SPARCL1通过MEK/ERK信号通路调节非小细胞肺癌细胞的增殖、凋亡和侵袭

摘要 目的：分析富含半胱氨酸的酸性分泌蛋白类似蛋白1（SPARCL1）对非小细胞肺癌（NSCLC）细胞增殖、凋亡、侵袭的影响,并探讨分裂原活化抑制剂（MEK）/细胞外调节蛋白激酶（ERK）通路在其中发挥的作用。方法：收集2019年9月~2021年6月期间本院接受手术治疗的84例NSCLC患者癌组织与相应癌旁组织,实时定量逆转录聚合酶链反应（qRT-PCR）法测定并比较各组织以及正常肺上皮细胞HBEpiC、NSCLC细胞A549、HCC827、H1299、H292中SPARCL1 信使RNA（mRNA）表达水平,选取A549、HCC827培养并分组,分为对照组、NC siRNA组、SPARCL1 siRNA组、U0126组（MEK/ERK特异性抑制剂）、SPARCL1 siRNA加U0126组,细胞计数法（CCK8）以及平板克隆法测定A549、HCC827细胞增殖,流式细胞仪测定A549、HCC827细胞凋亡,Transwell小室法测定A549、HCC827细胞侵袭能力,蛋白质印迹法（western blot）检测SPARCL1、p-MEK、MEK、p-ERK1/2、ERK1/2蛋白表达。结果：SPARCL1在NSCLC组织中mRNA表达水平低于癌旁组织（P＜0.05）;与HBEpiC细胞相比,NSCLC细胞A549、HCC827、H1299、H292细胞中SPARCL1 mRNA表达水平降低（P＜0.05）;与对照组相比,SPARCL1 siRNA组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率降低（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达升高（P＜0.05）,U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）;与SPARCL1 siRNA组相比,SPARCL1 siRNA加U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）。结论：SPARCL1可能通过调控MEK/ERK通路影响NSCLC A549、HCC827细胞增殖、侵袭与凋亡。     

基于JAK2/STAT3信号通路探讨白藜芦醇对骨肉瘤MG-63细胞凋亡及迁移的影响

摘要 目的：研究白藜芦醇(RES)通过蛋白酪氨酸激酶2/信号转导子与激活子3（JAK2/STAT3）信号通路对人骨肉瘤体外细胞株MG-63细胞凋亡、侵袭和迁移的影响。方法：体外培养MG-63细胞,以不同浓度的RES作用于MG-63细胞。Annexin V-FITC/PI双染流式细胞术检测不同时间和不同浓度的RES对MG-63细胞凋亡的影响。划痕实验和Transwell实验检测不同时间和不同浓度的RES对MG-63细胞侵袭和迁移能力的影响。免疫印迹实验检测不同时间和不同浓度的RES对MG-63细胞磷酸化蛋白酪氨酸激酶2（p-JAK2）、磷酸化信号转导子与激活子3（p-STAT3）、凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2家族促凋亡蛋白（Bax）及基质金属蛋白酶（MMP）-2、MMP-9表达的影响。结果：RES浓度越高,时间越久,MG-63细胞凋亡率越高（P<0.05）。RES浓度越高,MG-63细胞迁移和侵袭能力越弱(P<0.05)。RES处理MG-63细胞后其p-JAK2、p-STAT3、Bcl-2以及MMP-2、MMP-9的表达明显降低,而Bax蛋白表达明显升高,且p-JAK2、p-STAT3、Bax、Bcl-2以及MMP-2、MMP-9的表达水平变化具有RES浓度依赖性（P<0.05）。结论：RES可能通过调控JAK2/STAT3信号通路促使人骨肉瘤MG-63细胞凋亡,并抑制MG-63细胞侵袭和迁移。     

