Research progress of manganese exposure on abnormal GnRH release in hypothalamus affecting reproductive function / 环境与职业医学

Manganese plays an important physiological role in the organism, and excessive manganese exposure can cause impairment of neurological and reproductive functions. Gonadotropin-releasing hormone secreted by the hypothalamus acts as an initiator to regulate reproductive functions, such as gonadal development, onset of puberty, and gonadal hormone release. But the mechanism by which manganese damages the hypothalamus leading to abnormal gonadotropin-releasing hormone release is still unclear yet. Kisspeptin, prostaglandin E2, and nitric oxide may act as stimulators to increase the release of gonadotropin-releasing hormone, while the stimulatory or inhibitory effect of γ-aminobutyric acid on the release of gonadotropin-releasing hormone is controversial. Based on current research, manganese has been less studied with Kisspeptin, and studies with prostaglandin E2, nitric oxide, and γ-aminobutyric acid mainly focused on inflammation, oxidative stress, and neurotransmitter transmission. Therefore, taking Kisspeptin, prostaglandin E2, γ-aminobutyric acid, and nitric oxide as the breakthrough points, this paper introduced the mechanism of manganese affecting the release of gonadotropin-releasing hormone in the hypothalamus through the above four pathways, and proposed that the abnormal release of gonadotropin-releasing hormone in the hypothalamus may be one of the mechanisms by which manganese regulates reproductive function, providing a new direction for the prevention and treatment of manganese-induced reproductive damage in the future.

Construction of iPSC-derived Inhibitory Neural Network Tissue with Synaptic Transmission Potentials / 中山大学学报(医学科学版)

ObjectiveDirected differentiation of human induced pluripotent stem cells （hiPSCs） into spinal cord γ-aminobutyric acid （GABA）-ergic progenitor cells were implanted into an decellularized optical nerve （DON） bioscaffold to construct a hiPSC-derived inhibitory neural network tissue with synaptic activities. This study aimed to provide a novel stem cell-based tissue engineering product for the study and the repair of central nervous system injury. MethodsThe combination of stepwise directional induction and tissue engineering technology was applied in this study. After hiPSCs were directionally induced into human neural progenitor cells （hNPCs） in vitro， they were seeded into a DON for three-dimensional culture， allowing further differentiation into inhibitory GABAergic neurons under the specific neuronal induction environment. Transmission electron microscopy and whole cell patch clamp technique were used to detect whether the hiPSCs differentiated neurons could form synapse-like structures and whether these neurons had spontaneous inhibitory postsynaptic currents， respectively， in order to validate that the hiPSC-derived neurons would form neural networks with synaptic transmission potentials from a structural and functional perspective. ResultsThe inhibitory neurons of GABAergic phenotype were successfully induced from hiPSCs in vitro， and maintained good viability after 28 days of culture. With the transmission electron microscopy， it was observed that many cell junctions were formed between hiPSC-derived neural cells in the three-dimensional materials， some of which presented a synapse- like structure， manifested as the slight thickness of cell membrane and a small number of vesicles within one side of the cell junctions， the typical structure of a presynatic component， and focal thickness of the membrane of the other side of the cell junctions， a typical structure of a postsynaptic component. According to whole-cell patch-clamp recording， the hiPSC-derived neurons had the capability to generate action potentials and spontaneous inhibitory postsynaptic currents were recorded in this biotissue. ConclusionsThe results of this study indicated that hiPSCs can be induced to differentiate into GABAergic progenitor cells in vitro and can successfully construct iPSC-derived inhibitory neural network tissue with synaptic transmission after implanted into a DON for three-dimensional culture. This study would provide a novel neural network tissue for future research and treatment of central nervous system injury by stem cell tissue engineering technology.

Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

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.

The role of γ-aminobutyric acid in tumor immunity / 药学学报

italic>γ-Aminobutyric acid (GABA) is a crucial inhibitory neurotransmitter found in various cells in the human body. While the GABAergic system is typically associated with the nervous system, recent research has revealed that immune cells and tumor cells also express components of this system. In the tumor microenvironment (TME), GABA is secreted to act extracellularly on other cells. GABA is metabolized via the GABA shunt and is involved in the tricarboxylic acid (TCA) cycle by generating succinate, which can provide energy for tumor cells. Activation of GABA receptors (GABARs) is a major pathway through which GABA participates in the regulation of antitumor immune responses. The activation of GABA type A receptors (GABAARs) can inhibit the activation and proliferation of T cells, elicit anti-inflammatory macrophages, and promote tumor cell growth and migration, while activation of GABA type B receptors (GABABRs) is generally considered to inhibit cancer cell migration and induce cancer cell apoptosis. In general, receptor activation inhibits immune cells, but the effect on tumor cells varies. Additionally, the downregulation of the expression levels of GABA transporters (GATs) is involved in tumor progression. Although antagonists of GABA metabolism and drugs that act on GABA receptors are considered therapeutic drugs for tumors, there have been few clinical studies conducted on them.

