Shen Yuan extract exerts a hypnotic effect via the tryptophan/5-hydroxytryptamine/melatonin pathway in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sleep plays a critical role in several physiologic processes, and sleep disorders increase the risk of depression, dementia, stroke, cancer, and other diseases. Stress is one of the main causes of sleep disorders. Ginseng Radix et Rhizoma and Polygalae Radix have been reported to have effects of calming the mind and intensifying intelligence in Chinese Pharmacopoeia. Traditional Chinese medicine prescriptions composed of Ginseng Radix et Rhizoma and Polygalae Radix (Shen Yuan, SY) are commonly used to treat insomnia, depression, and other psychiatric disorders in clinical practice. Unfortunately, the underlying mechanisms of the SY extract's effect on sleep are still unknown. AIM OF THE STUDY: This study aimed to investigate the hypnotic effect of the SY extract in normal mice and mice with chronic restraint stress (CRS)-induced sleep disorders and elucidate the underlying mechanisms. MATERIALS AND METHODS: The SY extract (0.5 and 1.0 g/kg) was intragastrically administered to normal mice for 1, 14, and 28 days and to CRS-treated mice for 28 days. The open field test (OFT) and pentobarbital sodium-induced sleep test (PST) were used to evaluate the hypnotic effect of the SY extract. Liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay were utilized to detect the levels of neurotransmitters and hormones. Molecular changes at the mRNA and protein levels were determined using real-time quantitative polymerase chain reaction and Western blot analysis to identify the mechanisms by which SY improves sleep disorders. RESULTS: The SY extract decreased sleep latency and increased sleep duration in normal mice. Similarly, the sleep duration of mice subjected to CRS was increased by administering SY. The SY extract increased the levels of tryptophan (Trp) and 5-hydroxytryptamine (5-HT) and the expression of tryptophan hydroxylase 2 (TPH2) in the cortex of normal mice. The SY extract increased the Trp level, transcription and expression of estrogen receptor beta and TPH2 in the cortex in mice with sleep disorders by decreasing the serum corticosterone level, which promoted the synthesis of 5-HT. Additionally, the SY extract enhanced the expression of arylalkylamine N-acetyltransferase, which increased the melatonin level and upregulated the expressions of melatonin receptor-2 (MT2) and Cryptochrome 1 (Cry1) in the hypothalamus of mice with sleep disorders. CONCLUSIONS: The SY extract exerted a hypnotic effect via the Trp/5-HT/melatonin pathway, which augmented the synthesis of 5-HT and melatonin and further increased the expressions of MT2 and Cry1.

Anti-inflammatory effect and pharmacokinetics of dehydroandrographolide, an active component of Andrographis paniculata, on Poly(I:C)-induced acute lung injury.

Acute lung injury (ALI) is a common and critical respiratory disorder caused by various factors, with viral infection being the leading contributor. Dehydroandrographolide (DAP), a constituent of the Chinese herbal plant Andrographis paniculata, exhibits a range of activities including anti-inflammatory, in vitro antiviral and immune-enhancing effects. This study evaluated the anti-inflammatory effects and pharmacokinetics (PK) profile of DAP in ALI mice induced by intratracheal instillation of Poly(I:C) (PIC). The results showed that oral administration of DAP (10-40 mg/kg) effectively suppressed the increase in lung wet-dry weight ratio, total cells, total protein content, accumulation of immune cells, inflammatory cytokines and neutrophil elastase levels in bronchoalveolar lavage fluid of PIC-treated mice. DAP concentrations, determined by an LC-MS/MS method, in plasma after receiving DAP (20 mg/kg) were unchanged compared to those in normal mice. However, DAP concentrations and relative PK parameters in the lungs were significantly altered in PIC-treated mice, exhibiting a relatively higher maximum concentration, larger AUC, and longer elimination half-life than those in the lungs of normal mice. These results demonstrated that DAP could improve lung edema and inflammation in ALI mice, and suggested that lung injury might influence the PK properties of DAP, leading to increased lung distribution and residence. Our study provides evidence that DAP displays significant anti-inflammatory activity against viral lung injury and is more likely to distribute to damaged lung tissue.

