Effect of Suanzaoren Decoction on expression of ionotropic glutamate receptors and synaptic plasticity in hippocampus of anxiety rats / 中国中药杂志

This study investigated the effect of Suanzaoren Decoction on the expression of N-methyl-D-aspartate receptors(NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors(AMPAR) in the hippocampus and synaptic plasticity in rats with conditioned fear-induced anxiety. The effect of Suanzaoren Decoction on rat behaviors were evaluated through open field experiment, elevated plus maze experiment, and light/dark box experiment. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of glutamate(Glu) and γ-aminobutyric acid(GABA) in the rat hippocampus. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to assess the gene and protein expression of ionotropic glutamate receptors in the hippocampal region. Transmission electron microscopy was utilized to observe the changes in the ultrastructure of synaptic neurons in the hippocampal region. Long-term potentiation(LTP) detection technique was employed to record the changes in population spike(PS) amplitude in the hippocampal region of mice in each group. The behavioral results showed that compared with the model group, the Suanzaoren Decoction group effectively increased the number of entries into open arms, time spent in open arms, percentage of time spent in open arms out of total movement time, number of entries into open arms out of total entries into both arms(P<0.01), and significantly increased the time spent in the light box and the number of shuttle crossings(P<0.01). There was an increasing trend in the number of grid crossings, entries into the center grid, and time spent in the center grid, indicating a significant anxiolytic effect. ELISA results showed that compared with the model group, the Suanzaoren Decoction group exhibited significantly reduced levels of Glu, Glu/GABA ratio(P<0.01), and significantly increased levels of GABA(P<0.01) in the rat hippocampus. Furthermore, Suanzaoren Decoction significantly decreased the gene and protein expression of NMDAR(GluN2B and GluN2A) and AMPAR(GluA1 and GluA2) compared with the model group. Transmission electron microscopy results demonstrated improvements in synapses, neuronal cells, and organelles in the hippocampal region of the Suanzaoren Decoction group compared with the model group. LTP detection results showed a significant increase in the PS amplitude changes in the hippocampal region of Suanzaoren Decoction group from 5 to 35 min compared with the model group(P<0.05, P<0.01). In conclusion, Suanzaoren Decoction exhibits significant anxiolytic effects, which may be attributed to the reduction in NMDAR and AMPAR expression levels and the improvement of synaptic plasticity.

TAT-GluA2CT interferes with the protective effect of GluA2/TARPγ-8 coupling on nerve injury in status epilepticus model of rats / 中华实用儿科临床杂志

