The Mechanism And Role Of Pericytes Senescence In Radiation-induced Brain Injury

Objective:Radiation-induced brain injury（RBI）is a prevalent complication of radiotherapy for head and neck tumors.Investigating the specific mechanisms of RBI to prevent and treat it is vital for enhancing patient quality of life.Pericytes,as a component of the neurovascular unit,contribute to blood-brain barrier（BBB）disruption and central nervous system（CNS）deterioration when they undergo aging and damage.This study aims to explore the precise mechanism of RBI from the perspective of pericyte senescence,determine whether head radiotherapy can induce pericyte senescence,investigate the role of pericyte senescence in RBI,and assess the effect of senolytic drugs on pericyte senescence and cognitive function in RBI.Furthermore,as autophagy inhibition is a critical factor leading to senescence,the present study also examines the exact link between autophagy and radiation-induced pericyte senescence.Methods:In the first part of the study,mice were exposed to whole-brain radiation therapy（WBRT）at a dose of 15 Gy to detect changes in cognitive function,and senescence-related markers were examined in vitro by constructing an irradiation（IR）-associated human brain microvascular pericytes（HBVP）model.In the second part of the study,a blood-brain barrier model was established,and permeability was examined in vitro using post-IR or non-IR treated HBVP co-cultured with human brain microvascular endothelial cells.Then,post-IR HBVP supernatants were collected and co-cultured indirectly with normal CNS cells and glioma cells in the brain to detect functional changes.In addition,changes in senescence and proliferation indicators were examined using dasatinib combined with quercetin（D+Q）or all-trans-retinoic acid（RA）administration in vitro.Next,D+Q or RA-treated WBRT mice were tested for changes in cognitive function and pericyte senescence-related markers.In the third part of the study,the autophagy inhibitor bafilomycin A1（Baf A1）was treated with IR in vitro,followed by m RFP-GFP-LC3transfection to analyze changes in HBVP autophagic flux.Then,the expression of senescence markers was analyzed.The expression of cognitive function,autophagy,and senescence-related indicators and the effect on CNS key cells were analyzed by constructing a pericyte autophagy gene Atg7 knockout WBRT mouse model(PDGFRβ-Cre-Atg7^flox/flox).Finally,changes in markers of autophagy,senescence,proliferation,and lysosomal function were examined using the autophagy inducer rapamycin in vitro on Baf A1-administered post-IR HBVP treatment.Results:In the first part of the study,mice after WBRT exhibited cognitive impairment,blood-brain barrier disruption,pericyte damage,increased whole-brain senescence markers,pericyte ultrastructure displaying senescent-associated changes,and a heightened proportion of P16-positive pericytes.Irradiated HBVP also showed a senescent-like cell phenotype in vitro.In the second part of the study,the senescence-associated secretory phenotype（SASP）secreted by IR-HBVP reduced the integrity of the BBB in vitro and was toxic to CNS cells,while simultaneously promoting the proliferation,migration,and invasion of glioma cells.Additionally,D+Q or RA treatment reduced HBVP senescence characteristics and partially restored proliferative capacity in vitro,increased new object recognition,and reduced the proportion of P16-positive pericytes in RBI mice.In the third part of the study,Baf A1 treatment of IR-HBVP further increased senescence phenotypic features in vitro.The PDGFRβ-Cre-Atg7^flox/flox WBRT mouse model impaired new object recognition and further exacerbated pericyte and parts of other CNS cell senescence phenotypes.Lastly,rapamycin attenuated the senescence of IR-HBVP after Baf A1treatment in vitro,partially restored proliferative activity,increased the expression of the lysosomal tracer Lysotracker,and decreased the co-localized expression levels of LAMP1-positive and Proteostat-positive cells.Conclusions:In this study,we successfully constructed a mouse model of RBI-related cognitive dysfunction and a pericyte radiation model in vitro,which clarified that WBRT induced pericyte senescence.SASP factors secreted by senescent HBVP exacerbated BBB destruction and had deleterious effects on normal CNS cells while promoting glioma progression.Senolytic treatment with D+Q or RA ameliorated RBI pericyte senescence and delayed cognitive impairment.Moreover,this study further revealed that defective pericyte autophagy was a key factor exacerbating WBRT-induced pericyte senescence and cognitive dysfunction in mice,which may further induce whole-brain senescence by impairing the function of some CNS cells.Rapamycin induced autophagy by activating lysosomal function to ameliorate IR-induced pericyte senescence and partially restore pericyte proliferation.Part I: WBRT Induces Blood-Brain Barrier Dysfunction-Associated Pericyte SenescenceObjectives: Radiation-induced brain injury（RBI）is the most common complication of radiation therapy for head and neck tumors.Investigating the specific mechanisms of RBI and developing prevention and treatment strategies are crucial to improving patient survival quality.Pericytes,as components of the neurovascular unit,undergo senescence and damage that can lead to blood-brain barrier（BBB）destruction and consequent central nervous system（CNS）disease deterioration.This part of the study aims to determine whether whole-brain radiation therapy（WBRT）can induce pericyte senescence.Methods: Mice underwent WBRT to create an RBI model for cognitive function of new objects,and changes in the blood-brain barrier and aging-related indicators were analyzed through transcriptome sequencing of brain tissue and Western blot experiments.Pericyte ultrastructure changes were detected using electron microscopy,and P16-positive