The Role And Mechanism Of Peripheral Cell Autophagy In Radiation Brain Injury

Part I Effect of irradiation on HBVP cells PURPOSE: Radiation brain injury is one of the serious complications after radiotherapy for head and neck cancer(HNC).Human Brain Vascular Pericytes(HBVP)plays an important role in maintaining the structural integrity of the Blood Brain Barrier(BBB).It has been shown that after head radiotherapy can cause BBB destruction by affecting the function of perivascular cells,which is one of the causes of radiation brain injury.In this study,we investigated the functional changes of HBVP cells after irradiation to provide theoretical support for BBB destruction after radiotherapy and to provide ideas for the treatment and improvement of radiation brain injury.METHODS: In this study,Western Blot was used to detect the expression of intracellular protein levels in HBVP cells after 0 h,6 h,12 h,24 h,48 h and 72 h of irradiation,mainly including the expression levels of autophagy,apoptosis and inflammatory factor-related proteins,reflecting the changes in cellular functions of HBVP cells after radiation irradiation.Meanwhile,cellular immunofluorescence assay was used to compare the mean immunofluorescence intensity of cysteine protease-3(Caspase3)in HBVP cells before and48 h after irradiation,as well as to detect the apoptotic ratio of HBVP cells after irradiation by flow cytometry,reflecting the changes of apoptosis level of HBVP cells before and after irradiation.RESULTS: The protein expression levels of autophagy-specific microtubule-associated protein 1A/1B Light Chain 3B Protein II(LC3BII)and P62/SQSTM1(hereafter referred to as P62)in HBVP cells before and after irradiation was compared in the study.The results showed that the expression of LC3 BII and P62 increased in HBVP cells after irradiation,indicating that P62 degradation was impaired,autophagic flux was blocked and autophagy was restricted in HBVP cells after irradiation.The apoptotic proteins of HBVP cells before and after irradiation were also examined,and the results showed that the expression of apoptotic representative proteins such as sheared cysteine protease-3(cleaved-Caspase3)increased after irradiation,suggesting that radiation treatment could induce apoptosis in HBVP cells.It was further confirmed that irradiation could induce apoptosis in HBVP cells by flow cytometry and cellular immunofluorescence assays.The expression of matrix metalloproteinase 9(MMP9)and CC motif chemokine ligand 2(CCL2)was also up-regulated in HBVP cells after irradiation,while radiation treatment was able to up-regulate the expression of the nuclear factor κB(NF-κB).NF-κB signaling pathway,suggesting that radiotherapy can activate inflammatory response-related pathways.CONCLUSION: Irradiation blocked HBVP cell autophagy,increased HBVP cell apoptosis,and may promote inflammatory factor expression by activating the NF-κB signaling pathway.Part II Effect of autophagy on apoptosis and senescence of HBVP cells after irradiationPURPOSE: Some progress has been made in the treatment of different diseases by activating autophagy or inhibiting autophagy.In this study,we intend to explore the effect of autophagy on apoptosis and senescence of HBVP cells after irradiation by interfering with the autophagic function of HBVP cells through drugs and si RNA.METHODS: HBVP cells were treated by pretreatment with autophagy inhibitors 3-Methyladenosine(3-ma),Bafilomycin A1(Baf A1),Earle’s Balanced Salt Solution(EBSS),Rapamycin and si RNA down-regulation of Autophagy-related genes 7(ATG7)(si-ATG7)were pretreated HBVP cells for 24 h,followed by irradiation treatment.The changes of intracellular protein levels in pretreated HBVP cells at 48 h after irradiation were detected by q RT-PCR,followed by Western Blot,which mainly detected the expression levels of autophagy,apoptosis and inflammatory factors.HBVP cells were stained for senescence associated beta-galactosidase(SA-β-gal)and the number of positive cells was counted by detecting the expression levels of senescence associated proteins in HBVP cells at 5 d after irradiation.The mean immunofluorescence intensity of intracellular Caspase3 in HBVP cells before and 48 h after irradiation after autophagic function intervention was detected by cellular immunofluorescence assay,and the effect of irradiation on the level of apoptosis was examined by flow cytometry to detect the proportion of apoptotic cells.RESULTS: By comparing the expression levels of intracellular autophagy-related proteins LC3 BII and P62 in HBVP cells before and after irradiation after intervention in autophagy function,the results showed that pretreatment of HBVP cells with autophagy activator or autophagy blocker resulted in increased expression of autophagy-related protein LC3 BII in HBVP cells after irradiation compared with before irradiation,along with impaired degradation of P62,indicating that under the premise of autophagy activation The autophagic impairment in HBVP