HPLC法测定雷公藤粗制品中雷公藤甲素的含量

目的：建立高效液相色谱法测定雷公藤粗制品中雷公藤甲素的含量。方法：雷公藤粗制品经洗脱提取后，采用十八烷基键合硅胶柱，以乙腈-水（30：70）为流动相，以218nm为检测波长，检测雷公藤粗制品中雷公藤甲素的含量。结果：雷公藤甲素在1．92～11．52μg／ml范围内线性良好，r=0．9999，平均回收率为95．32％，RSD为1．16％。结论：本法操作简便、结果准确，可用于雷公藤粗制品中雷公藤甲素的含量测定。     

雷公藤甲素对人子宫内膜癌细胞株HEC-1B的影响

目的:研究雷公藤甲素对人子宫内膜癌细胞株HEC-1B体外生长特性的影响。方法:观察雷公藤甲素作用后子宫内膜癌细胞株HEC-1B的生长情况;用MTT法检测雷公藤甲素抑制细胞增殖的作用;流式细胞仪观察雷公藤甲素作用后对细胞周期及凋亡的影响。结果:倒置相差显微镜下示雷公藤甲素作用后细胞生长明显受到抑制;MTT检测显示雷公藤甲素能抑制HEC-1B细胞生长且生长抑制作用呈剂量-时间依赖性;流式细胞仪检测示雷公藤甲素低浓度(5ng/ml)条件下,能诱导HEC-1B细胞发生细胞周期阻滞,主要阻滞在S期,高浓度(40,80ng/ml)雷公藤甲素使细胞主要阻滞在G2/M期,并诱导其凋亡。结论:雷公藤甲素对子宫内膜癌细胞株HEC-1B的生长有抑制作用,使其阻滞在S期和G2/M期,并诱导其凋亡。     

双藤巴布剂中青藤碱、雷公藤甲素的体外释放与体外透皮机制研究

目的:研究双藤巴布剂中青藤碱、雷公藤甲素的体外释放与体外透皮机制;比较以化学单体入药(青藤碱雷公藤甲素巴布剂,STP)与浸膏入药(双藤巴布剂,STEP)释放速率与透皮速率的差异。方法:通过释放度测定法测定体外释放度;通过Franze扩散池法测定药物的体外透皮性,皮肤为裸鼠背部皮肤。结果:STP与STEP中青藤碱、雷公藤甲素的体外释放以Higuchi方程拟合度较优(STP:r青=0.9955,r甲=0.9958;STEP:r青=0.9920,r甲=0.9963)。经皮渗透较好地拟合了零级动力学方程,青藤碱在STP、STEP中的r分别为0.9951与0.9926,透皮速率分别为21.729μg.cm-2.h-1与20.063μg.cm-2.h-1;雷公藤甲素在STP、STEP中的r分别为0.9942与0.9902,透皮速率分别为0.4783μg.cm-2.h-1与0.4168μg.cm-2.h-1。结论:青藤碱、雷公藤甲素在STP、STEP中的释放速率大于其经皮渗透速率,属于皮肤限速型经皮给药制剂。     

体外观察雷公藤内酯醇对乳腺癌细胞MCF-7增殖及凋亡的影响

目的:探讨雷公藤内酯醇(triptolide,TP)对人乳腺癌细胞MCF-7增殖及凋亡的影响。方法:MTT法检测不同条件下TP对MCF-7细胞的增殖抑制作用;倒置显微镜观察TP对MCF-7细胞形态学影响;流式细胞仪检测MCF-7细胞的凋亡情况;Caspase检测试剂盒测定Caspase-3,9的变化。结果:TP以剂量及时间依赖性方式明显抑制MCF-7细胞的增殖;TP作用MCF-7细胞后,细胞出现明显的形态学改变(形态不规则、脱落,细胞碎片等)。流式细胞仪检测5μg/mL的TP明显诱导MCF-7细胞凋亡;Caspase-3,9表达水平明显升高,和对照组相比差异有统计学意义(P<0.05)。结论:TP可能通过线粒体通路诱导MCF-7细胞凋亡而发挥其抑制作用。     