微小RNA-451a对胰腺癌BxPc3细胞增殖、侵袭及迁移的影响

摘要 目的：研究微小RNA（miR）-451a对胰腺癌BxPc3细胞增殖、侵袭和迁移的影响。方法：体外培养BxPc3细胞,将不同浓度（50、100 和 200 μmol/L）miR-451a模拟物（mimic）转染至胰腺癌细胞株BxPc3中,利用荧光定量聚合酶链式反应（ PCR）法检测miR-451a的表达,MTT增殖实验检测不同浓度miR-451a对细胞增殖的影响,细胞划痕实验检测不同浓度miR-451a对细胞迁移能力的影响,Transwell实验检测不同浓度miR-451a对细胞侵袭能力的影响。Western blot法检测不同浓度 miR-451a转染后BxPc3细胞p-PI3K、p-AKT、TGF-β和p-smad2/3 蛋白的表达情况。结果：转染不同浓度miR-451a mimic后,胰腺癌BxPc3细胞内miR-451a的相对表达量明显升高,差异有统计学意义（P<0.05）。过表达miR-451a后,BxPc3细胞的增殖能力明显减弱,细胞迁移能力明显减弱,细胞侵袭能力明显减弱,差异均有统计学意义（P<0.05）。相比于0 μmol/L 组,转染50、200 μmol/L miR-451a后BxPc3 细胞p-PI3K、p-AKT、TGF-β和p-smad2/3蛋白表达水平明显降低（P<0.05）,并且p-PI3K、p-AKT、TGF-β和p-smad2/3蛋白表达水平变化具有浓度依耐性。结论：miR-451a能抑制胰腺癌BxPc3细胞株的增殖、迁移、侵袭能力。     

肝癌细胞粘附和收缩调节对其Rho蛋白表达和迁移影响的实验研究

运用定量显微形态分析技术、免疫荧光实验技术和MTT分析法,分别对裱衬纤维连接蛋白(fibronectin,FN)和应用肌球蛋白轻链激酶(myosin light chain kinase,MLCK)抑制剂ML-7作用下,肝细胞L02与肝癌细胞HepG2细胞骨架装配和Rho蛋白表达变化,以及细胞迁移能力进行检测和定量表征,了解肝癌细胞侵袭转移的胞内分子基础。结果显示：1)L02 Rho蛋白表达水平明显低于HepG2,裱衬FN低浓度（1～5 μg/mL）使 L02 Rho蛋白表达进一步下调,HepG2 Rho蛋白表达水平却明显增高,而裱衬 FN高浓度（10～40 μg/mL）L02Rho蛋白升高超过对照组,HepG2 Rho蛋白表达下降,且远低于对照组水平;2）随着裱衬FN浓度的增加,HepG2增殖抑制明显;3）裱衬低浓度FN使HepG2迁移运动能力增强,而高浓度FN使细胞净位移和迁移轨迹离散度减少;4）ML-7低浓度（6 μmol/L）使L02 Rho蛋白表达下调,而对HepG2 Rho蛋白表达无明显影响,L02细胞骨架解聚较HepG2明显;ML-7高浓度（10 μmol/L）HepG2Rho蛋白表达减少,且迁移速率降低;L02 Rho蛋白表达无进一步下调。说明：1)细胞粘附状态对调节HepG2 和L02Rho蛋白表达呈相反趋势;2)HepG2对骨架收缩抑制的响应较L02迟缓;3)HepG2Rho蛋白表达水平与其迁移能力呈正相关。     

L-瓜氨酸对子痫前期中的氧化应激和滋养层细胞侵袭的调控机制研究

摘要 目的：研究L-瓜氨酸（L-Cit）对子痫前期（PE）大鼠模型的治疗作用,及其对氧化应激和滋养层细胞侵袭的影响。方法：建立N-硝基-L-精氨酸甲酯（L-NAME）诱导的PE大鼠模型后,将大鼠分为Control组、PE组、PE+低、中和高剂量L-Cit组（依次为L-L-Cit、M-L-Cit和H-L-Cit组,剂量依次为100、200和500 mg/kg）,n=6,连续给药7 d。在孕第21 d时,检测各组大鼠收缩压（SBP）、24 h尿蛋白、血清超氧化物歧化酶（SOD）和丙二醛（MDA）水平,并对胎盘组织进行苏木精伊红（HE）染色。将HTR-8/Svneo细胞分为Control组、缺氧/复氧（H/R）组、H/R+L-Cit组和H/R+L-Cit+sc-221593组,分别对细胞进行H/R处理,并使用L-Cit（200 μg/mL）和特异性ERK1/2抑制剂sc-221593（10 μmol/L）培养细胞48 h。通过Transwell测定细胞侵袭。通过Western blotting检测total-ERK1/2、p-ERK1/2、total-p38、p-p38、基质金属蛋白酶（MMP）-2和MMP-9的蛋白表达。结果：与PE组比较,L-L-Cit组、M-L-Cit组和H-L-Cit组的SBP和24 h尿蛋白水平均降低（P<0.05）;胎盘组织形态明显改善;血清SOD升高,MDA降低（P<0.05）;胎盘组织中ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高（P<0.05）。与H/R组比较,H/R+L-Cit组的侵袭细胞数量升高（P<0.05）;ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高（P<0.05）。与H/R+L-Cit组比较,H/R+L-Cit+sc-221593组的上述变化均被逆转（P<0.05）。结论：L-Cit可能通过激活ERK/JNK通路减轻PE中的氧化应激并促进滋养层细胞侵袭,从而减轻PE症状。     

