The Role Of Connexin43 In Secondary Injury After Intracerebral Hemorrhage And The Theraputic Effect Of Ecto-mesenchymal Stem Cell Transplantation

Objective:To investigate the mechanism of connexin43(Cx43)in secondary injury after cerebral hemorrhage and the effect of EMSC transplantation.Methods:1.Astrocytes were treated with hemin,the degradation product of hematoma,to establish a cell model of oxidative stress injury.TAT-Gap19 was used to block Cx43 HC,and CBX to block Cx43 GJIC,observe the effect on hemin-induced apoptosis and ferroptosis,and explore the possible mechanism of action.2.The expression of Cx43 in ASC was knocked down by interfering lentivirus,and the sensitivity of cells to hemin-induced apoptosis and ferroptosis was observed.The possible mechanisms of action and signaling pathways were predicted by GSEA gene set enrichment analysis.3.ASCs were treated with cytokines IL-1α,TNF and complement C1 q to establish a stable A1 phenotype.The EMSCs were co-cultured in A1 to observe the reversal effect of EMSC on the A1 phenotype.4.A rat model of ICH was established,and EMSCs were intravascularly transplanted through the internal carotid artery on the same side of the hemorrhage to observe the improvement of neurological function in rats,and to observe the migration and transformation of transplanted cells by immunofluorescence staining.Return,ASC activation and A1 phenotype transformation around the hematoma,and the degree of neuronal and white matter fiber damage.5.The single-cell transcriptome sequencing was used to characterize the purified and cultured EMSCs in vitro,and the possible mechanisms of EMSCs’ immunomodulatory effects were predicted by means of bioinformatics analysis.Results:1.In a physiological state,ASC highly expresses Cx43,HC is in a closed state,and a syncytium-like structure is formed between cells through GJIC,which is essential for maintaining ASC homeostasis.After being stimulated by hemin,ASC was activated,Cx43 was down-regulated,HC was abnormally opened,GJIC function decreased,and the cells underwent apoptosis and ferroptosis.Blockade of Cx43 HC with TAT-Gap19 enhanced the resistance of ASC to hemin-induced apoptosis and ferroptosis,while blocking of Cx43 GJIC with CBX increased the sensitivity of ASC to hemin-induced apoptosis and ferroptosis.2.ASC knocked down Cx43 expression enhanced the resistance to hemin-induced apoptosis and increased sensitivity to ferroptosis.GSEA gene set enrichment analysis found that Cx43 was significantly associated with Hippo signaling pathway,iron binding gene set,lipid peroxidation pathway,hypoxia stress and other pathways,suggesting that CX43 may be involved in the regulation of lipid metabolism and ferroptosis.Transcription factor regulatory network analysis suggested that TEAD3 may be involved in the signaling pathway of Cx43 regulating ferroptosis.3.EMSC can reverse the A1 phenotype in both in vitro and in vivo experiments.Transplantation of EMSC through the internal carotid artery on the ipsilateral side of the hematoma can orientately migrate to the injured area,reduce brain edema and improve neurological function in ICH rats.Immunofluorescence staining showed that EMSC transplantation could inhibit ASC activation and A1 phenotype transformation,and protect neurons and white matter fibers.The aforementioned neuroprotective effects of EMSCs were attenuated after knockdown of Cx43 expression in EMSCs.The results of immunofluorescence co-localization showed that the transplanted EMSCs were diffusely distributed in the brain parenchyma around the lesion,but could not differentiate into neurons or glial cells.After transplantation,Cx43-knockout EMSCs failed to survive in the hematoma environment,eventually necrotic disintegration and encased by a glial scar.4.The single-cell transcriptome sequencing results of EMSCs showed that EMSCs expressed both mesenchymal stem cell and fibroblast marker proteins,and expressed cell proliferation-related marker proteins Mki67 and Top2 a.GSVA analysis of the cell function-related gene sets of EMSCs showed that EMSCs were enriched in gene sets related to DNA replication,damage repair,intercellular tight junctions,and TGF-β signaling pathways.Conclusions:1.Cx43 GJIC is essential for maintaining ASC homeostasis and protects cells from apoptosis and ferroptosis.Cx43 HC is in a closed state under physiological state,and abnormally opens after being stimulated by hemin,resulting in increased cell membrane permeability,apoptosis and ferroptosis.Targeted protection of Cx43 GJIC and blockade of Cx43 HC after ICH contributes to neuroprotection and attenuates secondary oxidative stress damage.2.In addition to its channel function,Cx43 can also regulate cell phenotypic transformation,apoptosis and ferroptosis through YAP/TEAD3 signaling pathway.3.Cx43 has various functions such as gap junction channel,hemichannel and signal transduction,and plays important roles in the oxidative stress injury of the cells after ICH.In the context of ICH,maintaining Cx43 expression and GJIC,blocking abnormally open HC can help stabilize the cell phenotype and reduce apoptosis and ferroptosis.4.ASC activation and A1 phenotype transformation play an important role in the secondary injury after ICH.EMSCs can inhibit ASC activation and reverse the A1 phenotype in a Cx43-dependent manner,exerting neuroprotective effects.TGF-β signaling pathway may be involved.5.Transplantation of EMSC through the internal carotid artery can efficiently deliver stem cells to the injured area to play a neuroprotective role.EMSC transplantation can inhibit ASC activation and A1 phenotype after ICH,reduce brain edema,protect neurons and white matter fibers,and this effect is dependent on the normal expression of Cx43.And the expression level of Cx43 in EMSC can affect the fate of transplanted EMSC.6.The neuroprotective effect of EMSC transplantation on ICH rats mainly depends on Cx43-mediated immunomodulatory function,rather than direct differentiation into neurons or glial cells to replace damaged cells.