Use of an UHPLC-MS/MS Method for Determination of Kuraridin and Characterization of Its Metabolites in Rat Plasma after Oral Administration.

Kuraridin is an active natural prenylated flavonoid ingredient originating from the well-known traditional Chinese medicine Sophora flavescens Ait., that possesses various bioactivities, such as antitumor activity, PLCγ1 inhibitory activity, glycosidase inhibitory activity, etc. However, there is no report on the plasma metabolic profile and pharmacokinetic study of kuraridin. The current study was designed to use an ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method for the quantification and characterization metabolites in rat plasma after oral administration of kuraridin. A liquid-liquid extraction method with ethyl acetate-acetonitrile (1:3) was used to extract the kuraridin from rat plasma samples. The chromatographic separation was carried out on a Hypersil GOLD UHPLC C18 column equipped with a C18 guard cartridge using a gradient elution with organic solvent-water as mobile phase. Based on comparing the retention times with reference standards or on the basis of MS2 fragmentation behaviors, a total of 19 metabolites were identified or tentatively characterized from rat plasma. Under the optimized conditions, the method showed good linearity (r² > 0.99) over the ranges of 1-500 ng/mL for kuraridin. The inter- and intra-day precisions were less than 8.95%, and the accuracy was in the range of -6.27-6.48%. The recovery of kuraridin ranged from 90.1% to 100.4%. The developed UHPLC-MS/MS method was thus successfully applied in the qualitative of metabolites and quantitative analysis of kuraridin in rat plasma.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration.

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS: Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Rhynchophylline inhibits methamphetamine dependence via modulating the miR-181a-5p/GABRA1 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria rhynchophylla (Miq.) Miq. ex Havil. is a plant species that is routinely devoted in traditional Chinese medicine to treat central nervous system disorders. Rhynchophylline (Rhy), a predominant alkaloid isolated from Uncaria rhynchophylla (Miq.) Miq. ex Havil., has been demonstrated to reverse methamphetamine-induced (METH-induced) conditioned place preference (CPP) effects in mice, rats and zebrafish. The precise mechanism is still poorly understood, thus further research is necessary. AIM OF STUDY: This study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence. MATERIALS AND METHODS: A rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence. RESULTS: Rhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro. CONCLUSION: This finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.

[Comparative study on macroscopic and microscopic identification of Celastrus aculeatus and Kadsura heteroclite].

OBJECTIVE: To compare and identify Celastrus aculeatus and Kadsura heteroclita with pharmacognosy methods for analyzing the quality of the crude drug. METHODS: Pharmacognosy study on Celastrus aculeatus and Kadsura heteroclite was carried out through plant identification, crude drug identification and microscopic characteristics identification. The characteristics of Celastrus aculeatus and Kadsura heteroclite were compared. RESULTS: There were significant differences between Celastrus aculeatus and Kadsura heteroclite in appearance of cork, attachments on internal surface of cork,shape of leave edge, number of lateral vein, type of stoma and vessel, and the crystals, the stone cells and the fibers in the same part of both drugs. CONCLUSION: The pharmacognosy characteristics of both crude drugs can be used for identification and quality control on Celastrus aculeatus and Kadsura heteroclite.

[Experimental research on prevention and treatment of fatty liver rats with Abrus cantoniensis].

OBJECTIVE: To investigate the effects of Abrus cantoniensis (AC) on blood lipid metabolism, pathomorphological change of the liver and fenestrae of liver sinus endothelial cell (LSEC) in fatty liver disease rats. METHODS: SD rats were divided into 7 groups: blank control group,fatty liver model group, simvastatin group (7.2 mg/kg), Gynostemma pentaphyllum group (16.2 mg/kg), high dose (40 g/kg), middle dose (20 g/kg) and low dose (10 g/kg) of AC groups. All rats except blank control group were fed with high fat diet for the first 3 weeks, then treated with different conditions as previously mentioned for the next 3 weeks while keep on feeding with high fat diet. At the 43rd day,the abdominal aortic blood was collected for measuring the serum concentration of AST, ALT, TC, TG, HDL-C, LDL-C, and liver tissues were taken to make pathological sections for observation by optical microscope or were prepared for scanning electronic microscope. RESULTS: The levels of AST, ALT, TC, TG, LDL-C were obviously decreased while HDL-C were increased in fatty liver rats by AC high dose. Meanwhile the cell morphology of liver tissues and the fenestraes of LSEC were improved as well. CONCLUSION: AC can ameliorate the levels of blood lipid in fatty liver rats and improve the pathological change of liver tissues. To some extent AC has the function of prevention and treatment of fatty liver.

