BMSC-derived Exosomes Reduce CCN1-mediated Mitochondrial Damage And Apoptosis Of Hypoxic Liver Cells

The liver is one of the most important organs of the human body,and has extensive functions such as metabolism,glycogen storage,drug detoxification,and bile secretion.Liver function is very important for homeostasis.Surgery,trauma,drugs,etc.can cause liver damage and lead to liver function damage or even death.And 80%of the liver cells in the liver tissue structure are the main performers of liver function and the target cells of ischemic hypoxia stress injury.Hepatocytes are rich in mitochondria.When the cells are damaged by oxidative stress,mitochondrial damage will occur,changing mitochondrial permeability and membrane potential,which in turn induces apoptosis.Therefore,mitochondrial damage and apoptosis of hepatocytes play a key role in liver diseases caused by ischemia and hypoxia.Bone marrow derived mesenchymal stem cells（BMSC）are multifunctional stem cells derived from bone marrow and have the potential to differentiate into a variety of cells.They can be used to treat liver diseases,but stem cell therapy can cause embolism,abnormal differentiation,tumor formation and other potential risks.Recently,many studies believe that BMSCs mainly regulate the immune response through the paracrine mechanism and down-regulate the local inflammatory injury.Among them,BMSC-derived exosomes are important components of paracrine and play an important role in cell communication.Exosomes are cystic membrane vesicles with a diameter of 40-150nm secreted by cells,which are rich in proteins and RNA,especially miRNAs,have the advantages of low immunogenicity,biocompatibility,etc.,can proliferate cells,migration,apoptosis and other biological processes to regulate.Cysteine-rich protein 61（Cellular Communication Network Factor 1,CCN1）as a cell matrix protein can participate in cell proliferation,differentiation,apoptosis and other processes.When cell damage occurs,it is often accompanied by increased levels of CCN1,different CCN1 can activate apoptosis through mitochondrial pathway and TNF-αreceptor pathway.However,the mechanism of action of BMSC-derived exosomes in hypoxic liver cell injury,especially the role of CCN1 in hepatocyte mitochondrial damage and apoptosis has not been elucidated,so this article will focus on the study of BMSC-derived exosomes to reduce oxidation by reducing CCN1 The specific mechanism of liver cell mitochondrial damage and apoptosis under stress provides a new therapeutic strategy for the treatment of hypoxic liver damage such as ischemia.We used hydrogen peroxide（H₂O₂）to induce liver cell line L02 to establish a model of liver hypoxia injury.First determine whether the exosomes secreted by BMSC have a protective effect on liver cells.The BMSC medium supernatant was collected and incubated with L02 cells under hypoxia.It was found that BMSC culture supernatant can reduce apoptosis,protect mitochondrial membrane potential,and reduce reactive oxygen species level.Subsequently,BMSC was treated with exosomes secretion inhibitor-GW4869 and the supernatant was collected.The above experiment was repeated.The results showed that the protective effect of BMSC supernatant on liver cells disappeared.Finally,the exosomes in the BMSC culture supernatant were extracted by ultracentrifugation and the above experiment was repeated.It was found that the exosomes also had a protective effect on the cells.To analyze the molecular mechanism of exosomes protecting hepatocytes,the differentially expressed genes in hypoxic hepatocytes treated with BMSC exosomes,purely hypoxic hepatocytes,and normoxia-cultured hepatocytes were screened by transcriptome sequencing.Bioinformatics analysis of differentially expressed genes will initially lock the best candidate gene for hepatocyte apoptosis in response to hypoxia induction and regulated by BMSC exosomes as CCN1.Further using Real time-PCR to confirm the sequencing results,it was found that the expression of CCN1 in normal cells was very low.When H₂O₂/hypoxia stimulated the cells,it significantly increased,and exosomes can significantly inhibit the increase of CCN1 caused by oxidative stress.To further investigate whether the protective function of exosomes is mediated by CCN1,we constructed a CCN1 stable transfected cell line and found that CCN1-over expression not only aggravates H₂O₂/hypoxia-induced hepatocyte apoptosis but also completely blocks The protective effect of hypoxic hepatocytes derived from BMSC-derived exosomes;reducing CCN1 expression（sh-CCN1）can significantly reduce H₂O₂/hypoxia-induced hepatocyte apoptosis,and the protective effect of BMSC-derived exosomes on low-expression CCN1 L02 cells is not obvious.The above results indicate that BMSC exosomes reduce hepatocyte apoptosis by reducing hypoxia-induced CCN1.We also conducted a preliminary analysis of the possible mechanism of miRNAs rich in BMSC exosomes to regulate the expression of CCN1 through bioinformatics.Second analysis of the published data of BMSC exosomes miRNAs sequencing data,screened miR-22-3p that may negatively regulate CCN1 and enrich in exosomes.However,the specific control mechanism needs to be verified by subsequent experiments.In order to explore the mechanism of increased CCN1 transcription levels during hypoxia,the ZBTB7B were screened through the intersection of Animal TFDB database and L02 cell transcriptome sequencing results.preliminary verification that hypoxia can promote the expression of ZBTB7B,which is consistent with the change of CCN1;and exosomes can reduce the m RNA and protein levels of ZBTB7B,suggesting that exosomes may regulate CCN1 expression by inhibiting ZBTB7B.In summary,BMSC exosomes can reduce the expression of CCN1 induced by hypoxia and reduce the damage and apoptosis of mitochondria of liver cells under oxidative stress.Bioinformatic analysis suggests that BMSC-derived exosomes may transfer miR-22-3p to hepatocytes on the one hand,the latter has the potential to inhibit the expression of CCN1;on the other hand,exosomes may also inhibit the expression of the hypoxic hepatocyte transcription factor ZBTB7B To reduce the expression of CCN1,the two work together to achieve the effect of reducing H₂O₂/hypoxia-induced mitochondrial damage and apoptosis.