Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis

Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin. Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTr assay. Fluorescent microscopic analysis of apoptotic cells stained with 4‘ , 6‘-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis. Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK) , inhibitor PD 98059. Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.     

正交实验法优化紫草素类脂囊泡的处方工艺

目的优选紫草素类脂囊泡的处方工艺。方法采用薄膜分散法制备紫草素类脂囊泡,以紫草素与司盘60的质量比、司盘60与胆固醇的用量比、磷酸盐缓冲液的用量以及水化时间作为考察因素,将包封率作为考察指标,进行正交实验优化制备工艺。用HPLC法测定包封率,用显微镜观察类脂囊泡的外观形态,并用粒径测定仪测定类脂囊泡的粒径。结果最佳制备工艺为:紫草素与司盘60的比例为1∶20;司盘60与胆固醇的比例为1∶1;磷酸盐缓冲液的用量为15mL;水化时间为30min。所得类脂囊泡的包封率为81.6%;形态为粒径均匀的球形和类球形,粒径为387.6nm,多分散指数PDI为0.476。结论该制备工艺稳定、可行、重复性好,可为工业化生产提供理论依据。     

PEG修饰的紫草素纳米结构脂质载体的制备和体外评价

[目的] 制备一种有长循环效果的聚乙二醇（PEG）修饰的紫草素纳米结构脂质载体,并对其进行理化表征和体外抗肿瘤效果评价。[方法] 采用乳化蒸发-低温固化法制备紫草素纳米结构脂质载体,通过超速离心法检测包封率;通过粒径、多分散指数（PDI）、Zeta电位、透射电镜、差示扫描量热、X射线等对制剂进行表征。采用CCK-8法考察乳腺癌MCF-7细胞的活性,以香豆素-6和Hoechst 33342为荧光探针定量考察细胞摄取行为。[结果] PEG修饰的紫草素纳米结构脂质载体粒径为（19.68±1.25）nm,多分散指数为（0.28±0.68）,Zeta电位为[-（20.27±1.27）]mV。平均包封率为98%,制剂外观圆整,分布均匀。紫草素以无定型物包载于制剂中。紫草素的抗肿瘤作用呈现浓度依赖性,中、高剂量的PEG修饰制剂组抗肿瘤活性明显高于未修饰制剂组和溶液组。细胞摄取实验结果与细胞毒性实验结果一致。[结论] 实验制备的PEG修饰的紫草素纳米结构脂质载体包封率较高,粒径小,体系稳定,具有良好的体外抗肿瘤和细胞摄取效果。     

紫草素及其衍生物抗妇科肿瘤作用研究进展

紫草素是从传统中药紫草中提取的萘醌类化合物,具有抗炎、抗氧化、抗肿瘤、促进伤口愈合等作用。近年来,紫草素及其衍生物的抗肿瘤作用得到广泛研究。多项体外和体内研究表明紫草素及其衍生物可有效抑制乳腺癌、宫颈癌等妇科恶性肿瘤的发生与发展。从阻滞细胞周期、促进肿瘤细胞凋亡、抑制癌细胞转移和侵袭、诱导自噬和坏死等方面综述近几年紫草素及衍生物在抗妇科肿瘤领域的研究进展,为紫草素类化合物在妇科肿瘤中的临床研究提供参考。     

Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo

Dysregulation of the ubiquitin‐proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell‐killing activity, and results from a clinical study using a shikonin‐containing mixture demonstrated its safety and efficacy for the treatment of late‐stage lung cancer. In this study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C₁ and C₄ of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin‐like activity of purified 20S proteasome (IC₅₀ 12.5 μmol/L) and tumor cellular 26S proteasome (IC₅₀ between 2–16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC‐3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB‐α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC‐3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin and inhibition of the proteasome activity by shikonin contributes to its antitumor property. © 2008 Wiley‐Liss, Inc.     

高效液相色谱测定条件的耐用性试验研究

目的：本研究以紫归敏康片中β,β′-二甲基丙烯酰紫草素含量测定确定的色谱条件为研究对象,考察其含量测定高效液相色谱（HPLC）条件的耐用性。方法：在不同的检测波长、流速、柱温、流动相配比、流动相PH、色谱柱等仪器条件下,对制剂中的指标性成分β,β′-二甲基丙烯酰紫草素进行含量测定。结果：在测试范围内,检测波长、流动相配比、流动相PH、色谱柱对含量测定无明显影响（RSD〈3.0%）,流速、柱温有较大影响（RSD〉3.0%）。结论：通过耐用性试验考察,确定了含量测定色谱条件的适宜性,保证了含量测定的准确性、重现性和可信性。     

