The flavonoid quercetin induces apoptosis and inhibits migration through a MAPK-dependent mechanism in osteoblasts

The present study was undertaken to evaluate effects of quercetin, a major dietary flavonoid occurring in foods of plant origin, on cell viability and migration of osteoblastic cells. Quercetin inhibited cell viability, which was largely attributed to apoptosis, in a dose-and time-dependent manner in osteoblastic cells. Similar cytotoxicity of quercetin was observed in adipose tissue-derived stromal cells. Quercetin exerted a protective effect against H₂O₂-induced cell death, whereas it increased TNF-α-induced cell death. Western blot analysis showed that quercetin induced activation of ERK and p38, but not JNK. Quercetin-induced cell death was prevented by the ERK inhibitor PD98059, but not by inhibitors of p38 and JNK. Quercetin increased Bax expression and caused depolarization of mitochondrial membrane potential, which were inhibited by PD98059. Quercetin induced caspase-3 activation, and the quercetininduced cell death was prevented by caspase inhibitors. Quercetin inhibited cell migration, and its effect was prevented by inhibitors of ERK and p38. Taken together, these findings suggest that quercetin induces apoptosis through a mitochondria-dependent mechanism involving ERK activation and inhibits migration through activation of ERK and p38 pathways. Quercetin may exert both protective and deleterious effects in bone repair.     

Clinical attachments: fond farewell or new beginning? A survey of the attitudes and practice of medical consultants and international medical graduates

Objectives

To analyse the experience of clinical attachment (CA) of international medical graduates (IMGs) and consultants.

Design

Analysis of questionnaires and CVs.

Setting and participants

573 IMGs applying for a house officer post and 102 consultant physicians working in North East England.

Results

IMGs had spent a mean of 16 months unemployed, of which 3.8 months was spent on CAs. The median number of CAs was two and the average number of applications sent before obtaining a CA was 73. 90% of IMGs found their CA helpful and 57% would not take up a post without a CA first. Criticisms related to lack of responsibility, isolation and poor job prospects. 90% would apply for honorary posts if advertised. 73% had received induction at the onset of placement, but only 32% had been assessed at the end. 50% of consultants took CAs and only 4% were thinking of stopping doing so. Those without CAs blamed work pressure (43%) and pressure from their employer (23%).

Conclusions

There are deficiencies in pastoral care, the application process and assessment, but CAs are valued by IMGs and offered by half the consultants surveyed. New immigration rules will mean fewer IMGs will come to the UK, but CAs will be needed by those that do, as well by refugees and European Economic Area (EEA) graduates. The tradition of CAs for international graduates could be used to accommodate those coming to the UK on exchanges and scholarships and form part of the recently announced Medical Training Initiative for IMGs.     

Reconstruction of basement membrane in skin equivalent; role of laminin-1

Abstract To reconstruct the basement membrane in a skin equivalent, the epidermodermal interface was coated with porcine type IV collagen and mouse laminin-1 at various ratios before keratinocyte seeding. Laminin-1, a component of the basement membrane, induced massive infiltration of keratinocytes into the dermal equivalent, while type IV collagen induced discrete demarcation between dermal and epidermal compartments without any infiltrating cells. Immunohistochemical staining indicated that the laminin-induced infiltrating cells expressed endogenous type IV collagens at the cell periphery, which were not incorporated into the basement membrane structure. The infiltrating cells did not express fibronectin receptor α₅β₁ integrin but showed MMP-9 secretion and cell surface associated MMP-2. However, when laminin-1 was preincubated with type IV collagen, laminin-1-induced keratinocyte infiltration as well as MMP-9 induction were almost completely suppressed to basal levels. Therefore, replenishment of the type IV collagen lattice seemed to cause laminin-stimulated cells to anchor to the lattice, in a similar manner to the basal cells on the basement membrane of normal skin. Our study suggests that the molar ratio of basement membrane components may determine the behavior of basal cells within the wound healing microenvironment, which is probably regulated either by extracellular matrix deposition or degradation. Received: 24 October 2000 / Revised: 9 February 2001 / Accepted: 31 March 2001     

Tumour necrosis factor-alpha-induced migration of human Langerhans cells: the influence of ageing

