Study On The Therapeutic Effect And Mechanism Of Tetrandrine On Microwave Radiation Brain Injury

Objective: The brain is one of the important target organs of microwave radiation,and its mechanism is unclear.Few drugs with single target are available for the prevention and treatment of microwave radiation injury.Previously,we have found that Tetrandrine(TET)can alleviate hippocampal injury induced by microwave radiation,but the mechanisms need further investigation.In this study,we further investigated the therapeutic effect of TET on microwave radiation-induced brain injury,and explored its mechanisms,with expectation to provide new insight for the development of preventive and therapeutic drugs.Methods:(1)Animal and cell models: C57BL/6 mice and in vitro cultured hippocampal neurons were used to establish animal and cellular models of microwave radiation with a SAR value of 9.4 W/kg and a radiation duration of 15 min.(2)TET administration method: 60 mg/kg/d TET was injected intraperitoneally immediately,1 d and 2 d after radiation,respectively,once per day for 3 consecutive days.(3)TET structure confirmation and drug concentration detection: Liquid-mass chromatography coupled with UV detector was used for drug structure verification,and drug concentration monitoring in blood and brain.(4)Neurobehavioral test: Open field and new object recognition experiments were performed to test the changes of anxiety and learning memory in mice.(5)Histopathological analysis: Light microscopy and transmission electron microscopy were used to observe the histopathological changes of hippocampus and striatum,followed by quantitative analysis.(6)Brain injury-related serological assays: The levels of ACTH,NE,CORT and S100β in serum were measured by radioimmunoassay.(7)Calcium activity detection of hippocampal neurons: Based on genetically encoded calcium imaging technique,AAV-GCa MP7 f virus was injected by brain stereotaxic localization.Fiber optic recording combined with behavioral paradigm was used to detect the changes of neuronal calcium activity in CA3 region of hippocampus.(8)Intracellular calcium concentration detection of hippocampal neurons: Fluo-4AM fluorescent probe loading was used to detect the changes of intracellular calcium concentration in hippocampal neurons by flow cytometry and fluorescence microscopy.(9)Examination of VGCC expression: Real-time fluorescence quantitative PCR technique was used to detect the expression of VGCC isoforms in hippocampus and striatum.Fluorescence in situ hybridization was used to detect the expression of Cav3.2 in hippocampal neurons.Results:(1)Effect of TET on neurobehavioral changes caused by microwave radiation: The results of open field experiments showed that the time and distance of exploring the central area of mice decreased significantly after microwave radiation,compared with pre-radiation.The results of the new object recognition experiment showed that the time of exploring new objects decreased significantly after radiation,compared with pre-radiation,and TET administration could significantly inhibit the above changes.(2)Effect of TET on the serological changes related to brain injury induced by microwave radiation: microwave radiation can lead to an increase in peripheral serum ACTH,CORT and S100β levels and a decrease in NE levels;TET administration significantly suppressed these changes.(3)Effect of TET on the histopathological changes of hippocampus and striatum caused by microwave radiation: HE staining results showed that 7 d after microwave radiation,the solidified and deeply stained nuclei of neurons were observed both in hippocampus and striatum,with glial edema and widened perivascular spaces,especially for the CA3 region of hippocampus and the pale bulb region of striatum.The ultrastructural observation showed the condensation and margination of nuclear chromatin,mitochondrial edema with focal cavitation,and endoplasmic reticulum expansion in neurons of hippocampal CA3 and striatal pallidum regions.Meanwhile,mitochondrial swelling and endoplasmic reticulum degranulation occurred in glial cells,accompanied with blurred synaptic cleft and widened perivascular gap.TET improved above pathological changes obviously.(4)Effect of TET on the changes of neuronal calcium activity in hippocampus caused by microwave radiation: The results of fiber optic recording combined with new object recognition and Y-maze experiment showed that,the neuronal calcium activity of hippocampal CA3 region significantly decreased,when mice explored new object or novel arm 7 d after microwave radiation,and TET administration significantly inhibited above changes.(5)Effect of TET on the changes of expression of VGCC subtype genes in hippocampus and striatum caused by microwave radiation: 7 d after microwave radiation,hippocampal Cav1.2,Cav1.3 and Cav3.2 subtype genes were significantly down-regulated,and TET administration significantly inhibited the down-regulation of Cav3.2 in hippocampus.No significant changes were observed for the expression of VGCC subtype genes in striatum.(6)Effect of TET on the changes of calcium concentration and Cav3.2 expression in hippocampal neurons caused by microwave radiation: 6 h after microwave radiation,the intracellular calcium concentration increased and the expression of Cav3.2 was downregulated significantly in hippocampal neurons.TET administration significantly inhibited the increase of calcium concentration and down-regulation of Cav3.2.Conclusion: The therapeutic effects of TET on microwave radiation brain injury were mainly manifested as follows:(1)TET inhibited the decrease in anxiety-like emotional behavior and learning and memory capacity in mice induced by microwave radiation.(2)TET inhibited the changes in serological HPA axis hormones ACTH,CORT,NE and brain injury-related cytokine S100β caused by microwave radiation.(3)TET significantly improved the histopathological changes in the hippocampus and striatum caused by microwave radiation,with the CA3 region of the hippocampus and the pallid bulb region of the striatum being more obvious.The mechanism of therapeutic effects includes:(1)TET enhances neuronal calcium activity in the CA3 region of the hippocampus to exert a therapeutic effect on microwave radiation-induced cognitive dysfunction.(2)TET inhibits the calcium overload of hippocampal neurons after microwave radiation.(3)TET exerts a therapeutic effect on microwave radiation brain injury by down-regulating hippocampal Cav3.2 gene expression.