Metabonomic study of biochemical changes in serum of PCPA-induced insomnia rats after treatment with Suanzaoren Decoction / 中国中药杂志

Suanzaoren Decoction(SZRD) is a classical formula for the clinical treatment of insomnia. This study analyzed the effect of SZRD on endogenous metabolites in insomnia rats based on metabonomics and thereby explored the anti-insomnia mechanism of SZRD. To be specific, DL-4-chlorophenylalanine(PCPA) was used to induce insomnia in rats. Then pathological changes of the liver and brain were observed and biochemical indexes such as 5-hydroxytryptamine(5-HT), dopamine(DA), glutamate(Glu), γ-aminobutyric acid(GABA), and norepinephrine(NE) in the hippocampus and prostaglandin D2(PGD2), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and IL-6 in the serum of rats were detected. On this basis, the effect of SZRD on PCPA-induced insomnia rats was preliminarily assessed. The metabolic profile of rat serum samples was further analyzed by ultra-performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS). Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were combined with t-test and variable importance in projection(VIP) to identify differential metabolites, and MetaboAnalyst 5.0 was employed for pathway analysis. The results showed that SZRD could improve the pathological changes of brain and liver tissues, increase the levels of neurotransmitters 5-HT, DA, and GABA in hippocampus and the level of PGD2 in hypothalamic-pituitary-adrenal axis(HPA axis), and reduce the levels of IL-1β and TNF-α in serum of insomnia rats. Metabonomics analysis yielded 12 significantly changed potential metabolites: 5-aminovaleric acid, N-acetylvaline, L-proline, L-glutamate, L-valine, DL-norvaline, D(-)-arginine, pyroglutamic acid, 1-methylguanine, L-isoleucine, 7-ethoxy-4-methylcoumarin, and phthalic acid mono-2-ethylhexyl ester(MEHP), which were related with multiple biochemical processes including metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of arginine and proline, arginine biosynthesis, glutathione metabolism. These metabolic changes indicated that SZRD can improve the metabolism in insomnia rats by regulating amino acid metabolism.

Regulation of Hypnotic Active Components of Cnidii Fructus on Melatonin Synthesis Rate-limiting Enzyme AANAT in Rats with PCPA-induced Insomnia / 中国实验方剂学杂志

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^-1， 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^-1）， CHC was high， medium and low （60， 30， 15 mg·kg^-1）， 10 mL·kg^-1， 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.

Effect of Anshen Navel Plaster on Spontaneous Activities And Levels of GABA, 5-HT in Insomnia Rats Caused by PCPA / 世界科学技术-中医药现代化

This experiment was aimed to mainly observe the effect of Anshen navel plaster on autonomic activities and to compare neurotransmitter gamma-aminobutyric acid (GABA) and monoamine neurotransmitter (5-HT) contents change of insomnia rats.The rats were treated by P-chlorophenylalanine (PCPA),which can make healthy rats lose sleep.The rat model of insomnia was prepared by intraperitoneal injection of PCPA.The healthy SD rats were randomly divided into the blank control group,model control group,low dosage of Anshen navel plaster group,high dosage of Anshen navel plaster group and Tongren Anshen pill group according to the random number table.EthoVision were used to record the rat's autonomic activities.Content changes of 5-HT and GABA of rats in each group were measured by enzyme-linked immunosorbent assay (ELISA).The results showed that rat's autonomic activities were decreased in the Anshen navel plaster group and Tongren Anshen pill group,which indicated that the Anshen navel plaster had a good sedative effect.Intraperitoneal injection of PCPA can remarkably change rat's serum neurotransmitter expression levels,especially GABA and 5-HT.After the intervention of experimental drugs,compared with the model group,the expression levels of GABA and 5-HT were increased significantly.It was concluded that Anshen navel plaster has obvious sedative effect.It can regulate the contents of sleep factors such as GABA and 5-HT.

Intervention effects of Jiaotai pills on PCPA-induced insomnia in rats / 中国中药杂志

To elucidate the intervention effects of Jiaotai pills(JTP) on p-chlorophenylalanine (PCPA)-induced insomnia in rats and its underlying mechanism, the insomnia model was established by single intraperitoneal injection with PCPA in rats. The locomotor activity of rats was observed, and the levels of nerve growth factor(NGF) in hypothalamus, hippocampus, prefrontal cortex and serum of rats were determined by using ELISA. Moreover, a proton nuclear magnetic resonance(¹H-NMR)-based metabonomic approach was developed to profile insomnia-related metabolites in rat serum and hippocampus and analyze the intervention effects of JTP on changes in underlying biomarkers related to locomotor activity, NGF and insomnia. According to the results, JTP could significantly suppress the locomotor activity of insomnia rats, and increase the NGF levels in hypothalamus, hippocampus, prefrontal cortex and serum of rats with insomnia. The disturbed metabolic state associated with PCPA-induced insomnia in rat serum and hippocampus could be intervened by JTP. Meanwhile, six and five potential biomarkers related to insomnia in rat serum and hippocampus were reversed by administration of JTP. In conclusion, the current study demonstrated that JTP had protective effects against PCPA-induced insomnia in rats, which was probably correlated with regulation of NGF level and metabolism of amino acids, lipids and choline.

Effects of p-chlorophenylalanine on the synthesis of pancreatic amylase in rats

Previously, we have reported that p-chlorophenylalanine (PCPA), a serotonin depletor, profoundly increased pancreatic fluid and bicarbonate secretion but remarkably inhibited pancreatic amylase secretion in anesthetized rats. The present study was performed to verify the detailed effects of PCPA on pancreatic amylase synthesis that is directly related to amylase exocrine secretion. PCPA significantly decreased pancreatic RNA and protein contents as well as the amylase activity. However, pancreatic DNA content, trypsin and chymotrypsin activities were not influenced by the treatment of PCPA. The rate of pancreatic amylase synthesis, which was assessed by the amount of incorporated (35S)-methionine into amylase for 1 h, was also significantly decreased by 44% in PCPA-treated rats. In order to determine whether the PCPA-induced decrease of amylase synthesis resulted from change in the level of amylase mRNA, Northern blot analysis was performed. The mRNA expression level of amylase was also decreased by 48% in the PCPA-treated rats, indicating that the inhibitory effect of PCPA on the synthesis of pancreatic amylase was mainly regulated at a step prior to translation. It was also revealed in SDS-polyacrylamide gel electrophoresis that the qualitative change of amylase was induced by PCPA. The 54 KDa amylase band seems to be degraded into small molecular weight protein bands in PCPA-treated rats, suggesting that the PCPA-induced decrease of amylase may be partly attributed to the degradation of synthesized amylase.