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OBJECTIVE To study the sedative and hypnotic effects of zolpidem and the content of amino acid neurotransmitters in the thalamus and hypothalamus after treatment with zolpidem.METHODS Experiments on the loss of righting reflex(LORR)induced by the upper-threshold dose pentobarbital sodium(50 mg·kg-1,ip)were conducted to establish a hypoxic insomnia model in mice by simulating an altitude of 5500 m.Based on this model,the synergistic effect of zolpidem(0.33,1,3,9 and 27 mg·kg-1,ip)and the subthreshold(20 mg·kg-1,ip)and upper-threshold pentobarbital sodium,as well as the sedative hypnotic effect of zolpidem(10,13,17,20,23,30 and 40 mg·kg-1,ip)were evaluated via the LORR in normoxic and hypoxic environments.One hour after ip given zolpidem,the levels of glutamic acid(Glu)and γ-aminobutyric acid(GABA)in the thalamus and hypothalamus of mice in either environment were determined by the high-performance liquid chromatography(HPLC)with fluorescence detection.RESULTS One-day treatment with hypoxia significantly shortened the duration of LORR induced by the upper-threshold dose pentobarbital sodium.Compared with normoxia vehicle and hypoxia induced insomnia vehicle groups,zolpidem 9 and 27 mg·kg-1 significantly shortened the latency to LORR(P<0.01,P<0.05)and prolonged duration of LORR induced by subthreshold and upper-threshold pentobarbital sodi-um(P<0.01,P<0.05).The median effective dose(ED50)of LORR induced by zolpidem was 16.21 and 20.55 mg·kg-1 in normoxic and hypoxic environments,respectively.The results of neurotransmitter level detection showed that Glu contents in the thalamus and hypothalamus and the ratio of Glu/GABA in the hypothalamus were decreased after treatment with zolpidem 40 mg·kg-1 in a normoxic environment(P<0.01,P<0.05).Compared with the normoxia control group,Glu content and the ratio of Glu/GABA in the hypothalamus were significantly increased after treatment with hypoxia(P<0.01,P<0.05),and zolpidem 40 mg·kg-1 could reverse their elevation.CONCLUSION The sedative-hypnotic effect of zolpidem is weakened in a hypoxic environment,and the effect of zolpidem on the levels of Glu and GABA in the hypothalamus may play an important role in the sedative-hypnotic effect of zolpidem.
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Objective:To investigate the effect of aucubin on behaviors and excessive activation of astrocytic in attention deficit/hyperactivity disorder (ADHD) model mice.Methods:Twelve wild-type C57BL/6 pregnant mice (female, clean grade) were intraperitoneally administered with esketamine (15 mg/kg) to establish an ADHD model in offspring mice. The offspring mice were divided into control+ saline group, control+ aucubin group, Ketamine+ saline group and Ketamine+ aucubin group according to the nest matching principle with 15 in each group.At 14 days after birth, mice in the control+ aucubin group and Ketamine+ aucubin group were administered with aucubin (5 mg/kg, once a day) by gavage for 5 days. Mice in control+ saline group and Ketamine+ saline group were administered with equal volume of 0.9% sodium chloride solution. The offspring mice were housed with their mothers in the same cage until 21 days after birth. Twenty-one days after birth, the offspring mice were evaluated by open field test and elevated plus maze tests. Immunofluorescence assay was used to detect the expression of glutamate decarboxylase 2 (GAD2), γ- aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP) in the amygdala. The morphological changes of astrocytes were quantitatively analyzed by Sholl analysis. GraphPad Prim 9.0.1 software was used for statistical analysis. The comparison of multiple groups was conducted by one-way ANOVA or Kruskal-Wallis test.Results:(1)The results of behavioral experiments showed that the total distance traveled in the open field test and the residence time in open arm of the elevated plus maze were statistically significant ( F=236.90, H=39.92, both P<0.001). The total distance ((7 044±249)mm, (22 891±2 175)mm, P<0.05) and the residence time in open arm(12.69(9.86, 17.24)s, 2.72(0.57, 3.87)s, P<0.05) of mice in Ketamine+ saline group were both higher than those in control+ saline group.The total distance((22 891±2 175)mm, (8 252±839)mm, P<0.05) and the the residence time in open arm(5.45(1.13, 10.99)s, 12.69(9.86, 17.24)s, P<0.05) of Ketamine+ aucubin group were both lower than those of Ketamine+ saline group.