Human Umbilical Cord MSC Derived Exosome Promote HSC Ferroptosis To Inhibit Liver Fibrosis Via Exosomal Beclin1 Transfer

Objective:A large number of patients suffer from chronic liver diseases.We found that human umbilical cord mesenchymal stem cell derived exosomes（hucMSC-Ex）played an important role in inhibiting liver fibrosis,but the detailed underlying mechanism is unknown.The activation of hepatic stellate cells（HSCs）to secrete a large amount of collagen is an important cause of liver fibrosis.This study aims to observe the effect of hucMSC-Ex on promoting HSCs ferroptosis and inhibiting liver fibrosis,and to explore the regulating mechanism of hucMSC-Ex transporting Beclin1in HSCs ferroptosis,which provides a new basis for hucMSC-Ex to repair liver fibrosis.Methods:Huc MSCs were isolated and cultured,and then hucMSC-Ex were separated and purified by ultracentrifugation method.The morphology of hucMSC-Ex were analyzed using transmission electron microscope（TEM）and atomic electron microscope.And the particle size of hucMSC-Ex were analyzed using nanoparticle tracking analysis（NTA）.The expressions of exosomal marker proteins CD9,CD63,CD81,Alix and TSG101 were analyzed by western blot and imaging flow cytometry.LX-2 were co-cultured with hucMSC-Ex.And we observed ferroptosis of LX-2 after hucMSC-Ex treatment including cell viability,reactive oxygen species（ROS）,lipid peroxidation malondialdehyde（MDA）,glutathione（GSH）and so on.Then the protein expression level of Beclin1 in hucMSC-Ex were detected by western blot.The protein and m RNA expression levels of Beclin1 in LX-2 treated with hucMSC-Ex were detected by real-time fluorescent quantitative PCR and western blot.Cell immunofluorescence staining and immunohistochemical staining were used to observe the expression levels of CD9 and Beclin1 in LX-2 and liver tissues treated with hucMSC-Ex.By overexpression or knockdown of Beclin1 in LX-2,we detected the Beclin1/System Xc-/GPX4 signaling pathway related to ferroptosis by western blot and real-time fluorescence quantitative PCR.Ferroptosis of LX-2 was observed by detection of the changes in ROS and mitochondrial membrane potential.Construct a CCl₄-induced ICR mice hepatic fibrosis model,and inject hucMSC-Ex and hucMSC-Ex^{sh Beclin1} by the tail vein.HE staining,sirius red staining and other histoimmunochemical staining were used to evaluate collagen deposition of liver tissues.And western blot and real-time fluorescent quantitative PCR were used to detect Beclin1/System Xc-/GPX4 signaling pathway to clarify the roles and mechanisms of hucMSC-Ex.Results:（1）The extracted hucMSC-Ex had the typical characterization of exosomes.（2）Huc MSC-Ex could promote ferroptosis of LX-2 and HSCs in mice fibrotic liver.（3）Beclin1 regulates System Xc-transcription to promote LX-2 ferroptosis.（4）Huc MSC-Ex could promote ferroptosis of HSCs by transporting Beclin1.（5）Huc MSC-Ex could promote the ferroptosis of HSCs,inhibit the activation of HSCs,and then alleviate liver fibrosis.Beclin1 knockdown in hucMSC-Ex weakened the inhibitory effect of liver fibrosis.Conclusion:Beclin1 is the key molecule for hucMSC-Ex to promote ferroptosis.Huc MSC-Ex regulates System Xc-/GPX4 signaling pathway by transporting Beclin1to promote HSCs ferroptosis,inhibit the activation of HSCs and collagen deposition,and then delay liver fibrosis.