[Study on purification of polysaccharides in root of Salvia miltiorrhiza by macroporous adsorption resin and ion-exchange resin methods].

OBJECTIVE: To study the purification and isolation of polysaccharides from Salvia miltiorrhiza. METHODS: The root was extracted by water purified preliminarily by alcohol precipitation, and then four different types macroporous adsorption resin and one ion-exchange resin were comparatively investigated in the purification and isolation of Salvia polysaccharides. RESULTS: The total polysaccharides of crude extracts was 40.35%, and protein content amounted to 8.96%. Compared with the traditional methods, AB-8, DB-301 type of resin used in purification of polysaccharides could simplify the working process and obtain better effect. Then the obtained crude polysaccharides through AB-8 resin were purified by ion-exchange resin DEAE-52. Three portions of powder were obtained through lyophilization and named as SMP1, SMP2, SMP3. CONCLUSION: Purification of Salvia polysaccharides can be conducted by adoping AB-8, DB-301 type of resin and DEAE-52 ion-exchange resin.

[Study on the detoxification of alcohol extracts from orientvine and its effective component on withdrawal syndromes of morphine].

OBJECTIVE: To observe the effect of alcohol extracts from orientivne and its effective component sinomenine on withdrawal syndromes and neurotransmitter of morphine-abstinent mice and on intracellular calcium level in morphine-dependent neuronal-cells line. To study the detoxification of alcohol extracts from orientvine and sinomenine on morphine-dependent animal and explore the mechanism of its effect. METHODS: The effect of alcohol extracts from orientivne and sinomenineon on abstinent syndromes was observed by experiment study on morphine-dependent ex vivo ileum from guinea pigs and morphine-dependent mice. The morphine-dependent model mice were established by injection on dosage increasing by degrees. The hypothalamic monomine neurotransmitters such as NA, DA, 5-HT were tested by fluorospectrophotometry. Morphine-dependent cell line was established by administering morphine at different doses into the culture medium. The cells were stained with fluo-3 and the intracellular calcium level was measured by flow cytometry. RESULTS: Alcohol extracts from orientvine and sinomenine could alleviate withdrawal contractile response of morphine-dependent ex vivo ileum from guinea pigs and withdrawal syndromes of morphine-dependent mice, decrease the concentration of the neurotransmitters, and elevate the intracellular calcium level and inhibit the decreasing of Ca2+ induced by naloxone. CONCLUSIONS: Alcohol extracts from orientvine and sinomenine have some effects on withdrawal syndromes which may be related to inhibiting neurotransmitters and the regulation of intracellular calcium concentration.

