| PART ONE: The expression of miRNA-Let-7i and TLR4 in temporal lobe epilepsy patients and epileptic rat modelsObjective: To investigate the expression profiles of miRNA-Let-7i and Toll-like receptor 4(TLR4)in brain tissues of patients with temporal lobe epilepsy and epileptic rat modelsMethods:Temporal neocortex tissues from 6 intractable temporal lobe epilepsy patients were randomly selcected from the brain tissue bank of TLE patients.And temporal neocortex tissues from 5 clinical-data-matched patients with traumatic brain injury underwent surgery were collected as control.Adult male Sprague-Dawley(SD)rats were used to establish acute elithium-pilocarpine epilepsy model and chronic PTZ-kindled epilepsy model.In both epileptic rat models,fully kindled rat were defined as the epilepsy group,and those without fully kindling were defined as the control group.Cortex and hippocampus tissues were obtained from the rat of epilepsy and control groups.Real-time quantitative PCR(q-PCR)and western-blot were used to investigate the expression levels of miRNA-Let-7i and TLR4 in brain tissues of epilepsy group and control group.Results:1.The expression levels of miRNA-Let-7i in temporal neocortex of patients with temporal lobe epilepsy(TLE)was downregulated than that in control patients(P<0.05);2.In both acute elithium-pilocarpine epilepsy model and chronic PTZ-kindled epilepsy model,the expression of miRNA-Let-7i in hippocampus and cortex were significantly lower in fully kindled group than that in control group(P<0.05);3.The expression levels of TLR4 in temporal cortex tissue of patients with temporal lobe epilepsy(TLE)was downregulated than that in control patients(P<0.05);4.In both epileptic rat models,the TLR4 expression in fully kindled group were upregulated while not in the controls(P<0.05).Conclusion:The miRNA-Let-7i expression were decreased in TLE patients and both two epileptic rat models.While its downstream target TLR4 expression levels in TLE patients and both two epileptic rat models were significantly increased than that in control groups.These results showed that miRNA-Let-7i and its downstream target TLR4 may be closely related to the occurrence and development of epilepsy.PART TWO: The effect of miRNA-Let-7i intervention on expression of TLR4 and animal epileptic behaviors in vivo.Objective: To evaluate the intervention efficiency on expression of TLR4 and animal epileptic behaviors of mi RNA-Let-7i,the specific agonist(agomir)and antagonist(antagomir)of miRNA-Let-7i were bilaterally injected into hippocampus of rats.TAK-242 was used to specific blockade TLR4 after antagomir intervention for assessing the effects of antagomir.We also used western blot to detect the expression of inflammatory factors IL-6 and TNF-? for reappraisal the inhibitory effect of TAK-242.Methods:1.Health and adult male SD rat were divided into 3 groups randomly: control group,agomir group,antagomir group.The control group was not intervened,agomir group and antagomir group were injected in bilateral hippocampus of agomir 0.2nmol/side or antagomir 0.4nmol /side.Real-time quantitative PCR and confocal laser scanning technique were used to assess the intervention efficiency of agomir and antagomir;Using miRNA-Let-7b to check the specificity of agomir and antagomir' intervention;Expression of TLR4 in rats' brain tissue was detected by Western-Blot after intervention of agomir and antagomir.2.Health and adult male SD rat were divided into 5 groups randomly: control group,sham group,agomir group,antagomir group,antagomir+TAK-242 group.Sterilization ddH2 O 4?L /side injected in bilateral hippocampus in sham group,and TAK-242(0.5mg/kg)was caudal vein injected in antagomir+TAK-242 group.The rest groups treatment were the same as above.3.Pilocarpine(320mg/kg)was intraperitoneally injected in rats to induced acute epilepsy.The Racine score of epileptic rats at sequential points within 1 hours after injected(10 min,20 min,30 min,40min,50 min,60min)were recorded and analyzed;Rats were daily intraperitoneal injected of subthreshold dose of PTZ(35mg/kg)to establish chronic-kindled model.We recorded the latency to fully-kindled and the highest Racine score of daily epileptic seizures of each group.4.Western blot was used to to detect the expression of IL-6 and TNF-?,downstream inflammatory factors of TLR4 in the sham group,antagomir group and antagomir+TAK-242 group.Results:1.In agomir group,the expression levels of miRNA-Let-7i was higher than that in control group at the 3rd day,the 1st week,the 2nd week,and