SIRT1/eNOS/NO通路在人参皂苷Rb1抗内皮细胞复制性衰老中的作用

目的:观察人参皂苷Rb1延缓人脐静脉内皮细胞(HUVECs)复制性衰老的作用,并探讨SIRT1/e NOS/NO通路在其中的作用机制。方法:建立原代HUVECs复制性衰老模型,根据细胞形态的变化、衰老相关β-半乳糖苷酶(SA-β-Gal)染色阳性率和纤溶酶原激活物抑制剂1(PAI-1)的表达水平评估HUVECs衰老情况;采用real-time PCR方法和Western blot法检测沉默SIRT1前后衰老细胞中e NOS和PAI-1的mRNA和蛋白表达,并检测细胞上清NO的含量。结果:累积细胞群体倍增水平(CPDL)为16的HUVECs可作为复制性衰老模型; 80μmol/L人参皂苷Rb1处理后衰老细胞内SIRT1和eNOS的mRNA及蛋白表达水平增加,NO含量增加(P 0. 05),而PAI-1的mRNA和蛋白表达水平下降(P 0. 05);沉默SIRT1后,衰老细胞内e NOS的mRNA及蛋白表达减少,PAI-1的mRNA及蛋白表达增加,NO含量减少(P 0. 05);与SIRT1沉默组比较,在沉默SIRT1基础上加用人参皂苷Rb1后,e NOS和PAI-1表达水平及NO的含量未见明显变化。结论:人参皂苷Rb1可通过调控SIRT1/e NOS/NO通路延缓HUVECs复制性衰老。     

木犀草素对STZ诱导的糖尿病肾脏的保护作用

目的:探讨木犀草素对糖尿病肾脏的保护作用及其可能机制。方法:雄性SD大鼠随机分为5组,即正常对照组、糖尿病模型组、木犀草素低、中、高剂量组,采用腹腔注射链脲佐菌素法建立糖尿病大鼠模型,生化方法测定血糖、尿蛋白、血及肾组织超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性及肾组织丙二醛(MDA)的含量;W estern blotting检测肾皮质转化生长因子β1(TGF-β1)、纤溶酶原激活物抑制物-1(PAI-1)蛋白质水平;光镜观察肾组织形态改变。结果:与糖尿病模型组相比,木犀草素治疗组大鼠血糖水平明显降低(P0.01)、24 h尿蛋白明显减少(P0.01),血清及肾组织SOD、CAT活性升高(P0.01),肾组织MDA含量显著降低(P0.01),木犀草素治疗组肾皮质TGF-β1及PAI-1蛋白表达明显低于糖尿病模型组。结论:木犀草素对STZ诱导的糖尿病大鼠肾脏具有一定的保护作用,其机制可能与其抗氧化作用及降低肾组织TGF-β1、PAI-1的蛋白表达有关。     

Improved fibrinolysis by an intensive lifestyle intervention in subjects with impaired glucose tolerance. The Finnish Diabetes Prevention Study

Aims/hypothesis The aim of this study was to investigate the effects of lifestyle intervention on the levels of plasminogen activator inhibitor (PAI-1) and fibrinogen in subjects participating in the Finnish Diabetes Prevention Study (DPS).Methods In five DPS centres, 321 subjects with impaired glucose tolerance (intervention group, n=163; control group, n=158) had their PAI-1 and fibrinogen levels measured at baseline and at the 1-year follow-up. Additional 3-year follow-up assessments were carried out in a sample of 97 subjects in one of the DPS centres (Turku). The intervention programme included an intensive lifestyle intervention aiming at weight reduction, healthy diet and increased physical activity.Results During the first intervention year, PAI-1 decreased by 31% in the intervention group but showed no change in the control group (p<0.0001). In the Turku subgroup, the decrease in PAI-1 persisted throughout the 3-year follow-up. Changes in PAI-1 were associated with the number of lifestyle changes made during the first year (p=0.008). Weight reduction was the most important factor explaining the decrease in PAI-1. Changes in fibrinogen levels did not differ between the groups.Conclusions/interpretation In addition to the previously reported reduction in the risk of type 2 diabetes in DPS participants with impaired glucose tolerance, the intensive dietary and exercise intervention had beneficial long-term effects on fibrinolysis as indicated by the reduced levels of PAI-1. These results suggest that elevated PAI-1 levels in obese subjects with impaired glucose tolerance are mostly reversible by lifestyle changes, especially those geared to weight reduction.     

