异甘草素对小儿脑胶质瘤细胞增殖、凋亡的影响及相关机制研究

目的 研究异甘草素对脑胶质瘤U251细胞增殖、凋亡的影响,并探讨其相关分子机制.方法 取对数生长期人脑胶质瘤U251细胞,用不同浓度(0、10、20、40、60、80μmol/L)异甘草素处理,每个浓度设5个复孔.异甘草素处理24h、48h、72h后,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖能力,采用流式细胞术检测细胞凋亡的变化,逆转录-聚合酶链反应(RT-PCR)法检测FoxM1 mRNA的表达水平.结果 U251细胞经过浓度为0、10、20、40、60、80μmol/L的异甘草素处理24h、48h及72h后,细胞增殖均不同程度地受到抑制.在相同作用时间,不同浓度之间细胞增殖情况比较差异有统计学意义(F值分别为38.5、98.9、108.1,均P＜0.05);在相同异甘草素浓度,不同作用时间(24h、48h、72h)细胞增殖情况比较差异均有统计学意义(F值分别为11.4、20.8、43.1、53.9、30.4,均P＜0.05).不同浓度异甘草素作用于U251细胞24h后,随着异甘草素浓度的增加,细胞凋亡率增加(F=120.3,P＜0.05).不同浓度异甘草素处理U251细胞48h后,FoxM1 mRNA相对表达量分别为(71.2±5.1)％、(54.6±3.3)％、(35.4±4.1)％、(20.8±4.0)％,比较差异有统计学意义(F=83.5,P＜0.05).结论 异甘草素体外可有效抑制U251细胞的增殖并诱导其凋亡,其分子机制可能与下FoxM1基因的表达有关.     

异甘草素对人宫颈癌细胞增殖的抑制作用

目的鉴于异甘草素(ISL)对不同肿瘤细胞系的不同作用,研究ISL对人宫颈鳞状上皮癌细胞(CaSki)癌细胞的影响及有关机制。方法体外研究采用MTT法测定细胞增殖;流式细胞仪检测细胞周期;逆转录聚合酶链反应(RT-PCR)分析细胞周期蛋白B1(cyclin B1)mRNA表达;Western免疫印迹分析cyclin B1,P34cdc2和磷酸化P34cdc2(phospho-cdc2,酪氨酸15)蛋白表达;体内研究通过建立人宫颈癌CaSki细胞裸鼠移植瘤模型观察抑瘤率。结果ISL浓度依赖性(10~80μmol.L-1)抑制CaSki细胞增殖,IC50为(19.3±3.3)μmol.L-1,且呈时间依赖性。ISL 20μmol.L-1作用3 d对细胞抑制率为82.3%;ISL浓度依赖性(10~80μmol.L-1)干扰CaSki细胞周期,细胞周期阻滞于S和G2/M期,S和G2/M期的百分率分别从23.1%和8.2%上升到43.2%和32.1%;同时G0/G1期细胞百分率从68.7%下降到24.7%;RT-PCR分析显示,ISL(10~80μmol.L-1)显著抑制cyclin B1 mRNA表达,抑制率最高可达61.7%,且呈时间依赖性;Western免疫印迹分析显示,ISL作用24 h cyclin B1蛋白表达开始不同程度下降,48 h下降明显,P34cdc2变化较小,ISL作用16 h后磷酸化P34cdc2(酪氨酸15)蛋白水平明显改变;ISL(20~500 mg.kg-1.d-1)剂量依赖性抑制CaSki细胞裸鼠皮下移植瘤的生长,抑瘤率分别为36.8%,51.2%和84.6%,且对动物体重和外周血白细胞数均无明显影响。结论ISL可以显著抑制人宫颈癌细胞体内外增殖,其机制与将细胞周期阻滞于S和G2/M期及影响细胞周期因子cyclin B1的表达和改变P34cdc2的磷酸化水平有关。     