(2)The immunofluorescence results showed that the levels of GAD2, GABA and GFAP intensity in amygdala of mice in the four groups were statistically significant ( F=145.50, 50.08, 53.83, all P<0.05). Compared with control+ saline group, the fluorescence intensities of GAD2 ((100.00±9.60)%, (24.86±4.14)%, P<0.05) and GABA ((100.00±16.84))%, (25.48±5.70)%, P<0.05) of Ketamine+ saline group were down-regulated, and the GFAP((100.00±18.02)%, (223.80±25.85)%, P<0.05) was up-regulated. Compared with Ketamine+ saline group, the fluorescence intensities of GAD2 ((24.86±4.14)%, (56.08±6.55)%, P<0.05) and GABA((25.48±5.70)%, (52.59±15.74)%, P<0.05) in Ketamine+ aucubin group were up-regulated, but the fluorescence intensity of GFAP ((223.80±25.85)%, (157.10±22.10)%, P<0.05) was down-regulated.(3)Sholl analysis indicated that the number of the intersections between the astrocyte processes or the branches of astrocyte processes was statistically significant in the 4 groups ( F=12.47, P<0.05). Compared with control+ saline group, the number of the intersections in Ketamine+ saline group((2.07±0.48), (1.67±0.72), P<0.05) increased. While the number of the intersections in Ketamine+ aucubin group was lower than that of Ketamine+ saline group ((1.20±0.78), (2.07±0.48), P<0.05). Conclusion:Aucubin administration can alleviate ADHD-like behaviors in offspring mice, and the mechanism may be associated with the inhibition of excessive astrocytic activation.
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Objective:To evaluate the effect of sleep fragmentation on postoperative cognitive dysfunction (POCD) and hippocampal glutaminergic metabolism in aged mice anesthetized with isoflurane.Methods:Forty healthy SPF-grade male C57BL/6J mice, aged 18 months, weighing 20-30 g, were divided into 4 groups ( n= 10 each) by the random number table method: normal control group (group C), sleep fragmentation group (group SF), isoflurane anesthesia/surgery group (group I/S), and sleep fragmentation plus isoflurane anesthesia/surgery group (group SF+ I/S). Group C did not received any treatment. Group SF received sleep fragmentation for 24 h. The right carotid artery exposure was performed under isoflurane anesthesia in group I/S. Group SF+ I/S received isoflurane anesthesia/right carotid artery exposure at 24 h after sleep fragmentation. The metabolic levels of glutamate (Glu), glutamine (Gln), Glu/Gln complex (Glx), and N-acetylaspartate (NAA) and their ratio to creatine (Cr) were measured by in vivo 9.4T hydrogen proton magnetic resonance spectroscopy at 2 h after anaesthesia. Y maze and Morris water maze tests were used to evaluate the cognitive function at 1-7 days after surgery. The mice were sacrificed after the behavioral testing, brain tissues were immediately obtained, and the number of Nissl bodies and density of dendritic spines in the hippocampal CA1 region were measured by Nissl staining and Golgi staining, respectively. Results:Compared with group C, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr, Gln/Cr and Glx/Cr in the hippocampal CA1 region were increased, and the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in SF, I/S and SF+ I/S groups ( P<0.05). Compared with group SF and group I/S, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr and Glx/Cr in hippocampal CA1 region was increased, the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in group SF+ I/S ( P<0.05). Conclusions:Sleep fragmentation exacerbates POCD in aged mice anesthetized with isoflurane, and the mechanism is related to nerve injury induced by abnormality in hippocampal glutaminergic metabolism excitability.
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Ischemic stroke is a common cerebrovascular disease with high mortality and disability rates. Astrocytes, as the most abundant glial cells in the brain, have the function of maintaining the homeostasis of the central nervous system, which requires precise coupling between neurotransmission and energy metabolism. Therefore, the metabolic crosstalk between astrocytes and neurons plays an important role in the occurrence and development of ischemic stroke. On the one hand, ischemic stroke leads to neuronal excessive excitation, metabolic disorders, and even death, simultaneously promoting changes in astrocyte metabolism profile and increased heterogeneity. On the other hand, the metabolic crosstalk between astrocytes and neurons can also affect the outcome of ischemic stroke. This article reviews the role of the metabolic crosstalk between astrocytes and neurons in ischemic brain injury, and looks forward to potential research directions and treatment targets in the future, in order to provide theoretical reference for the treatment of ischemic stroke.