Effects of Yudian Decoction （愈癫汤） on the Metabolic Balance of Glu/GABA in the Brain Tissue of Schizophrenia Model Rats / 中医杂志

ObjectiveTo explore the possible mechanism of Yudian Decoction （愈癫汤） in the treatment of schizophrenia. MethodTwenty male offspring from 5 normal female 17-day-pregnant SD rats were selected as blank group. Fifteen female 17-day-pregnant SD rats were injected intraperitoneally with methyl azomethine acetate （MAM） 25 mg/kg， and the male offspring simulated the neurodevelopmental abnormality to establish a rat model of schizophrenia. Sixty successfully-modeled rats were randomly divided into 20 rats in the model group， 20 rats in the Yudian Decoction group and 20 risperidone group. After 3 days of adaptive cage feeding， the rats in the Yudian Decoction group were gavaged with 1.54 g/（kg·d） of Yudian Decoction， the risperidone group was gavaged with 0.24 mg/（kg·d） of risperidone capsule， while the blank group and the model group were gavaged with 6.7 ml/（kg·d） of distilled water， once a day， for 14 consecutive days. Sample was collected on the day after the last gavage. The expression of glutamate receptor （GluR） and γ-aminobutyric acid receptor subunit α1 （GABAARα1）-positive neurons in the hippocampus and prefrontal cortex were detected by immunofluorescence， and the positive rate was calculated； the expression of small clear proteins （PVs） in the hippocampal CA1 region and the medial prefrontal cortex was detected by immunohistochemistry； The expression of glutamic acid decarboxylase 65 （GAD65） and glutamic acid decarboxylase 67 （GAD67） proteins and mRNAs in the hippocampus and prefrontal cortex were detected by immunoblotting and reverse transcription PCR. ResultCompared with the blank group， the positive rate of GluR in hippocampal area and prefrontal cortex of rats in the model group increased， the positive rate of GABAARα1 in hippocampal area decreased， the PV optical density value in hippocampal CA1 area and medial prefrontal cortex decreased， and the expression of GAD65， GAD67 proteins and mRNA in hippocampal area and prefrontal cortex decreased （P＜0.05 or P＜0.01）. Compared with the model group， GluR positivity rate in hippocampus and prefrontal cortex of risperidone group and Yudian Decoction decreased， GABAARα1 positivity rate in hippocampus increased， PV optical density value in hippocampus CA1 area and medial prefrontal cortex increased， and GAD65， GAD67 proteins and mRNA expression in hippocampus and prefrontal cortex increased （P＜0.05 or P＜0.01）. Compared with the risperidone group， the positive rate of GluR in hippocampus and prefrontal cortex and GABAARα1 in hippocampus in the Yudian Decoction group was reduced， the PV optical density value of hippocampal CA1 area was increased， the protein and mRNA expression of GAD67 in hippocampus area was elevated， and the protein expression of GAD65 in prefrontal cortex was reduced （P＜0.05）. ConclusionYudian Decoction may improve the pathological process of schizophrenia by regulating key regulators of glutamate/γ-aminobutyric acid （Glu/GABA） metabolic balance in the hippocampus and prefrontal cortex and maintaining the balance between neuronal excitation and inhibition.

Role of neurotransmitters in the pathogenesis of myopia / 国际眼科杂志(Guoji Yanke Zazhi)

With complex pathogenesis, myopia is a common ophthalmology disease and a major causation for visual impairment in children. For years, studies found that neurotransmitters, such as dopamine, nitric oxide, acetylcholine, γ-aminobutyric acid, 5-hydroxytryptamine, insulin and prostaglandins, are associated with children's refractive development and axial length growth. However, there are still many disagreements in their mechanisms of action. This article makes a systematic review on the roles of neurotransmitters in the pathogenesis of myopia including neurotransmitter receptors and antagonists to clarify the influence of different neurotransmitters on the occurrence and development of myopia, thus giving a comprehensive insight into its pathogenesis, building a basis for further research on the changes of neurotransmitters and providing new ideas and directions for the prevention and treatment of myopia.

Improvement effects of aucubin on ADHD-like behaviors in mice via the inhibition of excessive astrocytic activation / 中华行为医学与脑科学杂志

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.

