Geraniin inhibits oral cancer cell migration by suppressing matrix metalloproteinase‐2 activation through the FAK/Src and ERK pathways

Geraniin has been reported to have numerous biological activities, including antiviral, antihypertensive, antihyperglycaemic, liver protective, antidiabetic, and apoptotic activities. However, the anti‐migration effects of geraniin on oral cancer remain elusive. In this study, we revealed the potential antitumor mechanisms of geraniin through the inhibition of the migration and invasion of human oral cancer cell lines SCC‐9 and SCC‐14. The results of gelatin zymography and Western blot assays revealed that geraniin significantly reduced the activity and expression of matrix metalloproteinase‐2 (MMP‐2) of oral cancer cells in a concentration‐dependent manner. Furthermore, geraniin potently suppressed the phosphorylation of focal adhesion kinase (FAK), Src, and extracellular signal‐regulated kinase (ERK)1/2 but did not affect the phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and c‐Jun N‐terminal kinase 1/2. Moreover, blocking the MAPK/ERK1/2 pathway significantly enhanced the anti‐migration ability of geraniin in oral cancer cells. In conclusion, we demonstrated that geraniin inhibits the motility of SCC‐9 and SCC‐14 cells in vitro through a molecular mechanism that involves the attenuation of MMP‐2 expression and activity mediated by decreased FAK/Src and ERK1/2 pathways.     

Lead alters intracellular protein signaling and suppresses pro-inflammatory activation in TLR4 and IFNR-stimulated murine RAW 264.7 cells,in vitro

Lead (Pb) is a persistent environmental pollutant that has a structure and charge similar to many ions, such as calcium, that are essential for normal cellular function. Pb may compete with calcium for protein binding sites and inhibit signaling pathways within the cell affecting many organ systems including the immune system. The aim of the current study was to assess whether the calcium/calmodulin pathway is a principal target of environmentally relevant Pb during pro-inflammatory activation in a RAW 264.7 macrophage cell line. RAW 264.7 cells were cultured with 5 μM Pb(NO₃)₂, LPS, rIFNγ, or LPS+rIFNγ for 12, 24, or 48 hr. Intracellular protein signaling and multiple functional endpoints were investigated to determine Pb-mediated effects on macrophage function. Western blot analysis revealed that Pb initially modulated nuclear localization of NFκB p65 and cytoplasmic phosphorylation of CaMKIV accompanied by increased phosphorylation of STAT1β at 24 hr. Macrophage proliferation was significantly decreased at 12 hr in the presence of Pb, while nitric oxide (NO) was significantly reduced at 12 and 24 hr. Cells cultured with Pb for 12, 24, or 48 hr exhibited altered cytokine levels after specific stimuli activation. Our findings are in agreement with previous reports suggesting that macrophage pro-inflammatory responses are significantly modulated by Pb. Further, Pb-induced phosphorylation of CaMKIV (pCaMKIV), observed in the present study, may be a contributing factor in metal-induced autophagy noted in our previous study with this same cell line.     

己酮可可碱通过Hedgehog信号通路对日本血吸虫病肝纤维化形成的抑制作用

目的：探讨己酮可可碱（PTX）通过Hedgehog信号通路抑制日本血吸虫病肝纤维化的作用机制。方法：PMA诱导单核细胞THP-1分化成巨噬细胞,用可溶性日本血吸虫虫卵抗原（SEA）刺激活化THP-1源性巨噬细胞,留取培养上清用于检测其活化状况。CCK-8检测培养上清及PTX对肝星状细胞LX-2的活性影响。设空白对照组、上清刺激组（加入活化的巨噬细胞培养上清）、PTX干预组（加入活化的巨噬细胞培养上清和PTX）。收集以上3组LX-2培养上清及细胞,上清用于ELISA检测TGF-β蛋白表达,细胞用于提取总RNA后RT-PCR检测TGF-β,shh,gli-1的基因表达。结果：SEA可活化THP-1源性巨噬细胞,其活化后培养上清可活化LX-2细胞。上清刺激组TGF-β,shh,gli-1的基因表达空白对照组明显上调,而PTX干预组相对于上刺激组则明显降低。结论：Hedgehog信号通路可能在血吸虫肝纤维化形成过程中起重要作用,己酮可可碱可通过该通路对血吸虫肝纤维化的形成起抑制作用。     

知柏地黄汤通过调节ARE信号通路激活细胞抗氧化反应治疗阴虚火旺证的机制研究

[目的]通过研究知柏地黄汤对细胞抗氧化反应元件(antioxidant response element,ARE)信号通路的调节及其对大鼠血清代谢物的干预作用,探讨其治疗阴虚火旺证的可能机制。[方法]将人肝癌细胞系HepG2细胞分为对照组、知柏地黄汤低、中、高浓度组,分别以0、1、20和100mg·mL-1的知柏地黄汤干预24h,通过双萤光素酶报告基因实验筛选知柏地黄汤显著调控的信号通路;荧光定量PCR检测所筛选通路相关基因的表达,并利用磷钼酸比色法及氧化酶法检测细胞内三磷酸腺苷(adenosine triphosphate,ATP)含量、细胞耗氧率(oxygen consumption rate,OCR)、超氧化物歧化酶(superoxide dismutase,SOD)活性的变化。45只SD大鼠随机分成3组,空白组以0.9%氯化钠溶液灌胃21d,干姜组先以10g·kg-1干姜附子肉桂汤灌胃14d、再以0.9%氯化钠溶液灌胃7d,干知组先以10g·kg-1干姜附子肉桂汤灌胃14d、再以知柏地黄汤灌胃7d,灌胃体积为10mL/(kg·d),以液相色谱-质谱联用(liquid chromatography-mass spectrometry,LC-MS)技术检测血清中代谢物的改变。[结果]双萤光素酶报告基因实验显示,知柏地黄汤对ARE元件调控最为显著,荧光定量PCR结果显示知柏地黄汤可以促进核因子红细胞2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)、小肌腱膜纤维肉瘤(musculoaponeurotic fibrosarcoma,Maf)的转录(P0.001,P0.05),同时抑制Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein1,Keap1)的转录(P0.001)。与对照组比较,知柏地黄汤组细胞ATP含量(P0.05)、OCR(P0.001)及SOD活性(P0.05)均升高。动物实验证实,知柏地黄汤灌胃治疗可以改善阴虚火旺模型大鼠胆碱、甘胆酸及棕榈酰左旋肉碱(palmitoyl-L-carnitine,PALC)的紊乱,其中胆碱及PALC含量差异有统计学意义(P0.001)。[结论]知柏地黄汤能够通过ARE信号通路,抑制机体的氧化应激反应,进而改善阴虚火旺引起的代谢紊乱,这可能是知柏地黄汤治疗阴虚火旺证的作用机制之一。     

