This study aims to establish a rapid and sensitive UPLC-MS/MS method for simultaneously determining the content of strychnine and paeoniflorin in plasma and brain tissue of rats, and compare the pharmacokinetic behavior and brain tissue distribution of paeoniflorin combined with normal and toxic doses of strychnine in rats after percutaneous administration. Compared with those in the toxic-dose strychnine group, the AUC_(0-t), AUC_(0-∞), and C_(max) of strychnine decreased by 51.51%, 45.68%, and 46.03%, respectively(P<0.01), and the corresponding values of paeoniflorin increased by 91.41%, 102.31%, and 169.32%, respectively(P<0.01), in the compatibility group. Compared with the normal-dose strychnine group, the compatibility group showed insignificantly decreased C_(max), AUC_(0-t), and AUC_(0-∞) of strychnine, increased C_(max) and T_(max) of paeoniflorin(P<0.01), 66.88% increase in AUC_(0-t), and 70.55% increase in AUC_(0-∞) of paeoniflorin. In addition, the brain tissue concentration of strychnine decreased and that of paeoniflorin increased after compatibility. The combination of paeoniflorin with normal dose and toxic dose of strychnine can inhibit the percutaneous absorption of strychnine, and greatly promote the percutaneous penetration of paeoniflorin, whereas the interaction mechanism remains to be explored. The UPLC-MS/MS method established in this study is easy to operate and has good precision. It is suitable for in vivo study of pharmacokinetic behavior and brain tissue distribution of paeoniflorin and strychnine after percutaneous administration in rats, which provides reference for the safe and rational clinical use of strychnine and the combined use of drugs, and lays a solid foundation for the development of external preparations containing Strychni Semen.
Administration, Cutaneous ; Animals ; Brain ; Bridged-Ring Compounds/pharmacology* ; Chromatography, Liquid/methods* ; Glucosides ; Monoterpenes ; Rats ; Rats, Sprague-Dawley ; Strychnine ; Tandem Mass Spectrometry/methods* ; Tissue Distribution

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.
Animals ; Bicuculline ; pharmacology ; Disease Models, Animal ; Glycine ; metabolism ; Hyperalgesia ; drug therapy ; etiology ; metabolism ; Imidazoles ; pharmacology ; Inhibitory Postsynaptic Potentials ; drug effects ; physiology ; Male ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; Neurotransmitter Agents ; pharmacology ; Peripheral Nerve Injuries ; drug therapy ; metabolism ; Phenanthrolines ; pharmacology ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology ; Synaptic Transmission ; drug effects ; physiology ; Tissue Culture Techniques ; Touch

OBJECTIVETo examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis.

METHODSThe osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL (50 ng/mL) and macrophage-colony stimulating factor (50 ng/mL) were added to this system, followed by treatment with brucine (0.02, 0.04 and 0.08 mmol/L), or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1 (TGF-β1), nuclear factor-kappa B (NF-κB) and Hes1 were determined by enzyme-linked immunosorbent assay.

RESULTSCompared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells (P<0.01). Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1 (P<0.05 or P<0.01).

CONCLUSIONBrucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

Animals ; Bone Neoplasms ; metabolism ; prevention & control ; secondary ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Humans ; Jagged-1 Protein ; metabolism ; Macrophages ; drug effects ; physiology ; Mice ; Osteoclasts ; drug effects ; physiology ; Receptor, Notch1 ; metabolism ; Signal Transduction ; drug effects ; Strychnine ; analogs & derivatives ; pharmacology ; therapeutic use

This study was aimed to investigate the inducing-apoptosis effect of brucine on human monocytic leukemia cell line THP-1 cells and its possible mechanism. The inhibition effect of brucine on growth of THP-1 cells was measured by CCK-8 method. Morphological changes of THP-1 cells treated with brucine was detected by acridine orange/ethidium bromide (AO/EB)double staining. Annexin-V/PI double labeling method was used to assay the apoptosis rate of THP-1 cells. The effect of brucine on THP-1 cell cycle distribution was detected by PI single staining. RT-PCR was used to detect the expression of BCL-2 and BAX. The results showed that the brucine could inhibit the THP-1 cell growth in concentration and time-dependent manners at the range of 50 to 400 µg/ml. The cells stained with AO/EB revealed that the brucine induced the nuclear chromatin condensation. After the THP-1 cells were treated with brucine of 400µg/ml for 48 hours, most nucleic were stained as orange-red, and condensed, displaying the late apoptotic cell morphology. Annexin-V/PI detection showed that brucine could induce apoptosis of THP-1 cells in a concentration-dependent manner. Compared with the control group, more cells in brucine-treated group were arrested at G0/G1 phase in a concentration-dependent manner. RT-PCR detection revealed that the expression of BCL-2 was down-regulated strikingly and BAX was up-regulated. It is concluded that brucine can efficiently inhibit cell growth and block THP-1 cells in G0/G1 phase. The mechanism of THP-1 cell apoptosis induced by brucine may be related to the inhibition of BCL-2 and activation of BAX.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Strychnine ; analogs & derivatives ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate function of glycine receptors (GlyRs) at the hippocampal CA1 pyramidal cells and to characterize the pharmacological properties of these receptors at early postnatal stage. METHODS: We used whole cell patch clamp recording to study the current response in the acutely prepared hippocampal slices from postnatal day 11-13 rats induced by glycine applied in the artificial cerebrospinal fluid. RESULTS: Application of glycine to the pyramidal cells elicited strychnine sensitive chloride currents. EC50 for GlyRs respond to glycine was 123. 23 μmol/L and Hill coefficient was 1.24. Picrotoxin could partly blocked the currents. CONCLUSION Strychnine sensitive glycine receptors are functionally expressed in CA1 pyramidal neurons in rat hippocampal CA1 area at early postnatal stage, and some of GlyRs are αβ heteromeric receptors.
Animals ; CA1 Region, Hippocampal ; cytology ; Glycine ; pharmacology ; Patch-Clamp Techniques ; Pyramidal Cells ; drug effects ; Rats ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology

