雷公藤减毒研究进展

雷公藤为治疗类风湿性关节炎常用中药,具有清热解毒,祛风除湿,消肿止痛,杀虫止痒的功用,现代药理研究表明其具有抗炎、镇痛、抗肿瘤及免疫调节等作用,临床主要用于治疗免疫性疾病、肾脏性疾病、皮肤性疾病等,对类风湿性关节炎的疗效尤为显著。但严重的毒副作用,尤其是肝肾毒性,限制了其临床应用,成为迫切需要解决的难题,故雷公藤的减毒研究成为近年来的研究热点。本课题组主要研究雷公藤的经皮给药对其减毒增效的作用,但目前雷公藤的应用困境仍然没有得到改善,故对2006年至2016年近10年来雷公藤减毒研究的相关文献进行检索,包括传统的炮制减毒、配伍减毒和现代的制剂减毒、结构修饰减毒、生物技术减毒以及其相互的联合应用等,分析雷公藤的减毒现状和困境,对比现存减毒方式间的差异,为雷公藤减毒增效研究提供思路,促进其合理开发和临床安全应用,也为其他有毒中药的合理应用提供参考。     

从自噬角度研究雷公藤甲素引起HepG2细胞肝毒性的机制

目的:探讨雷公藤甲素(TP)对HepG2细胞的过氧化损伤及对自噬的调控作用。方法:将质量浓度为0.003 2,0.016,0.08,0.4,2 g·L~(-1)的TP作用于体外培养的HepG2细胞48 h,另设空白组做对比,采用噻唑蓝(MTT)法检测细胞生长活性,采用酶联免疫吸附测定(ELISA)法检测超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px)的活性,采用蛋白质免疫印迹(Western blot)法检测自噬相关蛋白微管相关蛋白轻链3(LC3Ⅱ)与Beclin1蛋白的表达情况,用免疫荧光法检测自噬相关蛋白LC3的表达情况。结果:与空白组比较,随着TP质量浓度的增加,HepG2活性下降(P0.05,P0.01);检测细胞上清中SOD,GSH-Px的活性,均较空白组降低(P0.05,P0.01);Western blot表明TP可上调LC3Ⅱ与Beclin1蛋白水平的表达(P0.05,P0.01),免疫荧光法同样表明TP能够引起自噬相关蛋白LC3表达的增多(P0.05,P0.01),均具有一定的浓度依赖性。结论:一定质量浓度的TP可造成肝细胞损伤,其损伤机制可能与过氧化损伤以及过氧化损伤导致的自噬过度激活有关。     

雷公藤甲素对RA患者T-bet/GATA3及CXCL10/CXCR3表达的影响

[目的] 考察雷公藤甲素（TP）对类风湿关节炎（RA）患者外周血单个核细胞（PBMC）中转录因子T-bet/GATA3和趋化因子（CXCL）10及CXCL受体3（CXCR3）表达的影响。[方法] 使用L929细胞考察TP的细胞毒性,确定实验药物浓度。CCK-8法检测TP对RA患者PBMC细胞增殖活性的影响,流式细胞仪分析TP对PBMC中Th细胞亚群比例及CXCR3受体表达调节,Luminex技术检测RA患者PBMC分泌干扰素-γ（IFN-γ）、白细胞介素（IL）-17/IL-17A、肿瘤坏死因子-α（TNF-α）、IL-4、IL-6、IL-10及CXCL10的表达水平。实时荧光定量聚合酶链反应（RT-qPCR）法分析T-bet、GATA3、CXCL10及CXCR3的mRNA表达水平。[结果] 当TP浓度<25 nmol/L,培养时间为48 h时,TP对L929没有显著的细胞毒性（P>0.05）。在此浓度范围内,当TP浓度为5 nmol/L时即表现出对PBMC细胞增殖具有显著抑制作用（P<0.05）。Th、Th1细胞亚群比例以及CXCR3受体表达均受到TP抑制（P<0.05）,但对Th2细胞亚群比例没有显著调节作用（P>0.05）。抗炎因子IL-4、IL-10,促炎因子IFN-γ、IL-6、TNF-α、IL-17/IL-17A,以及CXCL10表达均显著降低（P<0.05）。RT-qPCR结果显示,仅CXCL10表达显著降低,T-bet、GATA3以及CXCR3表达无显著变化（P>0.05）。[结论] TP对Th1细胞增殖及其相关细胞因子具有抑制作用,并且能显著降低CXCL10及CXCR3的表达,但未观察到对T-bet、GATA3表达的调节作用。     

