冬凌草甲素抑制人卵巢癌细胞恶性行为及机制研究

目的 研究冬凌草甲素（Oridonin）对人卵巢癌（Human ovarian cancer）SKOV3 细胞迁移和侵袭能力的影响及其潜在的分子机制。方法 采用CCK-8 法检测不同浓度（0、5、10、20、40、80μmol/L）的冬凌草甲素作用SKOV3细胞24、48 和72h 后细胞活力的变化,计算半数抑制浓度（IC50）。采用Annexin V-FITC/PI 双染法检测SKOV3细胞凋亡。采用划痕修复实验检测SKOV3细胞的迁移能力,Transwell小室实验检测SKOV3的侵袭能力。Western blot检测SKOV3细胞上皮细胞间充质转化（EMT）蛋白［E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）］和Wnt/β-连环蛋白（β-catenin）信号通路中β-catenin及下游靶分子原癌基因（C-myc）、细胞周期蛋白D1（Cyclin D1）蛋白的表达。结果 冬凌草甲素可抑制SKOV3 细胞活力,且具有时间-剂量依赖性,作用24、48 和72 h后IC50值为23.57、12.48和7.29μmol/L。与对照组比较,5、10和20μmol/L冬凌草甲素作用SKOV3细胞24h 后,细胞凋亡率明显升高,迁移率和侵袭率降低（P<0.05）。与对照组比较,5、10和20μmol/L冬凌草甲素可升高E-cadherin 蛋白表达（P<0.05）,降低Vimentin 表达（P<0.05）。与对照组比较,5、10和20 μmol/L冬凌草甲素可降低β-catenin、C-myc和Cyclin D1表达（P<0.05）。结论 冬凌草甲素具有抑制SKOV3细胞增殖、转移和侵袭能力的作用,该作用与其抑制Wnt/β-catenin 信号通路有关。     

冬凌草甲素对肿瘤细胞钠泵活性的影响

以（８６）Ｒｂ示踪法,观察３种肿瘤细胞的钠系转运活性及冬凌草甲素（Ｏｒｉｄ．）对其的影响。结果表明：（Ｏｒｉｄ．）腹腔注射及体外作用均能显著抑制艾氏腹水瘤细胞的钠系转运功能;体外作用１２小时,半数抑制浓度约为５μｇ／ｍｌ,在此浓度下,药物作用１小时的抑制率为２２％。同时测定人癌细胞系７９０１,Ｈｅｐ－２的钠泵活性高于人胚肺纤维母细胞系ＳＬ,且对Ｏｒｉｄ．较敏感。本文结果提示Ｏｒｉｄ．抗癌机理可能与钠泵活性抑制有关,从生物膜转运角度进一步证明Ｏｒｉｄ．的抗肿瘤作用。     

高效毛细管电泳法测定冬凌草片中冬凌草甲素的含量

目的:建立用高效毛细管电泳测定冬凌草片中冬凌草甲素的含量测定方法.方法:样品用乙醚超声提取,缓冲液为20 mmol·L-1磷酸盐缓冲液,pH=7.0,分离电压15 kM,石英毛细管33 cm×75 μm,操作温度25℃,紫外检测波长238 nm,压力进样6.0 kV×10 s,内标法定量.结果:线性范围为50～400 mg·L-1,平均回收率为99.5%,RSD为3.9%.结论:该方法简便、快速且消耗试剂少,适合于冬凌草片剂的质量控制.     

Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by oridonin nanosuspension

The mechanism for anti-tumor activity of oridonin (ORI) nanosuspension, prepared by the high pressure homogenization method, was studied using MCF-7 human breast carcinoma cells in vitro. MTT assay, observation of morphologic changes, flow cytometric analysis, and western blot analysis indicated that ORI nanosuspension could significantly intensify the in vitro anti-tumor activity to MCF-7 cells, as compared with ORI solution. Furthermore, ORI nanosuspension induced G₂/M stage proliferation arrest and apoptosis in MCF-7 cells depending on its concentration. In addition, western blot analysis indicated that the pro-caspase-3 protein was not cleaved into the activated form and the expression of anti-apoptotic Bcl-2 protein decreased, on the contrary, the expression of pro-apoptotic Bax protein increased in a dose-dependent manner in ORI nanosuspension-treated cells. These observations indicated that the anti-tumor activity of ORI nanosuspension was intensified by cell-cycle arrest and apoptosis induction.     

Oridonin induces ferroptosis by inhibiting gamma‐glutamyl cycle in TE1 cells

Oridonin (Ori) is a natural tetracyclic diterpenoid active compound with excellent antitumor activity, but the mechanism of Ori on esophageal cancer cell, TE1, remains unclear. In this study, we examined the levels of intracellular iron, malondialdehyde, and reactive oxygen species after Ori treatment, while interfering with the effects of Ori with ferroptosis inhibitor, demonstrating that Ori's inhibition of TE1 cell proliferation is associated with ferroptosis. To understand the molecular mechanism of Ori, we performed UPLC–MS/MS metabolomics profiling on TE1 cells, which show that gamma‐glutamyl amino acids (gamma‐glutamylleucine, gamma‐glutamylvaline), 5‐oxoproline, glutamate, GSH, and GSSG are changed significantly after Ori treatment. Meanwhile, the activity of gamma‐glutamyl transpeptidase 1 (GGT1) decreased. This revealed that Ori inhibited the gamma‐glutamyl cycle in TE1 cells. Furthermore, we found that Ori can covalently bind to cysteine to form the conjugate oridonin‐cysteine (Ori‐Cys), resulting in the inhibition of glutathione synthesis, which is consistent with the decrease in the enzymatic activity of glutamate cysteine ligase catalytic subunit (GCLC). Eventually, the value of intracellular GSH/GSSG was reduced, and the enzymatic activity of the glutathione peroxidase 4 (GPX4) was significantly decreased. In conclusion, our experiments indicated that Ori can inhibit the gamma‐glutamyl cycle, thereby inducing ferroptosis to exert anti‐cancer activity.     