Aloperine induces apoptosis and G2/M cell cycle arrest in hepatocellular carcinoma cells through the PI3K/Akt signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) ranks third among the most common causes of cancer-related deaths worldwide. The chemotherapy for HCC is still insufficient, so far. In searching for effective anti-HCC agents from traditional Chinese medicine, we discovered that aloperine (ALO), a quinolizidine alkaloid from Sophora alopecuroides L., exerts anti-HCC activities. However, the effects of ALO on HCC have been rarely studied, and its underlying mechanisms remain unknown. PURPOSE: This study aims to evaluate the anti-HCC activities of ALO and explore its underlying mechanisms. METHODS: MTT assay and colony formation assay were used to investigate the anti-proliferative effects of ALO on human HCC Hep3B and Huh7 cells. Hoechst 33258 staining was used to observe the morphological changes of cells after ALO treatment. Flow cytometry was used to analyze apoptosis induction, the collapse of the mitochondrial membrane potential and cell cycle distribution. Western blotting was used to examine the expression levels of proteins associated with apoptosis and cell cycle arrest, and key proteins in the PI3K/Akt signaling pathway. Small interfering RNA (siRNA) transfection was used to investigate the role of Akt in ALO-induced apoptosis and cell cycle arrest. Zebrafish tumor model was used to evaluate the anti-HCC effects of ALO in vivo. RESULTS: ALO inhibited the proliferation of Hep3B and Huh7 cells. ALO induced apoptosis in HCC cells, which was accompanied by the loss of mitochondrial potential, the release of cytochrome c into cytosol, as well as the increased cleavages of caspase-9, caspase-3 and PARP. Moreover, ALO induced G2/M cell cycle arrest by downregulating the expression levels of cdc25C, cdc2 and cyclin B1. In addition, ALO inhibited activation of the PI3K/Akt signaling pathway by decreasing the expression levels of p110α, p85, Akt and p-Akt (Ser473). Further study showed that inhibition of Akt by siRNA augmented ALO-mediated apoptosis and G2/M cell cycle arrest in HCC cells. Critically, ALO inhibited the growth of Huh7 cells in vivo. CONCLUSION: We first demonstrated that ALO induced apoptosis and G2/M cell cycle arrest in HCC cells through inhibition of the PI3K/Akt signaling pathway. This study provides a rationale for ALO as a potential chemotherapeutic agent for HCC.

Expression of miRNAs in Serum Exosomes versus Hippocampus in Methamphetamine-Induced Rats and Intervention of Rhynchophylline.

OBJECTIVE: To compare the expressions of miRNAs (microRNAs) in serum exosomes and in hippocampus and to provide insights into the miRNA-mediated relationship between peripheral and central nervous systems in the presence of methamphetamine. METHODS: Published results on conditioned place preference (CPP) in rats conditioned by methamphetamine were replicated. The expressions of miRNAs in serum exosomes and hippocampus were determined by gene-chip sequencing. We then predicted the potential target genes of selected, differentially expressed (DE) miRNAs and then carried out functional analysis of these target genes. We also verified our results by RT-qPCR. RESULTS: Methamphetamine reward could greatly increase the activity time and distance in the intrinsically nonpreferred side of the behavioral apparatus compared with control rats (P < 0.01). Rhynchophylline treatment significantly counteracted these changes (P < 0.01). Methamphetamine-induced CPP upregulated 23 miRNAs (log2 fold change [FC] > 1, P < 0.01) in serum exosomes, whereas rhynchophylline treatment could downregulate these miRNAs (log2 FC < -1, P < 0.01). Analysis of hippocampal miRNAs profiles found 22 DE miRNAs (log2 FC > 1 or <-1, P < 0.01). When methamphetamine induced CPP, 11 of those miRNAs were upregulated, whereas rhynchophylline treatment could downregulate these miRNAs. The other 11 miRNAs behaved in the opposite way. We selected six DE miRNAs from each of serum exosomes and hippocampus for target gene prediction and functional analysis. We found that, in both, the DE miRNAs and their target genes may be related to neuronal information transmission and synaptic transmission. CONCLUSIONS: Rhynchophylline blocked the alteration of behavior and the expression of some DE miRNAs induced by methamphetamine. The biological functions of these DE miRNAs target genes are correlated between serum exosomes and hippocampus. As to these biological processes and pathways which are involved in the development of addiction at multiple stages, we speculate that these DE miRNAs in serum exosomes and hippocampus are closely related to methamphetamine addiction.

[Effect of rhynchophylline on behaviors of methamphetamine-dependent zebrafish and the mechanism].

