Okicamelliaside targets the N-terminal chaperone pocket of HSP90 disrupts the chaperone protein interaction of HSP90-CDC37 and exerts antitumor activity.

Heat shock protein 90 (HSP90) has been recognized as a crucial target in cancer cells. However, various toxic reactions targeting the ATP binding site of HSP90 may not be the best choice for HSP90 inhibitors. In this paper, an ellagic acid derivative, namely, okicamelliaside (OCS), with antitumor effects was found. To identify potential anti-cancer mechanisms, an OCS photosensitive probe was applied to target fishing and tracing. Chemical proteomics and protein-drug interaction experiments have shown that HSP90 is a key target for OCS, with a strong binding affinity (KD = 6.45 µM). Mutation analysis of the target protein and molecular dynamics simulation revealed that OCS could competitively act on the key Glu-47 site at the N-terminal chaperone pocket of HSP90, where the co-chaperone CDC37 binds to HSP90, affect its stability and reduce the ∆Gbind of HSP90-CDC37. It was demonstrated that OCS destroys the protein-protein interactions of HSP90-CDC37; selectively affects downstream kinase client proteins of HSP90, including CDK4, P-AKT473, and P-ERK1/2; and exerts antitumor effects on A549 cells. Furthermore, tumor xenograft experiments demonstrated high antitumor activity and low toxicity of OCS in the same way. Our findings identified a novel N-terminal chaperone pocket natural inhibitor of HSP90, that is, OCS, which selectively inhibits the formation of the HSP90-CDC37 protein complex, and provided further insight into HSP90 inhibitors for anti-cancer candidate drugs.

Ursolic acid reduces hepatocellular apoptosis and alleviates alcohol-induced liver injury via irreversible inhibition of CASP3 in vivo.

Alcoholic liver disease (ALD) is one of the pathogenic factors of chronic liver disease with the highest clinical morbidity worldwide. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, has shown many health benefits including antioxidative, anti-inflammatory, anticancer, and hepatoprotective activities. We previously found that UA was metabolized in vivo into epoxy-modified UA containing an epoxy electrophilic group and had the potential to react with nucleophilic groups. In this study we prepared an alkynyl-modified UA (AM-UA) probe for tracing and capturing the target protein of UA from liver in mice, then investigated the mode by which UA bound to its target in vivo. By conducting proteome identification and bioinformatics analysis, we identified caspase-3 (CASP3) as the primary target protein of UA associated with liver protection. Molecule docking analysis showed that the epoxy group of the UA metabolite reacted with Cys-163 of CASP3, forming a covalent bond with CASP3. The binding mode of the UA metabolites (UA, CM-UA, and EM-UA) was verified by biochemical evaluation, demonstrating that the epoxy group produced by metabolism played an important role in the inhibition of CASP3. In alcohol-treated HepG2 cells, pretreatment with the UA metabolite (10 µM) irreversibly inhibited CASP3 activities, and subsequently decreased the cleavage of PARP and cell apoptosis. Finally, pre-administration of UA (20-80 mg· kg-1 per day, ig, for 1 week) dose-dependently alleviated alcohol-induced liver injury in mice mainly via the inhibition of CASP3. In conclusion, this study demonstrates that UA is a valuable lead compound for the treatment of ALD.

Effect of olive oil on the preparation of nanoemulsions and its effect on aroma release.

The present study focused on the effect of olive oil on Ostwald ripening of flavor nanoemulsions. The release of the aroma compounds from the nanoemulsion system was also investigated. The results showed that the droplets size of the nanoemulsions decreased sharply first and then kept stable with the increase of Tween 80. The optimum surfactant/cosurfactant (Km) ratio was determined at 7:1. The average particle size of nanoemulsion was 39.22 nm. The polydispersity index (PDI) was 0.242 nm, and the particle size distribution was in the range of 20-150 nm at the optimum Km. The stability of the nanoemulsions was improved after the addition of olive oil, and it increased noticeably with the increase of olive oil. The addition of olive oil could help to stabilize the emulsions and hamper Ostwald ripening. All the 11 aroma compounds in the nanoemulsions were detected after 24-h storage. While only 5 aroma compounds were found after 48-h storage, and α-pinene and ß-myrcene were the only two aroma compounds detected after 72-h storage with low contents of 1.41 and 0.5 mg/L. The addition of olive oil inhibited the release of the aroma compounds from the nanoemulsion system. The released ethyl acetate was reduced by 48% after the addition of 10% olive oil. Significant decrease on the release of α-pinene and nonanal was observed after the addition of 3% olive oil. And the decrease was also observed on the release of ß-myrcene, D-limonene, α-terpineol, decanal and eugenol when the olive oil content was ≥ 5%. However, benzyl alcohol, ß-ionone and 1-octanol showed no significant changes with the increase of olive oil. This indicated that the addition of olive oil could provide greater retention of the aroma compounds in the nanoemulsions.