Oridonin exhibits anti-angiogenic activity in human umbilical vein endothelial cells by inhibiting VEGF-induced VEGFR-2 signaling pathway

Oridonin has been found to be a potential anti-angiogenesis agent. However, its functional targets and the underlying mechanisms are still vague. In vitro studies we found that oridonin not only inhibited VEGF-induced cell proliferation, migration and tube formation but also caused G2/M phase arrest and triggered cellular apoptosis in HUVECs. In mechanistic studies revealed that oridonin exhibited the anti-angiogenic potency, at least in part, through the down-regulation of VEGFR2-mediated FAK/MMPs, mTOR/PI3K/Akt and ERK/p38 signaling pathways which led to reduced invasion, migration, and tube formation in HUVECs. Our results could provide evidence that oridonin exerts strong anti-angiogenesis activities via specifically targeting VEGFR2 and its signaling pathway.     

正交试验法优选地黄管食通颗粒的乙醇提取工艺

目的 优选地黄管食通颗粒乙醇提取的方法及制备工艺条件.方法 (1)建立了冬凌草中冬凌草甲素荧光暗斑的薄层定量新方法;(2)采用正交试验法,以冬凌草中冬凌草甲素的转移率为指标,优选地黄管食通颗粒的乙醇提取工艺条件.结果 最佳条件为10倍量95%乙醇回流提取3次,每次提取4h.结论 该工艺可以有效提高冬凌草的提取率.     

冬凌草甲素对微卫星不稳定结直肠癌的作用在体研究

目的探索冬凌草甲素对微卫星不稳定的结直肠癌的作用并对其机理进行研究。方法使用Lovo细胞(MSH2缺失结肠癌细胞株)建立裸鼠皮下接种模型,4周后使用该模型生长的肿瘤建立结肠造口的结肠癌原位移植模型,使用不同剂量的冬凌草甲素处理动物模型,观察肿瘤的生长曲线、肿瘤微卫星不稳定和TGFβ1、TGFβ2和IGF片段的大小变化。结果使用5 mg/kg7、.5 mg/kg和10 mg/kg 3个治疗组在注射冬凌草甲素后,在1、2、3、4、5周末,治疗组和对照组肿瘤抑制率差异有统计学意义(p<0.01),而3个治疗组之间差异无统计学意义,使用冬凌草甲素治疗后肿瘤微卫星标志仍呈不稳定状态。结论冬凌草甲素对错配修复基因缺失的结直肠癌有明显抑制作用,但LOVO细胞微卫星不稳定状态并未改变,发生的作用机理与抑制微卫星不稳定无关。     

阿糖胞苷联合冬凌草甲素诱导U937细胞株凋亡的初步研究

目的:探讨阿糖胞苷联合冬凌草甲素诱导白血病细胞株U937凋亡的作用。方法:用阿糖胞苷和冬凌草甲素单药或两药联合处理U937细胞后,采用细胞计数检测细胞增殖,瑞氏染色观察细胞形态,流式细胞术分析细胞磷脂酰丝氨酸外翻情况和线粒体膜电位变化,蛋白印迹法检测凋亡相关分子表达情况。结果:阿糖胞苷和冬凌草甲素单药均能抑制U937细胞增殖,而两药联合抑制作用更加明显,两者能协同诱导U937细胞凋亡,并明显促进细胞磷脂酰丝氨酸外翻和线粒体跨膜电位下降;两药联合较单药组还能明显诱导Caspase-9、Caspase-3活化及PARP的剪切。结论:阿糖胞苷与冬凌草甲素能够协同抑制U937细胞增殖、诱导细胞凋亡,其机制可能是主要通过线粒体途径介导。     

冬凌草甲素对肺动脉高压大鼠肺动脉压的调控作用及机制研究

目的:探讨冬凌草甲素对低氧高二氧化碳肺动脉高压大鼠肺动脉压的调控作用及机制研究。方法:清洁SD大鼠随机分为3组,正常对照组、低氧高二氧化碳组、低氧高二氧化碳加冬凌草甲素组。低氧高二氧化碳时间为4周。测定各组的平均肺动脉压(mPAP)、右心室重量比[LV/(RV S)]、管壁面积/管总面积(WA%)、管壁厚度/血管外径(WT%)、门冬氨酸特异半胱氨酸蛋白酶(caspase)3及9的活力、细胞色素C(Cyt-c)的表达。并用透射电镜观察其超微结构变化。结果:低氧高二氧化碳组mPAP、LV/(RV S)、WA%、WT%均高于正常对照组(P<0.01),caspase3、9活性,Cyt-c表达均低于正常对照组(P<0.01);冬凌草甲素干预组mPAP、LV/(RV S)、WA%、WT%均低于低氧高二氧化碳组(P<0.01),caspase3、9活性,Cyt-c高于低氧高二氧化碳组(P<0.01)。冬凌草甲素组可以发现肺小动脉平滑肌细胞核固缩,核膜间隙增大,线粒体浓聚,部分线粒体肿胀。结论:冬凌草甲素可以有效降低肺动脉压。其机制可能系通过诱导肺动脉平滑肌细胞线粒体途径凋亡,从而抑制肺动脉重构。