基于HPLC多指标成分、化学计量学和熵权优劣解距离法的苏木质量评价研究

目的 采用多指标成分、化学计量学联合熵权优劣解距离法综合评价苏木的质量。方法 采用HPLC法测定不同产地苏木中巴西苏木素、苏木精、原苏木素A、(±)原苏木素B、苏木酮A、鹰嘴豆芽素A、异甘草素和苏木查耳酮;对以上结果进行聚类分析、主成分分析、正交偏最小二乘法-判别分析,寻找影响苏木质量的主要潜在标志物;采用熵权优劣解距离法对不同产区苏木的质量优劣进行评价。结果 18批苏木聚为3类,不同产地苏木质量呈现一定的区域差异。苏木精、巴西苏木素、原苏木素A对苏木质量差异影响较大,是影响苏木质量的主要潜在标志物。广西、广东所产苏木质量最优,其次为四川、贵州和云南,福建地区产品质量较差。结论 本实验建立了苏木质量综合评价方法,操作便捷、可行性高、实用性强,为更加科学、系统地控制苏木质量提供技术支撑。     

Isoliquiritigenin attenuates septic acute kidney injury by regulating ferritinophagy-mediated ferroptosis

Defined differently from apoptosis, necrosis, and autophagy, ferroptosis has been implicated in acute kidney injury (AKI) such as ischemia-reperfusion injury induced AKI, folic acid caused AKI and cisplatin induced AKI. However, whether ferroptosis is involved in LPS induced AKI could be remaining unclear and there is still a lack of therapies associated with ferroptosis in LPS induced AKI without side effects. This study aimed to elucidate the role of isoliquiritigenin (ISL) in ferroptosis of LPS-induced AKI. We used LPS to induce renal tubular injury, followed by treatment with ISL both in vitro and in vivo. Human renal tubular HK2 cells were pretreated with 50 μM or 100 μM ISL for 5 h before stimulation with 2 μg/mL LPS. Mice were administered a single dose of either 50 mg/kg ISL orally or 5 mg/kg ferroptosis inhibitor ferrostatin-1 intraperitoneally before 10 mg/kg LPS injection. We found that LPS could induce mitochondria injury of renal tubular presented as the shape of mitochondria appeared smaller than normal with increased membrane density and are faction or destruction of mitochondrial crista through scanning electron microscope. Ferrostatin-1 significantly protected mice against renal dysfunction and renal tubular damage in LPS-induced AKI. ISL inhibited Fe²⁺ and lipid peroxidation accumulation in LPS-stimulated HK2 cells. It also increased the expression of GPX4 and xCT, reduced the expression of HMGB1 and NCOA4 then attenuated mitochondria injury in renal tubular following LPS stimulation. These results indicated the potential role of ISL against ferritinophagy-mediated ferroptosis in renal tubular following LPS stimulation.     

HPLC-DAD同时分析甘草中7种有效成分

 目的采用HPLC-DAD中梯度洗脱和检测波长时间序列采样的方法,建立甘草(Glycyrrhiza uralensis Fisch.)中4种黄酮成分(甘草苷、异甘草苷、甘草素、异甘草素)和3种甘草皂苷成分(甘草酸、甘草皂苷G₂、乌拉尔甘草皂苷B)的同时分析方法。方法采用C18(4.6 mm×250 mm,5μm)色谱柱。流动相:乙腈-(0.5%醋酸铵+1%冰醋酸),梯度洗脱0～12 min从6∶94到22∶78,12～25 min从22∶78到25∶75,25～45 min从25∶75到40∶60,45～50 min从40∶60到6∶94,保持5 min;检测波长:时间序列采样。结果7种成分的线性关系良好,平均加样回收率在98%～103%之间。结论该方法准确、灵敏度高,可用于甘草药材质量的评价。     

A Review: The Pharmacology of Isoliquiritigenin

Isoliquiritigenin (ISL) is one of the bioactive ingredients isolated from the roots of plants belonging to licorice, including Glycyrrhiza uralensis, Mongolian glycyrrhiza, Glycyrrhiza glabra, and so forth. Liquiritigenin is available in common foods and alternative medicine, and its derivative‐ISL is applied into food additives and disease treatment like cancer therapy, antibiotic therapy, and so on. This review aims at providing a comprehensive summary of the pharmacological activities of ISL. The information published between 1972 and 2014 from a number of reliable sources including PubMed, ScienceDirect, Springer, and Wiley‐Blackwell. The practical application of ISL on the various disease prevention and treatments may stem from its numerous pharmacological properties such as antiinflammatory, anti‐microbial, anti‐oxidative, anticancer activities, immunoregulatory, hepatoprotective, and cardioprotective effects. However, further studies are needed to verify the target‐organ toxicity or side effects investigation. Copyright © 2015 John Wiley & Sons, Ltd.     

