Study On The Repaired Mechanism Of HUCMSC-EXO On Raloxifene Injury In Endometrial Stromal Cells

Objective:To study the repair of damage to endometrial stromal cells by human umbilical cord mesenchymal stem cell-derived exosomes and its mechanism。Methods:Endometrial stromal cells were divided into control group,RAL groups with different concentrations（Raloxifene:0,20,30,40,50,60μmol/L）,30μmol/L RAL+human umbilical cord mesenchymal stem cell supernatant（RAL+h UCMSCs-Sup group）,30μmol/L RAL+Rapamycin（RAL+Rapa）,30μmol/L RAL+h UCMSCs-Sup+3-Methyladenine（RAL+h UCMSCs-Sup+3-MA group）,30μmol/L RAL+h UCMSCs-Sup+LY294002（RAL+h UCMSCs-Sup+LY294002 group）,30μmol/L RAL+human umbilical cord mesenchymal stem cell-derived exosomes（RAL+h UCMSCs-EXO group）,RAL+h UCMSCs-EXO+LY294002 group,different concentrations of RAL+exosome-free human umbilical cord mesenchymal stem cell supernatant(RAL+h UCMSCs-Sup^exo-group).（1）.MTT experiment was used to detect the viability of endometrial stromal cells.（2）.Flow cytometry was used to detect the relative amount of reactive oxygen species（ROS）in the endometrial stromal cell and the mitochondrial membrane potential level in the cell.（3）.Western Blot was used to detect the expression of m TOR,ULK1,p-ULK1,p-AKT,AKT,Bcl2,Bax,p-p62,p62 and LC3 and other related proteins.Results:（1）.With the increase of RAL concentration and the prolongation of action time,the survival rate of endometrial stromal cells decreased with significant differences（**p<0.01）.RAL significantly increased the level of intracellular ROS（**p<0.01）and decreased the level of intracellular mitochondrial membrane potential（**p<0.01）.After endometrial stromal cells were treated with 30μmol/L RAL,the expression of Bax protein in cells was higher than that of the control group,and the expression of Bcl2 protein was lower than that of the control group,and the difference was statistically significant（**p<0.01）.（2）.After the addition of h UCMSCs-Sup,the survival rate of endometrial stromal cells downregulated by RAL was significantly increased（**p<0.01）.Compared with the RAL group,h UCMSCs-Sup significantly reduced intracellular ROS levels（**p<0.01）and increased intracellular mitochondrial membrane potential levels（**p<0.01）.And h UCMSCs-Sup downregulated the expression of Bax protein in endometrial stromal cells and increased the expression of intracellular Bcl2 protein,and the difference was statistically significant（**p<0.01）。The detection of autophagy-related proteins showed that h UCMSCs-Sup upregulated the expression of intracellular p-ULK1,p-AKT,p-p62 and p62 proteins,and the expression ratio of LC3II/I proteins increased.After the addition of autophagy inhibitor 3-MA or LY294002,the survival rate of endometrial stromal cells restored by h UCMSCs-Sup,the down-regulated ROS level,and the up-regulated mitochondrial membrane potential were inhibited.The expression of p-ULK1,p-AKT,p-p62 and p62 proteins and the expression ratio of LC3II/I proteins decreased（**p<0.01）.（3）.With the addition of the autophagy activator Rapamycin,the survival rate of endometrial stromal cells was significantly increased（**p<0.01）,which had the same repair effect as h UCMSCs-Sup,while Rapa also reduced intracellular ROS levels（**p<0.01）and increased intracellular mitochondrial membrane potential levels（both**p<0.01）.The detection of autophagy-related proteins showed that Rapa upregulated the expression of p-ULK1,p-AKT,and p-p62 proteins in cells,and the expression ratio of LC3II/I proteins was also increased.（4）.After the addition of h UCMSCs-EXO,the survival rate of endometrial stromal cells downregulated by RAL was significantly improved（**p<0.01）,and h UCMSCs-EXO reduced intracellular ROS levels（both**p<0.01）and increased intracellular mitochondrial membrane potential levels（both**p<0.01）compared with the RAL group.Immunoprotein detection of autophagy-related proteins showed that h UCMSCs-EXO could increase the expression of p-ULK1,p-AKT and p-p62 proteins in endometrial stromal cells,and the expression ratio of LC3II/I proteins was also higher than that in RAL group（all**p<0.01）.After the addition of autophagy inhibitor LY294002,the survival rate of endometrial stromal cells（**p<0.01）,down-regulated ROS level,and up-regulated mitochondrial membrane potential were inhibited by h UCMSCs-EXO.The expression of intracellular p-ULK1,p-AKT,p-p62,p62 and p62 proteins and the expression ratio of LC3II/I proteins decreased（all**p<0.01）.Conclusion:（1）.RAL can induce an increase in the content of reactive oxygen species in endometrial stromal cells,reduce intracellular mitochondrial membrane potential levels,and inhibit cell survival.（2）.h UCMSCs-Sup can inhibit the increase of intracellular reactive oxygen species caused by RAL,increase the level of intracellular mitochondrial membrane potential,and activate the AKT/ULK1/p62 signaling pathway in endometrial stromal cells through the exosomes secreted by h UCMSCs to initiate autophagy,thereby improving the survival rate of endometrial stromal cells,while autophagy inhibitors 3-MA and LY294002 reverse the repair ability of h UCMSCs-Sup.