Effect of hydroxysafflor yellow A on depressive-like behavior and expression of GABAAR protein in hippocampus of chronic restraint stress model mice / 中华行为医学与脑科学杂志

Objective:To investigate the effects of hydroxysafflor yellow A (HSYA) on depressive-like behavior and expression of type A γ-aminobutyric acid receptor(GABAAR)in hippocampus of chronic restraint stress model mice.Methods:The SPF grade male C57BL/6C mice were divided into Control group, HSYA group, Model group, Model + HSYA group and Model + fluoxetine group according to random number table method, with 12 mice in each group.Mice model of depression was established by chronic restraint stress.Mice in HSYA group and Model+ HSYA group were intraperitoneally injected with HSYA(20 mg/kg), mice in Model+ fluoxetine group were injected intraperitoneally with fluoxetine (10 mg/kg), and mice in Control group and Model group administered with 0.9% sodium chloride solution intraperitoneally once a day for 14 days.Then, the forced swimming test (FST) and tail suspension test (TST) were performed to evaluate the depressive-like behavior of mice, and the protein expression levels of different subtypes of GABAAR in the hippocampus of mice were determined by Western blot.SPSS 19.0 and GraphPad Prism 8.0 software were used for data statistical analysis and mapping.One-way ANOVA was used for comparison among groups, and Tukey-HSD test was used for further pairwise comparison.Results:(1) In the behavioral tests, there were significant differences in swimming immobility time of FST and tail suspension immobility time of TST among the five groups ( F=21.59, 20.81, both P<0.05). The swimming immobility time ((143.91±9.97) s) and tail suspension immobility time (( 107.00±6.54) s) in Model group were higher than those in Control group ((52.92±6.70) s, ( 43.50±5.96) s, both P<0.05). There were no significant difference in swimming immobility time and tail suspension immobility time between Model+ HSYA group ((26.17±7.69)s, ( 20.17±7.89)s) and Model+ fluoxetine group ((61.60±16.22)s, (34.14±10.74)s)(both P>0.05), but the swimming immobility time and tail suspension immobility time in these two groups were lower than those in Model group (both P<0.05). (2) The Western blot results showed that there were significant differences in the expression of GABAARβ1 and GABAARβ2 protein in hippocampus among the four groups ( F=12.21, 11.40, both P<0.05). The expression levels of GABAARβ1(45.60±10.76) and GABAARβ2 (46.27±4.82) protein in hippocampus of Model group were lower than those in Control group ((100.00±3.44), (100.00±3.26), both P<0.05). Compared to Model group, the expression of GABAARβ1 (79.91±5.00) and GABAARβ2 (79.08±5.53) protein in hippocampus of Model+ HSYA group were higher (both P<0.05). In addition, the expression of GABAARα1 and GABAARγ1 proteins in hippocampus were not significantly different among the four groups( F=0.23, 0.10, both P>0.05). Conclusion:HSYA can effectively alleviate depressive-like behavior in depression model mice, which may be related with the upregulation of GABAARβ1 and GABAARβ2 of hippocampus tissue.

Caudate nucleus and thalamus in untreated patients with obsessive-compulsive disorder: a magnetic resonance spectroscopy study / 中华行为医学与脑科学杂志

Objective:To investigate the neurobiochemical metabolites of caudate nucleus and thalamus in patients with obsessive-compulsive disorder and their relationship with obsessive-compulsive symptoms.Methods:From April 2019 to January 2022 in Beijing Anding Hospital, totally 25 untreated patients with obsessive-compulsive disorder were recruited, and 20 healthy controls matched with gender, age and educational background were recruited for the study.The maps of neurobiochemical metabolites of patients and normal controls were collected by hydrogen proton magnetic resonance spectroscopy.With bilateral caudate nucleus and thalamus as brain regions of interest.The relative concentrations of N-acetylaspartic acid (NAA), glutamic acid (Glu) and γ-aminobutyric acid (GABA) were fitted by LCModel software.At the same time, the clinical symptoms of patients were evaluated with Yale-Brown obsessive-compulsive scale (Y-BOCS) and Hamilton anxiety scale (HAMA). SPSS 20.0 software was used for statistical analysis.Independent double sample t-test was used to compare the differences of different nerve biochemical metabolite concentrations between patients with obsessive-compulsive disorders and healthy controls.Pearson correlation analysis was used to explore the correlation between biochemical metabolite concentrations and clinical symptoms. Results:The Glu concentration in the left thalamus of patients with obsessive-compulsive disorder (3.97±0.41) was higher than that of the control group (3.66±0.55)( t=-2.11, P<0.05), while the NAA concentration was (4.87±0.47)lower than that of the control group (5.15±0.44)( t=2.05, P<0.05). The GABA concentrations in the right caudate nucleus (0.50±0.18) and thalamus (0.80±0.19) were lower than those in the control group ((0.63±0.23), (0.96±0.24))( t=2.08, 2.36, both P<0.05). Pearson correlation analysis showed that the Glu concentration in the left caudate nucleus of patients with obsessive-compulsive disorder was positively correlated with the total score of Y-BOCS( r=0.46, P<0.05). Spearman correlation analysis showed that Glu concentration in the right caudate nucleus was positively correlated with the total score of HAMA in patients with obsessive-compulsive disorder ( r=0.46, P<0.05). Conclusion:NAA, Glu and GABA metabolism in caudate nucleus and thalamus are abnormal in patients with obsessive-compulsive disorder, and Glu concentration is positively correlated with the severity of obsessive-compulsive and anxiety symptoms.