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Objective:To evaluate the role of extracellular signal-regulated kinase (ERK)1/2 in glutamate-induced ferroptosis in PC12 cells.Methods:PC12 cells were divided into 6 groups ( n=21 each) using a random number table method: control group (C group), glutamategroup (Glu group), glutamate+ ERK1/2 over-expression group (Glu+ ERK1/2-OE group), glutamate+ ERK1/2 plasmid empty vector group (Glu+ Vec group), glutamate+ ERK1/2 knockdown group (Glu+ si-ERK1/2 group)and glutamate+ ERK1/2 SiRNA negative control group (Glu+ si-NC group). Cells were treated with glutamate at a final concentration of 6 mmol/L for 72 h in Glu group and with the equal volume of PBS buffer for 72 h in C group. Glu+ ERK1/2-OE group was transfected with ERK1/2 overexpression plasmid, Glu+ Vec group was transfected with plasmid empty vector, and Glu+ si-ERK1/2 group was transfected with ERK1/2 siRNA, Glu+ si-NC group was transfected with siRNA negative control for 48 h, and then glutamate at a final concentration of 6 mmol/L was added and cells were treated for 72 h. The cell viability, lactic dehydrogenase (LDH)activity and contents of glutathione (GSH), ferrous ions and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Mitochondrial membrane potential (MMP) and lipid reactive oxygen species (Lip-ROS) were measured by flow cytometry. Results:Compared with C group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, ferrous ions content, MDA content and Lip-ROS levels were increased in Glu group ( P<0.05). Compared with Glu+ Vec group, the cell viability, GSH content and MMP were significantly increased, and the activity of LDH, contents of ferrous ions and MDA, and Lip-ROS levels were decreased in Glu+ ERK1/2-OE group( P<0.05). Compared with Glu+ si-NC group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, contents of ferrous ions and MDA, and Lip-ROS level were increased in Glu+ si-ERK1/2 group ( P<0.05). Conclusions:ERK1/2 is involved in glutamate-induced ferroptosis in PC12 cells.
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@#Objective To share a case of anti-glutamate decarboxylase 65 antibody stiff-person syndrome and autoimmune polyendocrinopathy syndrome type Ⅱ,to improve clinicians' understanding of this disease. Methods The clinical data of a 51-year-old woman who was admitted to the affiliated hospital of a Neurology Institute in Anhui Province in 2022 was retrospectively analyzed,and the relevant literature was reviewed. Results Patients' clinical to repeatedly lumbago,lumbar abdomen and the stiffness of lower limbs with unable to characteristics of early misdiagnosed as separate conversion disorder,after the inspection found that serum and cerebrospinal fluid resistance to glutamic acid decarboxylase antibody positive 65,thyroglobulin antibody and higher peroxidase antibody degrees,fasting and postprandial 2 hours blood sugar,glycosylated hemoglobin,Neuroelectrophysiology showed that the continuous motor unit potential was mainly distributed by body axis muscles in the resting state. The patient was diagnosed as anti-glutamic acid decarboxylase 65 antibody SPS and APS-Ⅱ (Hashimoto's thyroiditis,type 1 diabetes mellitus),and the condition improved after immunotherapy and symptomatic treatment. Conclusion Although anti-glutamate decarboxylase 65 antibody SPS complicated with APS-Ⅱ has certain clinical specificity,it is easy to be misdiagnosed and missed because of its rarity,especially in the early course of disease.