OBJECTIVE: To observe the effect of rhynchophylline on methamphetamine-dependent zebrafish and explore the possible mechanism. METHODS: Zebrafish were divided into control group, amphetamine group, low- (50 mg/kg) and high (100 mg/kg)-dose rhynchophylline groups, and ketamine (150 mg/kg) group. Conditioned place preference (CPP) was induced in zebrafish with methamphetamine, and the staying time in the drug box and the tracking map of the zebrafish were observed with Noldus Ethovision XT system. The protein expressions of TH, NR2B and GLUR2 in the brain of zebrafish with CPP were detected with Western blotting. RESULTS: Compared with the control group, zebrafish in methamphetamine group showed significant variations in the staying time and swimming distance in the drug box after conditioning (P<0.05) with obvious alterations of NR2B, TH and GLUR2 expressions in the brain (P<0.05). Treatment of methamphetamine-dependent zebrafish with high-dose rhynchophylline significantly reduced the variations in the staying time and swimming distance in the drug box (P<0.05) and in the expressions of NR2B, TH and GLUR2 in the brain (P<0.05). CONCLUSION: Rhynchophylline can inhibit methamphetamine dependence in zebrafish, the mechanism of which may involve the expressions of TH, NR2B and GLUR2 proteins in the brain.

[Effects of Emu eggshell on sexual function of male mice].

OBJECTIVE: To investigate the effects of Emu (Dromaius novaehollandiae) eggshell produced in Guangdong on sexual ability of male mice. METHODS: Mating test was performed in mature male mice after intragastric administration of Emu eggshell for 21 days. The relative weight of the sexual organs in immature male mice was measured after administration for 30 days. A model of sexual dysfunction of male mice was established by repeated hanging stress, and the mating ability of the male mice was examined. RESULTS: Emu eggshell administration significantly shortened the latency time and increased mating frequency in mature male mice, and the relative weight of the sexual organs of immature male mice was increased and sexual function recovery was promoted. CONCLUSION: Emu eggshell produced in Guangdong can enhance sexual function and promote the growth and development of the sexual organs of male mice to a certain extent.

[Changes in cAMP and cGMP levels in neonatal rat histaminergic neurons of tuberomammillary nucleus following 48-hour morphine exposure and effects of sinomenine intervention].

OBJECTIVE: To explore the effects of long-term morphine exposure on cAMP and cGMP levels in primary cultured tuberomammillary nucleus (TM) neurons of neonatal rats and the effects of sinomenine on morphine-dependent TM cells. METHODS: TM neurons after a 7-day primary culture were further cultured in the medium containing 100 micromol/L morphine for 48 h to prepare the cell model of morphine dependence. Serial doses of histamine or sinomenine were administered 30 min naloxone treatment, the cAMP and cGMP levels of the TM cells were determined by enzyme immunoassay. cAMP and cGMP levels were also determined in normal TM cells treated by histamine or sinomenine. RESULTS: After treatment with 100 micromol/L morphine for 48 h, cAMP and cGMP levels in the TM neurons were increased markedly. Treatment with 100 micromol/L naloxone added in the culture media caused an overshoot of cellular cAMP and a marked declination of cGMP, resulting in significantly increased cAMP/cGMP ratio. Sinomenine at 30 and 100 micromol/L and histamine at 40 micromol/L failed to obviously affect cAMP and cGMP levels in normal TM neurons, but sinomenine at 300 micromol/L and histamine at 80 micromol/L significantly increased the intracellular cAMP level. After pre-treatment with sinomenine at the above 3 doses or histamine at 40 micromol/L, the TM neurons with morphine dependence exhibited significant reduction in intracellular cAMP level but increment in cGMP level after naloxone treatment, with significantly reduced cAMP/cGMP ratio. CONCLUSION: Long-term morphine (100 micromol/L) exposure for 48 h can induce marked changes of cAMP and cGMP levels in the TM neurons. The central histaminergic nervous system may be responsible for the development of morphine dependence and withdrawal. Sinomenine can significantly reduce the cAMP level and enhance cGMP level of morphine-dependent TM neurons precipitated by naloxone, which results in a near-normal ratio of cAMP and cGMP.

Extract of Zuojin Pill ([characters: see text]) induces apoptosis of SGC-7901 cells via mitochondria-dependent pathway.