OBJECTIVETo study the effects of brucine on vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in a nude mouse model of bone metastasis due to breast cancer, and to assess the possible antitumor mechanism of brucine.

METHODSA syringe needle was used to directly inject 0.2 mL monoplast suspension (with 2×10(5) human breast cancer cells contained) into the bony femoral cortex of the right hind leg for modeling. Twenty-five nude mice were randomized into five groups and administered with an intraperitoneal injection of saline or drug for 8 consecutive days: model group (0.2 mL normal saline), low-dose brucine group (1.73 mg·kg(-1)), medium-dose brucine group (3.45 mg·kg(-1)), high-dose brucine group (6.90 mg·kg(-1)), and thalidomide group (200 mg·kg(-1)). Diet and activity were recorded, and the tumors were harvested 5 weeks later. The percentage of VEGF-positive cells was determined with hematoxylin and eosin staining and immunohistochemical staining, and MVD expression was determined by optical microscopy.

RESULTSThe VEGF expressions in brucine- or thalidomide-treated mice were significantly reduced as compared with mice in the model group (P <0.01). There were no significant difference between the high-dose brucine group and the thalidomide group (P >0.05). Significant difference was between the high- and low-dose brucine group P<0.05). Further, VEGF expression was significantly increased in the low- and medium-dose brucine groups compared with the thalidomide group (P <0.05). The MVD values in the three brucine and thalidomide groups were significantly lower than that in the model group (P <0.01). The MVD values in the medium- and high-dose brucine groups were not significantly different from those in the thalidomide group (P >0.05), while the MVD value showed a significant increase in the low-dose group compared with the thalidomide group (P <0.05).

CONCLUSIONBrucine could inhibit the growth of breast cancer to bone metastases, possibly by inhibiting tumor angiogenesis.

Animals ; Bone Neoplasms ; blood supply ; metabolism ; secondary ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Humans ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; drug effects ; pathology ; Strychnine ; analogs & derivatives ; pharmacology ; therapeutic use ; Vascular Endothelial Growth Factor A ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo study the molecular mechanism of cyclooxygenase-2 (COX-2), one of effective ingredient of brucine, in inducing non-small cell lung cancer cell apoptosis.

METHODCOX-2 promoter, transcription factor deletion mutants and COX-2 mRNA 3'-UTR-containing report plasmids were transfected with Renillia to non-small cell lung cancer A549 cell, in order to detect the activity of report gene luciferase and minimum cis-acting element of COX-2 promoter inhibited by brucine. The influence of brucine on IkappaB phosphorylation and the nuclear translocation of p65 were detected by immunoblotting assay.

RESULTBrucine significantly suppressed LPS-induced COX-2 promoter activation, but revealed minor impact on COX-2 mRNA stability. NF-kappaB in the vicinity of COX-2 promoter-262 was an important cis-acting element of brucine for inhibiting the activity of COX-2 promoter. Brucine was found to inhibit the phosphorylation of IkappaBalpha as well as the nuclear translocation of p65.

CONCLUSIONBrucine can improve A549 cells apoptosis by inhibiting the activity of NF-kappaB and the subsequent COX-2 gene expression.

Biological Transport ; drug effects ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Cell Line, Tumor ; Cyclooxygenase 2 ; genetics ; Humans ; NF-kappa B ; metabolism ; Phosphorylation ; drug effects ; Promoter Regions, Genetic ; drug effects ; genetics ; RNA Stability ; drug effects ; Strychnine ; analogs & derivatives ; pharmacology