Persistent pain maintains morphine-seeking behavior after morphine withdrawal through reduced MeCP2 repression of GluA1 in rat central amygdala.

As long-term opioids are increasingly used for control of chronic pain, how pain affects the rewarding effect of opioids and hence risk of prescription opioid misuse and abuse remains a healthcare concern and a challenging issue in current pain management. In this study, using a rat model of morphine self-administration, we investigated the molecular mechanisms underlying the impact of pain on operant behavior of morphine intake and morphine seeking before and after morphine withdrawal. We found that rats with persistent pain consumed a similar amount of daily morphine to that in control rats without pain, but maintained their level-pressing behavior of morphine seeking after abstinence of morphine at 0.2 mg/kg, whereas this behavior was gradually diminished in control rats. In the central nucleus of amygdala (CeA), a limbic structure critically involved in the affective dimension of pain, proteins of GluA1 subunits of glutamate AMPA receptors were upregulated during morphine withdrawal, and viral knockdown of CeA GluA1 eliminated the morphine-seeking behavior in withdrawn rats of the pain group. Chromatin immunoprecipitation analysis revealed that the methyl CpG-binding protein 2 (MeCP2) was enriched in the promoter region of Gria1 encoding GluA1 and this enrichment was significantly attenuated in withdrawn rats of the pain group. Furthermore, viral overexpression of CeA MeCP2 repressed the GluA1 level and eliminated the maintenance of morphine-seeking behavior after morphine withdrawal. These results suggest direct MeCp2 repression of GluA1 function as a likely mechanism for morphine-seeking behavior maintained by long-lasting affective pain after morphine withdrawal.

[Rapid identification and evaluation of Chinese materia medica quality by near infrared spectroscopy].

In recent years, the research object of traditional Chinese medicine(TCM) analysis has not just been limited to the common analysis technology, but focused on the key relationship between chemical ingredients and traditional functions, including Chinese material medica (CMM) attributes, chemical substance and biological function. Near infrared spectroscopy (NIR) analysis technology, due to its unique advantages, has been developed rapidly in the field of pharmaceutical analysis, especially in the TCM analysis. NIRS can reflect the global chemical information comprehensively, and this holistic approach can be used for the identification and cluster analysis of CMM. On the other hand, inspiring by the concept of TCM quality markers (Q-markers), by means of the activity screening assay of the key components from the CMM, multiple bioactive components quantitation can be achieved by the NIRS combined with chemometrics. Taking the full advantage of the NIR technology, a simple and reliable method for the fast evaluation of the quality of TCMs can be provided. Therefore, the progress and trend of modern TCM quality evaluation by NIR are discussed and prospected in the present review.

MeCP2 repression of G9a in regulation of pain and morphine reward.

Opioids are commonly used for pain relief, but their strong rewarding effects drive opioid misuse and abuse. How pain affects the liability of opioid abuse is unknown at present. In this study, we identified an epigenetic regulating cascade activated by both pain and the opioid morphine. Both persistent pain and repeated morphine upregulated the transcriptional regulator MeCP2 in mouse central nucleus of the amygdala (CeA). Chromatin immunoprecipitation analysis revealed that MeCP2 bound to and repressed the transcriptional repressor histone dimethyltransferase G9a, reducing G9a-catalyzed repressive mark H3K9me2 in CeA. Repression of G9a activity increased expression of brain-derived neurotrophic factor (BDNF). Behaviorally, persistent inflammatory pain increased the sensitivity to acquiring morphine-induced, reward-related behavior of conditioned place preference in mice. Local viral vector-mediated MeCP2 overexpression, Cre-induced G9a knockdown, and CeA application of BDNF mimicked, whereas MeCP2 knockdown inhibited, the pain effect. These results suggest that MeCP2 directly represses G9a as a shared mechanism in central amygdala for regulation of emotional responses to pain and opioid reward, and for their behavioral interaction.