脂联素抑制子宫内膜癌HEC-1B细胞增殖、迁移及侵袭的作用及机制研究

摘要 目的：探讨脂联素（APN）对子宫内膜癌HEC-1B细胞增殖、迁移及侵袭的抑制作用及分子机制。方法：分别采用磺酰罗丹明 B(SRB)实验、细胞迁移(Transwell)实验和划痕实验检测子宫内膜癌细胞HEC-1B的增殖、迁移和侵袭能力。采用蛋白免疫印迹（Western blot）法检测腺苷酸活化蛋白激酶(AMPK)信号通路相关蛋白、AdipoR1、AdipoR2、cyclinD1和cyclinE2蛋白表达水平。结果：与对照组相比,APN组HEC-1B细胞增殖、迁移及侵袭功能明显下降（P<0.05）。与对照组相比,APN组p-AMPK/AMPK比值明显提高,而p-mTOR/mTOR和p-4EBP1/4EBP1比值明显下降（P<0.05）。与对照组相比,APN组cyclinD1和cyclinE2蛋白表达水平明显下降（P<0.05）。APN组和对照组的AdipoR1、AdipoR2蛋白表达水平比较无统计学差异（P>0.05）。结论：APN能够激活AMPK信号通路并下调cyclinD1和cyclinE2蛋白表达,进而抑制子宫内膜癌细胞的增殖、迁移和侵袭功能。     

基于Wnt/β-catenin信号通路探讨miR-613对宫颈癌SiHa细胞增殖、迁移与侵袭的影响

摘要 目的：研究基于Wnt/β-catenin信号通路探讨微小RNA（miR）-613对宫颈癌SiHa细胞增殖、迁移与侵袭的影响。方法：体外培养宫颈癌SiHa细胞和正常宫颈上皮细胞H8,检测细胞中miR-613表达。根据转染miR-613 mimic浓度不同,将SiHa细胞分为0 μmol/L组,100 μmol/L和200 μmol/L组。MTT法检测细胞增殖情况,划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力。蛋白免疫印迹法检测miR-613表达对Wnt/β-catenin信号通路蛋白β-catenin、Vimentin、E-cadherin和MMP9表达的影响。结果：宫颈癌SiHa细胞中miR-613表达水平均显著低于正常宫颈上皮细胞H8,差异有统计学意义(P<0.05)。转染miR-613 mimic后,100 μmol/L、200 μmol/L 组SiHa细胞中miR-613表达水平显著上调,并且具有浓度依赖性(P<0.05)。与0 μmol/L组相比,100 μmol/L、200 μmol/L组的SiHa细胞增殖,迁移,侵袭能力均明显下降,并且具有浓度依赖性(P<0.05)。免疫印迹结果,与0 μmol/L组相比,100 μmol/L、200 μmol/L各浓度组的SiHa细胞Wnt/β-catenin信号通路蛋白β-catenin、Vimentin和MMP9表达显著下调,E-cadherin表达显著上调,并且具有浓度依赖性(P<0.05)。结论：miR-613能通过抑制Wnt/β-catenin信号通路抑制人宫颈癌细胞系SiHa细胞的增殖,迁移和侵袭。     