Effects of nucleus accumbens GABA-lateral hypothalamic area MCH neural pathway on rewarding feeding / 中华行为医学与脑科学杂志

Objective:To explore the effects of the γ-aminobutyric acid(GABA) neurons and melanin-concentrating hormone (MCH) neurons of the nucleus accumbens (NAc)-lateral hypothalamic area (LHA) neural pathway on the rewarding feeding(palatable food sweat condensed milk) in the obesity rats.Methods:Total 142 male Wistar rats of SPF grade were divided into normal diet (ND) group ( n=68) and high-fat diet induced obesity (DIO) group ( n=74) according to the principle of body mass matching. The rats in the two groups were given normal diet and high-fat diet for 8 weeks. Eight weeks later, 6 DIO rats were randomly selected to observe the nerve projection from GABA neurons in NAc to MCH neurons in LHA by fluorogold retrograde tracing combined fluorescence immunohistochemistry. And the expressions of c-Fos and MCH in LHA after ingestion of sweet condensed milk(rewarding feeding) were observed by fluorescence immunohistochemistry (6 rats in each group). GABA receptor agonist Musimol or GABA receptor antagonist Bicuculine was microinjected into the nucleus of LHA to observe the effect of GABA on rewarding food intake in ND and DIO rats ( n=8 in each group), and the changes of rewarding food intake after blocking MCH signal ( n=8 in each group). SPSS 17.0 was used for statistical analysis, two-way ANOVA and post hoc Bonferroni test were used for comparison among multiple groups, and t-test was used for comparison between two groups. Results:After 8 weeks of high-fat diet modeling, the intake of delicious food in DIO rats was significantly higher than that in ND rats((12.52±2.29) mL, (7.45±1.23) mL, t=4.778, P<0.01) after satiety.The results of fluorogold retrograde tracing combined with fluorescence immunohistochemistry showed that GABA neurons in NAc projected nerve fibers to neurons in LHA, and GABA A receptors in some neurons in LHA coexisted with MCH.The results of NAc-LHA pathway on delicious food intake showed that the interaction between rat group and drug intervention was significant( F=9.869, P<0.01). Simple effect analysis showed that the intake of delicious food after microinjection of Musimol into LHA nucleus of ND rats was significantly lower than that of microinjection normal saline ((4.25±1.38) mL, (7.29±1.49) mL, P<0.01), while the intake of delicious food after injection of Bicuculine was significantly higher than that of microinjection normal saline((10.72±2.11) mL, (7.29±1.49) mL, P<0.05). The intake of delicious food after microinjection of Musimol into LHA nucleus in DIO group was significantly lower than that of microinjection normal saline((3.51±1.77)mL, (13.68±2.95) mL, P<0.01), but there was no significant difference between microinjection Bicuculine and microinjection normal saline ((14.83±3.44) mL, (13.68±2.95) mL, P>0.05). The results of blocking MCH signal on delicious food intake showed that the interaction effect between SNAP-94847 and Bicuculine intervention was not significant ( F=1.468, P>0.05). The main effect of SNAP-94847 intervention was significant ( F=15.880, P<0.01)and the main effect of Bicuculine intervention was significant ( F=6.930, P<0.05). After intracerebroventricular injection of MCH receptor blocker SNAP-94847, the delicious food intake of ND rats was significantly less than that of injection normal saline((4.78±1.72) mL, (7.63±2.77) mL, P<0.05), and it was not affected by pre injection of Bicuculine in LHA ((6.24±2.18) mL, (4.78±1.72) mL, P>0.05). In the DIO rats, the interaction effect between SNAP-94847 and Bicuculine intervention was not significant( F=0.006, P>0.05). The main effect of SNAP-94847 intervention was significant ( F=18.46, P<0.01) and the main effect of Bicuculine intervention was not significant ( F=2.059, P>0.05). After intracerebroventricular injection of MCH receptor blocker SNAP-94847, the delicious food intake of DIO rats was significantly lower than that of injection normal saline((6.89±2.11) mL, (12.19±4.36) mL, P<0.05), and it was not affected by pre injection of Bicuculine in LHA ((8.72±2.26) mL, (6.89±2.11) mL, P>0.05). Conclusion:GABAergic signal in NAc can regulate the expression of MCH in neurons of LHA. In the DIO rats, the sensitivity of MCH neurons in LHA to satiety signal decreases and the hedonic feeding increases.