Interferon lambda in respiratory viral infection: immunomodulatory functions and antiviral effects in epithelium.

Type III interferon (IFN-λ), a new member of the IFN family, was initially considered to possess antiviral functions similar to those of type I interferon, both of which are induced via the JAK/STAT pathway. Nevertheless, recent findings demonstrated that IFN-λ exerts a nonredundant antiviral function at the mucosal surface, preferentially produced in epithelial cells in contrast to type I interferon, and its function cannot be replaced by type I interferon. This review summarizes recent studies showing that IFN-λ inhibits the spread of viruses from the cell surface to the body. Further studies have found that the role of IFN-λ is not only limited to the abovementioned functions, but it can also can exert direct and/or indirect effects on immune cells in virus-induced inflammation. This review focuses on the antiviral activity of IFN-λ in the mucosal epithelial cells and its action on immune cells and summarizes the pathways by which IFN-λ exerts its action and differentiates it from other interferons in terms of mechanism. Finally, we conclude that IFN-λ is a potent epidermal antiviral factor that enhances the respiratory mucosal immune response and has excellent therapeutic potential in combating respiratory viral infections.

Melatonin-related dysfunction in chronic restraint stress triggers sleep disorders in mice.

Stress may trigger sleep disorders and are also risk factors for depression. The study explored the melatonin-related mechanisms of stress-associated sleep disorders on a mouse model of chronic stress by exploring the alteration in sleep architecture, melatonin, and related small molecule levels, transcription and expression of melatonin-related genes as well as proteins. Mice undergoing chronic restraint stress modeling for 28 days showed body weight loss and reduced locomotor activity. Sleep fragmentation, circadian rhythm disorders, and insomnia exhibited in CRS-treated mice formed sleep disorders. Tryptophan and 5-hydroxytryptamine levels were increased in the hypothalamus, while melatonin level was decreased. The transcription and expression of melatonin receptors were reduced, and circadian rhythm related genes were altered. Expression of downstream effectors to melatonin receptors was also affected. These results identified sleep disorders in a mice model of chronic stress. The alteration of melatonin-related pathways was shown to trigger sleep disorders.

Chronic restraint stress induced changes in colonic homeostasis-related indexes and tryptophan-kynurenine metabolism in rats.

Chronic stressors represented risk factors for the etiology or exacerbation of several gastrointestinal diseases. The goal of the present study was to examine whether chronic restraint stress (CRS) could initiate and aggravate colonic inflammation, integrity damage and metabolic disturbance of rats. Firstly, increased inflammatory cytokines (interferon-γ (IFN-γ), tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10)) and decreased tight junction (TJ) proteins (occludin and zonula occludins-1 (ZO-1)) in rat colon were observed. Secondly, untargeted metabolomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass (UPLC-Q-TOF/MS) revealed that TRP metabolism was the most prominently affected. Thirdly, quantification of TRP and its metabolites via prominence ultrafast liquid chromatography coupled with a QTRAP 5500 mass (UFLC-QTRAP-5500/MS) showed that TRP, kynurenine (KYN), kynurenic acid (KA) and 3-hydroxykynurenine (3-HK) were significantly increased. At the same time, 5-hydroxytryptamine (5-HT) was unchanged and 5-hydroxyindolacetic acid (5-HIAA) was significantly decreased in the colon of CRS rats. Besides, TRP metabolic enzyme changes were with the same trends as the corresponding metabolites. Thus, our data showed that CRS could initiate colonic inflammation, integrity damage and colonic metabolism disturbance, especially TRP-KYN metabolism pathway of rats, which may provide an experimental background for future research on stress-related gastrointestinal dysfunction. SIGNIFICANCE: Chronic exposure to psychological stress could induce metabolic imbalance of the body, and stressful life events were intimately correlated with frequent relapses in patients with intestinal disorders. The present study showed that chronic restraint stress (CRS) could initiate and aggravate colonic inflammation, integrity damage and metabolic disturbance, especially tryptophan-kynurenine metabolism of rats. Tryptophan-kynurenine pathway may be involved in the initiation and development of diseases induced by chronic stress. This research may shed light on future research on stress-related gastrointestinal dysfunction.