the 4th week after agomir intrahippocampal injection;The expression of TLR4 was suppressed after agomir intrahippocampal injection compared with control group at the 3rd day,the 1st week,the 2nd week,and the 4th week(P<0.05).In antagomir group,the expression level of miRNA-Let-7i was decreased significantly than that in control group at the 3rd day,the 1st week,the 2nd week,and the 4th week after agomir intrahippocampal injection;The expression level of TLR4 was increased after agomir intervention compared with control group(P<0.05).2.In rats of pilocarpine induced acute epilepsy model,the latency of the first episode in agomir group was longer than control group and sham group;At the time points(30min,40 min,60min),the seizure score was lower than that of the control group and sham group.Compared with the control group and sham group,the latency of the first episode was significantly shorter in the antagomir group,and at the time points(10min,20 min,30min),the seizure score was higher than that of the control group and sham group(P<0.05);There was no significant difference in latency and seizure score between antagomir+TAK-242 group,control group and sham group(P>0.05).3.In the rat of PTZ chronic-kindled epilepsy model,the time needed for complete kindling in the agomir group was significantly longer than that in the control group and sham group(P<0.05);The level of seizure severity was lower in the 3rd-10 th days after PTZ intraperitoneal injection than that in control group and sham group(P<0.05).The latency for complete kindling was significantly shorter in the antagomir group than in the control group and sham group(P<0.05);Compared to control group and sham group,the seizure severity level was increased in the 3rd-10 th days after PTZ intraperitoneal injection(P<0.05).4.The expression of inflammatory factor IL-6 and TNF-? in the antagomir group was significantly higher than that in the sham group,and in antagomir+TAK-242 group it was downregulated compared to antagomir group and sham operation group.Conclusion:1.The expression level of miRNA-Let-7i and TLR4 can be specifically altered by miRNA-Let-7i agomir and antagomir intervention in hippocampus of rats;TLR4 is negatively regulated by miRNA-Let-7i.2.The latency and severity of seizure in epileptic rat were ameliorated by agomir intervention;Antagomir intervention could aggravate the seizure latency and severity in epileptic rat.3.The effect of antagomir on the behavior of epileptic model and the expression of IL-6 and TNF-? could suppressed by TAK-242 of TLR4 specific inhibitor.PART THREE: Effects of miRNA-Let-7i on hippocampal neurons apoptosis in epileptic rats following SEObjective:To observe the effect on hippocampal neurons apoptosis in rats following status epilepticus(SE)induced by pilocarpine,agomir and antagomir were used to specific upregulation and inhibit the expression of miRNA-Let-7i.Methods:1.Health and adult male SD rat were divided into 4 groups randomly:,control group,agomir group,antagomir group,antagomir +TAK-242 group.Sterilization dd H2O(4?L /side)was injected in bilateral hippocampus in control group,and TAK-242(0.5mg/kg)was caudal vein injected in antagomir+TAK-242 group;Agomir(0.4 nmol)and antagomir(0.8nmol)were injected in bilateral hippocampus.2.To observe the effect of miRNA-Let-7i on the neurons apoptosis,brain tissue in each group rats were obtained after 3 days of intervention and stained by Niss and Tunel.Results:1.In agomir group,we found the apoptosis in hippocampus CA1,CA3 and DG were significantly supressed compared with control group by Nissl and Tunel staining(P<0.05).2.Cells of CA3 and DG in hippocampus of rats were stained with Nissl and Tunel showed that the apoptosis was significantly lower in agomir group than that in control group(P<0.05).3.Nissl and Tunel staining of CA1,CA3 and DG cells in rats hippocampus of group antagomir showed that the apoptosis was significantly lower than that in control group(P<0.05)Tunel staining and Nissl staining showed no significant difference between the antagomir group and the control group in CA1(P>0.05).Conclusion:1.Up regulation of miRNA-Let-7i expression can significantly reduce the apoptosis of hippocampal neurons in epileptic rats.2.Down regulation of miRNA-Let-7i expression can significantly aggravated the apoptosis of hippocampal neurons in epileptic model rats,and TLR4's specific inhibitor TAK-242 can depressing the effect of increase neuronal apoptosis induced by antagomir. |
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