Systematic Effect of Modified Renshen Wumeitang in Regulation of GABA Signaling Pathway in Rats with Diarrhea / 中国实验方剂学杂志

ObjectiveTo investigate the effect of modified Renshen Wumeitang（MRWT） on the related regulatory factors of the γ-aminobutyric acid （GABA） signaling pathway in colon tissues of rats with diarrhea， and reveal the mechanism of MRWT in invigorating Qi， generating fluid， and checking diarrhea. MethodForty-eight SD immature rats were randomly divided into a blank group （n=12） and an experimental group （n=36）. The diarrhea model was induced in the experimental group by Sennae Folium combined with overstrain and improper diet for 14 days. Subsequently， the model rats were randomly divided into a model group （normal saline， 20 mL·kg-1）， a western medicine group （Medilac-Vita， 0.7 g·kg-1）， and a Chinese medicine group （MRWT， 35 g·kg-1）， with 12 rats in each group. The rats in the blank group received normal saline at 20 mL·kg-1， and those in the other groups were treated correspondingly， once a day for 7 days. The general condition， loose stool rate， and diarrhea index of the rats were observed daily. Immunohistochemistry was used to detect the optical density expression of GABA protein in the colon of rats. The content of phosphatidylinositol-3 kinase （PI3K）， protein kinase B2 （Akt2）， phosphorylated Akt （p-Akt）， and interleukin-1β （IL-1β） was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of PI3K， Akt2， and GABA type A receptor subunit β2 （GABRB2） in the colon of rats were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the blank group， the model group showed worsened general condition， The difference was not statistically significant of loose stool rate and diarrhea index， increased expression of GABA protein （P<0.05）， elevated expression of PI3K， Akt2， p-Akt， and IL-1β （P<0.05， P<0.01）， and up-regulated PI3K， Akt2， and GABRB2 mRNA and protein expression （P<0.01）. Compared with the model group， the western medicine group and the Chinese medicine group showed the improved general condition， decreased loose stool rate and diarrhea index （P<0.01）， and decreased content of PI3K， Akt2， p-Akt， and IL-1β （P<0.05）. The Chinese medicine group displayed decreased mRNA expression of PI3K， Akt2， and GABRB2 （P<0.05， P<0.01） and down-regulated protein expression of GABA， PI3K， and GABRB2 （P<0.05， P<0.01）. The western medicine group exhibited down-regulated mRNA expression of PI3K，Akt2，and protein of PI3K （P<0.05）. ConclusionMRWT can regulate the GABA signaling pathway， reduce Cl- flow in intestinal epithelial cells to the intestinal lumen， and improve the imbalance of colonic fluid metabolism in the colon of diarrhea rats， thereby exerting its effects of invigorating qi， generating fluid， and checking diarrhea.

Effect of electro-acupuncture therapy on limb spasm and excitability of motor neurons in stroke rats / 浙江大学学报·医学版

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.

Effects of Body Weight-supported Treadmill Training on Neuropathic Pain and Expression of Glutamate Decarboxylase (GAD)-65/67 in Spinal Dorsal Horn of Rats with Spinal Cord Injury / 中国康复理论与实践

Objective:To investigate the effect of body weight-supported treadmill training on neuropathic pain and expression of glutamate decarboxylase-65/67 (GAD-65/67) in spinal dorsal horn of rats with spinal cord injury (SCI). Methods:A total of 24 Sprague-Dawley rats were randomly divided into sham group, SCI-sedentary (SCI-Sed) group and SCI-Exercise (SCI-Ex) group, with eight rats in each group. Allen's method was used to make T10 incomplete SCI model. Seven days after SCI, SCI-Ex group was given body weight-supported treadmill training. Before SCI, and seven days, 14 days, 21 days, 28 days and 35 days after SCI, the von Frey filaments and thermal stimulation pain tester were used to evaluate the mechanical and thermal pain thresholds. Then, Western blotting and immunohistochemical analysis were performed on the spinal cord of all rats to detect the expression of GAD-65 and GAD-67. Results:The mechanical and thermal pain thresholds were higher in SCI-Ex group than in SCI-Sed group 21 days, 28 days and 35 days after SCI (P < 0.01). Compared with the sham group, the expression of GAD-65 and GAD-67 decreased in SCI and SCI-Ex groups (P < 0.05), and increased in SCI-Ex group compared with that of SCI-Sed group (P < 0.05). Conclusion:Body weight-supported treadmill training could increase the synthesis of GAD-65/67 in the distal spinal cord dorsal horn of incomplete SCI rats, and improve the pain thresholds of hind limbs in rats with SCI.