Central amygdala GluA1 facilitates associative learning of opioid reward.

GluA1 subunits of AMPA glutamate receptors are implicated in the synaptic plasticity induced by drugs of abuse for behaviors of drug addiction, but GluA1 roles in emotional learning and memories of drug reward in the development of drug addiction remain unclear. In this study of the central nucleus of the amygdala (CeA), which is critical in emotional learning of drug reward, we investigated how adaptive changes in the expression of GluA1 subunits affected the learning process of opioid-induced context-reward association (associative learning) for the acquisition of reward-related behavior. In CeA neurons, we found that CeA GluA1 expression was significantly increased 2 h after conditioning treatment with morphine, but not 24 h after the conditioning when the behavior of conditioned place reference (CPP) was fully established in rats. Adenoviral overexpression of GluA1 subunits in CeA accelerated associative learning, as shown by reduced minimum time of morphine conditioning required for CPP acquisition and by facilitated CPP extinction through extinction training with no morphine involved. Adenoviral shRNA-mediated downregulation of CeA GluA1 produced opposite effects, inhibiting the processes of both CPP acquisition and CPP extinction. Adenoviral knockdown of CeA GluA2 subunits facilitated CPP acquisition, but did not alter CPP extinction. Whole-cell recording revealed enhanced electrophysiological properties of postsynaptic GluA2-lacking AMPA receptors in adenoviral GluA1-infected CeA neurons. These results suggest that increased GluA1 expression of CeA AMPA receptors facilitates the associative learning of context-drug reward, an important process in both development and relapse of drug-seeking behaviors in drug addiction.

Optogenetic activation of brainstem serotonergic neurons induces persistent pain sensitization.

BACKGROUND: The rostral ventromedial medulla (RVM) is a key brainstem structure that conveys powerful descending influence of the central pain-modulating system on spinal pain transmission and processing. Serotonergic (5-HT) neurons are a major component in the heterogeneous populations of RVM neurons and in the descending pathways from RVM. However, the descending influence of RVM 5-HT neurons on pain behaviors remains unclear. RESULTS: In this study using optogenetic stimulation in tryptophan hydroxylase 2 (TPH2)- Channelrhodopsin 2 (ChR2) transgenic mice, we determined the behavioral effects of selective activation of RVM 5-HT neurons on mechanical and thermal pain behaviors in vivo. We found that ChR2-EYFP-positive neurons strongly co-localized with TPH2-positive (5-HT) neurons in RVM. Optogenetic stimulation significantly increased c-fos expression in 5-HT cells in the RVM of TPH2-ChR2 mice, but not in wild type mice. Behaviorally, the optogenetic stimulation decreased both mechanical and thermal pain threshold in an intensity-dependent manner, with repeated stimulation producing sensitized pain behavior for up to two weeks. CONCLUSIONS: These results suggest that selective activation of RVM 5-HT neurons exerts a predominant effect of pain facilitation under control conditions.

Actin polymerization-dependent increase in synaptic Arc/Arg3.1 expression in the amygdala is crucial for the expression of aversive memory associated with drug withdrawal.

Aversive memories associated with drug withdrawal may contribute to persistent drug seeking. Molecular mechanisms that are critical for aversive memory formation have yet to be elucidated. Recently, we showed in a rat conditioned place aversion (CPA) model that synaptic actin polymerization in the amygdala were required for aversive memory information. Here, we demonstrated that actin polymerization within the amygdala triggered transportation of activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) into amygdalar synapses. Increased synaptic Arc/Arg3.1 expression contributed to aversive memory formation by regulating synaptic AMPA receptor (AMPAR) endocytosis, as in vivo knockdown of amygdalar Arc/Arg3.1 with Arc/Arg3.1-shRNA prevented both AMPAR endocytosis and CPA formation. We also demonstrated that conditioned morphine withdrawal led to induction of LTD in the amygdala through AMPAR endocytosis. We further demonstrated that Arc/Arg3.1-regulated AMPAR endocytosis was GluR2 dependent, as intra-amygdala injection of Tat-GluR2(3Y), a GluR2-derived peptide that has been shown to specifically block regulated, but not constitutive, AMPAR endocytosis, prevented AMPAR endocytosis, LTD induction, and aversive memory formation. Therefore, this study extends previous studies on the role of actin polymerization in synaptic plasticity and memory formation by revealing the critical molecular events involved in aversive memory formation as well as LTD induction, and by showing that Arc/Arg3.1 is a crucial mediator for actin polymerization functions, and, thus, underscores the unknown details of how actin polymerization mediates synaptic plasticity and memory.