BACKGROUND: Langerhans cells (LCs) play essential roles in the initiation and regulation of cutaneous immune responses mediated through their successful migration from the epidermis to draining lymph nodes while carrying antigen. Tumour necrosis factor (TNF)-alpha, a keratinocyte-derived cytokine, has recently been shown to play an important role in the mobilization of LCs from human epidermis. Although it is known that with age the immune system changes, the influence of increasing age on the function of human LCs has not been defined clearly. OBJECTIVES: To examine the influence of age on the ability of TNF-alpha to induce LC migration. METHODS: Ten elderly (six men, four women; mean age 76 years, range 72-79) and 10 young (six men, four women; mean age 23 years, range 18-35) volunteers received intradermal injections of 200 U of human recombinant TNF-alpha diluted in sterile saline, and control injections of sterile saline alone, at each of two paired sites identified on photoprotected buttock skin. Two hours later, paired injection sites were excised by punch biopsy. One set of paired biopsies was processed for assessment of the frequency and morphology of epidermal LCs, following preparation of epidermal sheets and immunofluorescence staining for the LC marker CD1a. The remaining paired biopsies were processed in formalin and the inflammatory response to TNF-alpha was assessed by standard histological examination. RESULTS: Mean +/- SEM baseline values for LC frequency within epidermal sheets were significantly different between young (1156.3 +/- 38.5 cells mm(-2)) and elderly subjects (835.7 +/- 48.2 cells mm(-2); P < 0.01). Intradermal injections of 200 U of TNF-alpha caused a significant reduction in the frequency of LCs in both elderly and young subjects (P < 0.01). However, the extent of TNF-alpha-induced LC migration was substantially different between the two groups, with a mean 9% reduction in LC frequency in elderly volunteers compared with a mean 23% decrease in young subjects. Exposure to TNF-alpha was associated with a perivascular polymorphonuclear infiltrate at 2 h in all young subjects; in contrast, only 50% of the elderly individuals showed evidence of such a response. CONCLUSIONS: There are significant differences between young and old skin with respect to both resting LC numbers and their response to TNF-alpha. These age-related changes in LC frequency and function may contribute to the altered cutaneous immune function observed in the elderly.     

清热活血方对肿瘤坏死因子α诱导的类风湿关节炎成纤维样滑膜细胞迁移和黏附的影响

目的观察清热活血方治疗活动期类风湿关节炎的可能作用机制。方法分离类风湿关节炎患者的滑膜组织制备原代类风湿关节炎成纤维样滑膜细胞（RA-FLS）,采用肿瘤坏死因子α（TNF-α）诱导RA-FLS的迁移和黏附。设空白组（不加药物及TNF-α干预）、诱导组（10或20ng/mlTNF-α诱导）、清热活血方低、中、高剂量组（20、100、500ng/ml清热活血方+TNF-α诱导）。检测各组细胞迁移距离及面积、细胞黏附OD值和黏附抑制率,并检测细胞中PI3K/Akt信号通路相关蛋白（PI3K、p-Akt、Akt）表达。结果与空白组比较,诱导组细胞迁移距离及面积增加、黏附OD值增大、PI3K及p-Akt蛋白表达升高（P<0.01）;与诱导组比较,清热活血方各剂量组均可抑制细胞的迁移和黏附以及PI3K、p-Akt蛋白表达,并以500ng/ml效果最好（P<0.05或P<0.01）。结论清热活血方可能通过下调RA-FLS中PI3K、p-Akt蛋白表达,抑制RA-FLS的迁移和黏附,从而减缓滑膜炎症和关节破坏的进展。     

Prdx4蛋白表达改变对HeLa细胞迁移和侵袭的影响

目的:观察过氧化物还原酶4(peroxiredoxin4,Prdx4)蛋白表达的改变对人宫颈癌HeLa细胞迁移和侵袭的影响。方法:采用脂质体瞬时转染法将表达质粒pcDNA3.0-HA-Prdx4转染HeLa细胞;LV-Prdx4RNAi重组病毒感染HeLa细胞,构建Prdx4shRNAHeLa细胞系沉默Prdx4的表达;用蛋白质印迹法证实Prdx4蛋白表达水平的变化;应用划痕愈合实验和Transwell迁移及侵袭实验分别检测细胞迁移及侵袭能力的变化。结果:pcDNA3.0-HA-Prdx4质粒转染组HeLa细胞的Prdx4蛋白表达水平明显上调(P<0.05),而Prdx4shRNAHeLa稳定干扰细胞系Prdx4表达水平明显下调(P<0.05)。Prdx4过表达组HeLa细胞的迁移和侵袭能力明显增强,与空白对照组及空载体转染组比较,差异显著(P<0.01)。Prdx4表达干扰组HeLa细胞的迁移和侵袭能力明显受到抑制,与空白对照组及阴性对照组比较,差异显著(P<0.01)。结论:上调Prdx4蛋白表达水平可促进HeLa细胞的迁移和侵袭,在宫颈癌的发生和发展过程中可能起重要作用;下调Prdx4蛋白表达水平可以明显抑制HeLa细胞的迁移和侵袭能力,Prdx4有可能成为临床治疗宫颈癌的一个潜在靶点。     

