Effects Of Bone Marrow Mesenchymal Stem Cells-Derived MiR-30c-5p On Polarization Of Microglia In Cerebral Ischemia Reperfusion And Its Mechanisms

Objective:Ischemic stroke is a common clinical disease with high disability and fatality rate.The common clinical treatment strategy is to restore the blood perfusion in the cerebral ischemic area as soon as possible.Recovery of blood perfusion is beneficial to the recovery of tissue function in the ischemic area,however,it may also cause reperfusion injury and aggravate tissue damage.Microglia is important innate immune cells in the central nervous system and participate in regulating the immune balance in the brain.Studies have shown that microglia play an important regulatory role in nerve injury and repair after cerebral ischemia and reperfusion.Excessive polarization of proinflammatory microglia may be a significant cause of aggravated cerebral ischemia reperfusion（I/R）injury（IRI）,while anti-inflammatory microglia polarization can reduce IRI and promote functional repair of the nerves.Mesenchymal stem cells（MSCs）have potent capacity not only in immune regulation,but also in anti-inflammation,anti-oxidative stress and angiogenesis.These functions owe largely to their paracrine function.Our previous work has shown that bone marrow mesenchymal stem cells（BMSCs）conditioned medium（CM）can effectively alleviate viability and function of myocardiocytes after I/R both in vitro and in vivo.In this study will explore the effect of BMSCs-CM and exosomes from BMSCs on the polarization of microglia and cerebra IRI induced by I/R in vitro and in vivo,and the potential molecular mechanisms.Then further investigate the influence of different oxygen concentrations on the effects of BMSCs-CM and exosomes in order to obtain paracrine derivatives of BMSCs with better regulation of microglial cell polarization and brain IRI.This study will provide experimental basis and theoretical basis for the application of paracrine derivatives of BMSCs in the treatment of cerebral IRI.Methods:（1）Rat BMSCs isolated and purified by whole bone marrow and culture in normoxic culture（21%O₂）.The Normoxia BMSCs-CM（Nor-CM）were collected.OGD/R treatment of BV2 microglia was used to mimics I/R in vitro.BMSCs-CM was co-cultured with BV2cells at the reoxygenation stage.Cell counting kit-8（CCK-8）and LDH kit is used to detect cell viability and LDH level.Level of nitric oxide（NO）produced in BV2 cells were detected with a NO kit.Levels of inflammatory cytokines（IL-1β,IL-6,TNF-αand IL-10）secreted by BV2 cells were detected by ELISA.Levels of the apoptosis rate and level of phenotype-related proteins（CD86,CD206）in BV2 cells were detected by Flow cytometry.（2）BMSCs were cultured in three modes:normoxic culture（21%O₂）,continuous hypoxic culture（1%O₂）,and hypoxia pretreatment（1%O₂,24 h）.The corresponding BMSCs-CM（Nor-CM;Hypoxia BMSCs-CM,Hyp-CM.Hypoxia pre-conditioned BMSCs-CM,Hyp P-CM）were collected.Cell viability,LDH level and apoptosis rate were detected.Expression levels of apoptotic proteins（Bcl-2,Bax,Caspase-3,Cleaved-caspase-3）and expression levels of polarization-related proteins,inducible nitric oxide synthase（i NOS）and Arginase-1（Arg-1）,was detected by Western Blot.Levels and reactive oxygen species（ROS）were detected by Flow cytometry.Mitochondrial membrane potential（MMP）was detected by fluorescence microscope.Levels of inflammatory cytokines（IL-1β,IL-6,TNF-αand IL-10）and phenotype-related proteins（CD86,CD206）was detected by ELISA or Flow cytometry（3）BV2 cells were divided into four groups according to different time points of reoxygenation:OGD4h/R0h group,OGD4h/R3h group,OGD4h/R6h group,and OGD4h/R12h group.The expression levels of Notch signaling pathway proteins were detected by Western Blot.