Brucine抗肿瘤的分子水平研究进展

传统中药马钱子,为马钱科植物马钱子的干燥成熟种子,具有通络止痛、散结消肿的功效。现代研究表明其主要有效成分为生物碱类,含量约为生药的1.5%~5%,主要是士的宁、马钱子碱(Brucine)及其氮氧化物。其中Brucine是从马钱子中提取的吲哚型结构的生物碱,是一种白色晶体具有吲哚型结构的生物碱。研究显示Brucine的药理活性主要集中在镇痛作用、抗炎及免疫系统作用、心血管作用和抗肿瘤作用等几个方面。

Licorice Extracts Attenuate Nephrotoxicity Induced by Brucine Through Suppression of Mitochondria Apoptotic Pathway and STAT3 Activation

Licorice,one of the most widely used medicinal herbs in East Asia,has effects such as anti-inflammation,antioxidant,and detoxifying.This study aimed to evaluate the protective effect of licorice on brucine-induced nephrotoxicity.Sprague Dawley rats were administered with brucine intraperitoneally for 7 consecutive days with or without treatment with licorice.The content of blood urea nitrogen and creatinine in serum,the activities of superoxide dismutase and content of glutathione,malonaldehyde in kidney tissue were detected.Hematoxylin-eosin staining was employed to observe the histopathological changes of kidney.The expression and phosphorylation levels of protein were evaluated by Western blotting and immunohistochemical analysis.The results illustrated that treatment with licorice extracts(LE)significantly protected against the brucineinduced nephrotoxicity by reducing the content of blood urea nitrogen and serum creatinine,attenuating pathologic damage.The unbalance of oxidative stress was repaired by LE via increasing the level of glutathione,promoting the activities of superoxide dismutase and decreasing the content of malonaldehyde.In addition,LE overturned the influence of brucine on apoptosis-related protein and signal transducer and activator of transcription-3(STAT3)activation.Taken together,these data demonstrate that licorice may attenuate brucine-induced nephrotoxicity via inactivation of oxidative stress and mitochondrial-mediated apoptosis pathway.More importantly,the renoprotective effects may be mediated,at least partly,by preventing the activation of STAT3 protein.

Microstructure, Content and in vitro Release of Brucine and Strychnine in Strychnos Nux-Vomica Powder with Different Particle Sizes

To explore the effect of particle size on the quality uniformity and in vitro release performance of Strychnos nux-vomica powder, seven samples of Strychnos nux-vomica powder with different particle sizes were prepared.Microstructures and particle sizes were analyzed, and high performance liquid chromatography(HPLC) was used to test the contents and in vitro release performances of brucine and strychnine in the samples. Results showed that the contents and the in vitro release rates of brucine(or strychnine) in different samples were different since there are different proportions of endosperms to epidermal cells in Strychnos nux-vomica powder with different particle sizes. Brucine and strychnine in each sample were promptly released in the first ten minutes and their cumulative release rates were higher than 70% after ten minutes. Eighty minutes later, the cumulative release rate tended to be a constant. Considering the quality uniformity and safety of Strychnos nux-vomica powder used as traditional Chinese medicine, it would be better to control the particle size of Strychnos nux-vomica powder between 100 and 140 mesh in which the maximum cumulative release rate in vitro of brucine and strychnine can be relatively low within this range.

Brucine N-oxide reduces ethanol intake and preference in alcohol-preferring Fawn-Hooded rats

OBJECTIVE The alcoholism-related social problems have burdened the public health heavily.A better therapy for alcohol dependence as a chronic brain disease is highly required and interests the scientists worldwide. Our group has focused on screening the right drug with low toxicity and a sound curative effect from traditional Chinese medicine. METHODS Alcohol-preferring Fawn-Hooded(FH/Wjd) rat was used as an animal model of alcoholism to evaluate the effects of brucine N-oxide(BNO),an alkoloid naturally existing in the seeds of Strychnos nux-vomica L,on the alcohol-drinking behaviors.Furthermore,its adverse action and toxicity were investigated. RESULTS Treatment with BNO at the doses of 30,50 and 70 mg · kg^(-1)reduced the voluntary alcohol consumption and preference dosedependently and selectively without altering their water intake,total fluid intake,food consumption,body weight as well as sucrose preference. Remarkably,70 mg·kg^(-1)of BNO did suppress the deprivationinduced elevation of alcohol ingestion. Moreover,BNO used at the same doses as above had no influence on locomotion in an open field test and could not result in the place preference effect. CONCLUSION Taken together,BNO is of some significant pharmacological profiles to inhibit symptoms of alcohol dependence with high safety,and thence may be a potential pharmacotherapy.