红花苷对模拟微重力下细胞迁移的作用

目的观察红花苷修复模拟微重力下人脐静脉内皮细胞（HUVECs）细胞迁移抑制的作用并探讨其机制。方法MTT比色法选出红花苷干预HUVECs的最佳浓度;模拟失重-2D回转仪模拟失重培养HUVECs,分为正常重力对照组、模拟微重力组、红花苷微重力组。采用透膜侵袭小室模型检测细胞迁移;激光共聚焦显微镜观察红花苷干预后细胞骨架纤维状肌动蛋白（F-actin）的形态及对黏着斑（FAs）的影响。结果与正常重力对照组比较,模拟微重力组细胞迁移数下降（P<0.01）;红花苷微重力组较模拟微重力组细胞迁移数增加（P<0.05）。正常重力对照组的F-actin呈现明显极化、微丝集结成束,模拟微重力可导致F-actin骨架重构,极化下降并呈现弥散特点;而红花苷微重力组的F-actin排列有序,伪足较模拟微重力组增多,方向性显著增强。模拟微重力组较正常重力对照组FAs数量（VN）、面积（VA）及FAs到细胞边缘距离（DS）显著降低（P<0.05）;红花苷微重力组较模拟微重力组VN、VA、DS显著升高（P<0.05）。结论红花苷可改善模拟微重力作用导致的HUVECs迁移抑制,修复模拟微重力作用导致的HUVECs骨架F-actin的重构,改善FAs的生成及定位。     

格列本脲对U-251细胞增殖迁移的影响

目的:探讨格列本脲对U-251细胞增殖迁移的影响。方法:使用浓度为12.5¹600μmol/L的格列本脲（Glib）处理人胶质瘤细胞U-251,应用细胞增殖毒性检测试剂盒测定细胞增殖率并进行药物浓度筛选,划痕实验和Transwell法检测各实验组（使用浓度为100μmol/L、300μmol/L、500μmol/L的Glib处理细胞）及对照组（未加Glib）细胞迁移与侵袭情况。结果:CCK8法检测显示,在100⁸00μmol/L范围内,Glib可抑制U-251细胞增殖,并呈浓度依赖性,其48h的半数抑制浓度（IC50）为400μmol/L（F=150.30,P=0.000）;划痕实验显示Glib实验组细胞在24h及48h迁移宽度明显小于对照组,且随着药物浓度的增加迁移宽度比率降低（F=130.60,P=0.000）;Transwell结果显示,Glib抑制细胞迁移能力与划痕试验结果一致,Transwell显示实验组迁移细胞数比对照组明显减少（F=86.68,P=0.000）,实验组侵袭细胞数量比对照组明显下降（F=55.30,P=0.000）,随着Glib浓度的增加,抑制迁移和侵袭能力逐渐增强,具有浓度依赖性。结论:格列本脲能够抑制U-251细胞增殖迁移过程。     

山甲白花汤联合吉西他滨抗胰腺癌耐药机制研究

目的：探讨山甲白花汤联合吉西他滨抗胰腺癌耐药的作用机制。方法：将30只小鼠按随机数表法分为模型组、吉西他滨组和联合组,构建皮下移植瘤小鼠模型,分别使用山甲白花汤、吉西他滨及联合处理小鼠,观察肿瘤的体积、重量变化和类固醇受体辅激活因子/分化抑制蛋白1（Src/Id1）信号通路蛋白。将人胰腺癌细胞系1（PANC-1）细胞分为对照组、吉西他滨处理组、联合处理组、联合+微小核糖核酸-124-3p抑制剂（miR-124-3p inhibitor）组和联合+过表达信号转导蛋白2样1（oe-SDF2L1）组,比较各组细胞增殖与迁移能力、微核糖核酸-124-3p（miR-124-3p）、信号转导蛋白2样1（SDF2L1）及信使核糖核酸（mRNA）水平。结果：与吉西他滨组比较,联合组中肿瘤体积与重量降低、Id1和磷酸化非受体酪氨酸激酶/非受体酪氨酸激酶（p-Src/Src）、SDF2L1水平降低。与对照组比较,吉西他滨处理组细胞增殖与迁移率明显降低,微小核糖核酸-124-3p（miR-124-3p）水平升高且SDF2L1水平明显降低,与吉西他滨处理组比较,联合处理组细胞增殖与迁移率明显降低,miR-124-3p水平升高且SDF2L1水平明显降低,与联合处理组比较,联合+miR-124-3p inhibitor组和联合+oe-SDF2L1组细胞增殖与迁移率均明显升高,SDF2L1水平均明显升高。结论：山甲白花汤联合吉西他滨通过上调miR-124-3p抑制SDF2L1发挥抗胰腺癌耐药作用。