Bornyl caffeate induces apoptosis in human breast cancer cells in vitro via the ROS- and JNK-mediated pathways

Aim： To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms.
Methods： Human breast cancer cell line MCF-7 and other tumor cell lines （T47D, HepG2, HeLa, and PC12） were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential （MMP） was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species （ROS） were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis.

Results： Bornyl caffeate （10, 25, and 50 μmol/L） suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 μmol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 （p38 inhibitor）, SP600125 （JNK inhibitor）, z-VAD （pan-caspase inhibitor） or the thiol antioxidant L-NAC.

Conclusion： Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways.     

Abrus agglutinin suppresses human hepatocellular carcinoma in vitro and in vivo by inducing caspase- mediated cell death

Aim： Abrus agglutinin （AGG） from the seeds of Indian medicinal plant Abrus precatorius belongs to the class II ribosome inactivating protein family. In this study we investigated the anticancer effects of AGG against human hepatocellular carcinoma in vitro and in vivo.
Methods： Cell proliferation, DNA fragmentation, Annexin V binding, immunocytofluorescence, Western blotting, caspase activity assays and luciferase assays were performed to evaluate AGG in human liver cancer cells HepG2. Immunohistochemical staining and TUNEL expression were studied in tumor samples of HepG2-xenografted nude mice.
Results： AGG induced apoptosis in HepG2 cells in a dose- and time-dependent manner. AGG-treated HepG2 cells demonstrated an increase in caspase 3/7, 8 and 9 activities and a sharp decrease in the Bcl-2/Bax ratio, indicating activation of a caspase cascade. Co-treatment of HepG2 cells with AGG and a caspase inhibitor or treatment of AGG in Bax knockout HepG2 cells decreased the caspase 3/7 activity in comparison to HepG2 cells exposed only to AGG. Moreover, AGG decreased the expression of Hsp90 and suppressed Akt phosphorylation and NF-κB expression in HepG2 cells. Finally, AGG treatment significantly reduced tumor growth in nude mice bearing HepG2 xenografts, increased TUNEL expression and decreased CD-31 and Ki-67 expression compared to levels observed in the untreated control mice bearing HepG2 cells.
Conclusion： AGG inhibits the growth and progression of HepG2 cells by inducing caspase-mediated cell death. The agglutinin could be an alternative natural remedy for the treatment of human hepatocellular carcinomas.     

Puerarin inhibits angiotensin II-induced cardiac hypertrophy via the redox-sensitive ERK1/2, p38 and NF-κB pathways

Aim： To investigate the effects of puerarin （Pue）, an isoflavone derived from Kudzu roots, on angiotensin II （Ang II）-induced hypertrophy of cardiomyocytes in vivo and in vitro.
Methods： C57BL/6J mice were infused with Ang II and treated with Pue （100 mg·kg-1·d-1, po） for 15 d. After the treatment, systolic blood pressure （SBP） and left ventricular wall thickness were assessed. The ratios of heart weight to body weight （HW/BW） and left ventricular weight to body weight （LVW/BW） were determined, and heart morphometry was assessed. Expression of fetal-type genes （ANP, BNP and β-MHC） in left ventricles was measured using semi-quantitative RT-PCR. Mouse primary cardiomyocytes were treated with Pue （50, 100, 200 μmol/L）, then exposed to Ang II （1 μmol/L）. ROS level was examined with flow cytometry, the binding activity of NF-κB was determined using EMSA. Western blot was used to measure the levels of ERK1/2, p38 and NF-κB pathway proteins. [3H]leucine incorporation was used to measure the rate of protein synthesis.
Results： Oral administration of Pue significantly suppressed Ang II-induced increases in the myocyte surface area, HW/BW, LVW/BW, SBP and left ventricular wall thickness. Furthermore, Pue significantly suppressed Ang II-induced increases in ANP, BNP and β-MHC expression in the left ventricles in vivo. Treatment of cardiomyocytes with Pue （50–500 μmol/L） did not affect the viability of cardiomyocytes in vitro. Pretreatment of cardiomyocytes with Pue dose-dependently inhibited Ang II-induced increases in ROS production, NF-κB binding activity, protein synthesis and cell breadth. Furthermore, pretreatment with Pue significantly suppressed Ang II-induced activation of ERK1/2, p38 and the NF-κB pathway proteins and the expression of ANP and β-MHC in cardiomyocytes. The positive drug valsartan exerted similar effects on Ang II-induced cardiac hypertrophy in vivo and in vitro.
Conclusion： Pue attenuates Ang II-induced cardiac hypertrophy by inhibiting activation of the redox-sensitive ERK1/2, p38 and the NF-κB pathways.     

Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release

Aim： Paeonol （2＇-hydroxy-4＇-methoxyacetophenone） from Cortex moutan root is a potential therapeutic agent for atherosclerosis. This study sought to investigate the mechanisms underlying anti-inflammatory effects of paeonol in rat vascular endothelial cells （VECs） in vitro.
Methods： VECs were isolated from rat thoracic aortas. The cells were pretreated with paeonol for 24 h, and then stimulated with ox-LDL for another 24 h. The expression of microRNA-21 （miR-21） and PTEN in VECs was analyzed using qRT-PCR. The expression of PTEN protein was detected by Western blotting. TNF-α release by VECs was measured by ELISA.

Results： Ox-LDL treatment inhibited VEC growth in dose- and time-dependent manners （the value of IC50 was about 20 mg/L at 24 h）. Furthermore, ox-LDL （20 mg/L） significantly increased miR-21 expression and inhibited the expression of PTEN, one of downstream target genes of miR-21 in VECs. In addition, ox-LDL （20 mg/L） significantly increased the release of TNF-α from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose- and time-dependent manners. Moreover, paeonol （120 μmol/L） prevented ox-LDL-induced increases in miR-21 expression and TNF-α release, and ox-LDL-induced inhibition in PTEN expression. A dual-luciferase reporter assay showed that miR-21 bound directly to PTEN＇s 3＇-UTR, thus inhibiting PTEN expression. In ox-LDL treated VECs, transfection with a miR-21 mimic significantly increased miR-21 expression and inhibited PTEN expression, and attenuated the protective effects of paeonol pretreatment, whereas transfection with an miR-21 inhibitor significantly decreased miR-21 expression and increased PTEN expression, thus enhanced the protective effects of paeonol pretreatment.

Conclusion： miR-21 is an important target of paeonol for its protective effects against ox-LDL-induced VEC injury, which may play critical roles in development of atherosclerosis.     

不同剂量药物调控多囊卵巢患者诱导卵泡发育与排卵的阴道超声监测比较

目的 探讨经阴道超声监测在不同剂量调控下CC＋HMG诱导多囊卵巢患者卵泡发育与排卵结果 的比较。方法 选取南京大学医学院附属鼓楼医院2012年12月至2013年5月收治的75例需促排卵的多囊卵巢患者,根据药物剂量的不同随机分为常规加量组（40例）和低剂量加量组（35例）,通过CC联合HMG step—up—down方案进行卵泡诱导的临床资料进行分析。结果 两组超声监测表现为卵泡发育成熟并排卵、卵泡黄素化未破和小卵泡或闭锁等症状,所占比例分别为66．7％、21．3％、12．0％;常规加量组中9例多枚（≥3枚）卵泡发育成熟并排卵,比例为33．3％;低剂量加量组中3例多枚（≥3枚）卵泡发育成熟并排卵,比例为13．0％。结论 对于自然周期监测排卵卵泡发育不良及排卵异常的多囊卵巢患者进行CC联合HMG促排卵治疗,每次加量增加半支尿促性素（HMG）的组可以较好的控制优势卵泡发育个数,从而减少多枚（≥3）卵泡发育成熟并排卵的比例,降低多胎及腹水的发生风险。临床中对于药物诱导卵泡发育与排卵过程中实施超声检测,能够准确的观察卵泡生长,并指导临床合理的给药,促进卵泡的更好成熟与排卵。     