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Objective:To explore the clinical characteristics of neurological syndrome associated with anti-glutamic acid decarboxylase (GAD) antibodies (Abs).Methods:Six patients with neurological syndrome associated with anti-GAD-Abs admitted to Department of Neurology, Henan Provincial People's Hospital from January 2019 to October 2022 were chosen. The clinical manifestations, imaging and laboratory results, therapeutic schedules, and follow-up prognoses of these patients were collected and summarized.Results:Three females and 3 males were included, with onset age of (54.3±17.7) years. Three patients had stiff-person syndrome (SPS), 1 had limbic encephalitis+generalized epilepsy, 1 had extralimbic encephalitis+occipital epilepsy, and 1 had cerebellar ataxia who was diagnosed with paraneoplastic syndrome associated with small cell lung cancer. Four patients had elevated level of thyroid peroxidase antibodies, and 1 patient was positive for overlapping anti-gamma aminobutyric acid B receptor antibodies and Amphiphysin antibodies. Two patients with SPS had failed lumbar puncture; 1 had slightly increased white blood cells and proteins in cerebrospinal fluid (CSF); the remaining 3 patients were basically normal. Specific oligoclonal bands in CSF were observed in 2 patients. Brain MRI showed abnormal signals in the bilateral occipital lobes in 1 patient, and no specific inflammatory lesions in other patients. All patients accepted corticosteroids and intravenous immunoglobulin/plasma exchange therapies; except for the one with paraneoplastic syndrome associated with small cell lung cancer, the remaining 5 patients had improved modified Rankin scale (mRs) scores at discharge and received long-term immunotherapy. Two patients with SPS had gradually aggravated symptoms, and mRs scores reached 5 at the last follow-up (one for 3 years and the other one for 2 years).Conclusions:The clinical manifestations of patients with neurological syndrome associated with anti-GAD-Abs include SPS, limbic encephalitis, extralimbic encephalitis, epilepsy and cerebellar ataxia; some of these patients have paraneoplastic syndromes. Immunotherapies are effective except for these patients with paraneoplastic syndromes. Some patients with SPS tend to have a chronic course and a poor prognosis.
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γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.
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Glutamato Descarboxilasa/genética , Lactobacillus plantarum/genética , Catálisis , Ácido gamma-Aminobutírico , Concentración de Iones de Hidrógeno , Ácido GlutámicoRESUMEN
Depression is a complex mental disease with polygenic inheritance and a high incidence.Our understanding of the clinical manifestations and pathogenesis of depression has recently improved.Continuous progress in gene-editing technologies has increased the construction efficiency and reduced the cost of gene-knockout animals,leading to their increasing use in the fields of basic research and drug development for depression and providing a powerful tool for revealing the pathogenesis of depression.In this review,we summarize recent progress in understanding the roles and mechanisms of candidate genes in depression using knockout model mice.
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Objective @#To explore the mechanism of motor symptoms in Parkinson' s disease ( PD) aggravated by decreased glial cell line-derived neurotrophic factor ( GDNF) in striatum．@*Methods @#Male C57 / BL mouse (6 －8 weeks) ，were administered of PBS，AAV-GDNF or AAV-shGDNF in striatum by brain stereoscopic injection，com- binated with sub-acute PD model ，1-Methyl-4-phenyl-1，2，3，6-tetrahydropyridine ( MPTP) 30 mg / ( kg · d) was administered through intraperitoneal injection for five consecutive days ; subsequently，mice were randomly divided into PBS group，negative control ( NC) group，AAV-shGDNF group ，MPTP group ，MPTP + AAV-GDNF group， MPTP + AAV-shGDNF group．Behavior tests (rotarod，pole and field) were applied for assessing the motor ability of mice ; ELISA kit was used to detect striatal glutamic acid ( Glu) content ; Western blot and other techniques were carried out to detect the expression and distribution of GLAST，GLT-1 and GluN2B in striatum ; TUNEL stainingwas applied for observing the apoptosis of neurons in striatum. @*Results @#Compared with the NC group，the mice of the AAV-shGDNF group，with down-regulation of GDNF in striatum，had poor motor ability，decreased Glu trans- porter ( GLAST and GLT-1) ，and increased Glu content． Compared with the PBS group，the mice of the MPTP group had increased Glu content and decreased GluN2B in striatum．Compared with the MPTP group，the mice of the MPTP + AAV-GDNF group showed enhanced motor ability ，along with decreased Glu content ，increased GluN2B and less neurons apoptosis in striatum ; while，the mice of the MPTP + AAV-GDNF group showed worse motor ability，along with augmented Glu content，reduced GluN2B and more neurons apoptosis in striatum．@*Conclusion@#In PD pathological process ，decreased striatal GDNF may promote the neurons apoptosis by enhancing Glu excitotoxicity，thereby leading to the aggravation of motor symptoms.
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Bipolar disorder and obsessive-compulsive disorder comorbidity is becoming more and more common and has aroused clinical physicians' increasing interest. This review summarizes the epidemiology, clinical characteristics, neurobiochemistry, and treatment of comorbid bipolar disorder and obsessive-compulsive disorder and discusses the neurobiochemical mechanism. Findings from this review will provide evidence for identifying the clinical symptoms of bipolar disorder and obsessive-compulsive disorder comorbidity and for treatment of the comorbidity.
