The Study Of Neuroglobin Expression In The Temporal Cortex Of Bilirubin Encephalopathy Rats And Its Mechanism In Electroacupuncture Therapy

BackgroundBilirubin encephalopathy(BE)is a neurological syndrome that occurs in newborns and mainly caused by neuronal injury due to excessive oxidative stress produced by unconjugated bilirubin(UCB).In a variety of central nervous system diseases such as cerebral hemorrhage and cerebral ischemia,Neuroglobin(NGB)has been found to protect the brain tissue by inhibiting the apoptotic response caused by oxidative stress injury.However,the expression of NGB in BE model and its significance remain unclear,impeded the application of NGB in the treatment of BE.Electroacupuncture(EA),as a part of traditional Chinese medicine,has been proved to play a protective role in central nervous system diseases such as neurodegenerative diseases and hypoxic ischemic encephalopathy by upregulating the expression of NGB.The purpose of this study was to investigate the changes of NGB expression in the temporal cortex at different time points after the modeling of acute bilirubin encephalopathy and and explore the expression change of NGB and apoptosis-related proteins in the temporal cortex of rat pups after electroacupuncture stimulation in the BE model.To explore the protective mechanism of NGB on BE model rat pups and the application prospect of electrotherapy on BE.Methods75 7-day-old SD rats were divided into Sham group and BE model group.The neonatal BE model was established by injecting bilirubin solution(10μg bilirubin /g body weight)into cerebellar medulla cistrum.The BE model group was divided into 6 h,12 h,24 h,7 d and 28 d groups according to the different bilirubin action time.The 28 d group was only used for neurobehavioral score and Rotarod test,and the sham group was injected with the same amount of normal saline as control.5 rats in the Sham group and 5 rats in the model group were selected for weight monitoring at each time point,and the rat pups in the Sham group and the BE model group were fed to 28 days for neurobehavioral score and Rotarod test to verify the success of our experimental model.The pathological changes of temporal cortex and the ultrastructural changes of neurons and astrocytes in each group were observed by HE,Nissl staining and Transmission electron microscopy(TEM).Immunofluorescence staining was used to detect the expression sites and cell types of NGB.Western blotting and quantitative PCR were used to detect the expression of NGB and NGB m RNA in the temporal cortex of rats in each group.Western blotting was used to detect the the expression change of mitochondrial apoptosis signaling pathway related proteins Bax,Bcl-2,Cyto C and Caspase-3 in the model group and the Sham group at each time point.In addition,the SD rats with the highest NGB expression time point were selected as the research subjects,and the BE rats were treated with electroacupuncture.39 seven-day-old SD rats were divided into Sham,BE model and BE+EA group.The neonatal BE model was established by injecting UCB(10μg UCB/g Weight)into the cerebellomedullary cistern,Sham group was injected with the same amount of normal saline.BE rats were treated with EA at Baihui(GV20)and Quchi(LI11)acupoints with the frequency of 15 Hz for 15 min and treatment was performed 12 h before modeling,followed by treatment every 12 h,a total of three times.HE,Nissl staining and electron microscopy(TEM)were used to observe the pathological and ultrastructural changes of nerve cells in each group.Immunofluorescence staining was used to detect the expression sites and cell types of NGB.Westernblotting served to detect the expression of NGB,PI3 K,AKT,Bax,Bcl-2,Cyto c,Caspase-3 in the temporal cortex of rats in each group,the number of apoptotic cells was detected by Tunel staining..ResultsCompared with the Sham operation group,the body weight of the rat pups in the BE model group was lower than Sham group,the neurobehavioral score of the rats in the BE model group was significantly higher than that in the Sham group,and Rotarod experiment showed that the time of falling from the rotating rod in the BE model was significantly lower than that in the Sham group.HE and Nissl staining results showed that there were different degree of pathological damage and ultrastructural changes of temporal cortex neurons in the BE model group at each time point,and the damage was most severe at 24 h,HE and Nissl staining showed that a large number of neurons were blurred and the number of Nissl bodies decreased.Electron microscopy showed that the neuronal mitochondria swelling was more serious than the other groups;WB and q PCR results showed that the expression of NGB in the temporal cortex of the BE model group increased,and reached the peak at 12 h,and then decreased gradually.Immunofluorescence results showed that NGB was mainly expressed in the temporal cortex and only co-localized with neurons.WB detection of apoptosis-related proteins in each group confirmed that the apoptosis of temporal cortex neurons in BE model rats gradually upregulated with the increase of time and reached the peak at 24 h.After EA treatment,HE and Nissl staining show that EA treatment can reduce the damage of cortical neurons of BE rats and increase the number of Nissl bodies.TEM shows that EA treatment can alleviate the degree of mitochondria edema.Westernblotting shows that EA treatment can increase the expression of NGB,PI3K(p110 alpha),p AKT(Ser473)and the ratio of apoptosis-related protein Bcl-2/Bax,decerease the expression of Cleaved caspase-3.TUNEL staining showed that the number of apoptotic cells decreased after EA treatment.ConclusionThe neurotoxic effect of bilirubin in BE will promote the expression of NGB in the temporal cortex,and the expression level of NGB upregulated gradually with the increasing time of bilirubin exposure,reaching the peak at 12 h,and the peak of apoptosis at 24 h.This increased reactivity of NGB may not be able to effectively protect the brain tissue,and eventually lead to the inevitable occurrence of cell apoptosis.EA can inhibit the occurrence of apoptosis by promoting the expression of endogenous NGB and activating the PI3K/Akt pathway,And then play a protective role in nerve cells.Therefore,electroacupuncture may become a potential treatment method for BE.