白藜芦醇抑制胃癌细胞株MKN45增殖的机制研究*

目的 通过研究白藜芦醇抑制胃癌MKN45细胞增殖及对丝裂原活化蛋白激酶(MEK)/细胞外信号调节激酶（ERK）信号通路的影响,探讨白藜芦醇抗胃癌的作用机制。方法 体外常规培养胃癌MKN45细胞,将细胞分为对照组、MEK/ERK信号通路抑制剂PD98059组（PD98059组）、白藜芦醇低剂量组（100?μmol/L）、 白藜芦醇高剂量组（400?μmol/L）。各组细胞处理1、2和4?h后,MTT法检测细胞增殖抑制率,Western blotting法检测Ras、Raf、MEK、ERK1/2蛋白的表达,细胞免疫化学法检测Ras、Raf、MEK、ERK1/2蛋白荧光强度的变化。结果 与对照组比较,PD98059组、白藜芦醇低剂量组和白藜芦醇高剂量组细胞在处理1、2和4?h后,细胞增殖受到抑制（P?<0.05）,且呈时间剂量依赖性;PD98059组、白藜芦醇低剂量组和白藜芦醇高剂量组细胞Ras、Raf、MEK和ERK1/2表达下降,差异有统计学意义（P?<0.05）,且白藜芦醇的作用呈剂量依赖性（P?< 0.05）;MEK和ERK1/2蛋白主要定位于细胞质中,且PD98059组、白藜芦醇低剂量组和白藜芦醇高剂量组细胞ERK1/2的荧光强度均减弱。结论 白藜芦醇呈剂量依赖性抑制胃癌细胞株MKN45的增殖,可能与抑制MEK/ERK信号通路,减弱Ras、Raf、MEK和ERK1/2的表达有关。     

雷公藤红素对卵巢癌细胞增殖、凋亡的影响 及作用机制研究

目的 探讨雷公藤红素对人卵巢癌细胞株HEYa8 增殖、凋亡的影响及其作用机制。方法 采 用MTT 法检测不同浓度和时间雷公藤红素对HEYa8 细胞增殖的影响。将HEYa8 细胞随机分为：雷公藤 红素组（0.4 mg/L,培养24 h）和对照组（未处理）。检测两组细胞的生长迁移情况、细胞分裂指数及细胞 数量。利用Western blotting 检测雷公藤红素激活凋亡并抑制肿瘤细胞的现象及分子机制。结果 雷公藤红 素在浓度为0.4 mg/L 培养24 h 时效果最佳。雷公藤红素组侵袭细胞、细胞分裂指数、细胞数量及CD44 及GSDMS-N 双阳性细胞占总细胞百分比较对照组低（P <0.05）,凋亡相关蛋白相对表达量较对照组高 （P <0.05）。雷公藤红素组PI3K/Akt 信号通路关键分子的蛋白表达量较对照组低（P <0.05）。结论 雷公藤 红素能够抑制HEYa8 细胞生长并诱导细胞凋亡,其分子机制可能与PI3K/Akt 信号通路被抑制有关。     

车叶草苷对小鼠Lewis肺癌的抑瘤、抗炎作用及其机制

 目的 探讨车叶草苷对小鼠Lewis肺癌种植瘤的抑瘤和抗炎作用。方法 SPF级小鼠36只,制备荷瘤小鼠模型,然后将成瘤小鼠随机分为模型对照组、车叶草苷组、顺铂对照组。分别施加经口灌服车叶草苷或腹腔注射顺铂等干预因素,1次/d,连续3周。末次给药后,测量小鼠体质量、瘤体质量,检测血清中IL-1β、TNF-α含量;检测瘤组织中TLR4、MYD88、NF-κB的表达水平;检测瘤组织中p-NF-κB的表达定位及水平。结果 与模型对照组比较,车叶草苷组和顺铂对照组小鼠血清IL-1β、TNF-α含量显著降低,差异有统计学意义(P<0.01);车叶草苷组和顺铂对照组比较无统计学差异。与模型对照组比较,车叶草苷组和顺铂对照组小鼠瘤组织中TLR4、MYD88、NF-κB表达水平显著降低,差异有统计学意义(P<0.01);车叶草苷组和顺铂对照组比较无统计学差异。与模型对照组(0.261±0.026)比较,车叶草苷组(0.171±0.026)和顺铂对照组(0.207±0.020)小鼠瘤组织中p-NF-κB表达水平显著降低,差异有统计学意义(P<0.01);车叶草苷组和顺铂对照组比较无统计学差异。结论 车叶草苷对Lewis荷瘤鼠具有明显的抑瘤作用,能够降低荷瘤小鼠整体的炎性反应状态。