Network and Experimental Pharmacology to Decode the Action of Wendan Decoction Against Generalized Anxiety Disorder.

Objective: The mechanism of Wendan Decoction (WDD) against Generalized Anxiety Disorder (GAD) was predicted by network pharmacology and validated by in vivo and in vitro experiments. Methods: The targets of WDD for the treatment of GAD were obtained by a search of online databases. Further, PPI network and KEGG enrichment were used to identify the key targets and pathways. Ultimately, these key targets and pathways were validated by in vivo experiments on GAD mice modeled by repeated restraint stress (RRS) and in vitro experiments on inflammatory factor stimulated BV-2 cells. Results: Through searching the databases, the 137 ingredients of WDD that correspond to 938 targets and 4794 targets related to GAD were identified. Among them, 569 overlapping targets were considered as the therapeutic targets of WDD for GAD. PPI analysis showed that the inflammation-related proteins IL-6, TNF, SRC and AKT1 were the key targets, and KEGG enrichment suggested that PI3K/AKT and MAPK signaling pathways were key pathways of WDD in the treatment of GAD. In vivo experiments, RRS mice exhibited abnormality in behavioristics in open field test (OFT) and elevated plus maze (EPM) and increases in serum corticosterone and the percentage of lymphocytes positive for IL-6 in peripheral blood. These abnormal changes can be reversed by WDD and the positive control drug paroxetine. In vitro experiments, WDD can inhibit IL-6 induced activation of PI3K/AKT and MAPK signaling pathways in BV2 cells, and suppress the ensuing release of inflammatory factors TNF-α, IL-1ß and PGE2, and showed a dose-dependent effect. Conclusion: WDD is able to resist GAD by relieving inflammatory response in peripheral and central system.

The prescriptions from Shenghui soup enhanced neurite growth and GAP-43 expression level in PC12 cells.

BACKGROUND: Shenghui soup is a traditional Chinese herbal medicine used in clinic for the treatment of forgetfulness. In order to understanding the prescription principle, the effects of "tonifying qi and strengthening spleen" group (TQSS) including Poria cocos (Schw.) Wolf. and Panax ginseng C.A.Mey and "eliminating phlegm and strengthening intelligence" group (EPSI) composed of Polygala tenuifolia Willd., Acorus calamus L. and Sinapis alba L from the herb complex on neurite growth in PC12 cells, two disassembled prescriptions derived from Shenghui soup and their molecular mechanisms were investigated. METHODS: Firstly, CCK-8 kit was used to detect the impact of the two prescriptions on PC12 cell viability; and Flow cytometry was performed to measure the cell apoptosis when PC12 cells were treated with these drugs. Secondly, the effect of the two prescriptions on the differentiation of PC12 cells was observed. Finally, the mRNA and protein expression levels of GAP-43 were analyzed by RT-PCR and western blot, respectively. RESULTS: "Tonifying qi and strengthening spleen" prescription decreased cell viability in a dose-dependent manner, but had no significant effect on cell apoptosis. Meanwhile, it could improve neurite growth and elevate the mRNA and protein expression level of GAP-43. "Eliminating phlegm and strengthening intelligence" prescription also exerted the similar effects on cell viability and apoptosis. Furthermore, it could also enhance cell neurite growth, with a higher expression level of GAP-43 mRNA and protein. CONCLUSION: "Tonifying qi and strengthening spleen" and "eliminating phlegm and strengthening intelligence" prescriptions from Shenghui soup have a positive effect on neurite growth. Their effects are related to the up-regulating expression of GAP-43.

Sini powder () decoction alleviates mood disorder of insomnia by regulating cation-chloride cotransporters in hippocampus.

OBJECTIVE: To investigate the mechanism of Sini Powder () decoction (SND) in the treatment of insomnia. METHODS: The rats were randomly divided into four groups: control, model, SND-treated, and Estazolamtreated groups (n=15 in each group). Sleep deprivation (SD) rat model was established using the modifified multiple platform method for 14 h per day for 14 days, and the behavior of the rats were observed. Na-K-Cl-cotransporter (NKCC1) and K+/Cl- cotransporter (KCC2) in the hippocampus were tested by immunohistochemistry, real-time polymerase chain reaction, and western blot. RESULTS: SD rats displayed anxiety-like behavior, which was alleviated by SND. The protein expressions of NKCC1 and KCC2 in the hippocampus were signifificantly decreased in SD rats compared with those in control rats (P<0.05); these proteins were signifificantly increased by SND (P<0.05). The mRNA expression of KCC2 was signifificantly decreased in SD rats (0.62±0.35 vs. 2.29±0.56; P=0.044), while SND showed a tendency to increase the mRNA of KCC2 in SD rats (P>0.05). By contrast, the mRNA expression of NKCC1 was signifificantly increased in the hippocampus of SD rats (6.58±1.54 vs. 2.82±0.32; P=0.011), while SND decreased the mRNA expression of NKCC1 (6.58±1.54 vs. 2.79±0.81; P=0.016). CONCLUSIONS: Chinese medicine SND could alleviate mood disorder of SD rats by regulating cation-chloride cotransporters, such as NKCC1 and KCC2. These fifindings would have major implications in the mechanism of SND to relieve insomnia.

