Hippocampal pituitary adenylate cyclase-activating polypeptide mediates rapid antidepressant-like effects of Yueju pill.

Yueju pill, a classic Chinese Medicine formulated, was recently found to produce rapid antidepressant-like effects in a PKA-CREB signaling-dependent manner. In our study, we found that the Yueju pill induced a remarkable increase in PACAP. The intracerebroventricular injection of PACAP agonist induced a rapid antidepressant-like effect; conversely, the intrahippocampal infusion of a PACAP antagonist reversed the antidepressant response of the Yueju pill. Mice with hippocampal PACAP knockdown via viral-mediated RNAi displayed depression-like behavior. PACAP knockdown also blunted the antidepressant effect of the Yueju pill. PACAP knockdown resulted in down-regulated CREB and expression of the synaptic protein PSD95 at both baselines and after administration of the Yueju pill. However, administration of the Yueju pill in the knockdown mice promoted PACAP and PKA levels. Chronically stressed mice showed deficient hippocampal PACAP-PKA-CREB signaling and depression-like behavior, which were reversed by a single dose of the Yueju pill. In this study, we demonstrated that the up-regulation of PACAP induced activating of PKA-CREB signaling would play a part in the rapid antidepressant-like effects of the Yueju pill. We also identified iridoids fraction of Gardenia jasminoides Ellis (GJ-IF), a vital component of the Yueju pill, was identified to recapitulate rapid antidepressant-like behavior through increased hippocampal PACAP expression of the Yueju pill. The promotion of hippocampal PACAP may collectively represent a novel mechanism of rapid antidepressant-like effect.

Direct formation of the sesquiterpeonid ether liguloxide by a terpene synthase in Senecio scandens.

KEY MESSAGE: SsLOS directly catalyzed formation of the sesquiterpenoid ether liguloxide in the medicinal plant Senecio scandens. Terpene synthases determine the diversity of terpene skeletons and corresponding terpenoid natural products. Oxygenated groups introduced in catalysis of terpene synthases are important for solubility, potential bioactivity and further elaboration of terpenoids. Here we identified one terpene synthase, SsLOS, in the Chinese medicinal plant Senecio scandens. SsLOS acted as the sesquiterpene synthase and utilized (E,E)-farnesyl diphosphate as the substrate to produce a blend of sesquiterpenoids. GC-MS analysis and NMR structure identification demonstrated that SsLOS directly produced the sesquiterpenoid ether, liguloxide, as well as its alcoholic isomer, 6-epi-guaia-2(3)-en-11-ol. Homology modeling and site-directed mutagenesis were combined to explore the catalytic mechanism of SsLOS. A few key residues were identified in the active site and hedycaryol was identified as the neutral intermediate of SsLOS catalysis. The plausible catalytic mechanism was proposed as well. Altogether, SsLOS was identified and characterized as the sesquiterpenoid ether synthase, which is the second terpenoid ether synthase after 1,8-cineol synthase, suggesting some insights for the universal mechanism of terpene synthases using the water molecule in the catalytic cavity.

Yueju-Ganmaidazao Decoction confers rapid antidepressant-like effects and the involvement of suppression of NMDA/NO/cGMP signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Yueju-Ganmaidazao Decoction (YG) is a multiherbal medicine prescribed for treatment of mood disorder, consisting of two classical traditional Chinese herbal medicine Yueju and Ganmaidazao. Yueju and Ganmaidazao both are used for depression treatment. The combined decoction of Yueju and Ganmaidazao is prescribed to achieve optimal clinical outcomes by dealing with different symptoms of depression. Recent studies indicated ethanol extract of Yueju was capable to confer rapid antidepressant-like response. The antidepressant activity of YG decoction with fast-onset feature remains to be investigated. AIM OF THE STUDY: Rapid and safe antidepressant treatment is urgently needed. This study aimed to assess the rapid antidepressant-like activity of YG and the underlying mechanism, focusing on NMDA/NO/cGMP signaling. MATERIALS AND METHODS: The optimal doses for immediate and persistent antidepressant-like response were first screened using tail suspension test (TST) and forced swimming test (FST) post a single administration of YG. The rapid action was further confirmed by using the chronic mild stress (CMS) and learned helplessness (LH) paradigms. The expressions of NMDA receptor subunits were evaluated post stress and YG. The contributions of NMDA, NO, and cGMP signaling to the antidepressant effect of YG were investigated systematically using pharmacological interventions. RESULTS: The optimal dose for immediate and persistent antidepressant potential, evidenced with reduced immobility times in TST or FST from 30 min to 7 days, was determined. The rapid antidepressant-like effect was confirmed in CMS and LH paradigms, including instant normalization of sucrose preference behavior. The expression of NMDA subunit NR1 in the hippocampus was reduced from 30 min to 5 days post YG. In animals subjected to CMS and LH, hippocampal NR1 expression increased, reversed by YG. YG's antidepressant-like effect was blunted by pretreatment with the agonists along the signalings including NMDA (75 mg/kg), L-arginine (750 mg/kg) and sildenafil (5 mg/kg) in TST or FST. Conversely, administration of subeffective dose of individual antagonists, including MK-801 (0.05 mg/kg), 7-nitroindazole (30 mg/kg), methylene blue (10 mg/kg), in combination with a subeffective dose of YG, elicited antidepressant effects. CONCLUSION: YG conferred rapid antidepressant-like effects, and the antidepressant response was essentially dependent on suppression of NMDA/NO/cGMP signaling.

[Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens].

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize ß-macrocarpene synthase) into BL21 competent cells. The results showed that the content of ß-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

[Functional characterization of SsNES responsible for nerolidol biosynthesis in Senecio scandens].

