The Expression Of Genes And Protein Of ULBP2 And STRN In The Brain Of Drug-Refractory Epilepsy

Objective: To study the expression of genes and protein of UL16 binding protein 2 and STRN in the brain tissue of patients with drug-refractory epilepsy , and to explore the possible role in drug-refractory epilepsy and the clinical value.Materials and Methods: The 42 cases with refractory epilepsy included in this study were obtained from the files of the Departments of Neurosurgery of the following hospitals: the First Affiliated Hospital of Chongqing Univerisyt of Medical Sciences, Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, and Xinqiao Hospital of the Third Military Medical Univeristy. All the patients involved in our study group underwent the surgical resection of temporal lobe tissue. Before surgery, informed consents were obtained for the use of human brain tissues for research, and the study was approved by the local Ethic Committee. Two neuropathologists reviewed all the cases independently. The diagnosis of seizure type was confirmed according to the 1981 International Classification of Epileptic Seizures of the International League Against Epilepsy.The 20 controls were obtained from the files of the neurosurgery department of the first affiliated hospital of Chongqing University of Medical Sciences. These samples were comprised of temporal neocortical tissues adjacent of the lesion. All patients were diagnosed by pathological tests to have been suffered brain trauma. the two neurophthologists both reviewed these cases to confirm that they had no history of seizures or other neurological disorders as controls.Gene-chip, immunohistochemistry, immunofluorescence and Western blotting were used to test expression of ULBP2 protein and STRN protein in the surgically removed brain tissue of patients with drug-refractory epilepsy from the brain bank of our department(n=42), and the results were compared with that of normal controls (n=20)。The Computer Graphic Analysis System is used to test the average optical density (AOD) which is expressed as mean±SD( x±s)and tested via t-test. P value less than 0.05 as the level of significance.Results1.Gene chip: There was significant difference between the control group and the pharmacoresistance group (Ratio=4.180). The expression of SCN8A gene was shown to be increased in the brain tissues of the pharmacoresistance epilepsy group. There was significant difference between the control group and the pharmacoresistance group (Ratio=0.384). The expression of STRN gene was shown to be decreased in the brain tissues of the pharmacoresistance epilepsy group. 2. Immunohistochemistry : In human temporal cortex of patients with epilepsy, ULBP2 immunoreactivity was consistently observed in all cases and was confined mainly to neurons and glial cells, whereas faint immunoreactivity was apparent in the control brain tissues. Four visual field images were obtained randomly in every section by TC-FY-2050 pathology system acquisi-tioned image and were automatically analyzed by Motic Med 6.0 CMIAS pathology image analysis system. AOD value in epileptic temporal cortex tissue tissues was determined 0.0518±0.0232 ( x±s), and 0.0206±0.0011 ( x±s) in the control. AOD value in hippocampus of pharmacoresistance epilepsy group was determined 0.0509±0.0048( x±s), and 0.0198±0.0114 ( x±s) in the control. ANOVA analysis shows significantly up-regulated ULBP2 in epileptic tissue than the control (P<0.05).In control brain tissues, STRN immunoreactivity was consistently observed in all cases and was confined mainly to neurons and glial cells, whereas faint immunoreactivity was apparent in control brain tissues. Four visual field images were obtained randomly in every section by TC-FY-2050 pathology system acquisi-tioned image and were automatically analyzed by Motic Med 6.0 CMIAS pathology image analysis system. AOD value in epileptic temporal cortex tissue tissues was determined 0.3108±0.1087 ( x±s), and 0.4210±0.1238 ( x±s) in the control. AOD value in hippocampus of pharmacoresistance epilepsy group was determined 0.2706±0.0038 ( x±s), and 0.3952±0.0107 ( x±s) in the control. ANOVA analysis shows significantly down-regulated STRN in epileptic tissue than the control (P<0.05).3. Immunofluorescence analysis: In the temporal lobe cortex and hippocampus, strong immunoreactivity for the staining of ULBP2 protein was observed. There were more obvious in the pharmacoresistance epilepsy group than in control group.In the temporal lobe cortex and hippocampus, faint immunoreactivity for the staining of STRN protein was observed. There were less obvious in the pharmacoresistance epilepsy group than in control group.4. Western blot: Immunoblot was performed with ULBP2 antibody, the protein was detected by immunoblot as a band at 29kDa. Strongly stained bands were present in all samples of epileptic tissue whereas relative faint expression of the ULBP2 was observed in the control group. Another band ofβ-actin as positive control at 42 kDa also was observed correspondently in every channel. The ratio of optical density of ULBP2 andβ-actin was taken as variance calculated between two groups. Optical density value in Epileptic temporal cortex tissue was 0.9804±0.1401 and 0.5914±0.2197 ( x±s) in the control tissue. Optical density value in Epileptic hippocampus tissue was 0.9832±0.1508 and ( x±s)0.5866±0.1974 in the control tissue.The result shows a significant difference between epileptic tissue and the control group (P<0.05) .Immunoblot was performed with STRN antibody, the protein was detected by immunoblot as a band at 110kDa. faint stained bands were present in all samples of epileptic tissue whereas relative strongly expression of the STRN was observed in the control group. Another band ofβ-actin as positive control at 42 kDa also was observed correspondently in every channel. The ratio of optical density of STRN andβ-actin was taken as variance calculated between two groups. Optical density value in Epileptic temporal cortex tissue was 0.3318±0.1101 and 0.5846±0.1024 ( x±s) in the control tissue. Optical density value in Epileptic hippocampus tissue was 0.2786±0.0308 ( x±s)and 0.5686±0.1214 in the control tissue.The result shows a significant difference between epileptic tissue and the control group (P<0.05) .Conclusions:Our work showed that a increase in ULBP2 cDNA and protein level may be involved in the pathophysiology of refractory epilepsy which suggested that the ULBP2 may play a key role in the epilepsy immune disorder,so bring some promising immune targets toward the therapy of refractory epilepsy. Additional studies will be required to elucidate the mechanism by which ULBP2 affects brain function in refractory epilepsy.Our work also showed that a decrease in STRN cDNA and protein level may be involved in the pathophysiology of refractory epilepsy which suggested that the STRN may be associated with impairment of the brain protection cased by endogenous NO in refractory epilepsy,so bring some promising targets toward the therapy of refractory epilepsy. Additional studies will be required to elucidate the mechanism by which STRN affects brain function in refractory epilepsy.