雷公藤甲素叶酸-壳聚糖纳米粒的制备及其释药性能考察

目的:制备具有靶向性的雷公藤甲素叶酸-壳聚糖纳米粒,并考察其体外释药性能。方法:以粒径和PDI为指标,采用单因素试验和正交试验法,考察溶液pH值、反应温度、壳聚糖和多聚磷酸钠的比例及质量浓度对壳聚糖纳米粒的制备工艺的影响;通过雷公藤甲素与壳聚糖的偶联比及雷公藤甲素、叶酸活性酯和壳聚糖上的氨基反应确定最佳制备工艺,采用离子交联法制备雷公藤甲素叶酸-壳聚糖纳米粒。采用HPLC考察其体外释药特性。结果:最佳制备工艺为反应温度25℃,溶液pH 3.5,壳聚糖-多聚磷酸3∶1,壳聚糖与多聚磷酸钠的质量分数均为0.3%,制得的纳米粒平均粒径约170 nm,粒子分散指数(PDI)约0.21。载药纳米粒的释放率于4 h后达平衡,最大释药率约68%。结论:按优选工艺制备的雷公藤甲素叶酸-壳聚糖纳米粒质量稳定可靠,优选的工艺简便易行。     

雷公藤单体抑制神经胶质瘤的研究进展

雷公藤单体是从传统中草药雷公藤中分离出的化学单体成分,主要包括二萜、三萜、倍半萜、生物碱等,具有免疫抑制、抗炎、抗肿瘤等活性.近年来的文献报道雷公藤单体中雷公藤甲素和雷公藤红素以剂量-效应方式抑制颅内恶性肿瘤神经胶质瘤生长.其作用与调控抗凋亡蛋白Bcl-2,促凋亡蛋白Bax、核因子NF-κB的表达及下调Ras/Akt和Ras/ERK信号转导通道诱导肿瘤细胞凋亡;降低RNA聚合酶转录活性,抑制肿瘤细胞的增殖分化;抑制血管内皮细胞(EC)的血管内皮生长因子(VEGF)受体降低EC的增殖、迁移及小管的形成能力抑制肿瘤血管生成等机制相关.深入研究雷公藤单体的生物学效应及其分子机制将有助于阐明其药理机制.寻找高效低毒的单体将是今后研究的重点.     

微针条件下雷公藤甲素不同剂型对类风湿性关节炎大鼠皮肤损伤的研究

 目的 研究不同长度微电子机械系统（ MEMS）微针条件下,雷公藤甲素（TP）不同剂型对大鼠类风湿性关节炎（RA）模型皮肤损伤的影响。方法 制备TP丙二醇水溶液、脂质体、脂质体水凝胶贴剂3种剂型,采用Ⅱ型胶原诱导的大鼠RA模型,对大鼠左右侧皮肤分别作100和200 μm MEMS微针处理,用紫外分光光度法检测皮肤乳酸脱氢酶（LDH）活性。结果 给予MEMS微针处理后,大鼠皮肤LDH活性没有显著变化（P＞0.05）;TP不同剂型均使皮肤LDH活性明显升高（P＜0.05）,其中溶液组升高最大（P＜0.001）,脂质体组次之（P＜0.01）,脂质体水凝胶贴剂组升高最小（P＜0.05）,但是左右两侧皮肤比较无明显差异（P＞0.05）。结论 100和200 μm MEMS微针均可安全地用于RA大鼠的经皮给药促透;TP制备为脂质体水凝胶贴剂后可显著地减小对RA大鼠皮肤的损伤,是其透皮给药的理想剂型。
     

雷公藤配伍白芍对雷公藤提取物透皮吸收的影响

目的:考察雷公藤与白芍配伍后对雷公藤甲素和2种生物碱类成分(雷公藤吉碱、雷公藤次碱)透皮吸收的影响,从经皮转运的角度探讨雷公藤与白芍配伍减毒的机制。方法:采用改良Franz扩散池,以小鼠离体腹部皮肤为透皮屏障,以20%乙醇生理盐水为接收介质,通过HPLC测定接收液中雷公藤甲素(流动相乙腈-水梯度洗脱,检测波长220 nm)和雷公藤吉碱、雷公藤次碱[流动相乙腈-0.1%磷酸水溶液(57∶43),检测波长230 nm]的含量,计算其累积渗透量和稳态透皮速率等渗透动力学参数。结果:与雷公藤乳膏相比,雷公藤-白芍配伍乳膏均能降低雷公藤吉碱、雷公藤次碱的累积通过量,但对雷公藤甲素的累积透过量无影响;其中雷公藤-白芍以比例1∶2和1∶3配伍的乳膏中雷公藤吉碱和雷公藤次碱的24 h累积透过量呈明显的降低趋势。结论:雷公藤与白芍配伍经皮给药能降低毒性成分的透过量,同时不影响主要活性物质雷公藤甲素的透过量,从而达到了减毒增效的作用。     