A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.

[Identification of stable reference gene by qRT-PCR in Senecio scandens].

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.

Involvement of NMDA-AKT-mTOR Signaling in Rapid Antidepressant-Like Activity of Chaihu-jia-Longgu-Muli-tang on Olfactory Bulbectomized Mice.

Background: Fast-onset antidepressants are urgently needed. Chaihu-jia-Longgu-Muli-tang (CLM), a classic Chinese herbal medicine, has been used for antidepressant treatment with long history. Olfactory bulbectomization (OB) model is validated for identification of rapid antidepressant efficacy. Here we used OB model for investigating the rapid onset activity of CLM in mice, and also tested the involvement of prefrontal Akt-mTOR and associated AMPA/NMDA receptors as well as hippocampal BDNF in the rapid antidepressant-like effect of CLM. Methods: The OB model was first characterized with depression-like behaviors and the time course changes of the behaviors. The fast onset of antidepressant effect of CLM was evaluated using sucrose preference test, tail suspension test and forced swim test in OB mice after a single administration. The expression of synaptic proteins of AMPA and NMDA subunits as well as Akt/mTOR signaling in the prefrontal cortex, and hippocampal BDNF was evaluated with the immunoblotting method. Results: A single dose of CLM significantly improved the deficiency in the sucrose preference and decreased the immobility time in the tail suspension test in OB mice. In the prefrontal cortex (PFC) in OB mice, there was lower expression level of the AMPA receptor subunit GluR1, rescued by a single dose of CLM. Additionally, the expression of NMDA subunit NR1 was up-regulated in OB mice, whereas mTOR and its upstream Akt signalings were both down-regulated. These deficiencies were reversed by a single dose of CLM. The CLM treatment also attenuated the expressions of NMDA receptor subunits NR2A and NR2B, which did not change in OB mice. In the hippocampus, expressions of GluR1 and brain derived neurotrophic factor (BDNF) were both up-regulated in OB mice, although CLM increased GluR1, but not BDNF. Conclusion: CLM elicited rapid antidepressant-like effects in the OB model mice, and CLM reversal of the abnormality in PFC expression of AMPA and NMDA receptors and associated Akt-mTOR signaling may underlie the effects.

Neural Plasticity Associated with Hippocampal PKA-CREB and NMDA Signaling Is Involved in the Antidepressant Effect of Repeated Low Dose of Yueju Pill on Chronic Mouse Model of Learned Helplessness.

Yueju pill is a traditional Chinese medicine formulated to treat syndromes of mood disorders. Here, we investigated the therapeutic effect of repeated low dose of Yueju in the animal model mimicking clinical long-term depression condition and the role of neural plasticity associated with PKA- (protein kinase A-) CREB (cAMP response element binding protein) and NMDA (N-methyl-D-aspartate) signaling. We showed that a single low dose of Yueju demonstrated antidepressant effects in tests of tail suspension, forced swim, and novelty-suppressed feeding. A chronic learned helplessness (LH) protocol resulted in a long-term depressive-like condition. Repeated administration of Yueju following chronic LH remarkably alleviated all of depressive-like symptoms measured, whereas conventional antidepressant fluoxetine only showed a minor improvement. In the hippocampus, Yueju and fluoxetine both normalized brain-derived neurotrophic factor (BDNF) and PKA level. Only Yueju, not fluoxetine, rescued the deficits in CREB signaling. The chronic LH upregulated the expression of NMDA receptor subunits NR1, NR2A, and NR2B, which were all attenuated by Yueju. Furthermore, intracerebraventricular administration of NMDA blunted the antidepressant effect of Yueju. These findings supported the antidepressant efficacy of repeated routine low dose of Yueju in a long-term depression model and the critical role of CREB and NMDA signaling.

Functional characterization of ent-copalyl diphosphate synthase from Andrographis paniculata with putative involvement in andrographolides biosynthesis.

OBJECTIVES: To characterize the ent-copalyl diphosphate (ent-CPP) synthase involved in the biosynthetic pathway of andrographolides in a medicinal plant, Andrographis paniculata. RESULTS: The ent-CPP synthase (ent-CPS) gene was cloned from A. paniculata and its encoded ApCPS was demonstrated to react with (E,E,E)-geranylgeranyl diphosphate to form ent-CPP through recombinant expression in Escherichia coli. Site-directed mutagenesis of the Asp to Ala in the conserved DXDD motif of ApCPS resulted in loss of function. One Arg is located in the conserved position close to DXDD motif indicating the involvement of ApCPS in specialized metabolism. In addition, RT-PCR analysis revealed that ApCPS was expressed in all tissues of A. paniculata at all growth stages, which is consistent with andrographolides accumulating in these organs. Methyl jasmonate induced ApCPS gene expression, matching inducible accumulation of andrographolides in vivo. CONCLUSIONS: ApCPS is the first ent-CPS characterized in A. paniculata and is suggested to be involved in biosynthesis of andrographolides that have high pharmaceutical values.

[Cloning and functional characterization of phytoene desaturase in Andrographis paniculata].

A full-length cDNA of phytoene desaturase (PDS) gene from Andrographis paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp (GeneBank: KP982892), encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD ( H) -binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves. Virus induced gene silencing (VIGS) was performed to characterize the functional of ApPDS in planta. Significant photobleaching was not observed in infiltrated leaves, while the PDS gene has been down-regulated significantly at the yellowish area. To the best of our knowledge, this represents the first report of PDS gene cloning and functional characterization from A. paniculata, which lays the foundation for further investigation of new genes, especially that correlative to andrographolide biosynthetic pathway.