| Epileptic seizures have long been known to cause neuronal damage. In particular, convulsive status epilepticus can result in extensive injury and cell death to hippocampus. However, the precise mechanisms underlining this brain neurodegeneration are still not fully understood. A increasing evidences supports that brain inflammation induced by Epileptic insults is involved in the pathogenesis of epilepsy, which play a critical role in epileptogenic brain injury. Nuclearfactor-kappaB (NF-κB), a key regulator of immune and inflammatory response, has been implicated in the neuropathological processes of seizure and epilepsy. Our previous studies have demonstrated that inhibit the expression of NF-κB could ameliorate neuronal degeneration in brain of seizure rats, which has the brain protective effect. NF-κB activation is responsible for neuronal cell death, although mechanisms are still to be unclear. Each seizure event, particularly general tonic-clonic seizures, causes transient brain hypoxia/ischemia. During seizure activity, the cerebral blood flow in the hippocampus showed significant decreases compared with that in the cortex. HIF-la is a kind of nuclear transcription factor, which is widely existed in mammals and human body. Recent studies have demonstrated that hypoxia markers (including HIF-1α) are expressed in neurons exposed to recurrent seizures. But, the beneficial or detrimental role of HIF-la in epileptogenic brain damage remains unknown. Studies of tumor and digestive system have shown that NF-κB is a critical transcriptional activator of HIF-la and NF-κB activation controls HIF-la expression. Moreover, Inhibition of the activation of NF-κB/HIF-la pathway could suppress growth of tumor cells and attenuate intestinal epithelial barrier dysfunction. Then, could the aberrant activation of NF-κB/HIF-la pathway induce epileptogenic neuronal degeneration? This is no relevant reports. This in-vivo study was aimed to investigate the effect of activation of NF-κB/HIF-la pathway on the hippocampal neurodegeneration provoked by status epilepticus in rats, which might provide a new perspective to further reveal the mechanism of epileptogenic brain damage.Part I Effect of overexpression of HIF-la on hippocampal neurodegeneration provoked by status epilepticusObjective:To explore whether overexpression of HIF-la can cause hippocampal neurodegeneration provoked by status epilepticus.Methods:A total of 125 male SD rats were randomly divided into 4 groups:(1) the saline control group (Control group, n=25), (2) the pilocarpine-induced SE group (SE group, n=50), the SE group was further dibided into four subgroups according to observation time, (3) the SE with vehicle (dimethyl sulfoxide (DMSO)) pretreatment group (SE+DMSO group, n=25), (4) the SE with 2ME2 pretreatment group (SE+ 2ME2 group, n=25). SE was induced by injecting lithium chloride and pilocarpine. The control group was injected with the same dose of 0.9% saline instead of pilocarpine. The SE+2ME2 group received 2ME2 (15 mg/kg, i.p.) which was dissolved in DMSO (10mg/ml) 15 min before the pilocarpine injection. Rats in the SE+DMSO group received equivalent volume DMSO at the same time point. The seizure of rats was observed. The protein expressions of HIF-la and Cleaved caspase-3 in hippocampus of rats were examined by Western blotting and Immunohistochemistry. The degenerating neurons in hippocampus were detected by Fluoro-Jade C (FJC) staining.Results:No statistical differences were observed in the SE、 SE+DMSO and SE+2ME2 groups with regard to success rate of animals with status epilepsy(90%, 88%,92%; P>0.05), mortality during status epilepsy(33%,36%,30%; P>0.05), latency to grade 4 (SOT4)(32.36±10.05,33.95±9.668,34.87±11.28; P>0.05), latency to grade 5(40.80±9.855,41.73±10.34,43.17±10.40; P>0.05); the protein expressions of HIF-la and Cleaved caspase-3 in hippocampus of rats were increased in SE 24h subgroup (1.017±0.080,0.284±0.0535) than those in control group (0.151±0.083,0.060±0.017; P＜0.05); Compared with SE 24h subgroup, the protein expression of HIF-la and Cleaved caspase-3 (0.259±0.071,0.210± 0.051; P<0.05) in SE+2ME2 group was significantly decreased, and the integrated optical density (IOD) of HIF-la and Cleaved caspase-3 (CA1:201658 ±40978, 87308±14710 VS 64535±16278,38360±5173; P＜0.05. CA3:339091+24972, 148662±27198 VS 114860±25150,58596± 19940; P<0.05) in SE+2ME2 group was also decreased; in SE+2ME2 group the numbers of FJC positive cells in CA1 area (43.330±5.508) and CA3 area (36.67±6.028) were decreased than there in SE 24h subgroup (CA1:95.33±11.68, CA3:88.67±7.767; P＜0.05)Conclusions Overexpression of HIF-1α has detrimental effect on hippocampal neuro in pilocarpine-provoked status epilepsy, and underlying mechanism may be due to the suppression of apoptotic protein Cleaved caspase-3.Part II Aberrant activation of NF-kB contributes hippocampal neurodegeneration in status epilepticus rat via up-regulating expression of HIF-laObjective:To explore the correlation between NF-κB and HIF-la in hippocampal neurodegeneration of status epilepticus ratsMethods:A total of 110 male Sprague-Dawley rats were randomly divided into seven groups:(1) Control group treated with saline (Control, n=15), (2) sham group implanted cannula into lateral ventricle and treated with saline (sham, n=15), (3) SE group treated with pilocarpine (SE, n=15), (4) NF-κB activity inhibitor pyrrolidine dithiocarbamate (PDTC) group treated only with PDTC (PDTC, n=15), (5) SE+PDTC group treated with pilocarpine plus PDTC (SE+PDTC, n=15), (6) SE+HIF-la siRNA group implanted cannula into lateral ventricle and treated with pilocarpine plus HIF-1α siRNA (SE+HIF-la siRNA, n=15), (7) SE+Control siRNA group implanted cannula into lateral ventricle and treated pilocarpine plus with Control siRNA. SE was induced by injecting lithium chloride and pilocarpine. The seizure of rats was observed. The protein expressions of NF-κB and HIF-la in hippocampus of rats were examined by Western blotting. The degenerating neurons in hippocampus were detected by Fluoro-Jade C (FJC) staining.Results:Twenty-four hours after termination of SE, the nuclear protein expressions of NF-κB and HIF-la in hippocampus of rats were increased in SE group (0.57±0.06,0.47±0.07) than those in control group (0.23±0.03,0.20±0.03; P<0.05); and compared with SE group PDTC significantly decreased the nuclear protein expressions of NF-κB and HIF-la in SE+PDTC group (0.227±0.029, 0.136±0.026; P＜0.05); in SE+PDTC group the numbers of FJC positive cells in CA1 area (28.33±5.03) were decreased than there in SE group (76.67±13.32); HIF-la siRNA injected into lateral ventricle of rats significantly decreased the expression of HIF-1 a in hippocampus (0.22±0.03) and the number of FJC positive cell in CA1 area (27.34±7.02) in SE+HIF-la siRNA group than those in SE group (0.±0.06,76.67±13.32; P＜0.05).Conclusion:These data suggest that SE can result in activation of NF-icB/HIF-1α pathway in brain. Inhibition of the pathway can attenuate hippocampal neurodegeneration caused by SE, which has the brain protective effect. |
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