RNA-seq Reveals Underlying Mechanisms Of Bone Marrow-derived Mesenchymal Stem Cells In The Regulation Of Microglia-mediated Neuroinflammation After Subarachnoid Hemorrhage

Subarachnoid hemorrhage(SAH)is a cerebrovascular disease characterized by high mortality and high disability.Microglia,the innate immune cells in the central nervous system(CNS),is associated with the inflammatory response by releasing a variety of inflammatory cytokines after SAH.Therefore,the regulation of activated microglia-mediated neuroinflammation has become a potential therapeutic strategy for SAH.Recent studies suggested that bone marrow mesenchymal stem cells(BM-MSCs)have potential therapeutic effects on SAH,resulting from the regulation of microglial activation and production of inflammatory cytokines after SAH.However,the underlying molecular mechanisms of BM-MSCs in targeting microglia-mediated neuroinflammation after SAH are still unclear.Therefore,in this study,oxyhemoglobin(OxyHb)was used to stimulate mouse-derived BV2 microglia to simulate a SAH model in vitro.The first part investigates the effect of BM-MSCs on OxyHb-induced BV2 microglial activation;the second part explores the specific mechanism by which BM-MSCs regulates OxyHb-induced BV2 cell activation.This study will provide a theoretical basis for clinical application of BM-MSCs in the treatment of SAH.I.The effect of BM-MSCs on OxyHb-induced BV2 microglia activationObjective: To demonstrate the regulating effect of BM-MSCs on OxyHb-induced BV2 microglia activation.Method: 1.BV2 cells were treated with OxyHb of 10?M concentration at different time points and the NO release amount was detected respectively.The time point in which maximum NO was released was determined as the treatment time of OxyHb for BV2 cells;2.BV2 cells were divided into the control group,oxyhemoglobin stimulated group(OxyHb)and bone marrow mesenchymal stem cells co-culture group(OxyHb+BM-MSCs).OxyHb and OxyHb+BM-MSCs groups were treated with 10 ?M concentration of OxyHb,and the control cells were treated with the same amount of normal culture medium.The effect of BM-MSCs on OxyHb-induced BV2 microglia activation was clarified by morphological observation combined with qRT-PCR,WB and immunofluorescence techniques.Results: BV2 cells release the maximum NO at 24 h of OxyHb stimulation(P <0.001).Compared with OxyHb group,the NO production of OxyHb+BM-MSCs group was decreased(P <0.001).qRT-PCR results suggested that the expression of TNF-?,IL-1? and M1 markers(CD16,CD32,iNOS,CD80 and CD86)in OxyHb+BM-MSCs groups was down-regulated compared with that in OxyHb group,while the anti-inflammatory factor IL-10 was up-regulated(P<0.01).However,no significant changes were observed in IL-6 and M2 markers(CD206,Arg-1 and TGF-?).WB suggested that theexpression of M1 marker CD16 and CD86 in OxyHb+BM-MSCs group was down-regulated compared with that in OxyHb group(P <0.001),and there was no significant change in M2 marker CD206(P >0.05).The results of immunofluorescence showed that the expression of M1 marker CD16/32 in OxyHb+BM-MSCs group was lower than that in OxyHb group(P <0.01),and there was no significant difference in M2 marker CD206(P >0.05).Conclusion: BM-MSCs can regulate M1 activation of BV2 microglia induced by OxyHb.II.The mechanism of BM-MSCs modulates BV2 microglia activation induced by Oxy HbObjective: To explore the mechanism of BM-MSCs modulate BV2 microglia activation induced by Oxy Hb.Method: BV2 cells were classified into three groups: control,Oxy Hb and Oxy Hb+BM-MSCs.Oxy Hb group and Oxy Hb+BM-MSCs group were treated with 10?M concentration Oxy Hb for 24 h,and the control cells were treated with the same amount of normal culture medium.Results: GO analysis of DEGs results suggest that BM-MSCs coculture induced an overall change of cellular component and signal transduction in BV2 cells which stimulated by Oxy Hb.DEGs were highly clustered in several inflammation-relating signaling pathways,especially FoxO,TNF and PI3K-Akt signaling pathways.The network analysis inflammatory response-related DEGs suggests that AKT1 and IL1 b are important hub nodes in the network diagram of inflammatory response-related DEGs interactions.Futhermore,DEGs analysis results suggest that the expression of Kdm6 b,Kdm7a,Dnmt3l-ps1,Hdac5 and Setd7 genes in Oxy Hb+BM-MSCs group were up-regulated compared with that in Oxy Hb group,which related to epigenetic regulation mechanism.the BM-MSCs coculture significantly increased the expression of Kdm6 b which decreased in Oxy Hb-stimulated BV2 cells.Conclusion: DEGs between OxyHb-stimulated BV2 cells cocultured with and without BM-MSCs were highly clustered in several inflammations relate to signaling pathways,especially Fox O,TNF and PI3K-Akt signaling pathways;Kdm6b might play a critical role in the process of BM-MSCs regulate BV2 microglia inflammation induced by Oxy Hb.