Characterization Of Hydrogen Sulfide Donor And Up-Regulation Of Kir6.2 And SUR1 Expression In Inhibition Of Epilepsy

OBJECTIVEFirst,observe the surface signs and absorption spectra of H₂S donor Donors,and examine the effects of H₂S donor Donors on the activity of acutely isolated hippocampal neurons and human laryngeal epithelial cell line Hep-2 cells and the optical imaging effects;At the cellular level,primary hippocampal neurons were cultured and identified,hydrogen sulfide donor Donors were co-cultured with hippocampal neurons,epileptic cell models were established without magnesium extracellular fluid treatment,and hydrogen sulfide donor Donors were used to observe the cells of hippocampal neurons.To investigate the effects of activity on the expression of Kir6.2 and SUR1 mRNA in hippocampal neurons,to further investigate the protective effect of intracellular hydrogen sulfide donor Donors on hippocampal neurons,and finally to establish animal epilepsy models at the animal level.The intracerebroventricular injection of H₂S donor Donor was used to observe the behavioral manifestations.EEG and immunohistochemistry were used to detect the expression of Kir6.2 in hippocampus and cortex.The expression of Kir6.2 and SUR1 protein and mRNA in cortex and hippocampus were detected.Investigate the inhibitory effect and mechanism of in vivo hydrogen sulfide donor Donor on epilepsy.METHODSPart 1:The characterization of hydrogen sulfide donor Donor was observed under transmission electron microscope,and the absorption spectrum of hydrogen sulfide donor Donor was measured.The hydrogen sulfide donor Donor was used to detect primary Hippocampal neurons and human laryngeal cancer epithelium with CCK-8 kit.The effect of cell activity of the cell line Hep-2 was selected,and the maximum safe concentration and action time of the Donor were selected.The absorption of the hydrogen sulfide donor Donor in both cells and the luminescence channel were detected by confocal laser scanning.Part 2:At the cellular level,the role and mechanism of hydrogen sulfide donor Donors in hippocampal neurons were further explored.Primary hippocampal neurons of SD rats were cultured and identified by NSE/DAPI double staining method.The epilepsy cell model was established by liquid treatment for 3 hours.We divided the hippocampal neurons into 5 groups:1.Control group,normal extracellular fluid treatment 3 h;2.Epilepsy cell model group（EPI）treated with magnesium-free extracellular fluid for 3 h;3.Hydrogen sulfide donor Donor intervention Group（H₂S Donor+EPI）,cultured for 12 h in medium supplemented with hydrogen sulfide donor Donor,and treated with magnesium-free extracellular fluid for 3 h;4.H₂S donor Donor blank control group（H₂S Donor）.Culture media of H₂S donor Donors were treated with normal extracellular fluid for 3 hours;5.Hydroxylamine control groups（HA+EPI）were cultured for 12 hours with hydroxylamine-equipped media and treated with magnesium-free extracellular fluid for 3 hours.RT-qPCR were used to detect Kir6.2,SUR1 mRNA expression in hippocampal neurons.Part 3:At the animal level,further explore the role and mechanism of H₂S donor Donors on epilepsy.We randomly divided rats into 5 groups:1.Control group,saline injection into lateral ventricle,intraperitoneal injection of physiology Saline;2.Status epilepticus group（SE）,intracerebroventricular injection of saline,intraperitoneal injection of pentylenetetrazol;3.Hydrogen sulfide donor Donor intervention group（H₂S Donor+SE）,lateral ventricle injection of hydrogen sulfide donor Donor,and then Intraperitoneal injection of pentylenetetrazol;4.H₂S donor Donor blank control group（H₂S Donor）,lateral ventricle injection of hydrogen sulfide donor Donor,intraperitoneal injection of saline;5.Hydroxylamine control group（HA+SE）,lateral ventricle injection of physiology Saline was injected intraperitoneally with hydroxylamine 12.5 mg/kg.After 30 mins,pentylenetetrazol was injected intraperitoneally.Observe the behavior of rats in each group after modeling.Record the latency and duration of the first epileptic-like discharge in rats.Use the BL-420 biological signal system to record the changes of EEG in each group of rats by immunohistochemistry.Methods The localization and expression of Kir6.2 in hippocampus and cortex were detected in each group.The expression of Kir6.2 and SUR1 protein and mRNA in cortex and hippocampus were detected by Western blot and RT-qPCR.RESULTSPart 1 results:1.Scanning electron microscopy:Scanning electron microscopy was used to observe the morphology of hydrogen sulfide donor Donors.The microscopic morphology of hydrogen sulfide donor Donors showed uniform micro-clusters composed of many micro-tubes.These tubes showed a high degree of crystallinity and regularity.2.Absorption spectrum:The characteristic peak of the absorption spectrum.The Donor absorbs light strongly from 230 nm to 310 nm.As a reference,the fluorescence wavelength of the hydrogen sulfide donor Donor is selected