Characterization of tetrandrine,a potent inhibitor of P-glycoprotein-mediated multidrug resistance

Multidrug resistance (MDR) is one of the main obstacles in tumor chemotherapy. A promising approach to solving this problem is to utilize a nontoxic and potent modulator able to reverse MDR, which in combination with anticancer drugs increases the anticancer effect. Experiments were carried out to examine the potential of tetrandrine (Tet) as a MDR-reversing agent. Survival of cells incubated with Tet at 2.5 mol/l for 72 h was over 90%. Tet at 2.5 mol/l almost completely reversed resistance to vincristine (VCR) in KBv200 cells. Tet at a concentration as low as 0.625 mol/l produced a 7.6-fold reversal of MDR, but showed no effect on the sensitivity of drug-sensitive KB cells in vitro. In the KBv200 cell xenograft model in nude mice, neither Tet nor VCR inhibited tumor growth. However, VCR and Tet combined inhibited tumor growth by 45.7%, 61.2% and 55.7% in three independent experimental settings. In the KB cell xenograft model in nude mice, Tet did not inhibit tumor growth, but VCR and the combination of VCR and Tet inhibited tumor growth by 40.6% and 41.6%, respectively. Mechanism studies showed that Tet inhibited [³H]azidopine photoaffinity labeling of P-gp and increased accumulation of VCR in MDR KBv200 cells in a concentration-dependent manner. The results suggest that Tet is a potent MDR-reversing agent in vitro and in vivo. Its mechanism of action is via directly binding to P-gp and increasing intracellular VCR accumulation.Abbreviations DMSO Dimethyl sulfoxide - FBS Fetal bovine serum - MDR Multidrug resistance - MTT 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide - PBS Phosphate-buffered saline - P-gp P-glycoprotein - SDS Sodium dodecyl sulfate - Tet Tetrandrine - VCR Vincristine     

汉防己甲素对血小板聚集的抑制作用

汉防已甲素(下简称汉甲)和异搏定在离体兔、猪血小板聚集试验中,均能明显地抑制由花生四烯酸(AA)、二磷酸腺苷(ADP)和血小板活化因子(PAF)诱导的聚集反应,剂量与效应相关,汉甲的作用约2～10倍强于异搏定.在兔静脉注射汉甲10mg/kg或异搏定20mg/kg,也明显抑制AA、ADP和PAF诱导的血小板聚集反应.抑制作用在给药后20～30min开始,60min达到高峰,120min后作用逐渐消失.汉甲的作用机理可能与钙拮抗有关.     

汉防己碱和1，6—二磷酸果糖对大鼠局部脑缺血模型的作用

以大鼠大脑中动脉阻塞24小时后的梗塞面积和体积为指标，试验了汉防己碱和1，6－二酸果糖对大鼠局部脑缺血的保护作用，在大脑中动脉阻塞后，汉防己碱7．5，12．0和15．0mg.kg^-1以及1，6－二磷酸果糖20和350mg.kg^-1分别立即腹腔给药，以剂量依赖方式明显减少大鼠脑的梗塞面积和体积，MK8012．0mg.kg^-1亦能减少梗塞面积和体积，汉防己碱和1，6－二磷酸果糖联合用药所产生的保护作用比任何一药物单用时的保护作用都好，提示汉防巳碱和1，6－二酸磷酸果糖有协同作用，在大脑中动脉阻塞后1小时和2小时给药，汉防己碱和1，6－二磷酸果糖仍有脑缺血保护作用，但在梗塞后3小时给药则未见任何保护作用。提示中风或脑缺血后，既非5分钟即不可救药，但亦应尽早用药，实验结果表明汉防己碱和1，6－二磷酸果糖可能有希望的脑缺血保护剂。     

