Mitochondrial-Targeting Fluorescent Small Molecule IR-780 Alleviates Radiation-Induced Brain Injury

Backgrounds:Radiation-induced brain injury(RIBI)is a common complication of brain tumor patients after radiotherapy.It can also be seen in many cases,such as the loss of radioactive sources,accidents of radioactive facilities or nuclear weapon explosions in wartime.The pathophysiological process of RIBI includes genome damage,tissue cell apoptosis,neurogenesis reduction,neuroinflammation and blood-brain barrier(BBB)damage.However,the specific mechanism leading to RIBI is still unclear.Vascular injury is one of the key factors closely related to the severity of RIBI.BBB dysfunction,especially the injury of vascular endothelial cells,is the late pathological feature of RIBI.BBB which can selectively exchange substances between blood and brain,is composed of brain microvascular endothelial cells,pericytes,astrocytes,and neurons.The tight junction(TJ)between vascular endothelial cells determines the main barrier function of BBB.Previous studies have shown that BBB dysfunction may be a key pathological process affecting brain function after brain injury caused by different causes.The increase of BBB permeability induced by irradiation and cerebral angiogenic edema are related to pericyte loss,astrocyte activation,neuronal and vascular dysfunction.After BBB dysfunction,inflammatory cells and inflammatory factors in peripheral blood can also cause neuroinflammation through damaged BBB into the brain,which together with radiation-activated astrocytes and microglial inflammatory factors contribute to the pathophysiological development of RIBI.However,there is still a lack of effective vascular targeted therapy strategy.Therefore,the development of RIBI prevention and control strategy with BBB as the target has a broad application prospect.Previous prevention and treatment strategies for RIBI mainly include inhibition of neuroinflammation,promotion of nerve regeneration and reduction of oxidative stress.However,the therapeutic effect of these drugs is largely dependent on the ability of drug delivery across BBB,drug dose and radiation dose,and effective BBB-targeting therapeutic strategies are still lacking.We have recently developed a fluorescent small molecule IR-780.IR-780,approximately 667.105 Da,is one of the heptamethrin cyanine fluorescent small molecules for NIR fluorescence imaging.In earlier studies,our research team found that IR-780 could accumulate in the injured tissue and protect the tissue from radiation damage.By regulating inflammatory response and oxidative stress,IR-780 has good application effects in repairing tissue and organ damage.Our previous research found that IR-780 could accumulate and reduce ionizing radiation damage in lung tissue after radiation injury.However,whether IR-780 has protective effect in RIBI and the type of IR-780 accumulating cells are unknown.In this study,we found that IR-780 could reduce cognitive dysfunction after radiation in the whole brain of mice,reduce neuroinflammatory reaction in brain tissue,reduce apoptosis in brain tissue,and restore BBB function.The mechanism of reducing RIBI may be attributed to IR-780 reducing oxidative stress damage of endothelial cells after radiation by accumulating in brain microvascular endothelial cells damaged by radiation.These results show that IR-780 is a potential drug for the treatment of RIBI by targeting BBB.Methods:1.The protective effect of IR-780 on radiation-induced brain injuryThe whole brains of 6-8-week-old C57BL/6 mice received a single dose of 15 Gy to establish RIBI models.First,two months after irradiation,the cognitive function changes of mice in each group were evaluated through the Morris water maze(MWM)experiment.Secondly,three months after irradiation,morphological changes of microglia and astrocytes were evaluated by immunofluorescence staining,the proteins expression of cell markers(IBA1 and GFAP)and major inflammatory factors(TNF-α,IL-1β,MMP3)were evaluated by Western blot(WB),the aspect of RNA transcription of major inflammatory factors(TNF-α,IL-1β,MMP3 and i NOS)was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Finally,the coverage of TJ proteins(ZO-1,OCLN,and CLDN5)in the brain was detected by immunofluorescence staining,the gene transcription of TJ proteins(ZO-1,OCLN)was detected by qRT-PCR,and the expression of TJ proteins(ZO-1,OCLN,and CLDN5)was detected by WB.The BBB function was tested by Near Infrared Fluorescence Imaging(NIR)and Evans Blue leakage experiment,the BBB damage was detected by electron microscope.2.The preliminary mechanism of IR-780 alleviating RIBIThe main accumulation cell types of IR-780 in the radiation damaged brain were detected by immunofluorescence staining on frozen sections,and the subcellular localization of IR-780 was detected by co-localization of IR-780 and mitochondria.TUNEL staining was used to detect the level of brain tissue apoptosis in mice 3 months after irradiation.To investigate the effect of IR-780 on the oxidative stress state of brain microvascular endothelial cells after irradiation,a 20 Gy irradiation model of hCMEC/D3 cells was constructed in vitro.Flow cytometry was used to detect the levels of cell apoptosis,intracellular total reactive oxygen species,and mitochondrial reactive oxygen species 24 hours after irradiation.Results:1.IR-780 ameliorated cognitive impairment,alleviated neuroinflammatory response and recovered BBB function in mice after WBIThe Control+IR-780 group and Radiation+IR-780 group received intraperitoneal injection of IR-780(0.2 mg/kg)2 and 4 days before irradiation,and every 3 days after irradiation for 1 month.Two months after whole brain irradiation(WBI),it was found that irradiated mice exhibited significant spatial learning dysfunction through MWM,and IR-780 could significantly improve the spatial learning abilities of mice after WBI.Three months after WBI,it was found that astrocytes and microglia were activated in the irradiation group alone,characterized by hypertrophy of the cell body and increased expression of GFAP and IBA1.IR-780 could reduce the activation of microglia and astrocytes.After irradiation,inflammatory factor was increased by qRT-PCR and WB experiments,and IR-780 could decrease of inflammatory factors induced by irradiation.In the evaluation of BBB function,it was found that IR-780 could reduce the loss of TJ proteins,reduce BBB leakage,and reduce BBB barrier damage after WBI.2.IR-780 reduced apoptosis by reducing the level of oxidative stress in cerebral vascular endothelial cellsImmunofluorescence staining on frozen sections revealed that IR-780 mainly accumulated in the irradiated brain microvascular endothelial cells.Further,it was found that IR-780 was mainly accumulated in mitochondria in hCMEC/D3 in vitro through MitoTracker and IR-780 co-location staining.TUNEL staining found that IR-780 could reduce irradiation-induced brain tissue apoptosis.In addition,it was found that IR-780 could reduce the apoptosis of vascular endothelial cells induced by irradiation,reduce the total intracellular reactive oxygen species and mitochondrial reactive oxygen species of endothelial cells after irradiation.Conclusion:IR-780 was mainly accumulated in brain vascular endothelial cells after radiation,which could improve learning ability in mice after radiation,reduce the activation of microglia and astrocytes,alleviate radiation induced neuroinflammatory reactions,increase the expression of tight junction proteins after radiation,and protect BBB function.IR-780 mainly accumulated in the mitochondria of brain microvascular endothelial cells.IR-780 could reduce the generation of mitochondria and intracellular reactive oxygen species after radiation in brain microvascular endothelial cells,reduce radiation-induced apoptosis of brain microvascular endothelial cells.