具有线粒体靶向性的雷公藤甲素TPP-PEG-PCL脂质体的制备及其促肝肿瘤细胞凋亡研究

目的 成功制备雷公藤甲素（3-丙羧基）三苯基溴化膦（TPP）-聚乙二醇-b-聚己内脂（PEG-PCL）脂质体（Tr@TPP/Lip）,评价其靶向性及促肝肿瘤细胞凋亡效果。方法 采用正交试验优选Tr@TPP/Lip的制备工艺,再研究该载药系统的粒径、Zeta电位、载药量、包封率和多分散系数及透射电镜微观形态,评价Tr@TPP/Lip的稳定性、溶血性、释放情况;采用荧光试验,研究脂质体与肝肿瘤细胞的融合情况、线粒体靶向性和肝脏靶向性;在等剂量给药条件下,评价Tr@TPP/Lip促肝癌细胞凋亡效果。结果 正交试验优选的Tr@TPP/Lip粒径为（113.5±17.6）nm,Zeta电位（12.6±0.7）mV,包封率为（71.3±3.2）%,载药量为（3.9±1.1）%,多分散系数为0.12±0.04;透射电子显微镜图片显示Tr@TPP/Lip呈规则圆球形,该脂质体稳定性良好,具有较小的溶血率和良好的缓释药物性能;荧光试验结果显示,TPP阳离子能促进脂质体与肿瘤细胞的融合,并靶向线粒体,还能提高药物在肝肿瘤部位的靶向和滞留效果;细胞药效结果显示,Tr@TPP/Lip具有良好的促肝肿瘤细胞凋亡效果,能明显降低线粒体膜Zeta电位、增加细胞内活性氧水平和Caspase-3的释放,显著增加促凋亡蛋白Bcl-2、减少抗凋亡Bax蛋白的表达,这些细胞凋亡试验结果均明显优于雷公藤甲素普通脂质体和雷公藤甲素。结论 Tr@TPP/Lip具有较好的线粒体靶向功能,能增强药物促肝肿瘤细胞凋亡效果。     

Glycyrrhetinic Acid Accelerates the Clearance of Triptolide through P‐gp In Vitro

Triptolide (TP) is an active ingredient isolated from Tripterygium wilfordii Hook. f. (TWHF), which is a traditional herbal medicine widely used for the treatment of rheumatoid arthritis and autoimmune disease in the clinic. However, its adverse reactions of hepatotoxicity and nephrotoxicity have been frequently reported which limited its clinical application. The aim of this study was to investigate the mechanism of glycyrrhetinic acid (GA) effecting on the elimination of TP in HK‐2 cells and the role of the efflux transporters of P‐gp and multidrug resistance‐associated proteins (MRPs) in this process. An ultra performance liquid chromatography–electrospray ionization–mass spectrometry (UPLC‐ESI‐MS) analytical method was established to determine the intracellular concentration of TP. In order to study the role of efflux transporters of P‐gp and MRPs in GA impacting on the accumulation of TP, the inhibitors of efflux transporters (P‐gp: verapamil; MRPs: MK571) were used in this study. The results showed that GA could enhance the elimination of TP and reduce the TP accumulation in HK‐2 cells. Verapamil and MK571 could increase the intracellular concentration of TP; in addition, GA co‐incubation with verapamil significantly increased the TP cellular concentration compared with the control group. In conclusion, GA could reduce the accumulation of TP in HK‐2 cells, which was related to P‐gp. This is probably one of the mechanisms that TP combined with GA to detoxify its toxicity. Copyright © 2017 John Wiley & Sons, Ltd.     

雷公藤内酯醇纳米脂质体温敏凝胶的制备与鼻腔黏膜渗透性考察

目的制备鼻用雷公藤内酯醇纳米脂质体温敏凝胶(TP-NLS-TG),并进行离体黏膜渗透性研究。方法采用高压均质法制备雷公藤内酯醇纳米脂质体(TP-NLS);以泊洛沙姆407(P-407)与泊洛沙姆188(P-188)质量比(A)、氮酮用量(B)和溶胀时间(C)为考察因素,胶凝温度(GT)及TP-NLS-TG的均匀度为评价指标,优选处方,制备TP-NLS-TG。以牛蛙腹部皮肤模拟人体鼻腔黏膜,建立离体黏膜渗透模型,考察TP-NLS-TG的黏膜渗透作用。结果 TP-NLS-TG最优处方中P-407、P-188和氮酮的用量分别为12%、10%、3%,溶胀4 h后,所制备的TP-NLS-TG的GT为32.0℃,均匀度值为0.005,前8.0 h单位面积累积渗透量(Q8)为(9.296 3±0.614 7)μg/cm~2,释放曲线符合Higuchi数学模型。结论按最佳工艺制备的TP-NLS-TG的GT准确,载药均匀,具有较好的黏膜渗透性,可通过蛙皮吸收。     

Triptolide inhibits matrix metalloproteinase-9 expression and invasion of breast cancer cells through the inhibition of NF-κB and AP-1 signaling pathways