Effects of leptin on GABA content and receptor expression in hypothalamus of mice with sleep deprivation / 解放军医学杂志

[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.

Effect of Modified Shaoyao Gancaotang， Electroacupuncture and Acupuncture Combined with Herb on γ-Aminobutyric Acid Related Receptor Proteins in Cortex in SCA Rats / 中国实验方剂学杂志

Object:To analyze the effect of muscular tension injury in spasticity of cerebral apoplexy(SCA) rats with spasticity and the expression of γ-aminobutyric acid receptor (GABAα), γ-aminobutyric acid transporter (GAT-1) protein and mRNA expression in cerebral cortex modified Shaoyao Gancaotang, electroacupuncture Quchi, Yanglingquan and acupuncture combined with herb therapies alleviate the effect of SCA. Method:Randomly divided into two groups, divided into blank group, sham operation group, model group, modified Shaoyao Gancaotang group, acupuncture combined with herb group, electroacupuncture group, selected qualified SD rats into the group, 9 rats in each group. The modified Zea-Longa thread embolism method+internal capsule injection of NMDA receptor method was used to prepare a rat model. After the behavioral score confirms the success of the model,dispose of each group separately,electroacupuncture group (electroacupuncture on both sides of Yangling Spring and Quchi,1 time/d for 5 days), modified Shaoyao Gancaotang group (with Shaoyao Gancaotang 1 time/d,10 mL·kg-1 for 5 days),acupuncture combined with herb (modified Shaoyao Gancaotang was given, then electroacupuncture treatment was continued for 5 days). After treatment, the muscle tension was detected by behavioral analysis. Real-time fluorescent quantitative PCR（Real-time PCR） and Western blot were used to detect the expression of GABAα and GAT-1 related mRNA and protein in the cortex. Result:Compared with normal group, the muscle tension of the model group increased (P<0.01), the expression of GABAα mRNA and protein decreased (P<0.01), and the expression of GAT-1 mRNA and protein increased (P<0.01), compared with model group, the muscle tone score of the each treatment group decreased (P<0.05), the expression of GABAα mRNA and protein increased (P<0.05，P<0.01), and the expression of GAT-1 mRNA and protein decreased (P<0.05，P<0.01), of which the corresponding expression in the acupuncture combined with herb group was the most significant. Conclusion:Electroacupuncture Quchi, Yanglingquan,modified Shaoyao Gancaotang and acupuncture combined with three therapies can alleviate the muscle tension of limbs spasm of stroke,and the combination of acupuncture and medicine treatment on the cortical γ-aminobutyric acid receptor the highest expression efficiency suggests that it has the best effect on improving the excitatory spasticity of stroke limbs.

Association analysis of single nucleotide polymorphisms in the promoter region of GAD1 and GABRB3 with schizophrenia in Chinese Han population / 上海交通大学学报（医学版）

Objective: To investigate the correlation between the polymorphisms of locus in the promoter region of glutamate decarboxylase 1 (GAD1) gene and γ-aminobutyric acid (GABA)A receptor β-3 gene (GABRB3) and schizophrenia (SZ) in Chinese Han population. Methods: SNaPshot genotyping technique was used to detect the polymorphisms of rs3791878 and rs3749034 in the promoter region of GAD1 and rs4906902 in the promoter region of GABRB3 in 545 SZ patients (case group) and 624 healthy controls (control group). The distribution of alleles and genotypes under different genetic models between the case group and the control group in all samples were compared by SNPstats online software. The above analysis was also performed after the subjects were stratified according to gender. The correlation of G/T risk genotype of rs3791878 with the age of the first onset of male SZ was investigated by survival analysis. Results: Under over-dominant genetic model, the distribution of G/T risk genotype of rs3791878 showed statistically difference between the male SZ cases and male controls (P=0.000), and the difference was still statistically significant after Bonferroni correction (P=0.000). However, there was no significant difference in the distribution of alleles and genotypes under different genetic models of rs3749034 and rs4906902 between the case group and the control group in all samples (P>0.05), and there was also no significant difference in the distribution of alleles between the case group and the control group after them being stratified according to gender (P>0.05). Kaplan-Meier analysis showed that there was no significant difference between the age of onset of male SZ who carried G/T genotype in rs3791878 locus and that of male SZ who did not carry it (P=0.603). Conclusion: The polymorphism of rs3791878 in the promoter region of GAD1 is significantly associated with the incidence of male SZ in Chinese Han population.