Abstract: The activities of four CYP450 enzymes (CYP3A, 1A2, 2El and 2C) and the mRNA expression levels of CYP1A2, 2El, 2Cll and 3A1 in rat liver were determined after Wistar rats were orally administered with brucine (BR) at three dosage levels (3, 15 and 60 mg.kg-1 per day) and the high dose of BR combined with glycyrrhetinic acid (GA, 25 mg.kg-1 per day) or liquiritin (LQ, 20 mg.kg-1 per day) for 7 consecutive days. Compared with the control, brucine caused 24.5% and 34.6% decrease of CYP3A-associated testosterone 6beta-hydroxylation (6betaTesto-OH) and CYP2C-associated tolbutamide hydroxylation (Tol-OH), respectively, and 146.1% increase of CYP2El-associated para-nitrophenol hydroxylation (PNP-OH) at the high dose level. On the other hand, (BR+GA) caused 51.4% and 33.5% decrease, respectively, of CYP2El-associated PNP-OH and CYP1A2-associated ethoxyresorufin-O-de-ethylation (EROD) as compared with the high dose of BR group. Meanwhile, (BR+LQ) caused 41.1% decrease of CYP2El-associated PNP-OH and 37.7% increase of CYP2C-associated Tol-OH. The results indicated that the co-administration of BR with GA or LQ had effect on mRNA expression and activities of the CYP450 enzymes mentioned above to some extent, and the in vivo antagonism of LQ on BR-induced CYPs adverse effects and the in vivo inhibitory action of GA on CYP2E1 and 1A2 might play an important role in the detoxification of Radix Glycyrrhizae against Strychnos nux-vomica L.
Animals ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Cytochrome P-450 CYP1A1 ; metabolism ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; Cytochrome P-450 CYP2E1 ; genetics ; metabolism ; Cytochrome P-450 CYP3A ; genetics ; metabolism ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Cytochrome P450 Family 2 ; Flavanones ; pharmacology ; Gene Expression Regulation, Enzymologic ; Glucosides ; pharmacology ; Glycyrrhetinic Acid ; pharmacology ; Hydroxylation ; Liver ; enzymology ; metabolism ; Male ; Nitrophenols ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Steroid 16-alpha-Hydroxylase ; genetics ; metabolism ; Steroid Hydroxylases ; metabolism ; Strychnine ; analogs & derivatives ; isolation & purification ; pharmacology ; Strychnos nux-vomica ; chemistry ; Tolbutamide ; metabolism

OBJECTIVETo compare the pharmaceutical properties and the anti-tumor activities of three kinds of stealth liposomes prepared with different phospholipid composition containing brucine.

METHODStealth liposomes with different phospholipids composition, such as soybean phosphatidycholine (SPC), hydrogenated soybean phosphatidylcholine (HSPC) and the complex of SPC and HSPC, were prepared by ammonium sulfate transmembrane gradient method. Pharmaceutical properties such as shape, encapsulation efficiency and size of three stealth liposomes were compared intensively. Anti-tumor activity of SPC, HSPC and novel stealth liposomes composed of both SPC and HSPC were compared by established mouse liver cancer H22 model. Meanwhile, the mice body weight and immune organ weight were also compared.

RESULTThe encapsulation efficiency of novel, SPC and HSPC stealth liposomes were 77.7%, 64.8% and 74.8%, respectively. The mean diameters of them were less than 100 nm. The tumor inhibition rate of novel, HSPC and SPC stealth liposomes were 57.88%, 49.15%, 23.37%, respectively. The mice body weight, thymus gland index of three stealth liposomes group and spleen index of novel stealth liposomes group had no significant difference with the negative group while SPC and HSPC stealth liposomes group increased the spleen index.

CONCLUSIONPhospholipids composition is the key factor which determines the antitumor activity of brucine-loaded stealth liposomes.

Animals ; Antineoplastic Agents ; chemistry ; pharmacology ; Body Weight ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Liposomes ; adverse effects ; chemistry ; Mice ; Particle Size ; Phospholipids ; chemistry ; Strychnine ; analogs & derivatives ; chemistry

Glycine and gamma-aminobutyric acid (GABA) are localized and released by the same interneurons in the spinal cord. Although the effects of glycine and GABA on analgesia are well known, little is known about the effect of GABA in strychnine-induced hyperalgesia. To investigate the effect of GABA and the role of the glycine receptor in thermal hyperalgesia, we designed an experiment involving the injection of muscimol (a GABAA receptor agonist), baclofen (a GABAB receptor agonist) or glycine with strychnine (strychnine sensitive glycine receptor antagonist). Glycine, muscimol, or baclofen with strychnine was injected into the cisterna magna or lumbar subarachnoidal spaces of mice. The effects of treatment on strychnine-induced heat hyperalgesia were observed using the pain threshold index via the hot plate test. The dosages of experimental drugs and strychnine we chose had no effects on motor behavior in conscious mice. Intracisternal or intrathecal administration of strychnine produced thermal hyperalgesia in mice. Glycine antagonize the effects of strychnine, whereas, muscimol or baclofen does not. Our results indicate that glycine has anti-thermal hyperalgesic properties in vivo; and GABA receptor agonists may lack the binding abilities of glycine receptor antagonists with their sites in the central nervous system.
Animals ; Baclofen/*administration & dosage/pharmacology ; Drug Delivery Systems ; GABA Agonists/administration & dosage/pharmacology ; GABA Antagonists/administration & dosage/pharmacology ; Glycine/*administration & dosage/pharmacology ; Hot Temperature ; Hyperalgesia/chemically induced/*drug therapy ; Injections, Spinal ; Male ; Mice ; Mice, Inbred ICR ; Muscimol/*administration & dosage/pharmacology ; Pain Threshold ; Random Allocation ; Strychnine ; gamma-Aminobutyric Acid/metabolism