Triptolide is a diterpenoid epoxide that is endogenously produced by the thunder god vine, Tripterygium wilfordii Hook F. Triptolide has demonstrated a variety of biological activities, including anticancer activities, in previous studies. Invasion and metastasis are the leading causes of mortality for patients with breast cancer, and the increased expression of matrix metalloproteinase-9 (MMP-9) has been shown to be associated with breast cancer invasion. Therefore, the aim of the present study was to investigate the effect of triptolide on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced cell invasion and MMP-9 expression in breast cancer cells. The expression of signal molecules was examined by western blotting, zymography and quantitative polymerase chain reaction; an electrophoretic mobility gel shift assay was also used, and cell invasiveness was measured by an in vitro Matrigel invasion assay. The MCF-7 human breast cancer cell line was treated with triptolide at the highest concentrations at which no marked cytotoxicity was evident. The results demonstrated that triptolide decreased the expression of MMP-9 through inhibition of the TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK) and the downregulation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity. In addition, a Transwell assay revealed that triptolide reduced the ability of MCF-7 cells to invade Matrigel. These data demonstrate that the anti-invasive effect of triptolide is associated with the inhibition of ERK signaling and NF-κB and AP-1 activation, and suggest that triptolide may be a promising drug for breast cancer.     

Triptolide inhibits human breast cancer MCF-7 cell growth via downregulation of the ERα-mediated signaling pathway

Aim:

To investigate the anticancer mechanisms of triptolide, a diterpenoid isolated from the plant Tripterygium wilfordii Hook F, against human breast cancer cells and the involvement of the estrogen receptor-α (ERα)-mediated signaling pathway in particular.

Methods:

Human breast cancer ERα-positive MCF-7 cells and ERα-negative MDA-MB-231 cells were tested. PrestoBlue assay was used to evaluate the cell viability. The levels of ERα mRNA and protein were detected with real-time PCR and immunoblotting, respectively. Mouse models of MCF-7 or MDA-MB-231 xenograft tumors were treated with triptolide (0.4 mg·kg⁻¹·d⁻¹, po) or a selective estrogen receptor modulator tamoxifen (mg·kg⁻¹·d⁻¹, po) for 3 weeks, and the tumor weight and volume were measured.

Results:

Triptolide (5–200 nmol/L) dose-dependently inhibited the viability of both MCF-7 and MDA-MB-231 cells, with a more potent inhibition on MCF-7 cells. Knockdown of ERα in MCF-7 cells by siRNA significantly attenuated the cytotoxicity of triptolide, whereas overexpression of ERα in MDA-MB-231 cells markedly enhanced the cytotoxicity. Triptolide dose-dependently decreased the expression of ERα in MCF-7 cells and MCF-7 xenograft tumors. Furthermore, treatment of MCF-7 cells with triptolide inhibited the phosphorylation of ERK1/2 in dose- and time-dependent manners. In the mice xenografted with MCF-7 cells, treatment with triptolide or tamoxifen resulted in significant reduction in the tumor weight and volume. Similar effects were not obtained in the mice xenografted with MDA-MB-231 cells.

Conclusion:

The anticancer activity of triptolide against ERα-positive human breast cancer is partially mediated by downregulation of the ERα-mediated signaling pathway.     

经皮穴位电刺激不同时间对雷公藤甲素致大鼠急性肝损伤的影响

目的 观察经皮穴位电刺激（transcutanclus electrical acupoint stimulation,TEAS）不同刺激时间对雷公藤甲素致大鼠急性肝损伤的保护作用。方法 将50只SD大鼠随机分为空白组、模型组、TEAS Ⅰ组（刺激20 min）,TEAS Ⅱ组（刺激30 min）、TEAS Ⅲ组（刺激40 min）,以雷公藤甲素灌胃法复制急性肝损伤模型。TEAS各组刺激“足三里”穴,每日1次,连续5 d。观察肝脏组织形态学变化,检测血清天冬氨酸氨基转移酶（aspartate aminotransferase,AST）、丙氨酸氨基转移酶（alanine aminotransferase,ALT）及肝组织丙二醛（malondialdehyde,MDA）、肿瘤坏死因子- α（tumor necrosis factor- α,TNF- α）、白介素- 10（interleukin- 10,IL- 10）、硫化氢的含量。结果 模型组大鼠肝索排列紊乱,肝窦扩张,肝细胞水肿、脂肪变性、坏死;各TEAS组肝脏病理变化较模型组减轻。与空白组比较,模型组大鼠血清ALT、AST及肝组织MDA、TNF- α含量明显升高（P＜0.05）,硫化氢含量明显降低（P＜0.05）,肝组织IL- 10含量无明显变化（P＞0.05）。与模型组比较,TEAS Ⅰ、Ⅱ、Ⅲ组大鼠血清AST、ALT含量和肝组织MDA、TNF- α含量明显降低（P<0.05）或呈降低趋势（P>0.05）,硫化氢含量明显升高（P<0.05）。其中TEAS Ⅲ组与TEAS Ⅰ、Ⅱ组各指标比较,差异均具有统计学意义（P＜0.05）。结论 TEAS能减轻雷公藤甲素对肝组织的损伤,且较长时间（40 min）刺激的保护作用更为明显。