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Receptor and transporter binding and activity profiles of albiflorin extracted from Radix paeoniae Alba

Albiflorin, a traditional Chinese herb, is a main component of Radix paeoniae Alba, which has been used for the treatment of depressive disorders since ancient times. However, the mechanism of the antidepressant effect of albiflorin is poorly understood. Thus, we explored the binding profile of albiflorin at neurotransmitter receptors and transporters. We also characterised the in vivo effect of albiflorin on monoaminergic systems by using microanalysis to determine the extracellular levels of serotonin (5-HT) and norepinephrine (NE) in the hypothalamus of freely moving rats administered albiflorin. We found that albiflorin inhibited the uptake of 5-HT and NE and displayed robust binding affinities for the transporters of both neurotransmitters. By contrast, albiflorin (10 μM) showed no significant affinity to a wide array of central nervous system receptors. The results of our in vivo microdialysis studies showed that administration of albiflorin (3.5, 7.0, 14.0 mg/kg) significantly increased extracellular concentrations of 5-HT and NE in the hypothalamus of freely moving rats. Overall, the current study showed that albiflorin is a novel 5-HT and NE reuptake inhibitor with high selectivity.

Scientific RepoRts | 6:33793 | DOI: 10.1038/srep33793 The monoaminergic system is one of the most important targets in the pathophysiology and therapy of depression 6,7 . Monoamine reuptake inhibitors work by binding to their respective transporter proteins located presynaptically. This binding interaction induces an increase in the elimination time of the neurotransmitter cycle; thus, the extracellular concentration of neurotransmitters increases in the synaptic cleft. This increase in neurotransmitters in the cleft allows for increased downstream cellular signalling 8 . In the present study, we used a series of competition binding assays and transporter inhibition bioassays to determine the affinities of albiflorin to the rat and cloned human (h) transporters for the monoamines 5-HT, NE and dopamine (SERT, NET and DAT, respectively). To determine the selectivity of albiflorin, we evaluated the affinity of albiflorin at numerous other targets, including receptors for dopamine (D1, D 2 , D 3 , D 4 , D 5 ), 5-HT (5-HT 1A , 5-HT 1B , 5-HT 1D , 5-HT 2a , 5-HT 2C , 5-HT 3 , 5-HT 5A , 5-HT 6 , 5-HT 7 ,), NE (α 1A , α 1B , α 2A , β 1 ), acetylcholine (muscarinic M 1 , M 2 , M 3 , M 4 , M 5 ), opioids (μ , κ , δ ), adenosine (A 1 , A 2A ) and histamine (H 1 , H 2 , H 3 , H 4 ). In addition, we tested the effect of albiflorin on the transport activities of the three monoamines using transporter-transfected HEK293 cell lines as well as a rat brain synaptosomal preparation. Furthermore, to characterise the in vivo effect of albiflorin on monoaminergic systems, we used microdialysis to determine the extracellular levels of 5-HT and NE in the hypothalamus of freely moving rats administered albiflorin.

Results
Binding of albiflorin to rat monoamine transporters. To evaluate the ability of albiflorin to bind to monoamine transporters, competition binding assays were conducted using rat brain tissues. We found that albiflorin showed a high affinity for the rat SERT and NET (Table 1). For SERT, the binding of [ 3 H]-citalopram to rat cerebral cortical membranes was competitively inhibited by albiflorin and fluoxetine. Our results showed that albiflorin was 3-fold more potent than fluoxetine in binding to the rat SERT (K i values for albiflorin: 5.25 ± 0.17; fluoxetine: 16.32 ± 1.02 nM; Table 1; Fig. 1a). The affinity of albiflorin to the rat NET was 6-fold more potent than that for desipramine, a known specific inhibitor of NET. The K i values for albiflorin and desipramine were 1.25 ± 0.77 and 7.38 ± 0.67 nM, respectively (Table 1; Fig. 1b). To evaluate the ability of albiflorin to bind to DAT, we added [ 3 H]-WIN35,428 (1.0 nM) to the brain membrane preparation and used albiflorin or the DATspecific ligand nomifensine as the competitive compounds. We found that the affinity of albiflorin at the rat DAT was much lower than that for nomifensine (K i values for albiflorin and nomifensine were 3,656 ± 25.36 and 29.36 ± 2.36 nM, respectively; Table 1; Fig. 1c). Comparing the affinity of albiflorin to the three monoamine transporters, we found that albiflorin binds to SERT and NET with the highest affinity.