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Objective:To evaluate the therapeutic effect of hydroxysafflor yellow A (HYA) on rats with tinnitus and investigate its influence on γ-aminobutyric acid (GABA) and glutamic acid (Glu) levels of inferior colliculus.Methods:The model of rats with tinnitus received an injection of sodium salicylate and "water-drinking suppression" was extablished, and then were divided into four groups with random number table method: normal group, model group, positive control (carbamazepine 5 mg/kg) and HYA (20 mg/kg) groups. Animals were intraperitoneally injected for 15 days. The recovery time of water-drinking suppression of all groups were recorded. The threshold value of auditory brainstem response (ABR) under the different frequency (4, 12, 20 and 28 kHz) in each rat was measured. The levels of GABA and Glu in inferior colliculus in rats with tinnitus were detected by LC-MS/MS.Results:Compared with the model group, the recovery time of water drinking suppression [(3.55±0.69)d vs.(1.83±0.58)d] in HYA group was significantly prolonged ( P<0.01). Compared with the model group, the threshold value of ABR under different frequency (4, 12, 20 and 28 kHz) were significantly reduced in HYA group ( P<0.01). The GABA levels [(2.25±0.26) μmol/g vs.(1.96±0.19)μmol/g] in inferior colliculus of tinnitus rats in HYA group was significantly increased ( P<0.05) while the Glu levels [(2.95±0.34)μmol/g vs.(3.71±0.39)μmol/g] were significantly decreased ( P<0.01). Conclusion:HYA treatment could relieve tinnitus symptoms induced by sodium salicylate, which might be related to the recovery of excitatory/inhibitory neurotransmitter balance.
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@#To investigate the therapeutic effect of oral vaccine based on glutamate decarboxylase 65 (GAD65) on streptozotocin (STZ) -induced type 1 diabetic (T1D) mice, the mice model of T1D was established by intraperitoneal injection of low dose multiple STZ. CTB-GADIII encapsulated with calcium alginate (Ca-Alg-GADIII) was formulated using crosslinking technology with sodium alginate and calcium chloride, and was administered intragastric to T1D mice once a week for 5 consecutive weeks.Blood glucose and body weight of the mice were recorded weekly, and pharmacodynamics against T1D of Ca-Alg-GADIII were investigated by glucose tolerance assay (OGTT) and pancreatic histopathological analysis. The levels of glutamic acid decarboxylase antibody (GADA), and insulin autoantibody (IAA) and related cytokines (IL-4, IFN-γ, TGF-β1) in serum were detected by ELISA, and the CD4 + T cell subsets were detected by flow cytometry. The immunological mechanism of oral vaccine against T1D was preliminarily discussed. The results showed that the disease-related indicators improved in immunized mice: fasting blood glucose improved, glucose tolerance and insulin secretion increased, pancreatic injury decreased, autoantibodies like GADA and IAA titers significantly decreased, and CD4 + T cell immune balance in mesenteric lymph node (MLN) and pancreatic lymph node (PLN) improved to some extent. The results suggest that oral vaccine Ca-Alg-GADIII has some therapeutic effect on STZ-induced T1D mice.
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To investigate the effect of electro-acupuncture therapy on limb spasm and excitability of motor neurons in stroke rats. Ischemic stroke model was induced with middle cerebral artery embolization in SD rats. Thirty-three modeled rats were randomly divided into model group, electro-acupuncture group, and baclofen group with 11 rats in each group, and another 10 rats were taken as sham operation group. The electro-acupuncture group and the baclofen group were treated with electro-acupuncture and baclofen tablets respectively. The model group and the sham operation group had no intervention. The neural function was evaluated with Bederson's scale and balance beam test; the muscle tension was measured with electrophysiography; the pathological changes of brain tissue was examined with HE staining; the content of glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rat cerebral cortex was analyze with enzyme linked immunosorbent assay (ELISA) method, the expression of metabotropic glutamate receptor 1a () and γ-aminobutyric acid type B receptor subunit 1 () mRNA were detected with RT-qPCR. Compared with the model group, the neurological function scores of the electro-acupuncture group and the baclofen group showed a downward trend at d7 after operation (all >0.05), and the neurological function scores of the electro-acupuncture group and the baclofen group were significantly decreased at d12 after the operation (all 0.05). Compared with the model group, the electrophysiological results of the electro-acupuncture group and baclofen group were significantly increased after operation (all <0.05). The results of HE staining showed that there was no cell edema and degeneration in the sham operation group, no pyknosis of the nucleus, and no bleeding in the interstitium. Cell edema and degeneration and mesenchymal congestion appeared in the model group. Compared with the model group, the cytoplasmic edema and degeneration and the interstitial bleeding in the electroacupuncture group and the baclofen group were reduced. Compared with sham operation group, the Glu content and the relative expression of mRNA was increased in the model group, electro-acupuncture group and baclofen group, while the GABA content and the relative expression of mRNA decreased (all <0.05). Compared with model group, the Glu content and the relative expression of mRNA in the electro-acupuncture group and baclofen group decreased, and the GABA content and relative expression of mRNA increased (all <0.05). Electro-acupuncture may improve limb spasm after stroke through regulating the expression of Glu and GABA in the cerebral cortex and the excitability of motor neurons in rats.