The small GTPase RhoA, but not Rac1, is essential for conditioned aversive memory formation through regulation of actin rearrangements in rat dorsal hippocampus.

AIM: Actin rearrangements are induced in the dorsal hippocampus after conditioned morphine withdrawal, and involved in the formation of conditioned place aversion. In the present study, we investigated the mechanisms underlying the actin rearrangements in rat dorsal hippocampus induced by conditioned morphine withdrawal. METHODS: The RhoA-ROCK pathway inhibitor Y27632 (8.56 µg/1 µL per side) or the Rac1 inhibitor NSC23766 (25 µg/1 µL per side) was microinjected into the dorsal hippocampus of rats. Conditioned place aversion (CPA) induced by naloxone-precipitated morphine withdrawal was assessed. Crude synaptosomal fraction of hippocampus was prepared, and the amount of F-actin and G-actin was measured with an Actin Polymerization Assay Kit. RESULTS: Conditioned morphine withdrawal significantly increased actin polymerization in the dorsal hippocampus at 1 h following the naloxone injection. Preconditioning with microinjection of Y27632, but not NSC23766, attenuated CPA, and blocked the increase in actin polymerization in the dorsal hippocampus. CONCLUSION: Our results suggest that the small GTPase RhoA, but not Rac1, in the dorsal hippocampus is responsible for CPA formation, mainly through its regulation of actin rearrangements.

[Anti-inflammatory mechanism of qingfei xiaoyan wan studied with network pharmacology].

This study aims to clarify out the anti-inflammatory mechanism of Qingfei Xiaoyan Wan. Chemical constituents of Qingfei Xiaoyan Wan identified by UPLC Q-TOF, were submit to Molinspiration, PharmMapper and KEGG bioinformatics softwares for predicting their absorption parameters, target proteins and related pathways respectively; and the gene chip and real time-PCR were carried out to investigate the expression of inflammatory genes on lung tissue of guinea pigs or human bronchial epithelial cell lines. The predicted results showed that 19 of the 24 absorbable constituents affected at 9 inflammation-related pathways through 11 protein targets; Qingfei Xiaoyan Wan treatment can significantly reduce the infiltration of cytokines through ERK1 gene and 5 inflammatory pathways (Focal adhesion, Fc epsilon RI, Toll-like receptors, NK cell-mediated cytotoxic, and ERK/MAPK). The results of real time-PCR further confirmed that the anti-inflammatory effects of Qingfei Xiaoyan Wan were due to active ingredients such as arctigenin, cholic acid and sinapic acid intervened focal adhesion, Fc epsilon RI signaling and ERK/MAPK pathways. The novel approach of 'drug-target-pathway' will present an effective strategy for the study of traditional Chinese medicines.

Serine 363 of the {delta}-opioid receptor is crucial for adopting distinct pathways to activate ERK1/2 in response to stimulation with different ligands.

Distinct opioid receptor agonists have been proved to induce differential patterns of ERK activation, but the underlying mechanisms remain unclear. Here, we report that Ser363 in the δ-opioid receptor (δOR) determines the different abilities of the δOR agonists DPDPE and TIPP to activate ERK by G-protein- or ß-arrestin-dependent pathways. Although both DPDPE and TIPP activated ERK1/2, they showed different temporal, spatial and desensitization patterns of ERK activation. We show that that DPDPE employed G protein as the primary mediator to activate the ERK cascade in an Src-dependent manner, whereas TIPP mainly adopted a ß-arrestin1/2-mediated pathway. Moreover, we found that DPDPE gained the capacity to adopt the ß-arrestin1/2-mediated pathway upon Ser363 mutation, accompanied by the same pattern of ERK activation as that induced by TIPP. Additionally, we found that TIPP- but not DPDPE-activated ERK could phosphorylate G-protein-coupled receptor kinase-2 and ß-arrestin1. However, such functional differences of ERK disappeared with the mutation of Ser363. Therefore, the present study reveals a crucial role for Ser363 in agonist-specific regulation of ERK activation patterns and functions.