余甘子提取物调控LINC01772/miR-153对肺癌细胞A549生物学行为的影响

摘要 目的：探讨余甘子提取物对肺癌细胞A549增殖、迁移和侵袭的影响及机制。方法：体外培养A549细胞,分为对照组、不同剂量（低、中、高剂量）余甘子提取物组、si-NC组、si-LINC01772组、高剂量余甘子提取物+pcDNA组和高剂量余甘子提取物+pcDNA-LINC01772组,细胞计数试剂盒（CCK-8）法和克隆形成实验检测细胞增殖,划痕实验检测细胞迁移,嵌入式细胞共培养法（Transwell）检测细胞侵袭,免疫印迹法（Western Blot）检测细胞中上皮型钙黏蛋白（E-cadherin）和神经型钙黏蛋白（N-cadherin）蛋白表达水平,实时荧光定量PCR（RT-qPCR）检测LINC01772和miR-153表达水平。双荧光素酶报告基因实验验证LINC01772和miR-153调控关系。结果：与对照组相比,不同剂量余甘子提取物组A549细胞中LINC01772表达降低,且光密度值（OD值）、克隆形成数、迁移以及侵袭细胞数减少（P<0.05）,而miR-153含量与E-cadherin蛋白表达升高（P<0.05）,且呈剂量依赖性（P<0.05）。LINC01772在A549细胞中负调控miR-153表达。与si-NC组相比,si-LINC01772组A549细胞增殖,侵袭及迁移能力受到抑制（P<0.05）。与高剂量余甘子提取物+pcDNA组相比,高剂量余甘子提取物+pcDNA-LINC01772组A549细胞增殖,侵袭及迁移能力增强（P<0.05）。结论：余甘子提取物可能通过调控LINC01772/miR-153轴抑制肺癌细胞A549增殖、迁移和侵袭,其可能通过下调LINC01772进而上调miR-153表达发挥作用,具有开发为治疗肺癌药物的潜在价值。     

饱和脂肪酸诱导肝细胞系建立代谢相关脂肪性肝病细胞损伤模型

摘要 目的：探讨不同脂肪酸对肝细胞系脂质积累、细胞损伤的影响,选择合适诱导试剂及肝细胞系建立一种具有严重细胞损伤及炎症反应的晚期代谢相关脂肪性肝病（MAFLD）体外细胞模型。方法：以油酸（OA）或棕榈酸（PA）或其混合物分别处理HepG2和LO2细胞,以CCK8检测细胞存活率;以油红O染色及甘油三酯酶法检测细胞脂质积累程度;以qRT-PCR检测凋亡相关蛋白、纤维化相关蛋白、自噬相关蛋白、炎症因子的mRNA表达水平。结果：0.25 mmol/LPA作用HepG2细胞24 h可显著诱导甘油三酯（TG）和脂质积累,但对LO2细胞无明显影响;0.25 mmol/L PA处理两种细胞系可诱导显著的细胞损伤及炎症,OA可缓解PA对细胞的损伤作用。结论：利用PA处理HepG2细胞可引起一定程度的脂质积累,诱导显著的细胞损伤及炎症,是合适的MAFLD体外细胞模型。     

基于核因子相关因子2/血红素加氧酶-1信号通路探讨异荭草素对乳腺癌细胞恶性生物学行为的影响

摘要 目的：探讨异荭草素（ISO）对乳腺癌（BC）细胞恶性生物学行为及核因子相关因子2（Nrf2）/血红素加氧酶-1（HO-1）信号通路的影响。方法：体外培养人BC细胞系MDA-MB-231并分组：MDA-MB-231组、MDA-MB-231+ISO组（100 μmol/L ISO处理）、MDA-MB-231+ISO+OE-NC组（转染OE-NC后用100 μmol/L ISO处理）、MDA-MB-231+ISO+OE-Nrf2组（转染OE-Nrf2后用100 μmol/L ISO处理）。采用细胞计数试剂盒-8（CCK-8）检测MDA-MB-231细胞增殖;流式细胞术检测MDA-MB-231细胞周期和凋亡;Transwell实验检测MDA-MB-231细胞的侵袭和迁移能力;Western blot检测Nrf2/HO-1信号通路相关蛋白及凋亡蛋白表达。结果：与MDA-MB-231组相比,MDA-MB-231+ISO组、MDA-MB-231+ISO+OE-NC组细胞活力、S期和G2期细胞比例、迁移和侵袭能力、B淋巴细胞瘤-2（Bcl-2）、Nrf2、HO-1、基质金属蛋白酶-9（MMP-9）蛋白水平显著下降（P<0.05）,细胞凋亡率、G1/G0期细胞比例以及Bax、cleaved-Caspase-3蛋白水平显著上升（P<0.05）。与MDA-MB-231+ISO组相比,MDA-MB-231+ISO+OE-Nrf2组细胞活力、S期和G2期细胞比例、迁移和侵袭能力、Bcl-2、Nrf2、HO-1、MMP-9蛋白水平显著上升（P<0.05）,细胞凋亡率、G1/G0期细胞比例以及Bax、cleaved-Caspase-3蛋白水平显著降低（P<0.05）。结论：ISO可能通过抑制Nrf2/HO-1信号通路,抑制MDA-MB-231细胞恶性增殖、迁移和侵袭等行为。     