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Animales , Ratas , Terapia por Acupuntura , Neuronas Motoras , Ratas Sprague-Dawley , Espasmo , Accidente Cerebrovascular/terapiaRESUMEN
【Objective】 To investigate the central sedative and hypnotic effects and mechanisms of Shumian Capsule. 【Methods】 The blank control group, the positive drug diazepam group, and the low-, medium-, and high-dose groups of Shumian Capsule were designed in the experiments. Male Kunming mice were administered orally by gavage. We conducted the experiment of mouse autonomous activity, and assessed supra- and sub-threshold dose sleep with pentobarbital sodium, respectively. The levels of γ-aminobutyric acid (GABA) and glutamic acid (Glu) in the brain tissue of the mice were measured by enzyme-linked immunosorbent assay (ELISA) or colorimetric method. Male SD rats were injected intraperitoneally with p-chlorophenyl alanine [PCPA, 350 mg/(kg·d)] to establish an insomnia rat model, after which rats were continuously administered intragastrically for 7 days. The general status and body weight of the rats were observed. Total distance and standing times of rats were measured by open field test. Western blotting and immunohistochemistry assays were used to evaluate the protein expression of 5-HT1A receptor (5-HT1AR) in the rat hippocampus. 【Results】 Compared with the blank control group, the three dose groups of Shumian Capsule [1.6, 3.2, 6.4 mg/(kg·d)] for 14 consecutive days had significantly reduced the number of spontaneous activities and standing times in the mice (P<0.01). The high dose significantly prolonged the sleep duration of the mice induced by the supra-threshold dose of pentobarbital sodium (P<0.01). Compared with the blank control group, each dose group of Shumian Capsule had an increased GABA level in the mouse brain tissue in a dose-dependent manner (P<0.05); the medium- and high-dose groups had significantly reduced Glu levels in the hippocampus (P<0.05), while the high-dose group had significantly reduced Glu level in the cortex (P<0.05). In insomnia model rats, their activity was sluggish; their circadian activity rhythm disappeared; the hair became hard, rough and dull, with severe hair loss; and their weight reduced. After 7 consecutive days of the drug administration, the rats’ mental state in each Shumian Capsule dose group was improved. Compared with the blank control group, the rats’ body weight gains were significantly reduced after intraperitoneal injection of PCPA (P<0.01). Compared with the model group, each rat group of Shumian capsule treatment had significantly increased body weight gains (P<0.01). Compared with the blank control group, the distance of the rats in the model group increased in the open field test, and the expression level of 5-HT1AR protein in the hippocampus decreased. However, the distance of rats in diazepam group and each dose group of Shumian Capsule was significantly reduced (P<0.01), and the expression level of 5-HT1AR protein in the hippocampus of the high- and medium-dose groups of Shumian Capsule and diazepam group increased significantly (P<0.01). 【Conclusion】 Shumian Capsule has obvious effects of sedation, hypnosis and insomnia improvement, and its underlying mechanisms may be related to the increased GABA and decreased Glu contents in brain tissues, as well as up-regulated 5-HT1AR protein expression in the hippocampus.