Binding of albiflorin to human monoamine transporters. To determine whether albiflorin has affinities to the human monoamine transporters similar to those in the rat, we used HEK293 cells transfected with SERT, NET, or DAT and tested the ability of albiflorin to compete with specific inhibitors of these different transporters. As shown in Table 1, albiflorin showed the highest affinities to the human SERT and NET, consistent with our results from rats. Albiflorin and fluoxetine dose-dependently competed against the binding of the SERT radioligand [ 3 H]-citalopram in membranes from cells transfected with hSERT. The K i values for albiflorin and fluoxetine were 6.45 ± 0.48 and 14.23 ± 1.25 nM, respectively (  Fig. 2c). Similar to the rat monoamine transporter, the affinity of albiflorin for hSERT and hNET was higher than that for hDAT.
Albiflorin inhibits monoamine uptake in rat synaptosomes. To examine the effect of albiflorin on monoamine transport activity, rat brain synaptosomes were prepared and treated with various concentrations of albiflorin. Our results showed that albiflorin dose-dependently inhibited all three transporters (SERT, DAT and  Fig. 3c). Thus, consistent with its binding profile, albiflorin exhibited more potent inhibition of 5-HT and NE uptake at SERT and NET, respectively, than their corresponding selective inhibitors, but was less effective at inhibiting DA uptake at DAT ( Table 2).
Albiflorin inhibits uptake activity of human monoamine transporters. HEK293 cells were transfected with hSERT, hDAT, or hNET and were treated with albiflorin at various concentrations. Consistent with  Table 1.
the results from rat synaptosomes, we found that albiflorin also significantly inhibited the activities of NET and SERT in these transfected cell lines.  Fig. 4). These data demonstrate that albiflorin has a higher potency for inhibiting the transporter activities of SERT and NET than it does for DAT.  Table 1.
Scientific RepoRts | 6:33793 | DOI: 10.1038/srep33793 Albiflorin selectively binds to NET and SERT. To determine whether albiflorin might also bind to other potential targets, we tested the ability of albiflorin (10 μ M) to bind to the following protein targets, which included G-protein coupled receptors:  Table 3, albiflorin showed no significant affinity for any of these tested targets, suggesting that the binding of albiflorin is selective for SERT and NET.

Albiflorin administration induces an increase in monoamine levels in the microdialysate from the rat hypothalamus in vivo.
To evaluate the effect of albiflorin on central extracellular monoamine levels, we administered albiflorin orally to rats and determined monoamine levels using in vivo microdialysis. We found that acute administration of albiflorin led to a significant increase in hypothalamic 5-HT levels in dose-and time-dependent manners (two-way repeated-measures analysis of variance [ANOVA]: treatment, F (3,29) = 24.0, P < 0.0001; time, F (3,428) = 69.70, P < 0.0001). Post hoc Tukey's tests showed that, compared with vehicle, albiflorin

Discussion
Despite the wide use of Radix paeoniae Alba for treating mood disorders, there is a lack of scientific reports evaluating its pharmacological effects at the level of the receptor. In the present study, we found that albiflorin bound to NET and SERT with high affinity in both a rat synaptosomal preparation and intact cell lines expressing human transporters. Albiflorin showed higher affinity for SERT than for NET, whereas it showed only weak affinity for DAT. In addition, albiflorin (10 μ M) demonstrated no significant activity at numerous nontransporter targets. The results of in vitro bioassay experiments show that albiflorin exhibited selective inhibitory activity for neurotransmitter uptake at the 5-HT and NE monoamine transporters and increased extracellular levels of NE and 5-HT when compared with baseline levels of monoamines in the hypothalamus of rats. Taken together, these data support the view that albiflorin is a novel serotonin and norepinephrine reuptake inhibitor. In our present study, we conducted assays using both rat brain synaptosomes and a human transporter-transfected cell line to evaluate the effects of albiflorin and to clarify whether a species difference occurred. Previous studies have reported conflicting results regarding the affinities of SSRIs, including citalopram, paroxetine and fluoxetine, at rat and human SERTs [9][10][11] . These studies highlighted the importance of determining drug affinities in both species 12 . Our results demonstrated that albiflorin potently interacts with SERT and NET in both species. We found that albiflorin demonstrated equivalent and high affinity for both rat and human SERTs and showed a 17-fold higher affinity than fluoxetine in both rat SERT and human SERT binding assays. In addition, albiflorin potently bound hNET and rat NET with high affinity, showing a 4-fold higher affinity than desipramine in rat NET and hNET binding assays. Consistent with these results from our binding assays, the data from our functional studies exhibited a similar pattern in the ability of albiflorin to inhibit monoamine reuptake in rat synaptosomes and intact cells expressing human transporters. Albiflorin was approximately 10-fold more potent at inhibiting NE uptake than 5-HT uptake. Furthermore, assays examining the competition of albiflorin for the binding of [ 3 H]-WIN35,428, a selective DA reuptake inhibitor, showed inhibitory activity of albiflorin at only high micromolar concentrations. Together, our results indicate that this relatively weak binding interaction would produce no functional consequence on dopamine levels at the concentrations required to inhibit NET and SERT.