Objective:To investigate the protective effect of interfering peptide TAT-GluA2CT on hippocampal neurons in the Lithium chlorine-Pilocarpine status epilepticus model and the optimal time of administration.Methods:Male SD rats (72 cases) were induced to status epilepticus by using Lithium chlorine-Pilocarpine, while a control group ( n=12) was established.The 72 rats were divided into epilepsy group ( n=12), TAT-sham peptide group ( n=12), TAT-GluA2CT peptide group ( n=48) according to the random number table method, and the TAT-GluA2CT peptide group were further divided into the pre-1 h group ( n=12), the post-2 h group ( n=12), the post-4 h group( n=12), and the post-6 h group ( n=12) according to the administration time of the TAT-GluA2CT peptide.Nissl staining and terminal dUTP nick end labeling (TUNEL) assay were performed on 6 rats each from control group, epilepsy group, TAT-shampeptide group, pre-1 h group, post-2 h group, post-4 h group, and post-6 h group to observe the morphological changes and apoptosis of neurons in the CA1 region of the rat hippocampus.Western blot and co-immunopercipitation test were used to detect the expression of GluA2[second subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) recepter] and the coupling of GluA2/transmembrane AMPA receptor regulatory protein (TARP γ-8) complex in control group, epilepsy group, pre-1 h group, post-2 h group, post-4 h group and post-6 h group.The t-test was used to compare the data differences between 2 groups, and one-way ANOVA was adopted to compare the differences between the groups. Results:Compared with the epilepsy group, the number of neurons in each TAT-GluA2CT peptide group increased significantly, and the difference was statistically significant( epilepsy group 20.07±3.51, pre-1 h group 39.40±2.39, post-2 h group 38.43±2.42, post-4 h group 30.30±2.55, and post-6 h group 27.93±3.20, F=235.28, P<0.05). Compared with the epilepsy group, the number of apoptotic cells in each TAT-GluA2CT peptide group was significantly reduced, and the difference was statistically significant(epilepsy group 31.47±3.19, pre-1 h group 7.30±3.45, post-2 h group 9.27±3.81, post-4 h group 12.86±3.08, and post-6 h group 14.43±3.13, F=248.60, P<0.05). Compared with the control group, the expression of hippocampal GluA2 decreased after epilepsy induction, and the difference was statistically significant(control group 21 626.53±2 700.58, epilepsy group 14 578.16±2 917.02, pre-1 h group 13 375.47±3 180.54, post-2 h group 15 244.10±1 390.41, post-4 h group 15 799.16±4 559.49, post-6 h group 15 722.95±1 756.01, F=3.83, P<0.05). No statistical difference was observed in the expression of GluA2 between the TAT-GluA2CT peptide group and the epilepsy group( F=0.45, P=0.77). Compared with the epilepsy group, GluA2/TARPγ-8 complex coupling was decreased in each TAT-GluA2CT peptide group, and the difference was statistically significant(epilepsy group 24 509.80±3 718.54, pre-1 h group 12 055.18±5 847.11, post-2 h group 9 630.51±5 805.17, post-4 h group 12 749.35±7 108.45, post-6 h group 11 092.98±7 330.08, F=10.68, P<0.05). Compared with the epilepsy group, the incubation period of seizures in the pre-1 h group was prolonged and the seizure rating was decreased, with statistically significant differences[epilepsy group (18.58±3.99) min, pre-1 h group (103.25±9.21) min, t=29.23, P<0.05]. Conclusions:TAT-GluA2CT peptide can attenuate the neuronal damage in hippocampus of epileptic rats.The neuroprotective effect of TAT-GluA2CT peptide was most obvious at 1 h before or 2 h after administration of Pilocarpine.

Hippocampal neuronal damage and its possible mechanism in status epilepticus / 中华实用儿科临床杂志

Objective To investigate hippocampal neuronal damage and dynamic change of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR2 in status epilepticus, to find out whether GluR2/glyceral dehyde-3-phosphate dehydrogenase (GAPDH) interaction has any change.Methods Male Wistar rats (62 cases) were induced to status epilepticus by using LiC1-pilocarpine.The 62 rats were divided into 1 h (6 cases),6 h (12 cases), 24 h (12 cases),72 h (12 cases)and 7 d (20 cases)after status epilepticus.At the same time, the healthy control group (12 cases) was established.Morphologic changes of hippocampus and the amount of apoptotic cells in healthy control and SE model groups at different time points (6 h, 24 h, 72 h, 7 d) (6 cases each group) after status epilepticus were quantified by adopting Nissl staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining respectively.Expressions of GluR2 in healthy control and SE model groups at 1 h,6 h,24 h,72 h and 7 d (6 cases each group) after status epilepticus were detected by using Western blot.Co-precipitation and Western blot techniques were used to investigate whether the GluR2/GAPDH interaction in the hippocampus was increased.Results Compared with the healthy control group, the number of nerve cells in the hippocampal CA1 and CA3 regions was significantly reduced at all studied time points(F =30.866,24.043, all P ＜0.05).Apoptotic cells in hippocampal CA1 and CA3 regions were significantly increased at 24 h,72 h and 7 d after status epilepticus (F =84.762,52.574, all P ＜ 0.01).GluR2 at 1 h,6 h after status epilepticus was equal to that of the control group (P ＞ 0.05), but it was shown to be significantly down-regulated at other studied time points (F =76.506,P ＜ 0.01);when compared with the healthy control group,the GluR2/GAPDH interaction was significantly up-regulated in the hippocampus at 72 h after status epilepticus (t =7.029, P ＜ 0.05).Conclusions Status epilepticus can lead to neuronal damage in the hippocampus.Down-regulation of GluR2 and increase of the GluR2/GAPDH complex formation might be one of the mechanisms involved in hippocampal neuronal damage.