异甘草素抗食管癌细胞活性及其机制研究

目的:探讨中药单体异甘草素（isoliquiritigenin,ISL）对人食管鳞癌细胞系EC9706和KYSE450的影响及其作用机制。方法:利用四甲基偶氮唑盐（MTT）法检测ISL对上述2株人食管鳞癌细胞系的细胞毒性;流式细胞术分析ISL对细胞周期及凋亡的影响;Western-blotting技术分析IGF1/IGF-1R信号通路相关蛋白表达以了解ISL的可能作用机制。结果:MTT实验结果表明,ISL以剂量依赖性的方式抑制食管鳞癌细胞EC9706和KYSE450的增殖,IC50值分别为 25.56 μmol/L和27.78 μmol/L。此外,ISL可将2株细胞周期阻滞在G2/M期及以剂量依赖性的方式诱导细胞发生凋亡,并且显著下调IGF-1R表达及抑制其下游PI3K/AKT和RAS/MAPK信号通路的激活。结论:ISL具有很强的抗食管癌活性,其作用机制与其调节细胞周期和促使细胞凋亡有关。本研究首次证明中药单体ISL具有抗食管癌活性,为临床运用中药治疗食管癌提供了新思路。     

Drug Resistance Reversal Potential of Isoliquiritigenin and Liquiritigenin Isolated from Glycyrrhiza glabra Against Methicillin‐Resistant Staphylococcus aureus (MRSA)

Isoliquiritigenin (ISL) and liquiritigenin (LTG) are structurally related flavonoids found in a variety of plants. Discovery of novel antimicrobial combinations for combating methicillin‐resistant Staphylococcus aureus (MRSA) infections is of vital importance in the post‐antibiotic era. The present study was taken to explore the in vitro and in vivo combination effect of LTG and ISL with β‐lactam antibiotics (penicillin, ampicillin and oxacillin) against mec A‐containing strains of MRSA. Minimum inhibitory concentration (MIC) of both LTG and ISL exhibited significant anti‐MRSA activity (50–100 µg/mL) against clinical isolates of MRSA. The result of in vitro combination study showed that ISL significantly reduced MIC of β‐lactam antibiotics up to 16‐folds [∑ fractional inhibitory concentration (FIC) 0.312–0.5], while LTG reduced up to 8‐folds (∑FIC 0.372–0.5). Time kill kinetics at graded MIC combinations (ISL/LTG + β‐lactam) indicated 3.27–9.79‐fold and 2.59–3.48‐fold reduction in the growth of clinical isolates of S. aureus respectively. In S. aureus‐infected Swiss albino mice model, combination of ISL with oxacillin significantly (p < 0.05, p < 0.01, p < 0.001) lowered the systemic microbial burden in blood, liver, kidney, lung and spleen tissues in comparison with ISL, oxacillin alone as well as untreated control. Considering its synergistic antibacterial effect, we suggest both ISL and LTG as promising compounds for the development of novel antistaphylococcal combinations. Copyright © 2016 John Wiley & Sons, Ltd.     

Isoliquiritigenin treatment induces apoptosis by increasing intracellular ROS levels in HeLa cells

This study focuses on the relationship between the apoptosis induced by isoliquiritigenin (ISL) and the production of reactive oxygen species (ROS). Cell viability was evaluated using sulforhodamine B assay. The apoptotic rate was determined via flow cytometry. Intracellular ROS level was assessed using the 2,7-dichlorofluorescein probe assay. Poly-ADP-ribose polymerase (PARP) protein expression was examined using Western blot analysis. The results showed that ISL treatment inhibited cell proliferation by inducing apoptosis. The increased apoptotic rate and ROS production induced by ISL were inhibited by the co-treatment of ISL and free radical scavenger N-acetyl-cysteine (NAC), catalase (CAT), and 4,5-dihydroxyl-1,3-benzededisulfonic acid (Tiron). On the contrary, the increased apoptotic rate and the ROS production were compensated by the co-treatment of ISL and l-buthionine-(S,R)-sulfoximine (BSO). ISL treatment increased the degradation of PARP, which was counteracted by antioxidants (NAC or CAT), whereas the combination treatment of ISL and pro-oxidant (BSO) enhanced the PARP degradation induced by ISL. Our findings suggested that ISL treatment induced apoptosis by increasing intracellular ROS levels in HeLa cells.