A Connexin 3-YAP Axis Regulates Astroglial-Mesenchymal Transition In Hemoglobin Induced Astrocyte Activation After Intracerebral Hemorrhage

Objective:To observe the biological behavior of astrocytes after intracerebral hemorrhage(ICH)and to explore its biological significance and related molecular mechanisms.Methods:(1)We established collagenase-induced ICH mouse model and observed the differential xpression of GFAP and Vimentin during astrocyte activation in the peri-hematomal rain tissue by immunofluorescence staining and Western Blot analysis.Further,we lso observed the expression of epithelial marker E-Cadherin and mesenchymal arker N-Cadherin.(2)We established the intracerebral-hemoglobin-injection model and observed whether here is a similar change in the injection area as was observed in collagenase-induced CH model by immunofluorescence staining.(3)We established an in-vitro cellular model by exposing purified astrocyte cultures to emoglobin and then observed the expression of GFAP,E-Cadherin,Vimentin,N-adherin and SLUG by immunofluorescence staining and Western Blot analysis.The expression of Connexin 43 and YAP during the activation of astrocytes were tudied,the interaction between these two molecules were explored as well.(4)Astrocytes were activated by hemoglobin preconditioning and then exposed to emin,the cell viability and the cell death were evaluated by CCK-8 assay and LDH elease assay respectively.The cell apoptosis was detected by flow cytometry nalysis.The biological significance and the underlying mechanism were also xplored.Results:(1)The spatial and temporal differential expression of GFAP and Vimentin,E-Cadherin and N-Cadherin were observed in both collagenase-induced ICH and emoglobin-injection models,suggesting that hemoglobin produced by hemolysis fter ICH may induce the phenotypic change of astrocytes.(2)Hemoglobin can activate astrocytes in vitro and promote its proliferation and xpression of inflammatory cytokines.Activated astrocytes have decreased xpression of epithelial markers and increased mesenchymal markers,suggesting he de-differentiation and phenotypic change during the process of astrocyte ctivation.(3)Downregulation of Connexin 43 and nuclear translocation of YAP were observed n hemoglobin-treated astrocytes.Knocking down Cx43 triggered YAP nuclear ranslocation.Immunofluorescence co-localization analysis and co-immunoprecipitation showed that there was an interaction between Connexin 43 nd YAP.(4)Hemoglobin-preconditioned astrocytes acquired resistance to hemin-induced xicity.Hemoglobin treatment induced the expression of HO-1 and Nrf2 in strocytes and promoted Nrf2 nuclear translocation.Knocking down Nrf2 xpression by RNA interference attenuated the protective effect of hemoglobin-reconditioning on astrocytes.Conclusions:Hemoglobin produced after ICH can activate astrocytes,resulting in the down-regulation of Connexin 43.It liberates YAP and allows its nuclear translocation.Nuclear translocated YAP is transcriptionally active and promotes the expression of genes that are involved in cell proliferation and astroglial-mesenchymal transition.The Connexin 43/YAP axis mediates de-differentiation and phenotypic conversion during hemoglobin-induced astrocyte activation following ICH.Connexin 43-YAP signaling pathway can be developed as a target for modulating astrocyte activation.Hemoglobin-activated astrocytes acquired resistance to hemin-induced toxicity via Nrf2/HO-1 pathway.This phenomenon can be considered as the adaptive self-defense in the pathological process of ICH.Hemoglobin pre-warned astrocytes and enhanced their capability of handling the coming hemin “flood”.Nrf2/HO-1 may be employed as a target for neuroprotection after ICH.