Plasma and brain pharmacokinetics of ganoderic acid A in rats determined by a developed UFLC-MS/MS method.

Ganoderic acid A (GAA), an active triterpenoid of the traditional Chinese herbal medicine Lingzhi, has been reported to exhibit antinociceptive, antioxidative, and anti-cancer activities. The present study aims to establish a sensitive and rapid UPLC-MS/MS method for studying the plasma and brain pharmacokinetics of GAA in rats. The analytes were separated on a C18 column eluted with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at 0.3mL/min. The eluate was monitored by a mass detector using an MRM (m/z, 515.3-285.1) model in negative electrospray ionization. The calibration curve showed good linearity (r2>0.99), with limits of detection and quantification of 0.25 and 2.00 nmol/L, respectively. The intra- and inter-day precision and accuracy were less than 9.99% and ranged from 97.45% to 114.62%, respectively. The extraction recovery from plasma was between 92.89% and 98.87%. GAA was found to be stable in treated samples at room temperature (22°C) for 12h and in plasma at -20°C for 7d. The developed method was successfully applied to a pharmacokinetic study of GAA in rats. GAA could be rapidly absorbed into the circulation (Tmax, 0.15h) and eliminated relatively slowly (t1/2, 2.46h) after orally dosing, and could also be detected in the brain lateral ventricle (Tmax, 0.25h and t1/2, 1.40h) after intravenously dosing. The absolute oral bioavailability and brain permeability of GAA were estimated to be 8.68% and 2.96%, respectively.

Lotus leaf alkaloid fraction can strongly inhibit CYP2D6 isoenzyme activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herbal medicine He-Ye, the leaves of the lotus (Nelumbo nucifera) plant, is traditionally used in China for the treatment of sunstroke, thirst, diarrhea, and fever. Currently, the leaf is used not only as an herbal tea to reduce lipid level and control body weight, but also as a major ingredient in some lipid-lowering Chinese patented medicines. Our previous study demonstrated that the alkaloid fraction (AF) of the herb has a strong inhibitory effect on CYP2D6 isoenzyme activity in vitro. The present study aims to further verify this activity using the in vivo rat model and to explore the inhibitory mechanism on CYP2D6 using human liver microsomes (HLMs). MATERIALS AND METHODS: After a continuous 7-d oral dose of AF (50mg/kg) or a vehicle, Sprague Dawley rats received a single intravenous dose of dextromethorphan or metoprolol. Blood samples were collected at various time points, and the plasma concentrations of the relevant metabolites dextrorphan and hydroxymetoprolol were assayed by LC-MS/MS for evaluating the effect of AF on their pharmacokinetics and CYP2D6 activity. Dextromethorphan as a probe at different concentrations was incubated with HLMs in an incubation buffer system, in the presence or absence of AF at different concentrations. After incubation, the produced metabolite was assayed. RESULTS: After being pretreated with AF in rats, the plasma concentrations of dextrorphan and hydroxymetoprolol significantly decreased, with Cmax going from 79.44 to 29.96 and 151.18 to 83.39hng/mL (P<0.05), AUCall from 167.27 to 62.25 and 347.68 to 223.24hng/mL (P<0.05), and AUCinf from 183.39 to 84.76 and 350.59 to 234.57hng/mL (P<0.05), respectively, in comparison with those of untreated rats. The t1/2 of hydroxymetoprolol significantly increased from 1.14 to 1.99h (P<0.05). The in vitro incubation test showed that AF competitively inhibited the CYP2D6, with apparent Ki value of 0.64µg/mL. CONCLUSIONS: AF can strongly inhibit the activity of CYP2D6 enzyme, as confirmed by in vivo and in vitro models. Possible drug interactions may occur between AF and other medications metabolized by CYP2D6. Thus, caution should be paid when the lotus leaf and its preparations are concurrently administered with conventional medicines.