Preliminary analysis of primary and secondary factors of γ-aminobutyric acid enrichment in fermentation process of Sojae Semen Praeparatum / 中草药

Objective: To study the primary and secondary factors of γ-aminobutyric acid (GABA) enrichment in the processing of Sojae Semen Praeparatum (SSP), and lay the foundation for revealing the formation mechanism of high content of GABA in SSP. Methods: The dynamic changes of pH value, temperature, moisture, protease and glutamic acid decarboxylase (GAD) in the processing of SSP were determined by conventional methods. The GABA content of each sample was determined by pre-column on-line derivatization established by our laboratory. The correlation between each indicator and GABA was analyzed by SPSS software. Results: Correlation analysis and multiple regression analysis showed that the correlation coefficient between water, acid protease and GABA was 0.211 and -0.340, respectively, and the P values were 0.324 and 0.228, respectively. The correlation was small and there was no statistical difference. The absolute value of the regression coefficient showed that the primary and secondary status of other indicators in the formation of GABA was pH value (-0.375) > temperature (-0.284) > GAD (0.140) > alkaline protease (0.047) > neu-protease (-0.030), in which pH value, temperature and neutral protease had a negative correlation with GABA, and GAD activity and alkaline protease had a positive correlation with GABA. Conclusion: The temperature, pH value, GAD, neutral and alkaline protease are important factors affecting the formation of GABA in the fermentation process of SSP.

Correlation between blood oxygen level dependent fMRI signal and GABA content in anterior cingulate cortex after acupuncture of Hegu (LI4) / 针刺研究

OBJECTIVE: To explore the correlation between blood oxygen level dependent functional magnetic resonance imaging (BOLD-fMRI) signal and neurotransmitter γ-aminobutyric acid (GABA) concentration in the prefrontal cortex area after acupuncture or Von Frey filament stimulation (epidermal stimulation) at the right Hegu (LI4). METHODS: A total of 76 healthy volunteers (23 men and 53 women, 24.5±1.4 years in age) were recruited in the pre-sent study. Each volunteer received two sessions of fMRI magnetic resonance scanning (MRS) examinations, with an interval of one week between two sessions. The MRI scan sequences included pre-task MRS, resting state BOLD and task MRS, BOLD. A region of Interest (ROI) of 35 mm×30 mm×25 mm was located at the bilateral medial prefrontal cortex areas. In the two sessions of examinations, the right LI4 point was stimulated by manual acupuncture or Von Frey filament-pressing. The tasks were designed as the block design. Each block contained 3 intermittent acupoint stimulations, lasting 30 s in each stimulation and with two minutes' pause between two stimulations. The MRS data were processed by using Linear Combination (LC) Model software (for assessing GABA content), and the BOLD data of fMRI was analyzed by using SPM12 software (comparison within each group), REST1.8 (comparison between two groups), separately. RESULTS: Extensive deactivations were induced by both stimulations, mainly involving the midline regions as the medial prefrontal lobe, and limbic lobe. The deactivation effect of manual acupuncture stimulation was more extensive and intensive than that of Von Frey filament stimulation, especially in the medial prefrontal lobe. Data from 66 volunteers (after exclusion of 10 participants due to bigger standard deviation of GABA/Glx) showed no marked correlation between the GABA concentration and BOLD activation in the anterior cingulate cortex area in both groups(manual acupuncture stimulation group: r=－0.07, －0.08, 0.04; P=0.57, 0.88, 0.74; Von Frey filament epidermal stimulation group: r=－0.10, －0.09, －0.01; P=0.43, 0.46, 0.96). CONCLUSION: Acupuncture of LI4 elicits a stronger and broader negative activation effect in the limbic-paralimbic-neocortical network including the medial prefrontal cortex in comparison with Von Frey filament stimulation, but no apparent correlation was found between the GABA concentration and BOLD activation in the anterior cingulate cortex after manual acupuncture and Von Frey stimulation.