雷公藤甲素致小鼠肝损伤对转运体Mrp2和Oatp2的影响

目的：通过多药耐药相关蛋白2（multidrug resistance protein 2,Mrp2）和有机阴离子转运多肽2（organic anion transporting polypeptides 2,Oatp2）初步探究雷公藤甲素诱导小鼠肝损伤的机制。方法：雄性ICR小鼠单次灌胃给予雷公藤甲素（1.0 mg·kg^-1）24 h后称重,摘眼球采血后分离血清,测定血清生化指标;取肝组织做病理切片;采用免疫印迹法检测肝脏组织中Mrp2和Oatp2的蛋白表达量。结果：与对照组相比,雷公藤甲素组肝重指数显著增加（P<0.05）,部分血清生化指标显著上升（P<0.05）,且肝细胞发生核碎裂和脂肪变性。雷公藤甲素组Oatp2的表达较对照组显著升高了32.79%（P<0.05）,而Mrp2的表达较对照组显著下降了45.47%（P<0.01）。结论：雷公藤甲素可能通过上调肝细胞膜转运体Oatp2和下调Mrp2,扰乱肝内胆红素和胆汁酸代谢排泄平衡,可能是雷公藤甲素诱导肝损伤的机制之一。     

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。     

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.     

Suppression of the migration and invasion is mediated by triptolide in B16F10 mouse melanoma cells through the NF‐kappaB‐dependent pathway

Melanoma cancer is one of the major causes of death in humans worldwide. Triptolide is one of the active components of Tripterygium wilfordii Hook F, and has biological activities including induced cell cycle arrest and induction of apoptosis but its antimetastatic effects on murine melanoma cells have not yet been elucidated. Herein, we investigated the effect of triptolide on the inhibition of migration and invasion and possible associated signal pathways in B16F10 murine melanoma cancer cells. Wound healing assay and Matrigel Cell Migration Assay and Invasion System demonstrated that triptolide marked inhibiting the migration and invasion of B16F10 cells. Gelatin zymography assay demonstrated that triptolide significantly inhibited the activities of matrix metalloproteinases‐2 (MMP‐2). Western blotting showed that triptolide markedly reduced CXCR4, SOS1, GRB2, p‐ERK, FAK, p‐AKT, Rho A, p‐JNK, NF‐κB, MMP‐9, and MMP‐2 but increased PI3K and p‐p38 and COX2 after compared to the untreated (control) cells. Real time PCR indicated that triptolide inhibited the gene expression of MMP‐2, FAK, ROCK‐1, and NF‐κB but did not significantly affect TIMP‐1 and ‐2 gene expression in B16F10 cells in vitro. EMSA assay also showed that triptolide inhibited NF‐κB DNA binding in a dose‐dependent manner. Confocal laser microscopy examination also confirmed that triptolide inhibited the expression of NF‐κB in B16F10 cells. Taken together, we suggest that triptolide inhibited B16F10 cell migration and invasion via the inhibition of NF‐κB expression then led to suppress MMP‐2 and ‐9 expressions. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1974–1984, 2016.     

雷公藤内酯醇纳米脂质体涂膜剂制备研究

目的探讨制备雷公藤内酯醇纳米脂质体(TP-NLS)涂膜剂的最佳处方及工艺。方法以聚乙烯醇124(PVA124)、单硬脂酸甘油酯、甘油等基质材料的用量及搅拌时间为考察因素,以均匀性、成膜性、剥离程度为考察指标,采用U_7(7~4)均匀设计法优选TP-NLS涂膜剂的制备处方及工艺。以最佳工艺制备的涂膜剂进行体外模拟透皮实验,考察TP-NLS涂膜剂的经皮渗透过程。结果制备30 g TP-NLS涂膜剂的最佳处方及工艺为单硬脂酸甘油酯1.6g、甘油0.25g、PVA124 3.9g、卡波姆0.6g、CMC-Na 0.9 g,搅拌150 min。按该工艺制备的TP-NLS涂膜剂体外经皮渗透速率(Js)为0.350μg/(cm~2·h),24 h累积渗透量(Q_(24h))为11.534μg/cm2。结论按最佳制备工艺制备的TP-NLS涂膜剂均匀,易涂布,成膜时间短,膜剂成型后与皮肤的黏附性好,柔韧性强。药物经皮渗透速率快,24h累积渗透量增大。