cells could be caused by irradiation under the premise of either autophagy activation or inhibition;when the expression of ATG7 in HBVP cells was down-regulated,the autophagy impairment was more obvious after irradiation.Also after pretreatment with autophagy activator,autophagy inhibitor and si RNA,the expression of inflammatory factors increased and apoptosis was induced after irradiation,among which the autophagy inhibitor induced the most significant effect of apoptosis in HBVP cells.In addition,this study found that the administration of 10 Gy irradiation could induce HBVP cell senescence,and the effect of irradiation induced HBVP cell senescence was more significant after the addition of autophagy activator.The cellular expression levels of inflammatory factors were detected in the presence of dysfunctional autophagy,and the results showed activation of inflammatory pathways and increased expression of inflammatory factors both before and after irradiation.The PI3K/AKT and NF-κB signaling pathways were also examined,and the results suggested that the PI3K/AKT signaling pathway was down-regulated and the NF-κB signaling pathway was up-regulated after irradiation.CONCLUSION: Normal autophagy is the basis for maintaining normal function of HBVP cells.When the autophagy level of HBVP cells is disturbed,radiation may induce senescence of HBVP cells,promote apoptosis of HBVP cells and promote the expression of inflammatory factors by downregulating the PI3K/AKT signaling pathway while activating the NF-κB signaling pathway.Part III Conditional knockout of autophagy-associated gene ATG7 in mouse perivascular cells and its effect on radiation brain injuryPURPOSE: Cerebrovascular pericytes play an important role in maintaining the structural integrity and functional stability of the BBB,which is inevitably damaged after whole-brain radiotherapy.In this study,we investigated the effects of autophagy on autophagy,aging and BBB in irradiated mice by conditionally knocking down autophagy-related genes in mouse cerebrovascular pericytes,and also examined the cognitive function of mice before and after irradiation to investigate the role of autophagy and aging in radiation brain injury.METHODS: Atg7flox/flox;Pdgfrb-Cre transgenic mice were constructed,screened by PCR electrophoresis,and bred to obtain mice with genotype Atg7flox/flox;Pdgfrb-Cre as experimental mice and mice with genotype Atg7flox/flox as control mice.After a certain number of mice were obtained by breeding,they were irradiated with a whole brain at an irradiation dose of 15 Gy to construct a mouse model of radioactive brain injury.The expression levels of autophagy and senescence proteins were detected by extracting proteins from the brain tissue of mice 3 months after irradiation;the expression levels of CD31,CD68,Glial fibrillary acidic protein(GFAP)and Neun protein were detected to examine the effects of irradiation on endothelial cells,microglia,astrocytes and neurons in the brain tissue of mice.The effects of irradiation on endothelial,microglia,astrocyte and neuronal cell functions in mouse brain tissues were examined;immunofluorescence co-staining of senescence-associated protein P16 and platelet-derived growth factor receptor beta(PDGFRβ)was performed to explore the effects of irradiation on pericyte senescence in mice with defective pericyte autophagy;and the expression of tight junction(TJ)protein,which reflects the integrity of BBB,was examined.Immunofluorescence staining of ZO-1,a protein reflecting the integrity of BBB,was performed to investigate the changes of BBB structure before and after irradiation.The mice were also subjected to behavioral tests,mainly the Open Field Test(OFT)and New Object Recognition(NOR),to evaluate their cognitive function before and after irradiation.RESULTS: After irradiation,LC3 BII and P62 expression increased in mouse brain tissue,suggesting that P62 degradation was blocked and autophagy was impaired in mice,consistent with the results of in vitro cellular assays;P16 expression increased after irradiation in Atg7flox/flox;Pdgfrb-Cre transgenic mice compared with controls,indicating that defective pericyte autophagy can aggravate pericyte senescence due to irradiation;irradiation The structure of TJ-associated protein ZO-1 immunofluorescence staining was disrupted after irradiation,suggesting that irradiation could disrupt the integrity of BBB structure,and the BBB structure was more significantly affected by radiation in the presence of defective pericyte autophagy;in mouse behavioral assays,irradiation was shown to impair cognitive function in mice,and the impairment was more significant in Atg7flox/flox;Pdgfrb-Cre transgenic mice.The cognitive impairment was more pronounced in Atg7flox/flox;Pdgfrb-Cre transgenic mice.CONCLUSIONS: Head irradiation in mice can impair cerebrovascular pericyte autophagy,induce aging,disrupt BBB structure,and impair cognitive function,and defective pericyte autophagy can exacerbate these changes.