GSH-Responsive Drug Delivery System for Active Therapy and Reducing the Side Effects of Bleomycin.

The application of drug delivery system (DDS) has achieved breakthroughs in many aspects, especially in the field of tumor treatment. In this work, polyethylene glycol (PEG)-modified hollow mesoporous manganese dioxide (HMnO2@PEG) nanoparticles were used to load the anti-tumor drug bleomycin (BLM). When the DDS reached the tumor site, HMnO2@PEG was degraded and reduced to Mn2+ by the overexpression of glutathione in the tumor microenvironment, and the drug was released simultaneously. BLM coordinated with Mn2+ in situ, thereby greatly improving the therapeutic activity of BLM. The results of in vivo and in vitro treatment experiments showed that the DDS had excellent responsive therapeutic activation ability. In addition, Mn2+ exhibited strong paramagnetism and was used for T1-weighted magnetic resonance imaging in vivo. Furthermore, this therapeutic mode of responsively releasing drugs and activating in situ effectively attenuated pulmonary fibrosis initiated by BLM. In short, this DDS could help in avoiding the side effects of drugs.

Integrated molecular network and HPLC-UV-FLD analysis to explore antioxidant ingredients in camellia nitidissima Chi.

At present, screening of active ingredients from natural products for pharmacological and clinical research is mostly time-consuming and costly. In this study, a molecular network (MN) guided high performance liquid chromatography-ultraviolet-fluorescence detector (HPLC-UV-FLD) method was carried out to profile the global antioxidant activity compounds, including the trace amount ingredients in Camellia nitidissima Chi (CNC). Firstly, HPLC-UV-FLD postcolumn derivatization system was utilized to screen the antioxidants. Then the MN of CNC was established via mass spectrometry (MS) data for getting the connection between ingredient structures. As a result, HPLC-UV-FLD indicated three antioxidant ingredients: gallic acid (126.3 mg/g), catechin (564.8 mg/g), and salicylic acid (24.3 mg/g). Combined with the MN, the actives' precise location and connection relationship were clarified based on the structural similarities. A new antioxidant ingredient, okicamelliaside, was suggested and evaluated at free radical scavenging and enzymatic protection. The novel method of activity and structural correlation analysis based on MN could provide a useful guide for screening trace active ingredients in natural products. PRACTICAL APPLICATION: Three main ingredients were screened out from Camellia nitidissima Chi by HPLC-UV-FLD postcolumn derivatization system. Integrated molecular network and HPLC-UV-FLD analysis, a new type of antioxidant okicamelliaside was selected. The novel method of activity and structural correlation analysis based on molecular network could provide a useful guide for screening trace active ingredients in natural products.

Involvement of actin rearrangements within the amygdala and the dorsal hippocampus in aversive memories of drug withdrawal in acute morphine-dependent rats.

Aversive memories of drug withdrawal can generate a motivational state leading to compulsive drug taking. Changes in synaptic plasticity may be involved in the formation of aversive memories. Dynamic rearrangement of the cytoskeletal actin, a major structural component of the dendritic spine, regulates synaptic plasticity. Here, the potential involvement of actin rearrangements in the induction of aversive memories of morphine withdrawal was examined. We found that lesions of the amygdala or dorsal hippocampus (DH) but not nucleus accumbens (NAc) impaired conditioned place aversion (CPA) of acute morphine-dependent rats. Accordingly, conditioned morphine withdrawal induced actin rearrangements in the amygdala and the DH but not in the NAc. In addition, we found that conditioned morphine withdrawal also increased activity-regulated cytoskeletal-associated protein (Arc) expression in the amygdala but not in the DH, although actin rearrangements were observed in both areas. We further found that inhibition of actin rearrangements by intra-amygdala or intra-DH injections of latrunculin A, an inhibitor of actin polymerization, significantly attenuated CPA. Furthermore, we found that manipulation of amygdala beta-adrenoceptor activity by its antagonist propranolol and agonist clenbuterol differentially altered actin rearrangements in the DH. Therefore, our findings reveal that actin rearrangements in the amygdala and the DH are required for the acquisition and consolidation of the aversive memories of drug withdrawal and that the beta-noradrenergic system within the amygdala modulates aversive memory consolidation by regulating actin rearrangements but not Arc protein expression in the DH, which is distinct from its role in modulation of inhibitory avoidance memory.