near the absorption peak.3.CCK-8 results:Based on the experimental results,we chose an optimal concentration of 400μM and an optimization time of 12 h to detect in vitro optical imaging.4.Laser Confocalization results:Confocal observation of fluorescence emission of hippocampal neurons and Hep-2 after co-cultured with a hydrogen sulfide donor Donor for 12 hours.Hydrogen sulfide donor Donors still have high fluorescence emission properties after cell uptake,indicating that the cells have absorbed the Donors by specific immunoglobulin recognition.Part 2 results:1.NSE/DAPI double staining method:After staining with NSE antibody,it was further confirmed that the cultured cells were mainly neurons,accounting for about90%of the total cells.2.CCK-8 method:3 hours after magnesium-free extracellular fluid treatment,the activity of cells in EPI group,H2S Donor+EPI group and HA+EPI group was lower than that in Control group;compared with EPI group,H2S Donor The activity of cells in the+EPI group and H2S Donor group increased.3.RT-qPCR was used to detect the expression of Kir6.1 and SUR2 mRNA in hippocampal neurons:After treatment with magnesium-free extracellular fluid for3h,the expression of Kir6.2 and SUR1 mRNA in hippocampus of rats in the SE and HA+SE groups compared with the Control group All decreased;H₂S Donor+SE group and H₂S Donor group increased expression of Kir6.2 and SUR1 mRNA in hippocampus compared with SE.Part 3 results:1.Behavior:Rats in the Control and H₂S Donor groups had normal behavior and no seizures.Seizures occurred in rats of SE group,showing grades IV to V;the latency of epilepsy in the H₂S Donor+SE group was significantly prolonged,and the level of seizures was also decreased,showing grade I or grade II compared with the SE group.The incubation period was prolonged,and the duration of seizures was also shortened.The difference was statistically significant.2.EEG:Control,H₂S Donor group did not appear spike or spine-slow wave,as the normal EEG waveform.The sharp wave,spike wave or spine-slow wave in the SE group and HA+SE can be recorded,and the wave amplitude is larger.The amplitude of amplitude is significantly different from that in the control group.Occasionally observed in the H₂S Donor+SE group.Sharp wave,spike wave,or spine-slow wave were observed,amplitude decreased,and there was a difference compared with SE group.Hippocampus brain wave amplitude and control phase of Hippocampus donor Donor blank group.The difference was not statistically significant.3.Immunohistochemistry:Kir6.2 is mainly located in the cortex,hippocampal dentate gyrus,CA3 area.In the cortex,dentate gyrus,and CA3 regions,positive substances were observed in the Control group,but the particles were lightly stained and sparsely distributed;the immune response was gradually weakened in the SE and HA+SE groups,and the number of positive cells decreased,and the average optical density of the Control group decreased;Kir6.2 immunoreactivity increased gradually in H₂S Donor+SE group and H₂S Donor group,and the number of positive coloring cells was higher than that of SE group.4.RT-qPCR was used to detect the expression of Kir6.1 and SUR2 mRNA in the cortex and hippocampus:In the cortex of the SE model,the expression of Kir6.2and SUR1 mRNA in the hippocampus of the rats was decreased in the SE and HA+SE groups compared with the Control group;The expression of Kir6.2 and SUR1 mRNA in hippocampus of H₂S Donor+SE group and H₂S Donor group was higher than that of SE,and the difference was statistically significant.In the hippocampus of the SE model,the expression of Kir6.2 and SUR1 mRNA in the hippocampus of the rats of the SE and HA+SE groups was decreased compared with that of the Control group.The H₂S Donor+SE group and the H₂S Donor group of the hippocampus Kir6.2.The expression of SUR1 mRNA was increased compared with SE,and the difference was statistically significant.5.The expression of Kir6.2 and SUR1 protein in cortex and hippocampus was detected by Western blot:The contents of Kir6.2 and SUR1 protein in cortex of SE model and HA+SE group were significantly lower than that of Control after SE model.Significance.The protein levels of Kir6.2 and SUR1 in H₂S Donor+SE group and H₂S Donor group were significantly different from those in SE group.In the hippocampus of the SE model,the expression of Kir6.2 and SUR1 mRNA in the hippocampus of the rats of the SE and HA+SE groups was decreased compared with that of the Control group.The H₂S Donor+SE group and the H₂S Donor group of the hippocampus Kir6.2.The expression of SUR1 mRNA was increased compared with SE.CONCLUSIONS1.Hydrogen sulfide donors have good safety and cell imaging capabilities.2.Hydrogen sulfide donors have protective effects on primary hippocampal neurons;their protective effects may be related to the up-regulation of Kir6.2,SUR1expression.3.hydrogen sulfide donors can inhibit seizures in rats;its inhibition may be related to upregulation of Kir6.2,SUR1 expression.