积极心理学在血液透析诱导期的心理护理干预

目的：分析血液透析诱导期的心理问题,探讨研究积极心理学在血液透析诱导期的心理护理干预。方法：选取2012年1月-2013年6月在我院进行血液透析诱导期患者70例,根据诱导期普遍存在的问题,实施有针对性的护理干预。结果：血液透析诱导期不同程度存在心理问题,通过积极的护理干预,患者的心理状况有所改善,提高了患者的依从性和透析质量。结论：将积极心理学应用于血液透析诱导期的患者,有助于减轻患者的消极心理反应,促进患者顺利进入维持性血液透析阶段,减少血透并发症,提高生活质量。     

miR-124 靶向调控CMTM6 增强CIK 对胶质瘤细胞杀伤效应机制研究

目的 研究细胞因子诱导杀伤（cytokine induced killer ,CIK）细胞对胶质瘤细胞杀伤效应的影响以及微小核糖核酸（miRNA,miR）-124 在此过程中的调节作用及可能机制。方法 收集2017 年3 月～ 2019 年10 月鄂州市中心医院神经外科收治的30 例脑胶质瘤患者的癌组织及癌旁组织标本,通过qRT-PCR 法检测脑胶质瘤及癌旁组织中miR-124,含MARVEL 跨膜结构域的趋化素样因子家族基因6 (chemokine-like factor-like MARVEL transmembrane domaincontainingfamily member 6,CMTM6) mRNA 表达量及相关性。诱导培养CIK 细胞后,分别与胶质瘤细胞C6 和U87共培养,另转染miR-124 mimic,inhibitor 及阴性对照序列,通过CCK-8 实验检测CIK 细胞及转染对胶质瘤细胞的杀伤效应;双荧光素酶报告实验分析miR-124 与CMTM6 基因的靶向关系,qRT-PCR 和Western blot 检测胶质瘤细胞中miR-124,CMTM6 mRNA 及蛋白表达。结果 与癌旁组织比较,脑胶质瘤组织中miR-124 表达(0.325±0.031 vs1.004±0.086) 显著降低,CMTM6 mRNA 表达(3.167±0.324 vs 0.981±0.089) 显著升高,差异有统计学意义（t=39.051,26.337,均P ＜ 0.001）,二者呈负相关（r2=0.262）。与转染阴性对照比较,miR-124 mimic 转染及其与CIK 细胞共培养对C6(72.84%±3.81% vs 99.67%±3.45%,51.46%±3.18% vs 74.59%±3.47%),U87 细胞(71.93%±3.94% vs98.26%±3.59%,52.67%±3.24% vs 76.28%±3.64%) 增殖均有显著抑制作用,差异有统计学意义（q=16.340,15.486,14.087, 13.886,均P ＜ 0.001）。双荧光素酶报告实验显示,miR-124 和CMTM6 具有明显的靶向关系。Western blot 结果显示,与转染阴性对照+CIK 组比较miR-124 mimic 与CIK 细胞共培养的C6,U87 细胞CMTM6蛋白表达(0.435±0.042vs 0.897±0.061,0.354±0.029 vs 0.725±0.068) 显著降低,转染miR-124 inhibitor 与CIK 细胞共培养的C6,U87 细胞CMTM6 蛋白表达(1.142±0.121 vs 0.897±0.061,1.326±0.125 vs 0.725±0.068) 显著增加,差异均有统计学意义（q=13.817,10.839, 7.327,17.558,均P ＜ 0.001）。结论 CIK 细胞可有效杀伤胶质瘤细胞,miR-124 可通过靶向抑制CMTM6 增强CIK 细胞对胶质瘤细胞的杀伤效应。     