顺铂对HepG2细胞增殖、细胞周期及肝癌干细胞标志物的影响

目的:研究顺铂对HepG2细胞增、细胞周期及肝癌干细胞标志物(CD133、ICAM-1和ABCG2)的影响。方法:选取HepG2作为研究对象,分别采用MMT比色法、PI染色法及免疫荧光法检测不同浓度顺铂对其增殖、细胞周期、CD133、ICAM-1和ABCG2表达的影响。结果:每个浓度顺铂作用后均可以显著抑制HepG2细胞增殖力(F=12.23,P=0.004);顺铂对HepG2细胞增殖力的抑制作用和浓度可能与时间成正比。0 mg/L组静息期(G0/G1期)细胞比例为(50.25±0.79)%、2 mg/L组G0/G1期细胞比例为(89.24±0.41)%、4 mg/L组G0/G1期细胞比例为(29.54±3.02)%,2 mg/L组和4 mg/L组分别比0 mg/L组显著上升和下降,差异明显有统计学意义(t=6.53、-3.65,均P0.05)。0 mg/L组DNA合成期(S期)细胞比例为(47.13±0.74)%、2 mg/L组S期细胞比例为(5.65±0.42)%、4 mg/L组S期细胞比例为(67.46±3.24)%,2 mg/L组和4 mg/L组分别比0 mg/L组显著下降和上升,差异明显有统计学意义(t=-7.35、3.79,均P0.05)。结果提示2 mg/L组和4 mg/L组顺铂可让HepG2在G0/G1期与S期显著阻滞,差异有统计学意义(P0.01);顺铂处理后,剩余的HepG2细胞的CD133、ICAM-1和ABCG2呈现高表达水平。结论:HepG2细胞系中会有很少部分肝癌干细胞避开了顺铂的杀灭作用,是造成临床上肝癌反复发作的重要因素之一,临床上应予以重视。     

索拉非尼与重组腺病毒H101对肝癌细胞株HepG2的作用及机制研究

目的:研究索拉非尼与重组腺病毒H101对肝癌细胞株HepG2的作用并分析其具体机制。方法:选择索拉非尼、重组腺病毒H101分别组成重组腺病毒H101组、索拉非尼组、两药联合组以及空白对照组,并分别作用于购自中国科学院上海细胞生物研究所细胞库的肝癌细胞株HepG2。采用流式细胞技术检测HepG2细胞的凋亡情况;采用Western blot法检测细胞外信号调节激酶1/2(ERK1/2)、磷酸化ERK1/2(p-ERK1/2)以及髓样细胞白血病-1(Mcl-1)蛋白相对表达量;采用酶联免疫吸附法检测不同组别细胞培养上清液中的血管内皮生长因子(VEGF)水平。结果:重组腺病毒H101组、索拉非尼组、两药联合组的G0-G1期与G2-M期细胞均明显低于空白对照组,S期细胞均明显高于空白对照组,且两药联合组较重组腺病毒H101组与索拉非尼组更明显,差异均有统计学意义(均P0.05);重组腺病毒H101组、索拉非尼组、两药联合组的HepG2细胞凋亡率明显高于空白对照组,而两药联合组又明显高于重组腺病毒H101组与索拉非尼组,差异均有统计学意义(均P0.05)。重组腺病毒H101组、索拉非尼组、两药联合组的p-ERK1/2、Mcl-1蛋白相对表达量和VEGF表达均明显低于空白对照组,而两药联合组又明显低于重组腺病毒H101组与索拉非尼组,差异均有统计学意义(均P0.05)。结论:索拉非尼、重组腺病毒H101均可抑制肝癌细胞株HepG2的增殖,并诱导其凋亡,控制VEGF表达,联合应用具有更明显的效果,其主要机制可能与二者协同作用有效抑制p-ERK1/2、Mcl-1蛋白表达有关。     

Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro

Hypoxia is a hallmark of solid tumors, including breast cancer, and the extent of tumor hypoxia is associated with treatment resistance and poor prognosis. Considering the limited treatment of hypoxic tumor cells and hence a poor prognosis of breast cancer, the investigation of natural products as potential chemopreventive anti-angiogenic agents is of paramount interest. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the primary anthraquinone in the roots of Cassia alata L., is a naturally occurring quinone which exhibits a variety of biologic activities including anti-cancer activity. However, the effect of rhein on endothelial or cancer cells under hypoxic conditions has never been delineated. Therefore, the aim of this study was to investigate whether rhein inhibits angiogenesis and the viability of hormone-dependent (MCF-7) or -independent (MDA-MB-435s) breast cancer cells in vitro under normoxic or hypoxic conditions. Rhein inhibited vascular endothelial growth factor (VEGF₁₆₅)-stimulated human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and migration under normoxic and hypoxic conditions. In addition, rhein inhibited in vitro angiogenesis by suppressing the activation of phosphatidylinositol 3-kinase (PI3K), phosphorylated-AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) but showed no inhibitory effects on total AKT or ERK. Rhein dose-dependently inhibited the viability of MCF-7 and MDA-MB-435s breast cancer cells under normoxic or hypoxic conditions, and inhibited cell cycle in both cell lines. Furthermore, Western blotting demonstrated that rhein inhibited heat shock protein 90alpha (Hsp90α) activity to induce degradation of Hsp90 client proteins including nuclear factor-kappa B (NF-κB), COX-2, and HER-2. Rhein also inhibited the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF₁₆₅), epidermal growth factor (EGF), and the phosphorylation of inhibitor of NF-κB (I-κB) under normoxic or hypoxic conditions. Taken together, these data indicate that rhein is a promising anti-angiogenic compound for breast cancer cell viability and growth. Therefore, further studies including in vivo and pre-clinical need to be performed.     

Effects of ERK1/2 signal pathway on cardiomyocyte during glucose lowering

Objective: This study investigated whether the extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway affects cardiomyocyte apoptosis and the expression of tumor necrosis factor (TNF-α) at different glucose-lowering rates.

Methods: Cardiomyocytes of Wistar neonate rats were maintained in a medium supplemented with 25?mmol/L glucosamine for 72?h. Then the medium was changed to different concentrations of glucosamine, and all cells were divided into five groups. The survival rate of cardiomyocyte was measured using the Cell Counting Kit-8; cardiomyocyte apoptosis was measured using the flow cytometry instrument and laser confocal microscope; TNF-α was measured using the enzyme-linked immunosorbent assay; and ERK1/2 protein and phosphorylation were measured using the Western blot. Cardiomyocyte apoptosis and TNF-α were measured again after adding U0126.

Results: As the glucose-lowering rate increased, the survival rate of cardiomyocytes increased in group B and decreased in groups C, D, and E. The TNF-α concentration increased in groups B, C, and D and decreased in group E. After 24?h, the apoptosis rate decreased in group B and increased in groups C, D, and E. The expression of p-ERK1/2 increased in groups B, D, and E, and was the lowest in group C. After adding U0126, the survival rate of cardiomyocyte in all groups increased and TNF-α concentration decreased.

Conclusions: The influence of glucose-lowering rate on cardiomyocyte apoptosis and TNF-α was caused by the p-ERK1/2 pathway. During the glucose-lowering course, the p-ERK1/2 pathway promoted cardiomyocyte apoptosis, and TNF-α secretion was related to not only osmotic pressure but also ERK1/2 signal pathway activation.     

