SB203580对肾缺血/再灌注损伤时细胞凋亡及p38MAPK影响的实验研究

目的　探讨不同时间及不同浓度p38MAPK特异性抑制剂SB2 0 35 80对肾缺血 /再灌注损伤过程中肾功能、细胞凋亡及 p38MAPK活性、表达量、p38MAPK底物的影响。 方法　 4 9只大鼠按缺血 /再灌注及给药时间的不同 ,随机分为 7组 ,每组7只大鼠按正交拉丁方表的顺序 ,经尾静脉注射相同体积、不同剂量的SB ,使其在大鼠体内达到不同的浓度。测定BUN和Scr;用TUNEL试剂盒检测细胞凋亡情况 ;Westernblot技术用于蛋白定性及半定量分析。结果　SB可显著减轻大鼠肾缺血 /再灌注损伤造成的Scr和BUN的升高、肾小管上皮细胞的凋亡及 p38MAPK的激活 ,但存在模型、剂量及给药时机的差异 (P<0 .0 5 ) ,缺血前 3h之前给药 ,使其体内血药浓度达到 5 μmol/L左右可取得较好的效果。 结论　SB可显著减轻大鼠肾缺血 /再灌注损伤 ,在缺血前 3h之前给药 ,同时使其血药浓度达到 5 μmol/L左右可取得最佳效果。     

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.     

Intravenous Lipid Emulsions in the Prevention and Treatment of Liver Disease in Intestinal Failure

The development of intestinal failure-associated liver disease (IFALD) in pediatric and adult patients on parenteral nutrition is usually multifactorial in nature due to nutritional and non-nutritional causes. The role of lipid therapy as a contributing cause is well-established with the pathophysiological pathways now better understood. The review focuses on risk factors for IFALD development, biological effects of lipids, lipid emulsions and the mechanisms of lipid toxicity observed in laboratory animals followed by a synopsis of clinical studies in pediatric and adult patients. The introduction of fish oil-based lipid emulsions that provide partial or complete lipid replacement therapy has resulted in resolution of IFALD that had been associated with soybean oil-based therapy. Based on case reports and cohort studies in pediatric and adult patients who were at risk or developed overt liver disease, we now have more evidence that an early switch to partial or complete fish oil–based lipid therapy should be implemented in order to successfully halt and reverse IFALD.     

Endothelin receptors and calcium translocation pathways in human airways

Tension and phosphatidyl inositol (PI) turnover experiments were conducted to investigate the receptors and signal transduction pathways responsible for contractions elicited by endothelin (ET) ligands in human bronchus. Nicardipine (1 μM), the L-type calcium channel inhibitor, or incubation in Ca²⁺-free medium, produced marked inhibition of contractions to the ET_B receptor-selective agonist, sarafotoxin S6c, and especially those induced by KCl. In contrast, Ca²⁺-free medium was without appreciable effect against contraction produced by endothelin-1 (ET-1), the non-selective ET_A and ET_B receptor agonist. In Ca²⁺-free medium, ryanodine (10 μM), which inhibits intracellular calcium mobilization, reduced sarafotoxin S6c- and ET-1-induced responses, but was without effect on responses to KCl. Similarly, nickel chloride (Ni²⁺; 1 mM) caused marked inhibition of contractions induced by sarafotoxin S6c or ET-1, but had no significant effect on KCl concentration-response curves. The mixed ET_A/ET_B receptor antagonist SB 209670 (3 μM) inhibited responses to sarafotoxin S6c and ET-1 such that concentration-response curves were shifted rightward, at the 30% maximum response level, by 10.0- and 3.8-fold, respectively, whereas BQ-123 (3 μM), the ET_A receptor antagonist, was without effect on responses induced by either agonist. ET-1 (1 nM–0.3 μM) caused a concentration-dependent stimulation of PI turnover, whereas sarafotoxin S6c (0.3 nM–0.1 μM) induced only small and variable increases, except at the highest concentration. The increase in PI turnover evoked by ET-1 was inhibited by SB 209670 (3 μM), and also by BQ-123 (3 μM). This is consistent with linkage of ET_A receptors to activation of inositol phosphate generation in human bronchial smooth muscle cells. Collectively, the data suggest that differences exist in the relative contributions of intracellular and extracellular Ca²⁺ mobilization mechanisms elicited by ET_A and ET_B receptor activation. Thus, sarafotoxin S6c-induced, ET_B receptor-mediated contraction in human bronchial smooth muscle appears to be dependent, in part, upon extracellular Ca²⁺, although a significant component of the response was also mediated by intracellular Ca²⁺ release, including from ryanodine-sensitive stores. ET_A receptor-mediated contraction of human airway smooth muscle was activated largely via the release of intracellular Ca²⁺. Received: 21 July 1998 / Accepted: 26 January 1999     

Human Antibody Fragments Specific for the Epidermal Growth Factor Receptor Selected from Large Non-immunised Phage Display Libraries

Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2 nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (k_a and k_d). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.     