Nonselective interaction of uptake inhibitors with neuronal receptors may increase the potential of adverse effects. Previous studies have shown that citalopram and clomipramine displayed antagonist properties with modest affinities at 5-HT 2C sites 13,14 . Reboxetine was also found to have mild affinity for h5-HT 2C receptors. Although blockade of 5-HT 2C sites favourably influences mood, it may also elicit hyperphagia 15 . Moreover, antagonism by clomipramine of histamine H 1 receptors contributes to weight gain, and the actions of clomipramine on H 1 and acetylcholine muscarinic M 1 receptors cause cardiovascular autonomic and sedative side effects 10 . In our current studies, we screened a series of potential targets that albiflorin might interact with, but albiflorin displayed no or very weak affinity to all of them, including in particular, receptors for 5-HT, NE, DA, histamine and muscarine, suggesting that albiflorin may be a novel serotonin and norepinephrine reuptake inhibitor.
In support of the results from our in vitro studies, the results of our in vivo microdialysis studies showed that the administration of albiflorin significantly increased the levels of NE and 5-HT in the hypothalamus of rats at doses (3.5, 7.0 and 14.0 mg/kg) that were effective for antidepressant-like actions in the mouse forced-swim and tail-suspension tests 4 . These results suggested that the elevated extracellular monoamines were the consequence  of functional blockade of the monoamine transporters. The increases in monoamine levels were observed 60 min after administration of albiflorin, consistent with the results of the forced-swim and tail-suspension tests. Given the complicated etiology of depression, the important role of 5-HT and NE in antidepressant therapy, and the crosstalk between these neurotransmitters, it has been suggested that drugs that simultaneously increase the levels of 5-HT and NE may exert increased therapeutic efficacy 11,15 . Increasing 5-HT and NE levels in the local brain environment may result in increased levels of these key neurotransmitters in the circulation. These transmitters play important roles in the peripheral nervous system, as well as in the periphery 16 , and thus may have utility both centrally and peripherally in treating disorders associated with modified levels of 5-HT and NE, such as those involving various pain modalities 17,18 , inflammatory processes 19,20 and urinary incontinence 21 .  Table 2.
Scientific RepoRts | 6:33793 | DOI: 10.1038/srep33793 Compounds with dual-acting 5-HT and NE monoamine transporter inhibition have been shown to have multiple applications 22,23 . Therefore, as a novel 5-HT and NE reuptake inhibitor, the utility and precise functional activities of albiflorin warrant further study.    Human monoamine transporter binding assay. Membranes were prepared from HEK293 cells stably expressing hSERT, hNET, or hDAT as described previously 24,25   Nonspecific binding was determined by including separate samples of 1 μ M fluoxetine, 10 μ M desipramine, or 10 μ M nomifensine for SERT, NET and DAT, respectively.

