Role Of Zinc-?2-glycoprotein In Epileptogenisis And Its Molecular Mechanism

Part One The Expression of ZAG in Temporal Lobe Epilepsy in Patients and Rats Obejective: To investigate the expression and expression changes of ZAG in the neocortex of refractory TLE patients and in the hippocampus and cortex of PTZ-kindled rats compared to controls.Methods:1.14 temporal neocortex samples of patients with TLE and 20 histologically normal temporal neocortex samples of patients with brain trauma as controls were collected.SD rats were injected intraperitoneally with PTZ to establish chronic epilepsy model and the rats in control group were injected with normal saline(n=20);2.Double-labeled immunofluorescence analysis was used to identified the cellular distribution of ZAG in the brain tissues of patients and rats.The AZGP1 m RNA and ZAG protein expression in brain tissues of patients and rats was determined by RT-q PCR,immunohistochemical and western blot;3.ZAG level in brain tissues of rats received PTZ injection for 1day,1week,2 weeks,3 weeks and 4 weeks were measured using western blot.Results:1.ZAG was found in the cytoplasm of neurons in brain tissue from both patients and rats but not astrocytes;2.The level of AZGP1 m RNA was significantly decreased in refractory TLE patients and PTZ-kindled rats compared to controls(P<0.05);3.ZAG protein level was lower in TLE patients and PTZ-kindled rats compared to controls(P<0.05);4.ZAG level in brain tissues of PTZ-treated rats decreased as PTZ injection continued.Conclusion: ZAG may be synthesized in neurons,but not astrocytes.Both AZGP1 m RNA and ZAG protein levels were decreased in epilepsy patients and PTZ-kindled rat models.The reduction in ZAG may play an important role in the pathogenesis and pathophysiology of epilepsy.ZAG level in brain tissue of PTZ-treated rats was negatively correlated with PTZ injection time.Part Two The Role of ZAG in Epilepsy and Its Molecular Mechanism Obejective: To explore the potential mechanism of ZAG in epilepsy.Methods:1.Verification of the successful transfection of AAV: 60 SD rats were randomly divided into 4 groups: Controls,GFP group,AZGP1-3W group and AZGP1-9W group.Fluorescence scanning,RT-q PCR and western blot were used to detect the transfection effect at 3 weeks and 9 weeks after AAV injection.2.Exploring the potential mechanism of AZGP1 overexpression in PTZ-kindled rat model: Another 45 rats were randomly divided into 3groups: controls,GFP+PTZ group and AZGP1+PTZ group.Rats in AZGP1+PTZ group and GFP+PTZ group recovered for 3 weeks after AAV injection and then received intraperitoneal injection of PTZ for 28 days daily.Controls received equal amount of saline instead of AAV and PTZ.The behavioral and EEG differences of rats in each group were observed.To explore the potential biological role and molecular mechanism of ZAG in epilepsy,Co-IP of p-ERK,TGF-?1 and ZAG was performed.The expression of inflammatory cytokines TNF?,IL-6,TGF?,ERK and p-ERK in brain tissue of each group was detected by western blot.Results:1.The GFP-positive cells in the CA3 region of hippocampus werevisualized.The expression of AZGP1 m RNA and ZAG protein was significantly increased in rats of AZGP1-3W group and AZGP1-9W group compared to controls and AAV-GFP group(P<0.05);2.Co-IP identified that ZAG can bind to p-ERK and TGF-?1;3.Overexpression of AZGP1 suppressed seizures,prolonged the latency of PTZ kindling and alleviated the epileptiform discharge in PTZ-kindled rats;4.Overexpression of AZGP1 attenuated the increase of TNF?,TGF?and IL6 induced by PTZ kindling,and decreased the phosphorylation of ERK.Conclusion: ZAG may participate in epileptogenesis by interacting with p-ERK and TGF-?1.Overexpression of AZGP1 may suppress epilepsy via inhibiting TGF?-mediated ERK phosphorylation and decreasing TNF? and IL-6 mediated neuroinflammation.ZAG,as an anti-inflammatory cytokine,it may be a novel target for research and clinical treatment of epilepsy.