The synergistic anti-asthmatic effects of glycyrrhizin and salbutamol.

AIM: To investigate the efficacy of glycyrrhizin (GL) combined with salbutamol (SA) as an anti-asthma therapy. METHODS: Rat lung beta2-adrenergic receptor (beta(2)-AR) mRNA level was measured by real-time RT PCR. Intracellular cAMP accumulation was evaluated with a reporter gene assay. An in vitro acetylcholine-induced guinea pig tracheal strip contraction model was used to test the relaxing effects of GL and SA. The anti-inflammatory effects of GL and SA were tested using tumor necrosis factor-alpha-induced NF-kappaB transcriptional activation reporter assay, I-kappaB Western blotting and interleukin-8 ELISA. An in vivo guinea pig asthma model was used to prove further the synergistic effect of GL and SA. RESULTS: GL (0.3 micromol/L) increased mRNA levels of beta(2)-AR in vivo and the accumulation of cAMP in vitro. The combination of GL and SA also resulted in significant complementary anti-inflammatory effects via inhibition of NF-kappaB activation, degradation of I-kappaB and production of interleukin-8. A significant synergistic effect of the combination was detected both in vitro and in vivo in a guinea pig mode. CONCLUSION: The results demonstrate that GL and SA have synergistic anti-asthmatic effects and offer the possibility of a therapeutic application of GL in combination with beta(2)-AR agonists in the treatment of asthma.

Initial experience of heroin use under a two-chained operant schedule influences drug-seeking behavior after one month of abstinence.

AIM: To evaluate the influence of an initial heroin experience under a modified two-chained training schedule on drug-seeking behavior after a long abstinence period. METHODS: Rats were trained to respond for intravenous heroin (120 microg/kg) under a heterogeneous chained schedule of reinforcement using different responses in the first and second links of the chain. Animals received low-frequency drug administration training for four days and were then subjected to one month of abstinence in their home cages. Heroin-seeking behavior induced by re-exposure to the first chain associated context or discriminative stimuli was assessed after abstinence. RESULTS: Almost all animals could acquire operant skills quickly under the two-chained schedule training for four days, as measured in first active response latency, travel speed and goal-box enter latency. Both first chain associated context and discriminative stimulus could reinstate heroin-seeking behavior after one month abstinence. CONCLUSION: These observations suggest that an early experience of drug use is sufficient to maintain heroin-seeking behavior even after a one month abstinence.

GluA1 in Central Amygdala Promotes Opioid Use and Reverses Inhibitory Effect of Pain.

Increasing evidence suggests that long-term opioids and pain induce similar adaptive changes in the brain's reward circuits, however, how pain alters the addictive properties of opioids remains poorly understood. In this study using a rat model of morphine self-administration (MSA), we found that short-term pain, induced by an intraplantar injection of complete Freund's adjuvant (CFA), acutely decreased voluntary morphine intake, but not food intake, only at a morphine dose that did not affect pain itself. Pre-treatment with indomethacin, a non-opioid inhibitor of pain, before the pain induction blocked the decrease in morphine intake. In rats with steady MSA, the protein level of GluA1 subunits of glutamate AMPA receptors (AMPARs) was significantly increased, but that of GluA2 was decreased, resulting in an increased GluA1/GluA2 ratio in central nucleus of the amygdala (CeA). In contrast, pain decreased the GluA1/GluA2 ratio in the CeA of rats with MSA. Microinjection of NASPM, a selective inhibitor of homomeric GluA1-AMPARs, into CeA inhibited morphine intake. Furthermore, viral overexpression of GluA1 protein in CeA maintained morphine intake at a higher level than controls and reversed the pain-induced reduction in morphine intake. These findings suggest that CeA GluA1 promotes opioid use and its upregulation is sufficient to increase opioid consumption, which counteracts the acute inhibitory effect of pain on opioid intake. These results demonstrate that the CeA GluA1 is a shared target of opioid and pain in regulation of opioid use, which may aid in future development of therapeutic applications in opioid abuse.