Brucine Suppresses Breast Cancer Metastasis via Inhibiting Epithelial Mesenchymal Transition and Matrix Metalloproteinases Expressions

Objective： To examine the effect of brucine on the migration, invasion, adhesion and expressions of epithelial-to-mesenchymal transition （EMT） markers and matrix metalloproteinases （MMPs） in the highly metastatic breast cancer cell lines MDA-MB-231 and Hs578-T. Methods： MDA-MB-231 and Hs578-T cells were divided to 4 groups： the control group （0.1% DMSO）, and 25, 50 and 100 μmol/L brucine groups. The cell viability was determined using a CellTiter-Glo？ luminescent cell viability. The scratch wound healing assay and tanswell migration assay were used to determine the migration ability of these cells treated by different concentrations of brucine. The proliferation rate, invasive potential and adhesive ability were respectively performed by colony formation assay, transwell invasion assay and adhension assay. The protein and mRNA expressions of EMT biomarkers, MMP-2 and MMP-9 were investigated by real-time reverse transcription polymerase chain reaction and Western blot. Results： Compared with the control group, brucine had little effect on cell viability or proliferation （P〉0.05）, but led to a dose-dependent decrease on migration, invasion, adhension of MDA-MB-231 and Hs578-T cells （P〈0.01）. Furthermore, brucine increased the protein and mRNA levels of EMT markers such as E-cadherin and β-catenin in MDA-MB-231 and Hs578-T cells, and decreased the protein and mRNA levels of mesenychmal markers such as vimentin and fibronectin, as well as the expressions of MMP-2 and MMP-9 （all P〈0.01）. Conclusion： Brucine inhibited triple negative breast cancer cells metastasis potentially through EMT reversion and MMP-2 and MMP-9 inhibition.

Effects of Brucine on Vascular Endothelial Growth Factor Expression and Microvessel Density in A Nude Mouse Model of Bone Metastasis Due to Breast Cancer

Objective： To 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. Methods： A syringe needle was used to directly inject 0.2 mL monoplast suspension （with 2 × 106 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 mgokg^-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. Results： The VEGF expressions in brucineor 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）. Conclusion： Brucine could inhibit the growth of breast cancer to bone metastases, possibly by inhibiting tumor angiogenesis.

Effect of Baizhu(Rhizoma Atractylodis Macrocephalae)extract on intestinal absorption of brucine and strychnine in vitro and in situ

OBJECTIVE:To investigate the antagonistic effect of the extract of Baizhu(Rhizoma Atractylodis Macrocephalae)(RAM)on the intestinal absorption of brucine and strychnine in Strychnos nux-vomica(NUX)and propose the mechanism of these effects.METHODS:The apparent permeability value(Papp)and absorption rate constant(Ka)were chosen as indices.The everted intestinal sac model and in situ single-pass intestinal perfusion model were used to study the effects of the RAM extract on the absorption of brucine and strychnine.To confirm the results,the brucine and strychnine concentrations in hepatic portal venous blood were determined.Western blotting was used to study P-glycoprotein(P-gp)expression in the Caco-2 cell line.RESULTS:Papp and Ka of brucine and strychnine were significantly increased in the presence of a P-gp inhibitor,but no significant increase was noted in the presence of a tight junction regulator.The RAM extract inhibited the absorption of brucine and strychnine and enhanced P-gp expression.CONCLUSION:The primary absorption mechanism for brucine and strychnine is passive transport,which is affected by P-gp.

Brucine Inhibits Bone Metastasis of Breast Cancer Cells by Suppressing Jagged1/Notch1 Signaling Pathways

Objective： To examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand（RANKL）-induced osteoclastogenesis. Methods： The osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL（50 ng/m L） and macrophage-colony stimulating factor（50 ng/m L） 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. Results： Compared 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）. Conclusion： Brucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

Determination of strychnine and brucine in traditional Chinese medicine preparations by capillary zone electrophoresis with micelle to solvent stacking

A novel micelle to solvent stacking on-line sample preconcentration technique in capillary zone electrophoresis（MSS-CZE） has been developed to determine the strychnine and brucine in traditional Chinese medicine preparations.The optimal running buffer was 30 mmol/L H_3PO_4 containing 20%acetonitrile at pH 4.0.The sample matrix was 8 mmol/L H_3PO_4 containing 5 mmol/L sodium dodecyl sulfate（SDS） at pH 3.0.The established MSS-CZE method afforded more than 50-fold improvements in concentration sensitivity compared with typical CZE-UV analysis.The calibration curve was linear in the range from 0.2 to 15.0μg/ mL for both strychnine and brucine,with correlation coefficients of 0.9984 and 0.9976,respectively.The limits of detection（5/ N = 3：1） for strychnine and brucine were 0.02 and 0.05μg/mL,respectively.The MSS-CZE method has been successfully applied to the analysis of strychnine and brucine in Chinese medicinal preparations.

Tissue distribution and pharmacokinetics of brucine niosomal gels in rats after topical and oral application

Brucine has anti-inflammatory and analgesic effects and is the main active compound of the seeds of Strychnos nux-vomica L. To study brucine niosomal gels, a reliable and rapid LC-MS/MS method was established to quantify brucine levels in rats. Tissue distribution and pharmacokinetics of brucine were investigated after topical and oral application of brucine niosomal gels to rats. The plasma concentration versus time profiles suggested that systemic exposure of brucine for oral administration of brucine niosomal gels was higher than that for topical administration, and topical administration showed a relatively sustained release. There was a considerable amount of brucine distributed in the knee joint. These results provided a strong basis for the follow-up study of this preparation.