汉防已甲素对慢性低氧性肺动脉高压大鼠肺内bFGF表达的影响

目的　观察汉防已甲素 ( tetrandrine,Tet)对慢性低氧大鼠肺组织碱性成纤维细胞生长因子( b FGF)表达的影响 ,探讨 Tet降低低氧性肺动脉高压的机制。方法　建立低氧性肺动脉高压大鼠模型 ,检测血流动力学 ,观察肺腺泡内动脉 ( IAPA)结构的变化 ,用免疫组化法检测肺组织 b FGF表达。结果　 Tet能明显降低肺动脉压 ,抑制肺腺泡内动脉壁 b FGF表达 ,阻抑肺腺泡内动脉重建。结论　 Tet通过抑制 b FGF表达 ,从而阻抑肺腺泡内动脉重建是其降低肺动脉压的机制之一     

汉防己碱对大鼠皮层神经元钙通道的阻滞作用

在急性分离的大鼠大脑皮层神经元上,用膜片钳单通道记录技术研究汉防己碱（Ｔｅｔ）对电压依赖性钙通道的影响。结果表明,Ｔｅｔ７．５μｍｏｌ／Ｌ、１５μｍｏｌ／Ｌ和３０μｍｏｌ／Ｌ可浓度依赖性抑制大鼠皮层神经元膜Ｌ－和Ｎ－型电压依赖性钙通道,使其开放时间缩短,关闭时间延长,开放概率降低,从而降低钙内流,其对Ｌ－型钙通道的作用与钙通道阻滞剂Ｖｅｒａｐａｍｉｌ相似但较弱。Ｔｅｔ的多种药理作用可能与其阻滞通道     

粉防己碱预防包膜挛缩的实验研究

目的探讨粉防己碱预防包膜挛缩的作用。方法12只雌性成年新西兰兔,随机分为两组,每组6只,每只兔于肉膜下置入15ml光面硅胶假体4枚。粉防己碱组（实验组）术中于假体周围腔隙置入粉防己碱20mg,对照组仅置入假体,3个月后取假体包膜检测包膜张力、包膜重量、包膜厚度、包膜组织中胶原含量、α-平滑肌肌动蛋白免疫组织化学染色阳性细胞数量并进行包膜组织学检查。结果对照组包膜张力、包膜重量、包膜组织中胶原含量、α-平滑肌肌动蛋白免疫组织化学染色阳性细胞数量均高于实验组（P〈0.01）,两组包膜厚度无差异（P〉0.05）;两组包膜的显微结构形态基本一致,内层较平滑,中层是成层排列的纤维结构,外层由疏松结缔组织构成;对照组胶原纤维致密,中层较厚;两组包膜中均可见到典型的肌成纤维细胞,实验组可见肌成纤维细胞线粒体肿胀、内质网扩张、核染色质边集。结论粉防己碱有预防包膜挛缩的作用。     

粉防己碱抗氯化铯诱发家兔在体心脏早后除极及心律失常的作用

目的 :研究粉防己碱 (Tet)抗氯化铯 (CsCl)诱发触发活动及触发性心律失常作用。方法 :14只家兔分对照组和Tet组 ,Tet组给予Tet 0 5mg kg min共 10min ,对照组给予生理盐水 ,然后给予CsCl1 5mmol kg静注 ,给药前后记录心电图和右室心内膜MAP。结果 :Tet组和对照组均诱发EAD ,但Tet组EAD幅度低于对照组 ,室性心律失常发生率低于对照组。结论 :Tet有抗CsCl诱发EAD及室性心律失常作用。其机制可能是Tet抑制慢钙通道 ,减少内向电流。     

粉防己碱抗豚鼠再灌注早期心室颤动及其机制

目的：探讨粉防己碱抗豚鼠再灌注早期心室颤动作用及其机制。方法：利用心肌缺血再灌注损伤模型，研究粉防己碱对再灌注早期心脏去甲肾上腺素（ＮＡ）释放及心室颤动发生的影响。结果：①粉防己碱使心脏流出液中ＮＡ含量从每克心脏组织３．７２±０．９６ｐｍｏｌ／ｍｉｎ减低为０．７８±０．３６ｐｍｏｌ／ｍｉｎ（ｎ＝７，Ｐ＜０．００１）。②粉防己碱可明显降低再灌注早期心室颤动的发生（Ｐ＜０．０５）。结论：粉防己碱可明显减少再灌注早期心交感神经递质ＮＡ的释放，同时可明显减少心室颤动发生。该减少再灌注早期心交感神经递质ＮＡ释放的作用应是抗再灌注性心律失常—心室颤动作用机制之一。     