Anti‐proliferative effect of rhein,an anthraquinone isolated from Cassia species,on Caco‐2 human adenocarcinoma cells

In recent years, the use of anthraquinone laxatives, in particular senna, has been associated with damage to the intestinal epithelial layer and an increased risk of developing colorectal cancer. In this study, we evaluated the cytotoxicity of rhein, the active metabolite of senna, on human colon adenocarcinoma cells (Caco‐2) and its effect on cell proliferation. Cytotoxicity studies were performed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), neutral red (NR) and trans‐epithelial electrical resistance (TEER) assays whereas ³H‐thymidine incorporation and Western blot analysis were used to evaluate the effect of rhein on cell proliferation. Moreover, for genoprotection studies Comet assay and oxidative biomarkers measurement (malondialdehyde and reactive oxygen species) were used. Rhein (0.1–10 μg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco‐2 cells. Rhein (0.1 and 1 μg/ml) significantly reduced cell proliferation as well as mitogen‐activated protein (MAP) kinase activation; by contrast, at high concentration (10 μg/ml) rhein significantly increased cell proliferation and extracellular‐signal‐related kinase (ERK) phosphorylation. Moreover, rhein (0.1–10 μg/ml): (i) did not adversely affect the integrity of tight junctions and hence epithelial barrier function; (ii) did not induce DNA damage, rather it was able to reduce H₂O₂‐induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and reactive oxygen species (ROS) levels induced by H₂O₂/Fe²⁺. Rhein was devoid of cytotoxic and genotoxic effects in colon adenocarcinoma cells. Moreover, at concentrations present in the colon after a human therapeutic dosage of senna, rhein inhibited cell proliferation via a mechanism that seems to involve directly the MAP kinase pathway. Finally, rhein prevents the DNA damage probably via an anti‐oxidant mechanism.     

miR-195通过靶向调控趋化因子5促进胃癌细胞增殖、转移及侵袭的实验研究

目的:研究miR-195通过靶向调控趋化因子5促进胃癌细胞增殖、转移及侵袭的分子机制。方法:选取MGC803及NCI-N87细胞,根据转染不同分为:miR-NC组(空质粒),miR-195-mimics组(模拟序列)。实时荧光定量PCR法检测miR-195表达;MTT检测细胞增殖能力;Transwell侵袭实验检测细胞侵袭力;细胞划痕实验检测细胞转移能力;流式细胞术检测细胞凋亡情况;Western blot检测chemokine 5表达水平;Spearman相关分析miR-195及chemokine 5相关性。荧光素酶实验验证miR-195与chemokine 5的靶向关系。结果:miR-195-mimics组细胞miR-195水平高于miR-NC组(P0.05);miR-195 mimics组第1、2、3、4 d细胞活力低于miR-NC组(P0.05);miR-195 mimics组G1细胞高于miR-NC组,G2期、S期细胞低于miR-NC组,G2/S期细胞比值低于miR-NC组(P0.05);miR-195 mimics组划痕距离大于miR-NC组(P0.05);miR-195 mimics组细胞侵袭数低于miR-NC组(P0.05);miR-195-mimics组细chemokine 5蛋白含量低于miR-NC组(P0.05);miR-195 m RNA水平与chemokine 5蛋白含量负相关(r=-0.398,P=0.00);miR-195可直接靶向chemokine 5。结论:miR-195可通过靶向chemokine 5促进胃癌MGC803及NCI-N87细胞的增殖、转移及侵袭。     

Artematrolide A inhibited cervical cancer cell proliferation via ROS/ERK/mTOR pathway and metabolic shift

BackgroundArtematrolide A (AR-A), a guaianolide dimer isolated from Artemisia atrovirens, demonstrated significant inhibitory effect on three human hepatoma cell lines (HepG2, Huh7 and SMMC7721). The anti-cervical cancer effect and mechanism of this compound have yet to be explored. This study is to reveal the role and mechanisms of artematrolide A on cervical cancer cells, and provide the pharmacological understanding of artematrolide A.PurposeTo investigate the function and possible mechanism of artematrolide A on cervical cancer cells in vitro.MethodsHeLa S3 and SiHa cells were treated with artematrolide A at various concentrations. In this study, MTT, colony formation, cell migration and invasion, cell cycle analysis, cell apoptosis, reactive oxygen species (ROS) detection, western blotting, enzyme activity, and lactate production of artematrolide A were evaluated.ResultsArtematrolide A inhibited cell viability, proliferation, migration and invasion in a dose-dependent manner, caused cell cycle arrest in G2/M phase, and induced cell apoptosis via Bcl-2/PARP-1. The mechanism of action of artematrolide A included two aspects: artematrolide A suppressed cell proliferation by activating ROS/ERK/mTOR signaling pathway and promoted glucose metabolism from aerobic glycolysis to mitochondrial respiration by activating pyruvate dehydrogenase complex (PDC) and oxoglutarate dehydrogenase complex (OGDC) via inhibiting the activity of alkaline phosphatases (ALP).ConclusionArtematrolide A exhibited a significant cytotoxic activity on cervical cancer cells, induced G2/M cell cycle arrest and apoptosis by activating ROS/ERK/mTOR signaling pathway and promoting metabolic shift from aerobic glycolysis to mitochondrial respiration, which suggested artematrolide A might be a potential agent for the treatment of cervical cancer.     

