibrosis by MSC-ex.Methods:（1）MSCs were adherent culture,and the supernatant was collected to extract MSC-ex.The morphological structure of MSC-ex was observed by NTA and TEM.The expression of MSC-ex signature proteins CD63,TSG101,Alix and CD9 were analysed by Western blot and imaging flow.（2）Exosome uptake assay was used to detect the endocytosis of exosomes.TNF-αwas used to induce capillarization of LSEC,and MSC-ex was added to co-culture with LSEC.The level of angiogenesis markers were detected by q RT-PCR and Western blot,and the tube formation capacity of LSEC was detected by tubule formation assay.（3）Mice with early liver fibrosis induced by methionine choline deficiency diet（MCD）were constructed,and MSC-ex was injected by tail vein.The inhibitory effect of MSC-ex on LSEC capillarization was evaluated by immunohistochemical staining,sky wolf scarlet staining,transmission electron microscopy and scanning electron microscopy.（4）The expression of ubiquitin specific peptidase 9X（USP9X）in MSC-ex was detected.Then overexpressed and knocked down USP9X in LSEC,and the anti-capillarization effect of USP9X and possible regulated signaling pathways were observed by Western blot,q RT-PCR and cellular immunofluorescence.（5）MSCs were transfected with recombinant lentivirus to construct USP9X knockdown MSC-ex(MSC-ex^{sh USP9X}).Then,the effect of USP9X on LSEC capillarization under MSC-ex was observed.Results:（1）MSC-ex expressed exosome signature proteins like CD9,CD63,Alix and TSG101.Transmission electron microscopy showed that exosomes were disk-like vesicles with diameter of 100～150 nm,and had typical characteristics of exosomes.（2）MSC-ex attenuated LSEC tube formation in vitro in a concentration-dependent manner,and inhibit the expression of specific capillarization genes.（3）MSC-ex could reduce the collagen deposition and the expression of Ang-2 and CD34 in liver tissue and reduce LSEC“window loss”under MCD induced liver fibrosis.（4）LSEC overexpressed USP9X could inhibit the expression of LSEC capillarization specific genes Ang-2 and CD34,and inhibit tube formation capacity of LSEC,while LSEC knock-down USP9X were the opposite.（5）MSC-ex knock-down USP9X(MSC-ex^{sh USP9X})weakened the inhibitory effect of MSC-ex on LSEC capillarization.Conclusion:MSC-ex may inhibit the capillarization of LSEC and liver fibrosis by transporting USP9X,which is expected to be a possible therapeutic method against LSEC capillarization and be applied in the therapy of liver fibrosis.