γ-secretase inhibitor DAPT was used to inhibit Notch signaling pathway.The experiment was divided into four groups:Control group,OGD4h/R6h group,OGD4h/R6h+DAPT group,OGD4h/R6h+DMSO group.The expression levels of Notch signaling pathway in each group were detected by Western Blot.Viability,LDH release and oxidative stress levels of BV2 cells were detected.Expression levels of inflammatory cytokines,i NOS and Arg-1 were detected by ELISA or Western Blot.（4）The exosomes derived from Nor-CM,Hyp P-CM and Hyp-CM were collected by gradient ultra-high-speed centrifugation.The experiment was divided into 3 groups:Nor-Exos group,Hyp P-Exos group and Hyp-Exos group.The morphology of exosomes was observed by transmission electron microscopy.Phagocytosis of Dil-stained exosomes by BV2 cells was detected by super-resolution confocal microscopy.The expression levels of exosomal surface marker CD9,CD63 and TSG101 was detect by Western Blot.The concentration of exosomal protein was determined by Bradford protein assay（BCA）kit.BMSCs were pretreated by GW4869,BMSCs-CM was collected and cocultured with BV2cells stimulated by OGD4h/R6h.The experiment was divided into 5 groups:Control（control group）,OGD4h/R6h group,OGD4h/R6h+Hyp P-CM group,OGD4h/R6h+Hyp P-CM+GW4869 group and OGD4h/R6h+GW4869 group.Cell viability,LDH level the apoptosis rate were detected The expression levels of apoptotic proteins（Bcl-2,Bax,Caspase-3,Cleaved-caspase-3）and polarization-related proteins（i NOS,Arg-1）was detected by Western Blot.ROS,NO and MMP were detected.Expression levels of Notch signaling pathway proteins were detected.（5）According to bioinformatics methods,miRNA targeting Notch protein was searched.Their expression in BV2 cells treated with OGD/R was detected by Real-time Quantitative polymerase chain reaction（RT-q PCR）to screen the target miRNA.The expression of Notch1 was detected by Western Blot after transfection with miR-30c-5p mimics and inhibitor into BV2 cells.The SD rat middle cerebral artery occlusion/reperfusion（MCAO/R）model was constructed,and 100μg Nor-Exos or Hyp P-Exos were injected into the lateral ventricle.The experiments was divided into 5 groups:Sham group（sham operation group）,MCAO/R group,Nor-Exos（MCAO/R+Nor-Exos）group,Hyp P-Exos（MCAO/R+Hyp P-Exos）group,and miR-30c-5p inhibitor Hyp P-Exos^{miR-30c-5p（-）}(MCAO/R+Hyp P-Exos^{miR-30c-5p inhibitor})group.The Longa score was used to score the neurological deficits in each group.The cerebral infarction volume of each group was observed by TTC staining.The pathological changes of brain tissue were observed by HE staining.TUNEL staining was used to detect the level of apoptosis in the brain.Levels and activity of malondialdehyde,superoxide dismutase and catalase in brain tissue were detected by malondialdehyde,superoxide dismutase and catalase kits.The expression levels of CD68 and CD206 in microglia were detected by immunofluorescence labeling.Levels of serum inflammatory factors were detected by ELISA.The expression level of Notch signaling pathway proteins were detected by Western Blot.Results:（1）BMSCs-CM can not only ameliorate BV2 cells damage induced by OGD4h/R6h,but also significantly inhibit OGD/R-induced polarization of pro-inflammatory microglia and promote polarization of anti-inflammatory microglia.（2）All three types of BMSCs-CM can significantly improve the viability of BV2 cells,and reduce the level of apoptosis stimulated by OGD4h/R6h.They can effectively reduce the level of ROS and NO production and inhibit the decrease of MMP induced by OGD4h/R6h,significantly inhibit the expression of pro-inflammatory factors（IL-1β,IL-6,TNF-α）,inhibit the expression of i NOS and CD86,promote the expression of IL-10 and CD206.The effect of Hyp P-CM is better than that of Nor-CM and Hyp-CM.