OBJECTIVE: To observe the effects of water extract of Zuojin Pill ([characters: see text], ZJP) on inhibiting the growth of human gastric cancer cell line SGC-7901 and its potential mechanism. METHODS: Effects of ZJP on SGC-7901 cells growth were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, cell apoptosis and cell cycle were determined by flow cytometry, and apoptosis induction was detected by means of DNA gel electrophoresis. The cellular mechanism of drug-induced cell death was unraveled by assaying oxidative injury level of SGC-7901 cell, mitochondrial membrane potentials, expression of apoptosis-related genes, such as B cell lymphoma/lewkmia-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cleaved caspase-3 and caspase-9. RESULTS: ZJP exerted evident inhibitory effect on SGC-7901 cells by activating production of reactive oxygen species and elevating Bax/Bcl-2 ratio in SGC-7901 cells, leading to attenuation of mitochondrial membrane potential and DNA fragmentation. CONCLUSIONS: ZJP inhibits the cancer cell growth via activating mitochondria-dependent apoptosis pathway. ZJP can potentially serve as an antitumor agent.

[Primary culture of histaminergic neurons in neonatal rat hypothalamus tuberomammillary nucleus].

OBJECTIVE: To establish primary cell culture conditions for tuberomammillary nucleus (TM) histaminergic neurons of neonatal rats. METHODS: The tuberomammillary nucleus was dissected from the posterior part of the hypothalamus of neonatal rats, and neurobasal medium supplemented with B27 was used for the primary cell culture. The cultured neurons were identified by immunocytochemical method (ICC). RESULTS: TM neurons from the hypothalamus of neonatal rats were successfully cultured under the experimental condition and tended to mature morphologically on the days 7 to 9 of culture. The cultured neurons were stained by ICC and the green immunoreaction product was seen in the neurons under the fluorescence microscope, indicating that the cultured neurons were histamine-positive. CONCLUSION: TM neurons in the hypothalamus of neonatal rats can be cultured in vitro, and the primary cultured ones may serve as a cell model in vitro for the researches of central histaminergic system.

Effect of sinomenine on morphine dependence in isolated guinea pig ileum.

OBJECTIVE: To investigate the effects of sinomenine on morphine withdrawal response and acetylchline (Ach)-induced contracture in isolated guinea pig ileum. METHODS: The withdrawal contracture was elicited by subjecting isolated ileum incubated with morphine (3 micromol/L) at 37.5 degrees Celsius for 4 h to naloxone (1 micromol/L) treatment. Sinomenine (10, 50, 250 micromol/L) and nimodipine (Nim, 0.1 micromol/L) were administered 1 min before and after naloxone in morphine-dependent ilea bathed in Krebs solution containing morphine, to observe the changes in the withdrawal contracture of the ileum. The effect of sinomenine (10, 50, 250 micromol/L) on the contracture of untreated ileum in Krebs solution elicited by acetylcholine was also observed. RESULTS AND CONCLUSION: Naloxone-induced withdrawal contracture or acetylcholine-induced contracture of the ileum was significantly decreased in a dose-dependent manner, indicating that sinomenine can inhibit morphine withdrawal symptoms in guinea pigs.

[Mechanism of quercetin as an antidiarrheal agent].

OBJECTIVE: To investigate the antidiarrheal mechanism of quercetin extracted from Psidium guajava L. METHODS: The effects of quercetin on the contractility of guinea pig ileum in vitro and on the peristaltic motion of mouse small intestine in vivo were observed, and the peak value of contractility and peristaltic distance recorded. The inhibitory effect of quercetin on the permeability of the abdominal capillaries was also observed. RESULTS: Quercetin inhibited the contraction of guinea pig ileum in vitro and the peristaltic motion of mouse small intestine, and reduced the permeability of abdominal capillaries. CONCLUSION: Quercetin can inhibit the intestinal movement and reduce capillary permeability in the abdominal cavity, which may be the antidiarrheal mechanism of Psidium guajava L extract.

Effect of rhynchophylline on the expression of p-CREB and sc-Fos in triatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference rats.

