Hypoxic Preconditioning Enhances The Protective Effects Of Olfactory Mucosa Mesenchymal Stem Cells In Intracerebral Hemorrhage Via MiRNA-326-Mediated Autophagy

Background and objective:The effects of existing strategies on intracerebral hemorrhage(ICH)are limited.Mesenchymal stem cells(MSCs)hold great potential for treating ICH.However,the quantity and quality of MSCs decline in the cerebral niche,limiting the potential efficacy of MSCs.Hypoxic preconditioning is suggested to enhance the survival and neuroprotection of MSCs.Thus,the aim of the present study is to study the neuroprotective effect of transplanted hypoxia-preconditioned olfactory mucosa mesenchymal stem cells(OM-MSCs)in a model of intracerebral hemorrhage(ICH),and to explore the mechanism by which hypoxia preconditioning improves the protective effect of OM-MSCs.Methods:(1)In the in vivo model,ICH was induced in mice by administration of collagenase IV.At 24 h post-ICH,saline or normoxia or hypoxia OMMSCs was administered intracerebrally.The neuro-inflammation was evaluated on 3 days post-ICH,the behavioral outcome,brain injury.neuronal apoptosis,and survival rate of OM-MSCs were evaluated on 7days,14 days,and 28 days post-ICH.(2)In the in vitro model,OM-MSCs were exposed to hemin mimicking the microenvironment of ICH.Cellular senescence of normoxia/ hypoxia OM-MSCs was examined.To study the mechanism of hypoxic preconditioning regulating cellular senescence,the RNA-seq was performed to investigate the differences in the miRNA expression profiles between the normoxia and hypoxia OM-MSCs.Among differentially expressed miRNAs with 2-fold,we choose miRNA-326 to conduct the following research.We compared the cellular senescence of normoxia OM-MSCs transfected with miRNA-326 mimics,and hypoxia OM-MSCs transfected with miRNA-326 inhibitors.(3)To explore whether miRNA-326 reduces the cellular senescence of OM-MSCs by upregulating autophagy,we compared the cellular senescence and autophagy of normoxia/hypoxia OM-MSCs transfected with miRNA-326 mimics/inhibitor and the treatment of 3-MA/Rapamycin.Moreover,we used target gene prediction software to predict the target genes of miRNA-326.After confirming the targeting relation between miRNA-326 and PTBP1,we examined the cellular senescence and autophagy of MSCs treated with the PTBP1 expression vector or control vector.Moreover,we estimated the levels of PI3 K,and the autophagy of MSCs under PI3 K agonist treatment.Results:(1)Compared with the saline group,both normoxia OM-MSC and hypoxia OM-MSCs showed significant improvements in neuroinflammation,neurobehavioral tests,brain injury,and neuronal apoptosis at 3,7,14 and 28 days after ICH.However,hypoxia OMMSCs exhibited greater survival and neuroprotective effect at 14 and 28 days after ICH(P < 0.05).(2)In the in vitro model,hypoxic preconditioning alleviated cellular senescence of hemin-treated OM-MSCs.Differentially expressed genes between the hypoxia and normoxia cultured group suggested that the expression of miRNA-326 was obviously upregulated after hypoxic preconditioning.miRNA-326 mimics decreased the cellular senescence in normoxic OM-MSCs with hemin treatment(P < 0.05),whereas miRNA-326 inhibitors presented the opposite effects in hypoxia OMMSCs in the condition of hemin treatment(P < 0.05).(3)In the in vitro model,the alleviation of senescence in normoxia MSCs induced by the miRNA-326 mimics was partially reversed by treatment with 3-MA(P < 0.05).The increased cellular senescence in hypoxia-MSCs induced by the miRNA-326 inhibitor was partially reversed by treatment with rapamycin(P < 0.05).Thus,we suggested that hypoxic preconditioning ameliorating OM-MSCs senescence partially attributes to the miRNA-326-mediated autophagy.The dual-luciferase reporter assay confirmed that the miRNA-326 mimics resulted in significant downregulation of PTBP1 wild-type luciferase reporter activity(P < 0.05).PTBP1 overexpression decreased autophagy in miRNA-326 mimics-treated normoxia OM-MSCs.Furthermore,PTBP1 overexpression increased normoxic MSC senescence(P < 0.05),which was decreased by miRNA-326 mimics transfection.Moreover,overexpression of miRNA-326 could down-regulated PI3 K phosphorylation,overexpression of PTBP1 upregulated PI3 K phosphorylation.Treatment with PI3 K agonist partially reversed the regulatory effect of miRNA-326/PTBP1 on autophagy(P < 0.05).Conclusion:Hypoxic preconditioning reduced the senescence of OM-MSCs by upregulating miRNA-326/PTBP1/PI3K-mediated autophagy,and increased the survival rate of OM-MSCs,thereby enhancing the neuroprotective effect of OM-MSCs in a model of ICH.