（3）The expression of Notch signaling pathway related proteins（Notch1,Jagged1,NICD1,Hes1）in BV2 cells significantly up-regulated after OGD/R treatment and the expression was highest at ODG4h/R6h.Compared with OGD/R group,treatment with DAPT,increased cell viability;reliefed apoptosis rate;downregulated the expression of IL-1,IL-6,TNF-αas well as i NOS,whileupregulated expression of IL-10 and Arg1.（4）TEM results showed that the exosomes collected by gradient ultra-high-speed centrifugation are typically cup-shaped with a diameter of about 30-150 nm.These exosomes highly expressed exosomes-related markers CD9,CD63 and TSG101.Moreover,BV2 cells can phagocytose Dil-stained exosomes.BCA results show that the concentration of exosomal protein in Hyp P-Exos group was significantly higher than that of Nor-Exos group and Hyp-Exos group.Compared with the OGD4h/R6h+Hyp P-CM group,cell viability decreased,LDH release and apoptosis rate increased;the level of ROS and NO production increased;MMP were decreased in OGD4h/R6h+Hyp P-CM+GW4869 group.GW4869 pretreatment also upregulated levels of pro-inflammatory factors,CD86 and i NOS,whereas downregulated levels of IL-10,CD206 and Agr-1.Moreover,the expression level of Notch1 signaling molecules increased in GW4869 pretreatment of BMSCs.（5）After OGD4h/R6h treatment,the expression of miR-30c-5p in BV2 cells was significantly down-regulated.Transfection of miR-30c-5p mimics into BV2 cells could significantly increase the expression level of miR-30c-5p in cells,while transfection of miR-30c-5p inhibitor could significantly reduce its expression.Western Blot results confirmed that transfection of miR-30c-5p mimics downregulated the expression of Notch1 in BV2 cells,whereas transfection of miR-30c-5p inhibitor upregulated the expression of Notch1.In MCAO/R model,both Nor-Exos and Hyp P-Exos can effectively improve neurological deficits,reduce brain tissue infarct volume and cell apoptosis as well as malondialdehyde production,and increase superoxide dismutase and catalase levels.They can also reduce the levels of IL-1β,IL-6 and TNF-α,and CD68,whereas increase levels of IL-10 and CD206 in microglia in the brain.Nor-Exos and Hyp P-Exos can also inhibit the expression of Notch1 signaling molecules.Moreover,compared with Nor-Exos,Hyp P-Exos has a more significant effect.Compared with Hyp P-Exos group,after injection of Hyp P-Exos transfected with miR-30c-5p inhibitor Hyp P-Exos^{miR-30c-5p（-）}group),the neurological deficit score,brain tissue infarct volume and brain cell apoptosis levels increased,level of malondialdehyde increased whereas levels of superoxide dismutase and catalase decreased.The expression of IL-1β,IL-6,TNF-αand CD68 increased,while the expression of IL-10 and CD206 decreased in microglia.In addition,the expression level of Notch1 signaling pathway proteins were increased.Conclusions:（1）BMSCs-CM can effectively inhibit oxidative stress of microglia stimulated by OGD/R,improve the viability and apoptosis of microglia,inhibit the polarization of pro-inflammat-ory microglia and promote the polarization of anti-inflammatory microglia,and the effect of Hyp P-CM was most effective.（2）The effects of BMSCs-CM were mainly mediated by exosomes.Hyp P-Exos can more effectively reduce MCAO/R-induced intracerebral oxidative stress,alleviate intracerebral cell apoptosis,promote polarization of anti-inflammatory microglia,and improve brain function and structural damage.（3）The therapeutic effects of Hyp P-Exo on cerebral IRI were mainly mediated through miR-30c-5p which negatively regulated Notch1 signaling pathway in microglia.