汉防己甲素对人胰腺癌耐药细胞株SW1990-GEM多药耐药性的逆转机制

目的探讨汉防己甲素(Tet)对人胰腺癌吉西他滨(Gem)耐药细胞株SW1990-GEM耐药逆转作用及其可能的机制。方法应用免疫细胞化学法检测SW1990-GEM细胞多药耐药(MDR)1基因编码的P-糖蛋白(P-gp)表达;应用四甲基偶氮唑蓝(MTT)法检测不同浓度Tet对SW1990-GEM细胞的增殖抑制效应,并计算半数抑制浓度(IC50)及耐药逆转倍数。应用流式细胞仪检测无毒剂量Tet对SW1990-GEM细胞内罗丹明123(Rh123)蓄积情况的影响;Western-blot检测Tet应用前后SW1990-GEM细胞P-gp的表达。结果 P-gp在SW1990-GEM细胞较SW1990细胞高表达;无毒剂量Tet通过增加SW1990-GEM细胞内化疗药物蓄积发挥耐药逆转作用;SW1990-GEM细胞对Gem的耐药指数为217.91,加入Tet(1.5mg/L)后耐药指数为24.32,其逆转倍数为8.96;SW1990-GEM细胞在应用Tet前后P-gp表达无明显变化。结论 Tet通过增加肿瘤细胞内化疗药物浓度逆转SW1990-GEM细胞多药耐药性从而增强化疗药物抗肿瘤作用,其逆转机制与抑制P-gp功能有关,而对P-gp表达无影响。     

Effect of antenatal tetrandrine administration on endothelin-1 and epidermal growth factor levels in the lungs of rats with experimental diaphragmatic hernia

Purpose

The aim of this study was to evaluate the effect of the traditional Chinese medicine tetrandrine (Tet) and to determine its possible mechanism on expression of endothelin-1 (ET-1) and epidermal growth factor (EGF) in the lung of a rat model of nitrofen-induced congenital diaphragmatic hernia (CDH).

Methods

A single oral dose (115 mg/kg) of nitrofen on day 9.5 of pregnancy was maternally administered to induce CDH. Pregnant rats were divided into 4 groups on day 18.5: control (n = 5), CDH (n = 5), CDH+dexamethasone (Dex) (n = 5), and CDH+Tet (n = 5). All fetuses were delivered by cesarean delivery on day 21.5. Accordingly, there were 4 groups of fetuses: control (n = 38), CDH (n = 25), CDH+Dex (n = 21), and CDH+Tet (n = 22). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic evaluations and ET-1 and EGF immunohistochemistry staining were performed, and image analysis was performed after lung processing.

Results

Five female rats in the control group produced 38 fetuses without CDH. CDH was observed in 68 of the 128 rat fetuses (53.1%) among the other 3 groups. The LW/BW ratio of the CDH group was significantly lower than those of the Dex and EGF groups (P < .05). The lungs of fetuses with CDH showed marked abnormal structure such as pulmonary hypoplasia and vascular remodeling, in contrast to improved pulmonary structure in lungs of fetuses in the CDH+Dex and CDH+Tet groups. Statistical differences in morphologic parameters (radial alveolar counts, percentage of alveoli, percentage of medial wall thickness, and vascular volume) were found (P < .05). The immunoreactivity of EGF and ET-1 in the CDH group was markedly stronger than that in the control, CDH+Dex, and CDH+Tet groups (P < .01). In addition, EGF and ET-1 expression in the CDH+Dex and CDH+Tet groups was stronger than that in the control group (P < .05). There was no difference in lung EGF and ET-1 immunoreactivity between CDH+Dex and CDH+Tet groups (P > .05).

Conclusion

Antenatal treatment with Tet may improve lung growth and vascular remodeling, and its mechanism seems to be involved in decreasing EGF and ET-1 expression. Tet administered maternally may be a hopeful new therapeutic option in the treatment of CDH and may be effective in helping to avoid the side effects of Dex.