To explore the effect of rhynchophylline (Rhy) on the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference (CPP) rat, methamphetamine (2 mg/kg) was injected to rats and the conditioned place preference was observed in these rats treated with or without Rhy. An immunohistochemistry assay was used to determine the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area. Methamphetamine induced significant behavior alteration in CPP, while after pretreatment with rhynchophylline or ketamine, the time of staying in methamphetamine-paired compartment of rats was significantly reduced. Methamphetamine also increased the number of p-CREB positive cells in the striatum and hippocampal CA1 zone, as well as p-Fos positive cells. However, the compound Rhy could attenuate the effect. These findings show that Rhy can suppress the acquisition of CPP in rats induced by methamphetamine and the action may be related with the reduced expression of p-CREB and p-Fos in the striatum and hippocampus.

Effects of rhynchophylline on GluN1 and GluN2B expressions in primary cultured hippocampal neurons.

N-methyl-d-aspartate (NMDA) receptor subunits GluN1 and GluN2B in hippocampal neurons play key roles in anxiety. Our previous studies show that rhynchophylline, an active component of the Uncaria species, down-regulates GluN2B expression in the hippocampal CA1 area of amphetamine-induced rat. The effects of rhynchophylline on expressions of GluN1 and GluN2B in primary hippocampal neurons in neonatal rats in vitro were investigated. Neonatal hippocampal neurons were cultured with neurobasal-A medium. After incubation for 6h or 48 h with rhynchophylline (non-competitive NMDAR antagonist) and MK-801 (non-competitive NMDAR antagonist with anxiolytic effect, as the control drug) from day 6, neuron toxicity, mRNA and protein expressions of GluN1 and GluN2B were analyzed. GluN1 is mainly distributed on neuronal axons and dendritic trunks, cytoplasm and cell membrane near axons and dendrites. GluN2B is mainly distributed on the membrane, dendrites, and axon membranes. GluN1 and GluN2B are codistributed on dendritic trunks and dendritic spines. After 48 h incubation, a lower concentration of rhynchophylline (lower than 400 µmol/L) and MK-801 (lower than 200 µmol/L) have no toxicity on neonatal hippocampal neurons. Rhynchophylline up-regulated GluN1 mRNA expression at 6h and mRNA and protein expressions at 48h, but down-regulated GluN2B mRNA and protein expressions at 48 h. However, GluN1 and GluN2B mRNA expressions were down-regulated at 6h, and mRNA and protein expressions were both up-regulated by MK-801 at 48h. These findings show that rhynchophylline reciprocally regulates GluN1 and GluN2B expressions in hippocampal neurons, indicating a potential anxiolytic property for rhynchophylline.

Individual and combined effects of rhynchophylline and ketamine on proliferation, NMDAR1 and GluA2/3 protein expression in PC12 cells.

Rhynchophylline is an active component of the Uncaria species, which is a member of the Rubiaceae family. Our studies show that the downregulation of N-methyl-d-aspartate (NMDA) receptor subunit GluN2B expression in the nucleus accumbens, amygdala, medial prefrontal cortex, and hippocampal CA1 area by rhynchophylline is beneficial for the treatment of psychological dependence on amphetamines. The individual and combined effects of rhynchophylline and ketamine on proliferation and GluN1 and GluA2/3 protein expression in PC12 cells were investigated. PC12 cells were differentiated into neuron-like cells by treatment with nerve growth factor (50 ng/mL). After treatment for 48 h, differentiated PC12 cell proliferation and GluN1 and GluA2/3 protein expression were analyzed. The viability of PC12 cells was reduced by ketamine at doses of 0.50, 1.00, 1.50, and 2.00 mmol/L, with the viability of cells treated with 1.50 and 2.00 mmol/L of ketamine significantly lower than that of the control cells. However, PC12 cells treated with rhynchophylline showed no toxicity at doses of 0.25, 0.50, 0.75, or 1.00 mmol/L. While GluA2/3 protein expression was upregulated by ketamine, it was not influenced by rhynchophylline. GluN1 protein expression was downregulated by rhynchophylline (1 mmol/L), while treatment with ketamine, either alone or with rhynchophylline, had no effect. These findings demonstrate that rhynchophylline suppresses GluA2/3 expression in ketamine-induced PC12 cells and downregulates GluN1 expression. Ketamine's lack of effect on GluN1 expression offers a partial explanation for ketamine addiction and the anti-addictive properties of rhynchophylline.