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Objective:To explore the mechanism of premenstrual dysphoric disorder (PMDD) caused by liver-qi depression from the aspect of Glu-GABA metabolic pathways.Methods:Thirty-six rats with similar open field scores and regular estrus cycles were divided into blank group, model group, fluoxetine group, Shuyu capsule group, saikosaponin group and inhibitor group according to the random number table method, with 6 rats in each group. Stereotactic hippocampus surgery was performed during the first estrous cycle reception period after the estrus cycle was determined. In the non-receiving period of the third and fourth estrus cycles, the restraint model was constructed, and from the first day of the modeling, rats of the fluoxetine group were given fluoxetine capsules 2.67 mg/kg, while rats of the Shuyu capsule group and saikosaponin group were given Shuyu capsules 0.408 g/kg and saikosaponin 0.72 mg/kg once a day for 5 consecutive days. Rats in the inhibitor group were injected with 20 μl L-malic acid with 5 mmol/L concentration, which is an inhibitor of glutamate decarboxylase (GAD), in the hippocampus on the last day of modeling. After the administration, weighed the rats and carried out open field experiments. During the second and fivth estrus cycles of rats, the extracellular fluid of the hippocampus was collected by microdialysis technology, and the content of Glu and GABA in the dialysate was detected by HPLC-FLD. Results:After 5 days of administration, compared with the model group, the body weight of rats in the Shuyu capsule group, the inhibitor group and the fluoxetine group increased ( P<0.05), and the total score of the open field experiment decreased ( P<0.05); compared with the model group, during the receiving period of the five estrus cycle, the Glu level of the Shuyu capsule group and the inhibitor group decreased ( P<0.05); In the non-receiving period of the fifth estrus cycle, the Shuyu capsule group, Glu level of the fluoxetine group and the saikosaponin group increased, GABA level of Shuyu capsule group, inhibitor group and fluoxetine group decreased ( P<0.05), Glu/GABA level of Shuyu capsule group, fluoxetine group and inhibitor group (1.49 ± 0.13, 1.32 ± 0.33, 3.92 ± 0.79 vs. 0.35 ± 0.48) was higher than that of the model group ( P<0.05). Conclusion:The therapeutic mechanism of Shuyu capsule in the treatment of PMDD caused by liver Qi depression rats may be ascribed to inhibiting GAD from Glu-GABA metabolic pathway.
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Objective:To observe the effect of Naringin on neuronal apoptosis in mice with memory consolidation disorderinduced by sodium nitrite.Methods:Fifty mice were randomly divided into blank group, model group, standardized protocol group, high-dose Naringin group and low-dose Naringin group, with 10 mice in each group. The standardized protocol group was given Donepezil 1 mg/kg, the Naringin high and low dose groups were gavaged with Naringin solution 100 and 50 mg/(kg·d), blank group and model group were gavaged with equal volume of distilled water once a day for 21 days. The model was established on the 22nd day. The blank group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with 100 mg/(kg·d) sodium nitrite solution for 7 days. The cognitive ability of mice in each group was evaluated by platform jumping test, and the hippocampal synaptic structure was observed by electron microscope. The contents of acetylcholine (ACh), SOD, MDA and NO in hippocampus and the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) was detected by ELISA. The expression of N-methyl-D-aspartate receptor (NMDAR), glutamine receptor 2 (GluR), calcium/calmodulin dependent protease Ⅱ (CaMK Ⅱ), Caspase-3, Bcl-2 and Bad proteins in hippocampus of model mice were detected by Western blot.Results:The number and morphology of hippocampal neurons were normal, nucleus, mitochondria, rough endoplasmic reticulum and synaptic membrane of hippocampal neurons in high-dose Naringin group were clear. Compared with the model group, the latency of mice in the high-dose Naringin group was prolonged and the number of errors was reduced ( P<0.01). The levels of MDA and NO in hippocampus of mice in the high-dose Naringin group significantly decreased ( P<0.01), and the activity of SOD significantly increased ( P<0.01). The content of ACh (23.682 ± 2.835 μg/mg prot vs. 14.939 ± 2.901 μg/mg prot), ChAT (163.302 ± 21.278 U/g vs. 89.612 ± 11.497 U/g) increased, AChE (0.367 ± 0.015 U/mg prot vs. 0.471 ± 0.014 U/mg prot) activity decreased ( P<0.01); The expression of Bad (0.441 ± 0.010 vs. 0.633 ± 0.010), Caspase-3 (0.425 ± 0.036 vs. 0.537 ± 0.024) significantly decreased, and the expression of Bcl-2 (0.890 ± 0.014 vs. 0.727 ± 0.009) significantly increased ( P<0.01); The expression of CAMKⅡ (1.043 ± 0.037 vs. 1.475 ± 0.043) significantly decreased ( P<0.01), and the expression of NMDAR1 (0.407 ± 0.037 vs. 0.345 ± 0.012), GluR2 (1.125 ± 0.033 vs. 0.664 ± 0.023) significantly increased ( P<0.01). Conclusion:Naringin could play the role of protecing the neuron and improving the cognition of mice with memory consolidation disorder by regulating the balance of ACh and glutamate system and reducing neuronal apoptosis and antioxidant stress.