紫草素对宫颈癌Hela细胞增殖、侵袭和迁移的影响

目的:研究紫草素(shikonin)对人宫颈癌Hela细胞增殖、侵袭和迁移能力的影响,并对其机制进行初步探讨。方法:体外培养Hela细胞,分为对照组和紫草素组(浓度分别为4,8,16,32,64μmol·L-1),采用MTT法检测紫草素对Hela细胞增殖的抑制作用;紫草素处理Hela细胞24h后,采用流式细胞术观察细胞凋亡率的改变;Transwell小室法检测细胞侵袭和迁移力的变化;Western blotting法分析黏着斑激酶(FAK)蛋白表达及FAK磷酸化水平的变化;实时荧光定量PCR技术检测FAK mRNA表达的情况。结果:紫草素对Hela细胞的增殖具有抑制作用,且具有浓度和时间依赖性;细胞凋亡率随着紫草素浓度的增加而逐步增高,其中32,64μmol·L-1的紫草素可明显抑制细胞增殖并诱导其凋亡(P<0.001)。4,8,16μmol·L-1的紫草素作用于Hela细胞后,能够显著降低Hela细胞的穿膜细胞数(P<0.05)。紫草素能够呈浓度依赖性的下调Hela细胞中FAK蛋白和mRNA的表达,使FAK磷酸化水平降低。结论:紫草素可显著抑制Hela细胞的增殖、侵袭和迁移能力,其机制可能与抑制FAK磷酸化水平有关。     

吴紫糊中左旋紫草素的含量测定

目的：建立吴紫糊中左旋紫草素的测定方法。方法：高效液相色谱法,色谱条件色谱柱为安捷伦C18柱（4.6mm×250mm,5μm）,流动相为甲醇-0.1%磷酸盐缓冲液（80∶20）,检测波长516nm,柱温25℃,流速10mL/min。结果：进样量在8.2∽41.0μg/mL范围内,线性关系良好（r=0.999 9）平均回收率为101.30%（RSD为1.55%,n=9）。结论：高效液相色谱法测定吴紫糊中左旋紫草素含量结果准确、重现性好,适用于吴紫糊的质量控制。     

Studies on in vitro metabolism of shikonin

Shikonin is one of the active components isolated from the root of Arnebia euchrona (Royle) Johnst. It has been shown to possess significant antibacterial, antiinflammatory and antitumour activities and has been used clinically. In this paper, rat liver microsomes were incubated in vitro to study the metabolism of shikonin and a reverse‐phase high performance liquid chromatography/photodiode array detector (DAD) method was used to separate and detect the metabolites. The effects of several factors on the metabolism were investigated and the metabolic system was optimized. The main metabolites were purified by RP‐HPLC and their structures were determined by UV, ¹H‐NMR and MS. S‐1 was dihydroxylated shikonin, S‐2 was 2‐OH shikonin and S‐3 was 6‐OH or 7‐OH shikonin. Copyright © 1999 John Wiley & Sons, Ltd.     

乙酰紫草素联合奥沙利铂对人结肠癌HT29细胞增殖及凋亡的影响

目的探讨乙酰紫草素联合奥沙利铂对人结肠癌HT29细胞增殖及凋亡的影响。方法将人结肠癌HT29细胞分为空白组、乙酰紫草素组(10μmol/L)、奥沙利铂组(1μmol/L)和联合给药组(10μmol/L乙酰紫草素+1μmol/L奥沙利铂),MTT法检测HT29细胞增殖抑制率,流式细胞术检测HT29细胞凋亡率、qRT-PCR法检测HT29细胞Ki-67、Bcl-2及Bax mRNA表达,Western blot法检测磷酸化磷脂酰肌醇-3-羟激酶(p-PI3K)、磷酸化蛋白激酶B (p-Akt)蛋白表达。结果人结肠癌HT29细胞经乙酰紫草素、奥沙利铂和联合给药处理24、48、72 h后,细胞增殖抑制率均增加,其中联合给药组细胞增殖抑制率最高(P<0. 05)。药物处理HT29细胞48 h后,与空白组比较,乙酰紫草素组、奥沙利铂组和联合给药组凋亡率、Bax mRNA表达增加(P<0. 05),而Ki-67、Bcl-2 mRNA表达及p-PI3K、p-Akt蛋白表达降低(P<0. 05);联合给药组的细胞凋亡率、Bax mRNA表达均高于乙酰紫草素组和奥沙利铂组,Ki-67、Bcl-2 mRNA表达及p-PI3K、p-Akt蛋白表达低于乙酰紫草素组和奥沙利铂组。结论乙酰紫草素和奥沙利铂对人结肠癌HT29细胞均具有抑制增殖及诱导凋亡作用,可能与调控PI3K/Akt信号通路有关,且两者联用时效果更好。