GSK-3β suppresses the proliferation of rat hepatic oval cells through modulating Wnt/β-catenin signaling pathway

Aim:

Glycogen synthase kinase 3β (GSK-3β) plays a crucial role in hepatic biology, including liver development, regeneration, proliferation and carcinogenesis. In this study we investigated the role of GSK-3β in regulation of growth of hepatic oval cells in vitro and in liver regeneration in partially hepatectomized rats.

Methods:

WB-F344 cells, the rat hepatic stem-like epithelial cells, were used as representative of oval cells. Cell viability was examined using a WST-8 assay. The cells were transfected with a recombinant lentivirus expressing siRNA against GSK-3β (GSK-3βRNAiLV) or a lentivirus that overexpressed GSK-3β (GC-GSK-3βLV). Adult rats underwent partial (70%) hepatectomy, and liver weight and femur length were measured at d 7 after the surgery. The expression of GSK-3β, phospho-Ser⁹-GSK-3β, β-catenin and cyclin D1 was examined with immunoblotting assays or immunohistochemistry.

Results:

Treatment of WB-F344 cells with the GSK-3β inhibitor SB216763 (5 and 10 μmol/L) dose-dependently increased the levels of phospho-Ser⁹-GSK-3β, but not the levels of total GSK-3β, and promoted the cell proliferation. Knockout of GSK-3β with GSK-3βRNAiLV increased the cell proliferation, whereas overexpression of GSK-3β with GC-GSK-3βLV decreased the proliferation. Both SB216763 and GSK-3βRNAiLV significantly increased the levels of β-catenin and cyclin D1 in the cells, whereas GSK-3β overexpression decreased their levels. In rats with a partial hepatectomy, administration of SB216763 (2 mg/kg, ip) significantly increased the number of oval cells, the levels of phospho-Ser⁹-GSK-3β, β-catenin and cyclin D1 in liver, as well as the ratio of liver weight to femur length at d 7 after the surgery.

Conclusion:

GSK-3β suppresses the proliferation of hepatic oval cells by modulating the Wnt/β-catenin signaling pathway.     

Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways

Aim:

To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action.

Methods:

Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting.

Results:

PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC₅₀ less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity.

Conclusion:

Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.     

Dual Antiplatelet Therapy Duration Determines Outcome After 2- But Not 1-Stent Strategy in Left Main Bifurcation Percutaneous Coronary Intervention

Objectives

The aim of this study was to investigate clinical outcomes after left main coronary artery (LM) bifurcation percutaneous coronary intervention (PCI) and the impact of the duration of dual antiplatelet therapy (DAPT) according to treatment strategy.

SB203580对急性肺栓塞后肺动脉高压及MCP-1表达的影响

 目的：观察单核细胞趋化蛋白1(monocyte chemoattractant protein-1, MCP-1)与急性肺栓塞（pulmonary thromboembolism, PTE）后肺动脉高压形成的关系;探讨p38丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）特异性抑制剂SB203580对急性PTE后肺动脉高压及MCP-1表达的影响。方法：采用自体血栓回输法复制Sprague-Dawley大鼠急性PTE模型;将大鼠随机分成5组,每组观察1 h、4 h和8 h 3个时点;急性PTE模型复制前1 h,分别对MCP-1中和抗体C1142组及SB203580组进行药物预处理;在1 h、4 h和8 h检测各组肺动脉平均压力（mean pulmonary artery pressure, MPAP）和MCP-1 mRNA及蛋白表达。结果：（1）在相同时点,急性PTE组MPAP和MCP-1 mRNA及蛋白表达均较溶剂对照组显著升高（P<005）;（2）在相同时点,C1142组及SB203580组MPAP和MCP-1 mRNA及蛋白表达均较急性PTE组显著降低（P<005）。结论：（1）急性PTE后MCP-1的大量表达参与急性PTE性肺动脉高压的形成;（2）SB203580可能通过p38 MAPK信号转导通路,下调MCP-1表达,降低急性PTE肺动脉压力。