Extract from Phyllanthus urinaria L. inhibits hepatitis B virus replication and expression in hepatitis B virus transfection model in vitro.

OBJECTIVE: To explore the effects of the extract from Phyllanthus urinaria L. on hepatitis B virus (HBV) replication and expression in HBV transient transfection model in vitro. METHODS: The eukaryotic expression plasmid pHBV1.1, which contains 1.1-fold-overlength genome of HBV, was transfected into the human hepatoma cell line, HepG2, to establish and assess the HBV transient transfection model. The extract from Phyllanthus urinaria L. was prepared in different concentrations and methyl thiazolyl tetrazolium was used to detect the maximum nontoxic concentration of the drug. The extract from Phyllanthus urinaria L. were added into the transfected cell, at the concentrations of 0.8, 0.2 and 0.05 g/L, respectively. Four days after drug application, enzyme-linked immuno sorbent assay was used to detect the concentration of HBsAg in the supernatants, Southern blot was applied to analyze HBV DNA level, and Western blot was used to detect the expression of HBcAg in cells. RESULTS: After the transfection of plasmid pHBV1.1 into HepG2 cells, the concentration of HBsAg in supernatants was increased obviously as compared with that of the normal cells (P<0.05), and all expected HBV replicative intermediates were confirmed by Southern blot analysis, which ensured the successful establishment of the HBV transient transfection model. After the application of drugs at the concentrations of 0.8 and 0.2 g/L, the level of HBsAg was obviously decreased in the supernatants, as compared with that of the virus group (P<0.05); Southern blot showed that the level of HBV rc DNA, ds DNA, ss DNA was obviously reduced compared with that of the virus group (P<0.01); Western blot revealed that the expression of HBcAg in the drug group was obviously inhibited, as compared with that of the virus group (P<0.01). CONCLUSIONS: The extract from Phyllanthus urinaria L. obviously inhibited replication and expression of HBV in HBV transfected cell lines in vitro, thus exerting distinctive anti-HBV effects.

[Effect of sihui mixture on the learning and memory ability, mRNA expressions of hippocampal NMDA subunit NR2A and NR2B, and EphB2 receptor in fatigue rats].

OBJECTIVE: To study the learning and memory ability, expressions of hippocampal N-methyl D-aspartate (NMDA) subunit NR2A and NR2B, and EphB2 receptor in fatigue rats, and to observe effects of Sini Powder, Shenghui Decoction, and Sihui Mixture on them. METHODS: The central nervous system fatigue model was duplicated by paradoxical sleep deprivation for 168 h using multiple platform method. Experimental rats were randomly divided into the normal control group, the model group, the Sini Powder group, and the Shenghui Decoction group, ten in each. Corresponding medicines and distilled water were given to them by gastrogavage at 6, 30, 54, 78, 102, 126, and 150 h after sleep deprivation. Changes of the learning and memory ability were observed using Y maze. mRNA expressions of NMDA subunit NR2A and NR2B, and EphB2 receptor in fatigue rats were quantitatively analyzed using Real-time PCR. RESULTS: Compared with the normal control group, the Y maze correct percentage in the model group obviously decreased (P<0.05), mRNA expressions of NR2B and EphB2 obviously decreased (P<0.901), with no obvious change in NR2A. Compared with the model group, Sihui Mixture could obviously improve Y maze results and mRNA expressions of NR2A and NR2B, and EphB2 (P<0 01). No statistical difference was found between the Sini Powder group and the Shenghui Decoction group. Compared with the Sini Powder group, mRNA expressions of EphB2 obviously increased in the Sihui Mixture group (P<0 01). mRNA expression of NR2A could be more obviously increased in the Shenghui Decoction group than in the model group (P <0 01). CONCLUSION: The central nervous system fatigue could result in decreased Y maze results and gene expressions of hippocampal NR2B and EphB2. Sihui Mixture could improve rats' learning and memory ability, which might be possibly achieved through up-regulating mRNA expressions of hippocampal EphB2 and NR2B.