The Expression Of PD-1 And PD-L1 In Epileptic Foci Of Refratory Temporal Lobe Epilepsy

BackgroundEpilepsy is a relatively high incidence of disease,often accompanied with nervous system dysfunction.It relapse suddenly and stop unexpected,which is seriously affecting the health of patients and increaseing the burden of the society.The pathogenesis may be related to a variety of factors,such as immune inflammatory reaction,glial cells,heredity,ion channels and abnormal transmission of synapses.The immunological response plays an important role in the treatment and prognosis of epilepsy.PD-1/PD-L1 signaling pathway plays a negative regulatory role in inflammatory response,and participates in regulating the function of inflammatory cells and the secretion of a variety of cytokines.Many studies of PD-1/PD-L1 signaling pathway focused in the areas of tumor,trauma,stroke,and autoimmune disease.But few studies have been done in epileptic diseases,especially in epileptic foci of refratory temporal lobe epilepsy.The purpose of this study is to investigate the expression and the roles of PD-1 and PD-L1 in epileptic foci of drug refratory temporal lobe epilepsy.ObjectTo detect the expression of PD-1 and PD-L1 in refractory temporal lobe epilepsy and to investigate the expression of HMGB1,IL-6 and STAT-3 which is closely associated with PD-1 / PD-L1 signaling pathway.Methods 24 cases of epileptic foci specimens were collected during intractable temporal lobe epilepsy surgery in the PLA 153 Center Hospital From January 2014 to January 2016.10 normal brain tissue samples were collected as control group during the process of cortical fistulas in the surgery of thalamic tumors.Nissl staining was used to observe the quantity and morphous damage of neurons in the tissue.H.E.staining was used to observe the infiltration of inflammatory cells in the tissue.Immunofluorescence staining and western blotting were used to detect the expression of PD-1,PD-L1,HMGB1,IL-6,STAT-3 and P-STAT3,then the results were statistically analyzed.Results1.Nissl staining results: abnormal neurons were observed in epileptic tissues.The Nissl bodies were lighter in color and decreased in number.And some Nissl bodies dissolved and disappeared.Some neurons showed edema and vacuolar degeneration.In the control group,the nuclei,axons and dendrites of neurons were easily differentiated.The nuclei of neurons were dark blue,Nissl bodies were purple blue,and Nissl bodies were massive or granular around the nucleus.2.H-E staining results: compared with the control group,the number of inflammatory cells in the epileptic tissues(3.875±1.72)was significantly increased(P < 0.05).3.Immunofluorescence staining results:3.1 In order to define the expression of PD-L1 in microglia,astrocytes and neurons in the epilepsy foci,we performed immunofluorescence staining.We stained PD-L1 with Iba-1,Neun,and DAPI respectively.The number of positive cells is counted.The results were as follows: 1.PD-L1 was expressed in epileptic tissues and the positive cell number is(26.69±6.28)higher than the control group(6.23±3.09)(P < 0.05);2.The number of PD-L1 positive microglia cells in experimental group(13.33±3.25)was higher than the control group(5.00±2.58)(P < 0.05);3.The number of PD-L1 positive neurons PD-L1 was higher in the experimental group(22.03±4.28)than in the control group(4.50±1.58)(P < 0.05).4.PD-L1 is also expressed on astrocytes.3.2 To investigate the expression of inflammatory cytokine HMGB1 in epileptic tissue,we performed immunofluorescence staining according to the epileptic status and found that: 1 compared with the control group(78.1 ± 25.90),the expression of HMGB1 in experimental group was significantly higher(134.1 ± 16.56).(P < 0.05).HMGB1 can migrate from the nucleus to the nucleus when stimulated,cell death,apoptosis,tissue hypoxia and ischemia/reperfusion.In epileptic focus,compared with the control group(22.3 ± 11.85%),the proportion of HMGB1 transferred from the nucleus to the nucleus(41.3 ± 4.60%)was significantly higher in experimental group(P < 0.05).2 The number of HMGB1 positive neurons HMGB1 was higher in the experimental group(48.9±9.85)than in the control group(13.9±2.75)(P < 0.05).At the same time,we calculated the proportion of HMGB1 transferred from nucleus to cytoplasm.The results showed that the proportion of experimental group(51.1 ± 7.47%)was significantly higher than that of the control group(11.9 ± 4.69%)(P < 0.05).4.Western blot results: Western blot analysis showed that PD-1 protein expression in the experimental group(0.709 ± 0.418)(P < 0.05),PD-L1 protein expression in the experimental group(1.461 ± 0.651 *)was also higher than that of the control group(0.694 ± 0.413),the difference was statistically significant(P <0.05);the expression of IL-6 was higher in the experimental group(1.065 ± 0.67)than that of the control group(0.804 ± 0.15),the difference was statistically significant(P <0.05);the expression of STAT3 protein in the experimental group(1.717 ± 0.11)was higher than that of the control group(1.307 ± 0.23)),The difference was statistically significant(P <0.05);the expression of P-STAT3 protein(1.461 ± 0.651)was also higher than the control group(0.627 ± 0.28),the difference was statistically significant(P <0.05).Conclusion 1.Seizures can cause inflammatory cell infiltration,neuronal degeneration and death.2.The expression of PD-1 and PD-L1 is increased in epileptic foci of drug refratory temporal lobe epilepsy..3.The expression of –HMGB1,IL-6 and STAT-3 in epilepsy tissues was significantly up-regulated,we speculate that the PD-1/PD-L1 signaling pathway may be involved in the pathophysiological evolution of epilepsy.