Independent validation of stromal uPA in ABCSG-08: Level 1b evidence for the prognostic value of uPA immunohistochemistry

PurposeTo validate the prognostic role of urokinase-type plasminogen-activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) protein expression in FFPE archived tumor samples when assessed by immunohistochemistry.Patients and methodsFresh-frozen, paraffin-embedded (FFPE) samples from 303 postmenopausal women with hormone receptor-positive, early breast cancer were investigated. The patients had received 5 years of endocrine therapy in the prospectively randomized ABCSG-8 trial. Immunohistochemistry for stromal uPA and PAI-1 protein expression was correlated with distant recurrence-free survival (DRFS) and overall survival (OS).ResultsWe detected stromal uPA in 132 of 297 tumors (44.4%) and stromal PAI-1 expression in 74 out of 299 samples (24.7%). Co-expression of uPA and PAI-1 was present in 48 of 294 (16.3%) cases. Neither uPA nor PAI-1 expression was associated with tumor size, age, nodal status, grading, or quantitative receptor status. Patients whose tumor stroma expressed uPA protein had a significantly shorter DRFS (adjusted HR for relapse: 2.78; 95% CI 1.31–5.93; p = 0.008 Cox regression analysis) than women without uPA expression. No such association was seen for PAI-1 and the uPA/PAI1 ratio. After a median follow-up of 5.6 years, women with uPA-positive tumors demonstrated significantly shorter DRFS (93.3% vs. 84.8%; p < 0.013 log-rank test), and tended to have a worse OS (83.0% vs. 77.3%; p = 0.106) compared to women with uPA negative tumors.ConclusionThis independent validation in archived tumor samples from a large prospective randomized trial confirms the clinical utility of stromal uPA evaluation by immunohistochemistry. This provides level 1b evidence for the prognostic role of stromal uPA in women with endocrine-responsive early breast cancer.     

Cell signalling associated with fibrinolytic ligand binding to human colorectal carcinoma cells

Summary Addition of purified plasmin or plasminogen (0.1 M) to serum-free culture media elevated cellular D-myo-inositol 1,4,5-trisphosphate (InsP ₃) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca²⁺ channels of the type blocked by 5 M nifedipine, and 100 M EGTA, a Ca²⁺ chelator, did not suppress plasmin's ability to elevate InsP ₃. Binding assays at 4° C with¹²⁵I-labelled plasmin indicated maximum binding within 1 h suggesting that the effects of plasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers.Abbreviations InsP ₃ inositol trisphosphate - Ptd phosphatidyl     

Impact of proteolytic enzymes in colorectal cancer development and progression

Tumor invasion and metastasis is a highly complicated, multi-step phenomenon. In the complex event of tumor progression, tumor cells interact with basement membrane and extracellular matrix components. Proteolytic enzymes (proteinases) are involved in the degradation of extracellular matrix, but also in cancer invasion and metastasis. The four categories of proteinases (cysteine-, serine-, aspartic-, and metalloproteinases) are named and classified according to the essential catalytic component in their active site. We and others have shown that proteolytic enzymes play a major role not only in colorectal cancer (CRC) invasion and metastasis, but also in malignant transformation of precancerous lesions into cancer. Tissue and serum-plasma antigen concentrations of proteinases might be of great value in identifying patients with poor prognosis in CRC. Our results, in concordance with others indicate the potential tumor marker impact of proteinases for the early diagnosis of CRC. In addition, proteinases may also serve as potential target molecules for therapeutic agents.     

纤溶酶原激活剂抑制物-1基因4G/5G多态性与特发性卵巢早衰的相关性

目的:探讨纤溶酶原激活剂抑制物-1(plasminogen activator inhibitor-1,PAI-1)基因启动子区4G/5G多态性与特发性卵巢早衰(premature ovarian failure,POF)发病的关系。方法:应用PCR-RFLP分析,检测65例特发性POF患者(POF组)和75例正常妇女(对照组)PAI-1基因4G/5G多态性。结果:特发性POF患者组PAI-1基因型频率分布,4G/4G型为29.2%,4G/5G型为55.4%,5G/5G型为 15.4%;POF患者组4G/4G基因型频率明显高于对照组(16.0%),但差异无显著性意义(x2=3.54,P>0.05);而4G等位基因频率(0.569)显著高于对照组(0.447)(x2=4.18,P<0.05),携带4G等位基因个体发生特发性POF的相对风险OR= 1.64,95%CI1.02～2.64。结论:PAI-1基因4G/5G多态性与特发性POF的发病有关,4G等位基因可能是特发性POF发病的危险因素之一。     