Rhein Protects against Obesity and Related Metabolic Disorders through Liver X Receptor-Mediated Uncoupling Protein 1 Upregulation in Brown Adipose Tissue

Liver X receptors (LXRs) play important roles in regulating cholesterol homeostasis, and lipid and energy metabolism. Therefore, LXR ligands could be used for the management of metabolic disorders. We evaluated rhein, a natural compound from Rheum palmatum L., as an antagonist for LXRs and investigated its anti-obesity mechanism in high-fat diet-fed mice. Surface plasmon resonance assays were performed to examine the direct binding of rhein to LXRs. LXR target gene expression was assessed in 3T3-L1 adipocytes and HepG2 hepatic cells in vitro. C57BL/6J mice fed a high-fat diet were orally administered with rhein for 4 weeks, and then the expression levels of LXR-related genes were analyzed. Rhein bound directly to LXRs. The expression levels of LXR target genes were suppressed by rhein in 3T3-L1 and HepG2 cells. In white adipose tissue, muscle and liver, rhein reprogrammed the expression of LXR target genes related to adipogenesis and cholesterol metabolism. Rhein activated uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) in wild-type mice, but did not affect UCP1 expression in LXR knockout mice. In HIB-1B brown adipocytes, rhein activated the UCP1 gene by antagonizing the repressive effect of LXR on UCP1 expression. This study suggests that rhein may protect against obesity and related metabolic disorders through LXR antagonism and regulation of UCP1 expression in BAT.     

RACK1对硼替佐米诱导的多发性骨髓瘤细胞凋亡及MAPK/ERK通路的影响

目的:探讨蛋白激酶C受体(Receptor for activated C kinase1,RACK1)对硼替佐米(Bortezomi,Bor)诱导的多发性骨髓瘤(Multiple myeloma,MM)细胞凋亡及MAPK/ERK通路的影响。方法:选取6例岳阳市第一人民医院收治的MM患者及6名正常体检者,用实时荧光定量PCR检测血浆及人MM细胞系中RACK1 m RNA的表达。将MM细胞分为3组:对照组(不干预)、Bor组(75n M的Bor干预12 h)和Bor+siRACK1组(RACK1 si RNA转染24 h后再行Bor干预)。CCK-8法检测各组细胞中的细胞存活率,Hoechest 33342染色检测细胞凋亡,Western Blot检测MAPK/ERK通路相关蛋白表达。结果:与正常体检者相比,MM患者血浆及MM细胞系中RACK1 m RNA表达显著增加(P0.05)。Bor作用12 h、24 h和48 h可显著降低MM细胞的存活率(P0.05)。与对照组相比,Bor组和Bor+siRACK1组细胞存活率显著降低,Bor+siRACK1组细胞存活率明显高于Bor组(P0.05)。Hoechest 33342染色显示对照组细胞核染色均一,未见凋亡小体,Bor组见少量凋亡小体,而Bor+siRACK1组细胞见大量凋亡小体,表现为核固缩或碎块状;与对照组相比,Bor组和Bor+siRACK1组细胞中多发性骨髓瘤细胞凋亡率显著增加(P0.05),Bor+siRACK1组多发性骨髓瘤细胞凋亡率明显高于Bor组(P0.05)。三组间多发性骨髓瘤细胞凋亡率对比差异有统计学意义(P0.05)。与对照组相比,Bor组和Bor+siRACK1组细胞中p-P38和p-ERK的表达显著降低,而Bor+siRACK1组p-P38和p-ERK的表达低于Bor组(P0.05),3组间P38和ERK的表达对比差异无统计学意义(P0.05)。结论:RACK1沉默可增强Bor诱导的MM细胞凋亡及生长抑制,其机制可能与MAPK/ERK途径抑制有关。