Effects Of Ethanol On The Morphology And The Expression Of BDNF,Sortilin And P75^NTR In Rat Hippocampal Neurons

Background and purposeWith the rapid growth of global ethanol（Et OH）consumption,Et OH-related mortality is also increasing.Alcoholism caused by excessive drinking has become an independent lethal factor,and the harm to society caused by alcoholism,drunk driving and acute alcoholism is unquestionable.In forensic identification,the role of Et OH in death cases and the division of legal responsibilities have always been hot topics of debate.In clinical cases,excessive Et OH intake causes severe damage to the digestive,respiratory,circulatory,and nervous system of patients.Therefore,the research on the mechanism of alcohol damage not only provides directions for the treatment of clinically related diseases,but also provides a theoretical basis for forensic identification.The hippocampus is one of the major structures of the limbic system,which is responsible for the storage of short-term memory and spatial location.Et OH changes the structure and function of hippocampal nerve cells,and affects their normal physiological functions,leading to memory,behavior,and long-term learning disabilities.In this study,the western blot（WB）and Immunofluorescence（IF）techniques were used to observe the changes in the morphology of primary rat hippocampal neurons by Et OH,and to investigate the effects of Et OH on brain-derived nerve growth factor（BDNF）,Sortilin and nerve growth factor receptor p75（p75NTR）expression,thus,providing a theoretical basis for clarifying the molecular mechanism of Et OH damage and giving directions for the treatment of Et OH-related diseases.MethodsMale and female Sprague–Dawley（SD）rats（born within 24h）were selected to dissect their brains in a sterile environment.Then the hippocampal tissues were isoslated.The tissues were digested by papain,and then blown by a high-temperature polished glass tube to prepare a cell suspension,which was collected for In vitro culture.After 7 days of in vitro incubation,cultures were treated with ethanol（Et OH）.Cells with better condition and higher purity were randomly divided into control group（PBS）,Et OH-treated low-dose group（1.5 g/L）and Et OH-treated high-dose group（3.0 g/L）.All cells were further cultured for 24h.Western Blot technology was used to detect the protein expression of BDNF,Sortilin and p75^NTR;IF technology was used to detect the effects of alcohol on the morphology of hippocampal neurons and the expression and location of BDNF,Sortilin and p75^NTR.Results1.The IF showed that compared with the control group,the number of hippocampal neurons in the Et OH-treated group was significantly reduced（1.5 g/L Et OH vs.Control,P<0.01;3.0 g/L Et OH vs.Control,P<0.01）.The cell body was swollen and the number of primary dendrites significantly decreased（1.5 g/L Et OH vs.Control,P<0.01;3.0 g/L Et OH vs.Control,P<0.01）,and the length of dendrites at various levels was shortened（1.5 g/L Et OH vs.Control,P<0.001;3.0g/L Et OH vs.Control,P<0.0001）.However,in the concentration of Et OH had no effect on the number of hippocampal neurons,the morphology and number of dendrites（P>0.05）.2.Western Blot results showed that compared with the control group,the protein expression of BDNF in hippocampal neurons in the Et OH-treated low-dose and high-dose groups was significantly reduced（1.5 g/L Et OH vs.Control,P<0.01;3.0 g/L Et OH vs Control,P<0.0001）,but the expression of Sortilin（1.5 g/L Et OH vs.Control,P<0.01;3.0 g/L Et OH vs.Control,P<0.001）and p75^NTR（1.5g/L Et OH vs.Control,P<0.05;3.0 g/L Et OH vs.Control,P<0.01）was significantly increased.However,compared with the low-dose group,the expression of BDNF,Sortilin and p75^NTR did not change significantly in the high-dose group（P>0.05）.3.The IF results showed that compared with the control group,the BDNF in the Et OH-treated low-dose group and the high-dose group was localized in the cytoplasm,and the protein expression was significantly reduced（1.5 g/L Et OH vs.Control,P<0.05;3.0 g/L Et OH vs.Control,P<0.01）.Sortilin was localized on the cell membrane with significantly increased expression（1.5 g/L Et OH vs.Control,P<0.05;3.0 g/L Et OH vs.Control,P<0.001）.p75^NTR was localized in the cell membrane with significantly increased expression（1.5 g/L Et OH vs.Control,P<0.01;3.0 g/L Et OH vs.Control,P<0.01）.However,compared with the Et OH-treated low-dose group,the expression of BDNF,Sortilin and p75^NTR was not changed significantly in the high-dose group（P>0.05）.Conclusion1.Et OH induces a decrease in the number of hippocampal neurons and primary dendrites and a shortening of the length of dendrites.2.Ethanol treatment decreased the level expression of BDNF and increased the expression level of sortilin and p75^NTR in hippocampal neurons.