雷公藤甲素对急性髓系白血病伴FMS样酪氨酸激酶3内部串联重复细胞株MV411增殖和凋亡的影响及其机制研究

目的 探讨雷公藤甲素(TP)对急性髓系白血病伴FMS样酪氨酸激酶3内部串联重复(FLT3-ITD)的细胞株MV411增殖和凋亡的影响,及其对PI3K-Akt-mTOR通路的作用.方法 四甲基偶氮唑盐(MTT)法测定不同浓度TP作用24、48、72 h时MV411细胞的增殖抑制率;流式细胞术检测48、72 h的细胞凋亡率;实时荧光定量聚合酶链反应(PCR)检测PI3K-Akt-mTOR通路相关基因FLT3、PTEN、PI3K、Akt、mTOR mRNA的表达.结果0、5、10、20、40、80 nmol/L TP作用24、48、72 h,MV411细胞增殖受到抑制,呈现时间-剂量依赖性.0、10、20 nmol/L TP作用48 h后,MV411细胞平均早期凋亡率分别为(3.30±0.20)%、(17.10±0.36)%、(35.67±0.61)%,作用72 h后分别为(7.37±0.32)%、(49.33±0.40)%、(68.92±0.11)%,同一时间各浓度组间差异均有统计学意义(均P=0.000).TP能明显降低FLT3、PI3K、Akt、mTOR mRNA的表达,升高PTEN mRNA的表达.结论TP可能通过影响PI3K-Akt-mTOR通路相关基因的表达发挥抑制MV411细胞增殖、诱导其凋亡的作用.     

Injectable thermo-responsive nano-hydrogel loading triptolide for the anti-breast cancer enhancement via localized treatment based on “two strikes” effects

The clinical application of triptolide (TPL) in tumor therapy has been greatly limited by its toxicity and inefficient delivery. Herein, a localized and sustained-release thermo-sensitive hydrogel was developed for the intra-tumor administration of TPL. Based on the amphiphilic structure of poly (N-isopropylacrylamide-co-acrylic acid)-g-F68 copolymer, it was able to form nano-micelles to efficiently encapsulate TPL, and then turn into a hydrogel at 37 °C. TPL@nano-gel exhibited a sustained drug release profile in vitro and a stronger anticancer effect caused by “two strikes”. The “first strike” was its enhanced cytotoxicity compared to free TPL, due to the enhanced pro-apoptosis effect observed in both MDA-MB-231 and MCF-7 cells caused by the regulation of endogenous mitochondrial pathways. Furthermore, TPL@nano-gel exhibited a “second-strike” through its anti-angiogenesis capabilities mediated through VEGFR-2 signaling inhibition. As expected, after intra-tumoral injection at a 0.45 mg/kg TPL-equivalent dose three times over 14 days in 4T1 tumor-bearing mice, TPL@nano-gel led to lower systemic toxicity and higher antitumor efficacy compared to multiple injections of TPL. In this regard, these findings indicate that this injectable thermo-responsive hydrogel carries great potential for TPL as a safe and effective cancer therapy.     

Triptolide mitigates radiation-induced pulmonary fibrosis via inhibition of axis of alveolar macrophages-NOXes-ROS-myofibroblasts