Lotus Leaf Alkaloid Extract Displays Sedative-Hypnotic and Anxiolytic Effects through GABAA Receptor.

Lotus leaves have been used traditionally as both food and herbal medicine in Asia. Open-field, sodium pentobarbital-induced sleeping and light/dark box tests were used to evaluate sedative-hypnotic and anxiolytic effects of the total alkaloids (TA) extracted from the herb, and the neurotransmitter levels in the brain were determined by ultrafast liquid chromatography-tandem mass spectrometry. The effects of picrotoxin, flumazenil, and bicuculline on the hypnotic activity of TA, as well as the influence of TA on Cl(-) influx in cerebellar granule cells, were also investigated. TA showed a sedative-hypnotic effect by increasing the brain level of γ-aminobutyric acid (GABA), and the hypnotic effect could be blocked by picrotoxin and bicuculline, but could not be antagonized by flumazenil. Additionally, TA could increase Cl(-) influx in cerebellar granule cells. TA at 20 mg/kg induced anxiolytic-like effects and significantly increased the concentrations of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and dopamine (DA). These data demonstrated that TA exerts sedative-hypnotic and anxiolytic effects via binding to the GABAA receptor and activating the monoaminergic system.

[Newly diagnosed abnormal glucose tolerance in patients with acute coronary syndrome and without known diabetes mellitus].

OBJECTIVE: To investigate the prevalence of newly diagnosed abnormal glucose tolerance in patients with acute coronary syndrome and without known diabetes mellitus (DM) and to access the correlation of glycometabolic abnormality with traditional risk factors of cardiovascular diseases. METHODS: The clinical data of 1328 patients with cute coronary syndrome (ACS), without previous DM, and with the admission blood glucose < 11.1 mmol/ who were consecutively admitted in 52 hospitals of 7 cities in China from June 1 to August 31 2005, were collected. A simple oral glucose tolerance test (OGTT) was conducted before they were discharged. RESULTS: Glycometabolic perturbation was seen in 887 of the 1328 patients (67%). The prevalence rates of newly diagnosed DM and impaired glucose regulation (IGR) were 22% and 45% respectively. Blood pressure, blood lipid, body mass index, waistline, and hemoglobin A1C were all significantly higher in those with glycometabolic perturbation than in those with normal glucose metabolism (all P < 0.05). Waistline and blood lipid were the risk factors of abnormal glycometabolic status (both P < 0.05). CONCLUSION: Newly diagnosed DM is common among the ACS patients without DM history. Newly diagnosed glycometabolic status is associated with waistline and blood lipid.

Construction of the High Throughput Technology for Screening Osmotic Stress Relevant Genes / 中国生物工程杂志

With the development of functional genomics, high throughput analysis of genes’ function has been the mainstream of research, and exogenous gene's over expression via Agrobacterium-mediated transformation is the most commonly used method in gene functional analysis.The versatile plant expression vector cassette named pBHT-5 was constructed by the method of site-specific mutagenesis based on pBI121. First of all, the restriction enzyme SfiI recognition site in trfA gene (X00713) which was relevant to plasmid replication and stability was replaced without changing its amino acid composition. And then the SfiIA,SfiIB sites were added between promoter CaMV35s and terminator NOS. The versatile plant expression vector cassette can be directly used to construct plant expression vector containing the full-length genes cloned by Clontech SMARTTM technology, which will raise the efficiency of vector construction. The result will provide basis of new genes’ high throughput screening and functional analysis, then get the new genes functioning in plant osmotic stress resistance.