Effect Of Salubrinal On Improving Oxidative Stress-Induced BMSCs Senescence And Senile Osteoporosis

Age-related aging diseases as a common disease of the elderly group are receiving more and more extensive attention and exploration.Studies have shown that different types of bone cells enduring cellular senescence in the bone microenvironment,which leads to an increased risk of osteoporosis.Osteoporosis is mainly manifested by decreased bone density and bone mass,resulting in increased bone fragility,thereby increasing the risk of fracture,while senotherapies therapy to remove senescent bone cells can improve the bone microstructure of senescent bone.However,the mechanisms underlie for the formation of senescent bone cells are still unknown.Hydrogen peroxide（H₂O₂）is an inducer of cellular oxidative stress that induces cellular mitochondria to produce oxidative radicals（ROS）,promote cell senescence and affect cell longevity.In this paper,the relationship between the unfolded protein response（UPR）signaling pathway branch activated by endoplasmic reticulum stress（ER stress）and the aging of bone marrow mesenchymal stem cells（BMSCs）induced by oxidative stress was studied.At first,we found that in H₂O₂-induced senescent bone marrow mesenchymal stem cells,PKR-like endoplasmic reticulum kinase（PERK）-eukaryotic initiation factor 2α（e IF2α）signaling branch is specifically activated.Blocking the PERK-e IF2αpathway with specific inhibitor AMG’44 did not improve senescence related phenotypes in aging BMSCS.After AMG’44 treatment,there was no significant change in the proportion ofβ-galactosidase stained positive cells of senescent bone marrow mesenchymal stem cells,and there was no significant change in the expression levels of P16 and P21 proteins of aging marker molecules.Furthermore,we treated senescent cells with the e IF2αdephosphorylation inhibitor salubrinal and found that after the PERK pathway was continuously activated,the expression of positive cells withβ-galactosidase positive staining,as well as the m RNA of cell senescent markers Cdkn1αand Cdkn2αand corresponding related proteins were down-regulated.In addition,salubrinal promotes apoptosis of senescent bone marrow mesenchymal stem cells that induced by H₂O₂.After salubrinal treatment,a significant increase in the trend of early apoptosis of senescent cells and upregulation of Chop and Bim expression of the apoptosis pathway were found.On this basis,further studies found that salubrinal could significantly improve the osteogenic differentiation ability of senescent BMSCs.After salubrinal treatment,m RNA expression of Alp,Runx2,and Osteocalcin,markers of osteogenic differentiation,was elevated,and a significant increase in alkaline phosphatase（ALP）and alizarin-stained positive cells were found.At last,in vivo experiments showed that salubrinal administration significantly restored the bone microstructure of senescent SAMP6 mice.After Salubrinal adminstration,the m RNA levels of Cdkn1αCdkn2αand senescence related inflammatory factors（SASP）in BMSCs cells extracted from SAMP6 mice were significantly decreased.The indicators reflecting bone microstructure were improved,as bone volume fraction（BV/TV）,trabecular number（Tb.N）,trabecular thickness（Tb.Th）,mean cortical thickness（Ct.Th）,and cortical area fraction（Ct.Ar/Tt.Ar）increased,and the trabecular space（Tb.Sp）.Taken together,our data revealed an unrecognized role of ER stress signaling in the maintenance of cellular senescent phenotype in BMSCs and demonstrated that the activation of e IF2asignaling with salubrinal is helpful for the clearance of senescent BMSCs and the improvement of bone integrity of aged mice.