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Article in Chinese | WPRIM | ID: wpr-906329


Objective:To observe the effects of Cnidii Fructus hypnotic active components (CHC) on the behaviors of rats with p-chlorophenylalanine (PCPA)-induced insomnia and melatonin (MT) synthesis rate-limiting enzyme arylalkylamine <italic>N</italic>-acetyltransferase (AANAT), and explore the protective mechanism of CHC on the pineal gland. Method:Male SD rats of SPF grade were randomly divided into a blank control group, a model group, a MT group, and high-, medium-, and low-dose CHC groups with 10 rats in each group. Except for the blank control group, other groups received 4.5% PCPA suspension at 10 mL·kg<sup>-1</sup>, intragastric administration, for two consecutive days. After PCPA model of insomnia was established, normal and model groups were gavaged at the same volume of 2% Tween-80, MT control group (10 mg·kg<sup>-1</sup>), CHC was high, medium and low (60, 30, 15 mg·kg<sup>-1</sup>), 10 mL·kg<sup>-1</sup>, once a day, for consecutive 7 days. Four days after administration, open field, elevated cross maze, and pentobarbital sodium-induced sleep tests were conducted, respectively. Serum MT was detected by enzyme-linked immunosorbent assay. The mRNA expression level of AANAT was determined by real-time fluorescence-based quantitative polymerase chain reaction (Real-time PCR). The expression of AANAT protein in the pineal gland was detected by Western blot. Result:Compared with the results in the blank control group, the total distance of open field activity and standing times and duration in the central area were increased (<italic>P</italic><0.05, <italic>P</italic><0.01), the proportions of open arm entry (OE%) and open arm time (OT%) were decreased (<italic>P</italic><0.05), and the sleep latency was prolonged (<italic>P</italic><0.01) in the model group. Compared with the model group, no significant difference was observed in the low-dose CHC group, while other groups exhibited reduced total distance of activity (<italic>P</italic><0.05, <italic>P</italic><0.01), elevated OE% (<italic>P</italic><0.05), shortened sleep latency, and prolonged sleep time (<italic>P</italic><0.05, <italic>P</italic><0.01). Compared with the serum MT in the blank control group, that in the model group was decreased (<italic>P</italic><0.01). Compared with the model group, no significant difference was observed in the low-dose CHC group, while other groups displayed increased serum MT (<italic>P</italic><0.05). The mRNA and protein expression of AANAT was decreased in the model group as compared with that in the blank control group (<italic>P</italic><0.01). Compared with the model group, the MT group and the high-dose CHC group showed up-regulated expression (<italic>P</italic><0.05). Conclusion:CHC improved the behavioral indexes of PCPA-induced insomnia, increased the synthesis and secretion of MT in pineal cells, and elevated the serum MT level, which was related to the up-regulation of the mRNA and protein expression of AANAT in the pineal gland.

Article in Chinese | WPRIM | ID: wpr-802304


Objective:To observe the effect of isopimpinellin on primary hippocampal neuron cells γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and receptor genes expressions, in order to explore its hypnotic mechanism. Method:The primary hippocampal neurons of neonatal Sprague-Dawley rats were cultured in vitro. And subsequent experiments were conducted in the optimal state of cell growth, and the purity was identified by immunohistochemistry of neuron-specific enolase. Hippocampal neurons were randomly divided into five groups, namely blank control group, diazepam group (25 mg·L-1), and low-dose (5 mg·L-1), moderate-dose (10 mg·L-1) and high-dose (20 mg·L-1) isopimpinellin groups. Early apoptosis of hippocampus neuron cells were detected using flow cytometry technique after 24 h administration, and the changes in the levels of GABA and 5-HT were detected using enzyme-linked immunosorbent. The changes in mRNA expressions of receptor genes relating to gamma-aminobutyric acid type A receptor(GABAA) genes GABRA1,GABRA5,GABBR1, gamma-aminobutyric acid type B receptor genes (GABAB) GABRB2, 5-hydroxytryptamine 1A receptor (5-HT1A)5-HT1A(A),5-HT1A(B),5-HT1A(C) were detected by real-time quantitative PCR(Real-time PCR). Result:On the 7th day, the hippocampal neurons grew in a good condition, and the purity was above 90%. Apoptosis rates of hippocampal neurons in the low-dose and moderate-dose groups were significantly lower than that in the blank control group (PP1,GABRA5,5-HT1A(A),5-HT1A(C) in the moderate-dose and high-dose isopimpinellin groups were significantly higher than those in the blank control group (PP1,5-HT1A(B) in the low-dose, moderate-dose and high-dose isopimpinellin groups were significantly higher than those in the blank control group (PPConclusion:The hypnotic mechanism of isopimpinellin may be related to the inhibition of hippocampal neuron apoptosis, the increase of the content of inhibitory neurotransmitter GABA, and the up-regulation of GABA and 5-HT-related receptor genes.

Article in Chinese | WPRIM | ID: wpr-327862


<p><b>OBJECTIVE</b>To explore the protective effect of baicalin against rotenone-induced injury on PC12 cells, and the po-tential mechanism of action action was also explored.</p><p><b>METHOD</b>PC12 cells were injured by rotenone and were treated with different concentrations (0.1, 1, 10 μmol x L(-1)) of baicalin at the same time. Cell viability was analyzed by MTT, and morphology was observed by phase-contrast microscopy. The cell apoptosis was detected by flow cytometry by Annexin V-FITC/PI staining. The intracellular ROS level was determined by fluorescence microscope with DCF-DA staining. The expression of Bcl-2, Bax and Caspase-3 was analyzed by Western blot.</p><p><b>RESULT</b>The viability of PC12 cells exposure to rotenone for 24 hour was gradually decreased with dose escalating and 1.5 μmol x L was adopted to do the following experiment. Baicalin increased cell viability, improved cell morphology and decreased intracellular ROS level. Moreover, FACS indicated baicalin attenuated the apoptosis induced by rotenone significantly. Western blot showed that Bcl-2, Bax and Caspase-3 expression in rotenone-induced PC12 cells was reversed by baicalin.</p><p><b>CONCLUSION</b>This study has demonstrated that baicalin protects PC12 cells against rotenone-induced apoptosis, at least in part, by scavenging excessive ROS and inhibiting the mitochondrion-dependent apoptotic pathway.</p>

Animals , Apoptosis , Caspase 3 , Metabolism , Cell Survival , Cytoprotection , Flavonoids , Pharmacology , Gene Expression Regulation , Intracellular Space , Metabolism , PC12 Cells , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Rats , Reactive Oxygen Species , Metabolism , Rotenone , Pharmacology , bcl-2-Associated X Protein , Metabolism