Anxiolytic Effect of Chanmu Anshen Granule and Its Bioactive Mechanism / 中国实验方剂学杂志

Objective:To assess the anxiolytic effect of Chaimu Anshen granules (CMASG) and investigate its bioactive mechanism. Method:ICR mice were randomly divided into normal group, diazepam group(0.002 g·kg-1),Jieyu Anshen granules group(0.001 4 g·kg-1), high, medium, and low-dose (0.001 98,0.000 99,0.000 495 g·kg-1)Chaimu Anshen granule groups, with 20 mice in each group. To detect the anxiolytic effect of CMASG, mice were intragastrically administered for 4 weeks in the morning, and light-dark box transition test and open field test were performed once the other day. After the behavior tests, blood samples were collected. Six mice of each group were perfused with formalin through heart, and then the brains were fixed for immunohistochemistry test. Hippocampus of the other mice in each group were collected and stored in liquid nitrogen. The content of γ-aminobutyric acid(GABA)and glutamic acid(Glu)in hippocampus and blood samples were detected by enzyme-linked immunosorbent assay (ELISA), and the ratio of GABA/Glu was calculated. The expression of GABAα1 receptor was evaluated by the immunohistochemistry method. To test the hypnosis effect of CMASG, mice were administered intragastrically for 7 days. The sub-threshold dose of pentobarbital sodium in the sleep experiment was tested. Result:Compared with normal group, the light-dark box transitions test demonstrated that low-dose and medium-dose CMASG groups significantly prolonged the duration in light box(PPPPPPPPPPα1 receptor protein in hippocampus showed that the medium-dose CMASG significantly increased the expression of GABAα1 protein. The sub-threshold dose of pentobarbital sodium on sleep experiments confirmed that the medium-dose CMASG significantly increased the rate of sleep in mice. Conclusion:CMASG showed an anxiolytic effect, and its bioactive mechanism was related with the increase of GABA content, and the decrease of Glu content in hippocampus. Furthermore, it increased the expression of GABAα1 protein in hippocampus. The changes in content of GABA and Glu in peripheral blood were positively correlated with the changes in hippocampal tissues, which provided reference for clinical diagnosis. CMASG also exhibited an effect in improvement of sleep.

Effects of γ-aminobutyric acid on feeding changes in rats and its potential mechanism / 中华行为医学与脑科学杂志

Objective To investigate whether γ-aminobutyric acid (GABA) receptor signaling pathway is involved in the regulation of thalamic undefined (ZI)-nucleus accumbens (NAc) neural pathways on gastric distraction (GD)-sensitive neuronal firing activity and the impact on food intake,the number of times and the frequency in rats.Methods Six rats were randomly selected and the neural pathway between Zl and NAc in rat thalamus was observed by fluorescent gold (FG) retrograde tracing method.Eighty-two rats were randomly selected,and the gastric balloon was placed in gastric cavity,the microelectrode was placed in the NAc,and the stimulating electrode was placed in the ZI.The single-cell discharge recording method was used to observe the effect of electrical stimulation ZI on the excitability of GD-sensitive neurons in rat NAc.Eighteen rats were randomly selected and were divided into three groups according to the random number table.They were NS group,GABA group,GABA + GABA receptor antagonist bicuculline (BIC) group with 6 in each group,and the rat NAc was used to embed the cannula.The method of GABA and BIC was injected to observe the changes of cumulative food intake in rats for 4 h.Eighteen rats were randomly selected and randomly divided into three groups:sham stimulation (SS) group,50 μA electrical stimulation group,50 μA electrical stimulation + BIC group with 6 in each group.The 4 h cumulative food intake of rats was observed by electro-stimulation of rat ZI and rat NAc injection of BIC.Results Fluorescent gold retrograde tracking combined with fluorescent immunohistochemical staining showed that there were visible GABA and fluorescent gold double labeled neurons in ZI.Electrical stimulation of ZI,the frequency of GABA-sensitive GD neurons in rat NAc increased significantly (GD-E increase:(78.8±8.4) ％,GD-I increase:(89.3±9.2) ％,P＜0.01),but the inhibitory effect was antagonized by BIC (GD-E increase:(113.8 ± 13.6)％,GD-I increase:(121.8± 14.2)％,P＜0.01).Microinjection of GABA in rat NAc significantly increased the cumulative food intake for 4 h ((155.72± 18.84) kcal,t=3.41,P＜0.05),which was antagonized by partial BIC (123.43± 15.11) kcal,t =3.28,P＜ 0.05).Electrical stimulation of ZI significantly increased the food intake in rats ((39.07± 11.27) kcal,t =2.96,P＜0.05),and this effect can be partially antagonized by BIC ((34.17 ± 10.85) kcal,t =2.33,P＜ 0.05).Conclusion The ZI-NAc neural pathway regulates the discharge activity of rat gastric distension (GD)-sensitive neurons and the feeding status of rats,and the GABA receptor signaling pathway may be involved in mediating the process.