Mechanism Of Conditional UTX Knockout Reducing SCMECs Senescence To Promote The Repair Of Spinal Cord Injury

ObjectiveSpinal cord injury（SCI）is a traumatic disease of the central nervous system,which often leads to motor,sensory and autonomic dysfunction of patients below the injury level.Until now,there is still no effective treatment.SCI usually causes destruction of microvascular network,and the limited regeneration ability of micro-vessels impair the recovery of neurological function after SCI.Whether endothelial cell senescence was involved in this process and the internal mechanism need to be further explored.The epigenetic factor ubiquitous transcribed tetratripeptide repeat,X chromome（UTX）is widely expressed in the eukaryotic nucleus,and plays an indispensable role in a variety of physiological and pathological processes.However,it is not clear whether UTX is involved in microvascular endothelial cell（SCMECs）senescence after SCI.This study aims to explore the role and mechanism of UTX in regulating endothelial cell senescence,which may provide potential therapeutic targets for the treatment of SCI.Method（1）8-week-old C57BL/6 mice were selected to establish a SCI model using the modified Allen’s method.Senescence-related beta-galactosidase（SA-β-gal）staining,immunofluorescence staining,q RT-PCR,and western blot（WB）were used to detect the senescence phenotype at different time points after SCI.The primary brain microvascular endothelial cells（BMECs）and mice brain microvascular endothelial cell line（b End.3）were intervened with hydrogen peroxide（H₂O₂）to induce senescence.Immunofluorescence staining,SA-β-gal staining,q RT-PCR,WB,and PCR-array were used to detect the expression of senescence related markers.Tube formation,transwell migration and scratch wound healing assay were applied to evaluate cellular biological activity.（2）Epigenetic zymogram PCR-array was used to screen the differentially expressed epigenetic factors,and verified by immunofluorescence staining,q RT-PCR and WB in vitro.After establishment of SCI model,spinal cord tissues were collected at 3,7,14and 28 days after the injury,respectively.WB,q RT-PCR,and immunofluorescence staining was used to detect the expression of UTX.BMECs from endothelial cells conditional knockout UTX mice(Tek-Cre:UTX^flox/flox,UTX^-/-)and the same litter control mice(UTX^flox/flox)were treated with H₂O₂to induce cell senescence.Immunofluorescence staining was used to detect the senescence related markers.UTX knockdown lentivirus（UTX-KD）and control lentivirus（UTX-NC）were constructed and transfect into b End.3 cells.SA-β-gal staining,q RT-PCR,WB,and immunofluorescence staining were performed to evaluate the effect of UTX on endothelial cell senescence.Tube formation,transwell migration and scratch wound healing assay were applied to evaluate the effect of UTX on cellular biological activity.Based on UTX^-/-mice and UTX^flox/floxmice,the SCI model was established.Immunofluorescence staining,SA-β-gal staining,BMS score,neuroelectrophysiological analysis and bladder diameter and detrusor thickness evaluation were applied to detect the effect of UTX on vascular endothelial cell senescence and neurological function repair.（3）BMECs from UTX^-/-mice and UTX^flox/flox mice were intervened with hydrogen peroxide（H₂O₂）to induce senescence.RNA-sequencing（RNA-seq）and chromatin immunoprecipitation-q PCR（Ch IP-q PCR）were used to screen the downstream target gene Calponin 1（CNN1）.q RT-PCR,WB and immunofluorescence staining were applied to verify whether UTX affects the expression of CNN1.CNN1 overexpression lentivirus（CNN1-OE）and corresponding negative control lentivirus（CNN1-NC）were co-transfected into b End.3 cells with UTX knockdown lentivirus.WB,immunofluorescence staining,SA-β-gal staining,tube formation test,transwell migration,scratch wound healing assay,etc.were performed to determine whether UTX affects b End.3 cell senescence and biological activity by regulating the expression of CNN1 in vitro.UTX^-/-mice and UTX^flox/flox mice were subjected to SCI models,and CNN1 overexpressed adeno-associated virus（AAV-CNN1）was injected locally into the injured spinal cord.Immunofluorescence staining,SA-β-gal staining,BMS score,neuroelectrophysiological analysis and bladder diameter and detrusor thickness were applied to explore whether UTX affects microvascular endothelial cell senescence and neurological function repair by regulating the expression of CNN1.Results（1）SA-β-gal staining and immunofluorescence staining showed that SCI induced the senescence of microvascular endothelial cell and reached the peak on the 14th day after injury.The results of q RT-PCR and WB also confirmed that the expression of senescence-related indicators（P53,P21,P16）increased significantly after SCI and reached the peak on the 14th day,while the expression of Lamin B1 decreased sharply after injury.In vitro SA-β-gal staining showed that 200μM H₂O₂ at concentration significantly induced the senescence of b End.3 cells.Immunofluorescence staining,q RT-PCR and WB showed that the b End.3cells induced by H₂O₂ had high expression of senescence-related indicators.PCR-array results showed that H₂O₂ induced b End.3 cells released many senescence-related secretory phenotypes（SASP）.Tube formation test,transwell migration,scratch wound healing assay indicated that the biological activity of senescent b End.3 cells decreased significantly.Similarly,immunofluorescence staining suggested that the primary BMECs treated with H₂O₂ had high expression of senescence-related indicators.（2）The results of epigenetic zymogram PCR-array showed that primary BMECs treated with H₂O₂highly expressed UTX.In vivo experiments also confirmed that UTX showed a trend of first increase and then decrease after SCI,reaching the peak on the 7th day after injury.After using lentivirus to knock down the UTX expression of b End.3 cells,the expression of senescence related indicators and SASP decreased significantly,while the cell biological activity increased.After SCI,compared with UTX^flox/flox mice,the overall senescence level of UTX^-/-mice and he expression of senescence-related indicators of endothelial cells were significantly reduced.The BMS score,neuroelectrophysiological analysis,bladder diameter and detrusor thickness evaluation,axon staining,etc.showed that the neurological function recovery of UTX^-/-mice was significantly better than that of the control group.（3）RNA-seq successfully screened the differentially expressed gene CNN1 after UTX knockout,and Ch IP-q PCR confirmed the targeted binding relationship between UTX and H3K27 of CNN1.Immunofluorescence staining,q RT-PCR and WB further showed that knockdown of UTX could significantly reduce the expression of CNN1.The UTX was successfully knocked down in b End.3 cells which was intervened with H₂O₂.The expression of senescence-related indicators was significantly reduced,and the cell biological activity was significantly increased after UTX knockdown.After transfection of CNN1overexpression lentivirus based on knockdown of UTX,the expression of senescence-related indicators was significantly increased,and the cell biological activity was decreased.In vivo experiments further confirmed that after the conditional knockout of vascular endothelial cell UTX,the spinal cord overall senescence level and endothelial cell senescence-related indicators were significantly reduced,and the recovery of neurological function was significantly improved.The use of AAV-CNN1 could significantly increase the vascular endothelial cell senescence-related indicators and inhibit the repair of neurological function.Conclusion（1）After SCI,cell senescence of spinal microvascular endothelial cells happened,and the biological activity of the senescent endothelial cells weakened.（2）The expression of UTX was increased after SCI,and conditional knockout of vascular endothelial cells UTX could reduce vascular endothelial cells senescence and promote the recovery of neurological function.（3）Conditional knockout of UTX reduced the senescence of vascular endothelial cells by down-regulating the expression of CNN1,thus promoting the recovery of neurological function in mice after SCI.