TREK-1激动剂亚麻酸对星形胶质细胞谷氨酸诱导电流和摄取谷氨酸的影响

目的观察双孔钾通道TREK-1激动剂亚麻酸对星形胶质细胞谷氨酸诱导电流和摄取谷氨酸的影响。方法应用大鼠海马脑片膜片钳技术研究TREK-1激动剂亚麻酸对星形胶质细胞谷氨酸诱导电流的影响;原代培养大鼠皮层星形胶质细胞,采用免疫荧光双标法检测TREK-1在星形胶质细胞的表达,采用谷氨酸浓度检测法观察TREK-1激动剂亚麻酸对星形胶质细胞摄取谷氨酸的影响。结果 TREK-1在星形胶质细胞上表达丰富,谷氨酸可诱导星形胶质细胞产生内向电流,与对照组相比,给予TREK-1激动剂亚麻酸干预后星形胶质细胞谷氨酸诱导电流显著增加(P0.05),并显著增强原代培养的星形胶质细胞摄取谷氨酸能力(P0.05)。结论 TREK-1在星形胶质细胞上表达,TREK-1激动剂亚麻酸显著增加星形胶质细胞谷氨酸诱导电流,增强原代培养的星形胶质细胞摄取谷氨酸能力,提示TREK-1活性改变与星形胶质细胞平衡缓冲功能密切相关。     

电针配伍穴位通过HIF-1α/VEGF通路对大鼠脊髓损伤后运动功能的影响

目的：观察电针配伍穴位对急性脊髓损伤（SCI）后大鼠运动功能的影响及脊髓神经元缺氧诱导因子1α(HIF-1α)、血管内皮生长因子（VEGF）表达变化,探讨电针促进大鼠运动功能恢复作用及其可能机制。方法：108只雌性SD大鼠,随机分为假手术组36只（Sham组）、模型组36只（SCI组）和电针组36只（SCI+EA组）。以T10为中心,用HI—0400脊髓打击器构建大鼠SCI模型;Sham组只去除椎板,不损伤脊髓。SCI+EA组术后1d开始电针干预,穴位配伍为大椎、至阳、命门、夹脊、足三里、三阴交,每日一次,每次30min,干预28d。于损伤后1d、3d、7d、14d、21d、28d 6个时间点,采用BBB评分评估大鼠后肢运动功能变化;HE染色观察损伤脊髓节段组织结构变化;免疫荧光法、Western Blot法检测各组大鼠脊髓组织中HIF-1α、VEGF蛋白定位及表达变化。结果：(1)BBB运动功能评分SCI+EA组14d后明显升高,与SCI组比较有差异（P<0.05）;(2)HE染色Sham组脊髓结构完整,神经元形态清晰。SCI组3d有打击空洞,出血。7d、14d可见坏死神经元,...     

不同浓度维甲酸诱导体外培养人羊膜上皮细胞分化为神经样细胞的研究

目的探讨维甲酸诱导人羊膜上皮细胞向神经样细胞分化。方法以DMEM/F12培养基培养原代人羊膜上皮细胞,并将细胞分为5组:对照组;1×10-8mol·L-1维甲酸组;1×10-7mol·L-1维甲酸组;1×10-6mol·L-1维甲酸组;1×10-5mol·L-1维甲酸组。各维甲酸组经维甲酸处理7 d后,用免疫组化检测神经干细胞巢蛋白(nestin)、微管相关蛋白2(MAP-2)、神经胶质纤维酸性蛋白(GFAP)和细胞全能性标记物Oct-4表达,并计数阳性细胞比例。结果与对照组比较,各维甲酸组显著提高人羊膜上皮细胞分化为神经样细胞的比例,其中1×10-6mol·L-1维甲酸组阳性细胞比例最高;结论维甲酸体外能诱导人羊膜上皮细胞向神经样细胞分化,最适诱导浓度为1×10-6mol·L-1。