纤溶酶原激活物抑制因子-1对肝星状细胞转化生长因子β1、透明质酸、基质金属蛋白酶抑制因子-1的影响

目的 探讨外源性纤溶酶原激活物抑制因子-1(PAI-1)对人肝星状细胞(HSC)转化生长因子β1(TGFβ1)、基质金属蛋白酶抑制因子-1(TIMP-1)及透明质酸(HA)表达的影响.方法 培养液中加入PAI-1,采用四甲基偶氮唑盐法检测人HSC株LX-2的增殖变化,确定PAI-1的最佳干预浓度.将LX-2培养液中加入PAI-1分别培养12、24、48 h,采用酶联免疫吸附法(ELISA)检测细胞上清液中TGFβ1、TIMP-1及HA的变化.结果 阴性对照组A492为0.473±0.035;PAI-1浓度为5、10、20、40、80μg/L时,A492分别为1.249±0.440、1.636±0.315、1.283±0.413、0.938±0.263、0.303±0.125,PAI-1浓度为10μg/L时刺激作用最为明显(F=11.697,P＜0.01).加入PAI-1培养12、24、48 h组较阴性对照组细胞上清液中TGFβ1、HA及TIMP-1表达增加(F=1566.752、P＜0.01,F=235.632、P＜0.01,F=67.359、P＜0.05).结论 PAI-1可促进肝星状细胞TGFβ1、TIMP-1及HA的表达,从而影响肝纤维化的发生发展.

Abstract：

Objective To study the effect of plasminogen activator inhibitor type-1 ( PAI-1 ) on the protein expression of transforming growth factor-beta 1 ( TGFβ1 ), hyaluronic acid ( HA ) and tissue inhibitor of metalloproteinase-1 (TIMP-1) of hepatic stellate cells(HSC). Methods Methyl thiazolyl tetrazolium (MTT)was performed to detect the effect of PAI-1 on the proliferation of LX-2 ,and to determine the perfect intervention concentration of PAI-1. After incubation with PAI-1 in LX-2 culture solution for 12 h,24 h,48 h,the protein expression of TGFβ1 ,TIMP-1 and HA was observed with enzyme linked immunosorbent assay (ELISA). Results A492 in the negative control was 0. 473 ± 0. 353, and 1. 249 ± 0. 440, 1. 636 ± 0. 315,1.283 ± 0. 413,0. 938 ±0. 263 and 0. 303 ±0. 125 when the concentration of PAI-1 was 5,10,20,40 and 80 μg/L,respectively,a concentration of 10 μg/L of PAI-1 showed the strongest stimulation remarkablely ( F = 11. 697, P ＜ 0. 01 ).After incubation with PAI-1 for 12 h,24 h,48 h,the protein expression of HA,TGFβ1 and TIMP-1 in the LX-2 supernatant significantly increased compared to negative control (F = 1566. 752,235. 632 and 67. 359,respectively,P ＜ 0. 01 or ＜ 0. 05 ). Conclusion PAI-1 may influence the development of hepatic fibrosis through promoting the protein expression of HA ,TGFβ1 and TIMP-1 of HSC.     

冠心病患者血浆纤溶酶原激活物抑制物-1水平的变化

目的 :探讨纤溶酶原激活物抑制物 1(PAI 1)与冠心病及其不同临床亚型的相关性及其与冠状动脉病变程度的关系。方法 :选择 345例非糖尿病的住院患者 (其中 2 95例已行冠状动脉造影 ) ,分为对照组和冠心病组 ,测定血浆PAI 1抗原水平。结果 :血浆PAI 1抗原水平在对照组与冠心病组间无统计学差异。与稳定型心绞痛组 (SAP)相比 ,不稳定型心绞痛 (UAP)患者PAI 1水平显著升高 [(2 5 .0± 7.2 ) μg/L比 (2 2 .3± 7.1) μg/L ,P <0 .0 5 ],经Logistic回归分析 ,PAI 1水平与UAP仍有显著性相关 ,调整后的OR值为1.83(P <0 .0 5 )。PAI 1血浆水平与冠状动脉病变支数无关。结论 :血浆PAI 1抗原水平升高可能与UAP有关 ,但与冠心病的发生无关 ,且对冠状动脉病变范围无预测价值。