Purpose: IR-induced pulmonary fibrosis is one of the most severe late complications of radiotherapy for lung cancer. It is urgently needed to discover a new drug for anti-IR lung fibrosis. Our previous studies have indicated that TPL exhibits both anti-IR lung fibrosis and anti-tumor activities. To reveal the mechanism of TPL on anti-IR lung fibrosis, alveolar macrophages (AMs) were examined for TPL effect on their axis of Nicotinamide adenine dinucleotide phosphate oxidase-reactive oxygen species (NOXes-ROS) and myofibroblast activation. Methods and Materials: The fibrosis-prone C57BL/6 mice were irradiated with 15 Gy on whole chest, then one day later, mice were treated without or with TPL (i.v. 0.25 mg/kg, qod for 1 month). The AMs were collected from bronchoalveolar lavage fluids and studied for the production of ROS and the levels of NOXes. The effect of AMs on myofibroblast activation as labeled with F4/80 or α-SMA (α-smooth muscle actin) were examined using flow cytometry, Western blotting, or immunohistochemical staining. Results: TPL effectively reduced the IR-induced lung fibrosis as evidenced by the less myofibroblasts, less collagen deposit and less ROS in the IR-lung tissues. We found that ROS which responsible for myofibroblasts activation was mainly from AMs and was NOX2 and NOX4 dependent. TPL significantly reduced the infiltrated AMs in IR-lung tissues, and in addition, down regulated the level of NOX2 and NOX4 in AMs both in vitro and in vivo. Furthermore, by inhibiting NOXes dependent ROS in AMs, TPL deprived AMs' paracrine activation of myofibroblasts. Conclusions: Our work demonstrated that the anti-fibrotic effect of TPL on IR-induced pulmonary fibrosis was related to its inhibition on the axis of alveolar macrophages-NOXes-ROS-myofibroblasts.     

Triptolide Inhibits Breast Cancer Cell Metastasis Through Inducing the Expression of miR-146a,a Negative Regulator of Rho GTPase

Triptolide, an extract of Tripterygium wilfordii, has been shown to have a potent anticancer activity. In the present study, it was found that triptolide could effectively induce apoptosis and inhibit proliferation and invasion in malignant MDA-MB-231 breast cancer cells. The study focused on its effect on inhibiting invasion, which has not been extensively reported to date. We predicted that triptolide may change invasion activity via microRNAs (miRNAs), which have been recognized as important regulators of gene expression. miRNAome variation in MDA-MB-231 cells with or without triptolide treatment demonstrated that miR-146a was upregulated following treatment with triptolide. Our previous studies have shown that miR-146a can inhibit migration and invasion by targeting RhoA in breast cancer. This time, we found that miR-146a can target Rac1, another key member of the Rho GTPase family. Luciferase reporter containing Rac1 3′-UTR was constructed to prove this hypothesis. In addition, following treatment with triptolide, the expression of RhoA and Rac1 was found to be decreased. These results indicated that triptolide exerts its anti-invasion activity through a miRNA-mediated mechanism, which indirectly regulates the expression of Rho GTPase. Triptolide combined with miR-146a could improve the effect of triptolide treatment on breast cancer.     

雷公藤内酯醇对致敏大鼠淋巴细胞凋亡的影响

目的 探讨雷公藤内酯醇（TP）体内外对致敏大鼠淋巴细胞淋巴细胞凋亡的影响。方法 采用卵蛋白（OVA）致敏并反复刺激建立过敏性气道炎症模型,24只SD大鼠随机分为正常组、阳性对照组和TP处理组,每组8只。用TUNEL原位末端标记法,DNA电泳及电镜等方法,观察体内外TP对致敏大鼠淋巴细胞凋亡的影响及机制。结果 致敏大鼠BALF中嗜酸性粒细胞（Eos)、淋巴细胞均较正常组明显增高（P＜0．05）。体内应用TP可减少致敏大鼠BALF中Eos、淋巴细胞数目,同时可增加其BALF中淋巴细胞凋亡百分率。体外实际显示,不同剂量TP呈剂量依赖性（10^-7-10^5g/ml）的促进OVA抗原刺激的脾淋巴细胞凋亡,该效应随作用时间凋亡,可能是其抗炎机制之一,并可能是通过Fas/FasL途径发挥作用;同时TP可明显增加DXM的促淋细胞凋亡作用。为阐明TP对抗哮喘气道炎症的作用机制和探讨激素依赖性哮喘治疗的新途径提供了有意义的实验资料。