The Protective Role Of Different Concentration Necrostatin-1 On Hippocampal Neurons After Status Epilepticus In Mice

Objective:The investigation was performed to examine the effects of the best concentration of specific necroptosis inhibitor Necrostatin-1 on hippocampal neurons after status epilepticus（SE）induced by kainic acid in mice.Methods:66 male ICR mice weighted 25 to 30 g were randomly divided into control group（Sham）,status epilepticus（SE）,intervention group of different doses of Nec-1(10μmol·L^-1,μmol·L^-140μmol·L^-1,80μmol·L^-1)group,a total of six groups by intraperitoneal injection of kainic acid 12 mg/kg status epilepticus in mice model was established,in the first 15 min after intraperitoneal injection of unilateral lateral ventricle intervention agent Nec-1 given different concentration at different time points after the success of the model（24 h and 48 h,72 h）by Western blot method programmed necrosis in hippocampus associated protein RIP1 RIP3 MLKL and apoptosis related proteins cleaved-caspase3 Bcl-2 expression of Bax,screening the most obvious point in time the protein expression of immunohistochemistry and Western blot method programmed necrosis in hippocampus associated protein RIP1RIP3 MLKL and apoptosis related proteins-B cell lymphoma（Bcl-2）Capases 2-3Bax expression Using immunohistochemical H E staining to observe the pathological morphological changes of hippocampus neuron cell in mice.The effect of Tunnel staining on the apoptosis of hippocampal neurons in mice was determined,and the optimal concentration of neurocytes was selected.Results:Seizures after 24 hours,Western blot showed that DMSO+KANICE ACID group MLKL RIP1 Bax cleave-caspase3 expression to peak and DMSO group and DMSO+KANIC ACID ratio highest seizures after 24 hours,Western blot results showed that 40μmol·L-1 intervention dose group can obviously cut RIP1 MLKL RIP3 cleaved-caspase3 Bax protein expression,raise the Bcl-2 expression in mice 24h after seizures IHC results show that the control RIP1 MLKL RIP3 cleaved-caspase3Bax protein expression quantity are less,in hippocampus after seizures RIP1 MLKL RIP3 cleaved-caspase3 Bax protein content increased significantly（P<0.05）after intervention for Nec-1,with the increase of concentration cell positive rate is on the decline,80μmol·L-1 treatment group compared with 40μmol·L-1 treatment group a slight upward trend,40μmol·L-1 Nec-1 treatment group can significantly cut RIP1MLKL RIP3 cleaved-caspase3 Bax protein expression（P<0.05）HE dyeing visible DMSO group of hippocampal CA3 area neat cells,form normal;The cell lines of DMSO+KANIC ACID group were scattered,some of the cytoplasm were vacuolated,and some of the neurons were unevenly distributed,and the nucleolus disappeared.40μmol·L-1 Nec-1 the treatment group can obviously reduce the neuron cell body swelling TUNEL staining nucleus pycnosis,cell morphology change visible DMSO group of hippocampal CA3 area of a small amount of brown TUNEL positive granular cell,and DMSO+KANIC group of hippocampal CA3 area visible large brown positive cells compared with DMSO+KANIC group,40μmol·L-1 Nec-1 set of CA3 area TUNEL positive cells decreased significantly（P<0.05）Conclusion:（1）Our results suggest that With the increase in the concentration of Nec-1 intervention,the expression of RIP1 MLKL RIP3 cleaved-caspase3 Bax protein was decreased,and the group of 80μmol·L-1 treated group had a higher trend than that of the 40μmol·L-1 treatment group.（2）The protective effect of 40μmol·L-1 Nec-1treatment group on KA-induced mice was more obvious than that of other treatment groups.