Drugs and reagents. Albiflorin
Other potential binding targets. Albiflorin (10 μ M) was tested for numerous other potential binding targets by using standard receptor binding procedures 24,25 . The tested targets included the following receptors: D 1 , D 2 , D 3 , D 4 , D 5 , 5-HT 1A , 5-HT 1B , 5-HT 1D , 5-HT 2a , 5-HT 2C , 5-HT 3 , 5-HT 5A , 5-HT 6 , 5-HT 7  Rat synaptosome uptake assay. Male Sprague-Dawley rats were killed by asphyxiation with carbon dioxide and cervical dislocation followed by decapitation. The brains were removed quickly and the cerebral cortex, hypothalamus and striatum were dissected on ice. All dissected tissues were homogenised using a Teflon-glass tissue homogeniser in 4 mL of ice-cold sucrose (0.32 M). The homogenised tissue was centrifuged at 2,000× g for 10 min at 4 °C. The synaptosomal pellets were resuspended in 8 mL of ice-cold assay buffer (10 mM HEPES, 133 mM NaCl, 4.85 mM KCl, 1.2 mM KH 2 PO 4 , 1.5 mM MgSO 4 , 1.5 mM CaCl 2 , 11.1 mM glucose, pH 7.4). Bio-Rad protein assay reagents (1:5 ratio) were used to analyse the protein concentration of the synaptosomal suspension. Uptake of 5-HT, NE and DA was determined as described previously 9,24,25 . The compounds to be tested were dissolved in DMSO, further diluted with assay buffer, and tested over a 10-log unit concentration range in duplicate. Samples of 30-50 μ g of synaptosomes (per sample in a total volume of 200 μ L) were preincubated at 37 °C in a shaking water bath for 10 min. Vehicle or modifiers were then added for 10-20 min. Uptake was initiated by adding  In vivo microdialysis studies. The effect of albiflorin on extracellular monoamine levels in frontal cortices was determined using methods previously described 1 . Microdialysis probes (CMA Microdialysis) with an active membrane surface of 2 mm were perfused with artificial cerebrospinal fluid (125 mM NaCl, 3 mM KCl, 0.75 mM MgSO 4 and 1.2 mM CaCl 2 , pH 7.4) in situ for at least 18 h before experimentation. The microdialysis probe was then implanted via the guide cannula into the hypothalamus and perfused with artificial cerebral spinal fluid at a flow rate of 1 μ L/min. A 3-h stabilisation period was allowed after probe implantation, and dialysate sampling was conducted according to previously described methods 1,28 . Briefly, five control samples (ficomole/20-μ L sample) were obtained prior to drug injection to demonstrate a steady baseline. All subsequent values were expressed as a percentage of the mean preinjection value (i.e., percentage of baseline). After baseline sampling was completed, animals were treated with albiflorin (3.5, 7.0, 14.0 mg/kg) or vehicle (0.2% Tween 80 and 0.5% methylcellulose dissolved in water) orally administered at the end of the sixth sample collection. For a minimum of 3 h after albiflorin administration, dialysis samples were collected every 20 min and analysed to determine the levels of NE, 5-HT and DA. At the end of each experiment, animals were euthanised, and the probe placement was verified by histology. Data from rats with incorrect probe placement were discarded. A 20-μ L sample from the hypothalamus was analysed for 5-HT, NE and DA content using methods described previously 1,28 . The HPLC system consisted of a microbore reverse-phase column (particle size 5 μ m, 150 × 4.6 mm; Model C-18, DIKMA Technologies Ltd., Beijing, China), an Agilent 1100 pump (flow rate 1.0 mL/min; Agilent Technologies, Palo Alto, CA, USA) and a Hewlett-Packard HP 1049A glassy carbon amperometric detector (Agilent Technologies, Palo Alto, CA, USA). The mobile phase consisted of 85 mM citrate, 100 mM sodium acetate, 0.9 mM octyl sodium sulphate, 0.2 mM EDTA and 15% methanol at pH 3.7. External standard curves were used to quantify the amounts of NE, 5-HT and DA in each sample, which was calculated using the area under the curve.
Statistical analysis. All data for inhibition curves were analysed by nonlinear regression using Prism software (GraphPad Software Inc., San Diego, CA) to determine IC 50 values. For the microdialysis experiments, two-way repeated-measures ANOVA followed by Tukey's test was used to compare the percentage increase over baseline between groups, time and treatment as independent factors, and time as the repeated factor.