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[Abstract] Objective To observe the effects of the adipocyte hormone leptin on GABA content and receptor expression in hypothalamus of mice with sleep deprivation, and explore the possible mechanisms. Methods Male C57BL/6 mice were randomly divided into three groups (8 each): control group, sleep deprivation (SD) group and leptin supplement (L-SD) group. Mice in control group were set up in a water environment without sleep deprivation, mice in SD group were set up in a "modified multi-platform water environment" to establish a sleep deprivation model, and mice in L-SD group were given leptin 1.3 mg/kg intraperitoneally twice daily in conjunction with sleep deprivation. Seven days after sleep deprivation, the general conditions of mice were observed, body weight was measured and hypothalamic tissues and plasma specimens were collected. ELISA was used to detect the plasma leptin levels, hypothalamic γ-aminobutyric acid (GABA) and glutamate (Glu) contents. Western blotting was performed to detect the expression levels of GABA key glutamate decarboxylase 67 (GAD67) and GABAA receptor α1 subtype protein (GABAARα1). Results Compared with control group, the weight of mice in SD group significantly reduced [(22.03±0.42) g vs. (17.75±0.75) g, P0.05). The hypothalamic Glu levels were obviously higher in SD group [(686.56±10.01) ng/g] and L-SD group [(668.64+9.93) ng/g] than that in control group [(577.11±16.36) ng/g] (P0.05). The expressive levels of GAD67 and GABAARα1 protein in the hypothalamus of mice in SD group [0.68±0.06, 0.69±0.07] were significantly lower than that in control group (1.09±0.13, 0.99±0.07) (P<0.05); While the expressive levels of GAD67 and GABAARα1 proteins in the hypothalamus of mice in L-SD group (1.39±0.19 and 1.33±0.14, respectively) were significantly higher than those in SD group and control group (P<0.05). Conclusion Leptin can up-regulate the expression of the key GABA synthase GAD67, increase the content of GABA and the expression of GABAARα1 protein in hypothalamus of sleep-deprived mice, which may be an important mechanism of leptin affecting sleep.
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OBJECTIVE：To desig n and sy nthesize poly （γ-glutamic acid ）-ampelopsin（γ-PGA-AMP），and to characterize it and evaluate its anti-tumor activity in vitro . METHODS ：Synthetic product was produced through an esterification reaction between γ-PGA and ampelopsin. The structure of synthetic product was characterized by the UV spectrophotometry ，Fourier transform infrared（FT-IR）spectroscopy，1H-NMR spectra and the quantitative elemental analysis. The content of ampelopsin in synthetic product was determined by UV absorption spectrometry at 292 nm. Using 5-FU as positive control ，MTT assay was used to determine inhibitory effects of γ-PGA-AMP and ampelopsin on human breast cancer cell MCF- 7，human liver cancer cell HepG 2 and human lung cancer cell A 549. The IC 50 was calculated. RESULTS ：The results showed that the free 7-hydroxyl group of ampelopsin and the a-carboxyl group of γ-polyglutamic acid had been esterified to obtain γ-PGA-AMP；the yield of γ-PGA-AMP was 55.7％，and the content of ampelopsin was 32.3％. The inhibitory effect of γ-PGA-AMP and ampelopsin on MCF- 7，HepG2 and A 549 cells was obvious. IC 50 of γ-PGA-AMP（to 3 above tumor cells ）were 40.19，28.29 and 55.23 μg/mL，those of ampelopsin were 105.30，81.23，130.10 μg/mL，those of 5-FU were 24.72，87.98，30.99 μg/mL，respectively. CONCLUSIONS ：γ-PGA-AMP with anti-tumor effect in vitro is synthesized successfully ，and its anti-tumor effect is stronger than that of ampelopsin.
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Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.