pericytes（PDGFRβ+）expression was analyzed by immunofluorescence.A human brain microvascular pericyte radiation model was constructed in vitro at a dose of 10 Gy,and changes in senescence-related markers were detected by SA-β-Gal staining,Western blot experiments,and immunofluorescence.Results: In the first part of the study,mice exhibited cognitive decline,blood-brain barrier disruption,pericyte damage,increased whole-brain senescence markers,pericyte ultrastructure with senescence-like changes,and an increased proportion of P16-positive pericytes after WBRT.Additionally,irradiated human brain vascular pericytes（HBVP）in vitro experienced induced cellular senescence.Conclusions: This study successfully demonstrated that WBRT induces pericyte senescence by constructing a mouse model of RBI-related cognitive dysfunction and a radiation-induced pericyte model in vitro.Part II: The Role of Pericyte Senescence in RBI and the Application of Senolytic TherapyObjectives: As the first part of the study has confirmed the ability of WBRT to induce pericyte senescence,the subsequent investigation aims to further explore the role of pericyte senescence.Specifically,the study aims to examine the impact of pericyte senescence on RBI and the effect of selectively eliminating senescent cells on pericyte senescence,as well as on cognitive function in RBI.Methods: An in vitro blood-brain barrier model was established using post-irradiation（IR）or non-IR-treated HBVP co-cultured with human brain microvascular endothelial cells,and permeability was examined by fluorescein isothiocyanate-bovine serum albumin（FITCBSA）leakage assay.Normal medium,HBVP supernatant,and post-IR HBVP supernatant were collected and treated with normal CNS cells and glioma cells to detect functional changes.Additionally,changes in senescence and proliferation-associated markers were detected using dasatinib and quercetin（D+Q）or rapamycin（RA）administration in vitro.Mice were administered D+Q or RA for two months after WBRT to detect changes in cognitive function using new object recognition,and survival was recorded in vivo.The expression of pericyte senescence-related markers was detected by immunofluorescence staining,Western blot assay,and brain tissue RNA sequencing.Results: Post-IR HBVP-secreted senescence-associated secretory phenotype（SASP）reduced the integrity of the BBB in vitro,increased the senescent phenotypic characteristics of non-senescent pericytes and astrocytes,inhibited endothelial cell angiogenesis,induced microglia M2 phenotype activation and DNA damage,oligodendrocyte damage and death,and hippocampal neuronal cell apoptosis.Meanwhile,the post-IR HBVP supernatant promoted the proliferation,migration,and invasion of glioma cells.In addition,D+Q or RA treatment reduced pericyte senescence characteristics and partially restored proliferative capacity in vitro,and intervention increased new object recognition and reduced the proportion of P16-positive pericytes in RBI mice.Conclusions: SASP secreted by senescent HBVP exacerbates BBB destruction and has detrimental effects on normal CNS cells,while promoting glioma cell proliferation.Senolytic treatment with D+Q or RA improves pericyte senescence and delays cognitive decline in RBI.Part III: The Role and Mechanism of Autophagy on Pericyte Senescence after WBRTObjectives: Since autophagy is closely related to senescence,this study further investigated the precise connection between autophagy and radiation-induced pericyte senescence.Methods: In this study,autophagic flux was analyzed using Bafilomycin A1（Baf A1）as an autophagy inhibitor and by employing the m RFP-GFP-LC3 reporter.Senescence indicators were then analyzed by SA-β-Gal staining.Additionally,mice were subjected to the construction of a PDGFRβ-Cre-Atg7flox/flox pericyte-specific autophagy gene Atg7 knockout transgenic WBRT mouse model,and new object recognition function was examined.The expression of autophagy and senescence-related indicators in transgenic mice after WBRT was analyzed by Western blot analysis,and the proportion of P21-positive pericytes was detected by immunofluorescence staining.The relationship between senescence markers and key CNS cells（neurons,astrocytes,microglia,oligodendrocyte progenitor cells）in brain tissue of transgenic mice after WBRT was detected by brain tissue transcriptome sequencing and immunofluorescence.Finally,the autophagy inducer,rapamycin,was tested for autophagy function,senescence markers,proliferation markers,and lysosomal function markers in irradiated HBVP after Baf A1 treatment.Results: Baf A1 treatment of HBVP after irradiation further increased senescent phenotypic characteristics in vitro.The PDGFRβ-Cre-Atg7flox/flox WBRT mouse model was successfully constructed,showing further exacerbation of new object recognition deficits and whole-brain senescence characteristics,with a further increase in the proportion of P21-positive pericytes.Meanwhile,some CNS cells（neurons,microglia,oligodendrocyte progenitors）exhibited an increased proportion of P16-positive cells.The activation of autophagic flux with rapamycin in vitro reduced senescence characteristics,partially restored proliferative activity,increased the expression of the lysosomal tracer Lysotracker,and decreased the co-localized expression levels of the lysosomal marker LAMP1 and the senescence-associated markers Proteostat-positive cells for Baf A1 treatment with irradiation in HBVP.Conclusions: The present study further revealed that defective pericyte autophagy is a key factor aggravating WBRT-induced pericyte senescence and cognitive dysfunction in mice,which may further induce whole-brain senescence by impairing the function of some CNS cells.Rapamycin-induced autophagy ameliorates radiation-induced pericyte senescence and partially restores pericyte proliferation after autophagic injury by activating lysosomal function.