The Study On The Inhibition Effect Of Probucol On Hypoxia-reoxygenation-induced Injury And Aggravation Action Of OxLDL In Hippocampal Neuron

Background and objective：There are many reasons to induce ischemic cerebrovascular disease.22%of themoccur because of carotid atherosclerotic stenosis. The most common primary site forcarotid atherosclerosis-induced ischemic injury is hippocampus in the brain. Carotidendarterectomy is a classical method for clinically treating carotid arteryatherosclerotic stenosis. However, this method usually results in cerebralischemia-reperfusion injury, including hippocampal tissue, and the occurrence ofhyperperfusion syndrome in the brain. Some correlative data also indicate that oxLDLplays an important role in leading to ischemic cerebrovascular disease. And it canstimulate the inflammatory cytokine secretion, produce peroxide, and result in tissueand cell injury. Probucol possesses the effect about lipid regulation, anti-oxidation,and anti-inflammatory. Herein, we construct a hypoxia-reoxygenation injury model ofHT22hippocampal neurons, observe the effect of oxLDL and Probucol onhypoxia-reoxygenation induced injury in hippocampal neuron, and discuss theprobable mechanism and potential application in antioxidant treatment.Method and results:1. According to the literatures, a closed device was prepared for hypoxic culture.After HT22hippocampal neurons reached80%confluency, to place the neuronsin the prepared device, and then introduce the gas mixture, including95%N2+5%CO2for6h. In succession, to restore the oxygen for18h to construct ahypoxia-reoxygenation injury model of HT22hippocampal neurons. The testing results indicate that hippocampal neurons suffered from hypoxia-reoxygenationinduced injury.In the control group, neuronal somata had stronger refractivity, and theconnections between dendrites and axons were close and orderly. In contrast withhypoxic cultured group for6h, in hypoxic cultured group for24h, the refractivityof neuronal somata diminished; The connections between dendrites and axonswere sparse; Apoptosis in neurons increased; MTT activity reduced1.2fold; LDHactivity increased1.2fold. But in contrast with control group, inhypoxia-reoxygenation group, neuronal refractivity disappeared; The connectionsbetween dendrites and axons were broken; Neurons shown strong fluorescentparticles-type apoptotic morphology; MTT activity reduced2fold; LDH activityincreased3.5fold; TLR2protein expression increased1.8fold; Moreover, incontrast with hypoxic cultured group for24h, in hypoxia-reoxygenation group,apoptosis of neurons increased; MTT activity diminished1.3fold; LDH activityincreased1.2fold; TLR2protein expression increased1.1fold.2. To divide the cultured HT22neurons into three groups, as follows: Control group:normally cultured for24h in25l PBS; model group: hypoxic cultured for6hand normally cultured for18h in moderate PBS; oxLDL group: hypoxic culturedfor6h and normally cultured for18h in50mg/L oxLDL. After treatment, everyrelative index was detected. The results show that in the hypoxia-reoxygenationprocess, in contrast to the model group, ROS level and red lipid droplet of neuronsin the oxLDL group was evidently higher; SOD activity was low1.3fold, HO-1protein expression was reduced1.5fold, the MDA level enhanced1.1fold;Cholesterol ester level rised1.5fold.3. To divide the cultured HT22neurons into four groups, as follows: model groupadded25l PBS, Probucol group added80μmol/L Probucol; oxLDL group added50mg/L oxLDL; Probucol+oxLDL group added80μmol/L Probucol and50mg/L oxLDL. After hypoxic cultured for6h and normally cultured for18h, everyindex was detected. The results show that: in contrast with oxLDL group,Probucol+oxLDL group had stronger refractivity; Their axons had less breakage; Apoptotic morphology was less; MTT activity increased1.3fold; LDH activitydecreased1.4fold; HO-1protein expression was more; TLR2protein expressionwas less; Total SOD activity increased1.5fold; The MDA level wasdown-regulated1.1fold; Cholesterol ester level decreased1.2fold; In addition,the nuclear NF-κB fluorescence intensity visibly decreased; Red lipid droplet inneural endochylema also reduced.Conclusions:1. Hypoxia-reoxygenation-induced injury in HT22neurons has the characteristic ofsoma swelling, the disappearance of somatic refractivity, axon breakage,increasement of apoptotic morphology and TLR2protein expression, anddecreasement of neural activity and function.2. In the hypoxia-reoxygenation process, oxLDL results in increasement of ROS andMDA level, reduction of SOD activity and HO-1protein expression, lipidaccumulation in HT22neurons, and aggravation of hypoxia-reoxygenation-induced injury.3. Probucol has inhibition effect on hypoxia-reoxygenation-induced injury andaggravation action of oxLDL. The protection of Probucol may be related to itseffect on enhancement of SOD activity and HO-1protein expression,decreasement of MDA and ROS level, down-regulation of TLR2proteinexpression, inhibition of NF-κB transcriptional activity, and depression of lipidlevel in HT22neurons.