Objective To observe the effects of δ-opioid receptor agonists D-Ala2-D-Leu5-enkephali （DADLE） on hepatocyte apoptosis and expressions of bcl-2 and caspase-3 in septic rat, and to investigate the possible mechanism by which DADLE protects the liver in sepsis. Methods Sepsis was reproduced in rats by cecum ligation and puncture (CLP). Fifty-four SD rats (either male or female) were randomly divided into CLP group (n=18), DADLE group (n=18) and sham operation (SO) group (n=18). The rats were respectively killed at different time (2 h, 4 h and 6 h after operation). Hepatocyte apoptosis was detected by TdT-mediated dUTP Nick End Labeling (TUNEL). The expressions of bcl-2 and caspase-3 protein were detected by immunohistochemistry. And the changes of pathology in hepatic tissue were detected by light microscope. Results The hepatic pathological lesion of rats in CLP group was obviously serious compared with SO group, while it was obviously improved in DADLE group. The apoptosis index of rat hepatocytes in CLP group significantly increased compared with SO group, and further it was prominent at 4 h （P＜0.01). The apoptosis index of rat hepatocytes at each time of DADLE group was significantly decreased compared with CLP group （P＜0.01）. Expression of caspase-3 protein in liver tissues of CLP group significantly increased compared with SO group （P＜0.01）, while the expression of bcl-2 protein significantly decreased （P＜0.05）. Expression of caspase-3 protein in liver tissues of DADLE group significantly decreased compared with the CLP group （P＜0.01）, while the expression of bcl-2 protein significantly increased （P＜0.05）. There was positive correlation between expression of caspase-3 in liver tissues and apoptosis index of hepatocyte (r=0.83, P＜0.01) and negative correlation between expression of bcl-2 in liver tissues and apoptosis index of hepatocyte (r=-0.65, P＜0.01). Conclusions The findings indicate that δ-opioid receptor agonists DADLE can obviously improve hepatic pathological changes of septic rats. And its protective mechanism contains down regulation of caspase-3 expression, upregulation of bcl-2 expression and thus the apoptosis of hepatocyte is repressed.
研究白介素6(IL-6)在过氧化氢诱导的肺泡上皮细胞凋亡中的作用及其机制。方法：以人Ⅱ型肺泡上皮细胞A549细胞株为实验对象，MTT法检测IL-6对A549细胞增殖活性的影响，TUNEL检测法检测IL-6对A549细胞凋亡率的影响，Western blot观察caspase-3和caspase-9蛋白量的变化。结果：IL-6能够增加A549细胞的增殖活性，减少其凋亡。与模型组相比，IL-6干预组的caspase-3和caspase-9的蛋白表达量明显降低,有统计学意义。结论：IL-6能够抑制过氧化氢诱导的肺泡上皮细胞凋亡，并且与caspase-3和caspase-9蛋白的表达量下降有关。提示IL-6对肺泡上皮细胞的保护作用可能是通过抑制细胞凋亡相关蛋白来实现的。
OBJECTIVE: To investigate apoptosis of chondrocytes cultured in vitro and related expression of caspase-3. METHODS: Apoptosis of chondrocytes were detected by flow cytometry analysis and TUNEL staining. The expression of caspase-3 was determined by RT-PCR and Western blot, and caspase-3 protein activity was determined by ELISA. RESULTS: Apoptosis was observed in chondrocytes cultured in vitro from passage 1 to passage 4 at various degrees. The percentage of apoptosis of chondrocytes on day 7 was much higher than that on day 3 (15.7% +/- 0.3% vs 8.9% +/- 0.6%, P lt; 0.01). caspase-3 mRNA and protein expressed in chondrocytes during whole culture process. Along with the culture time extension in vitro, caspase-3 expression and protein activity up-regulated, coincident with apoptosis of chondrocyte. caspase-3 was activated and a fragment of 20 kDa was detected after 7 days of culture. CONCLUSION: caspase-3 is involved in apoptosis of chondrocytes cultured in vitro.
【摘要】 目的 探讨铁螯合剂去铁胺（DFO）对诱导白血病细胞HL-60的分子机制。 方法 2003年7-12月用钙黄绿素（calcein）检测HL-60细胞LIP。台盼蓝活细胞拒染实验进行活细胞计数及细胞存活率测定；光镜形态学观察及流式细胞仪（FCM）等方法检测HL-60细胞凋亡；比色法检测caspase-3（基于pNA标记底物的比色法）活性。 结果 ①不同浓度的DFO作用于HL-60细胞后，随培养时间延长及DFO浓度的增加，动态铁池降低，细胞生存率逐渐下降，凋亡率增加，显示一定的时间剂量依赖性。②HL-60细胞在不同浓度的DFO作用下，caspase-3的活性逐渐升高。50、100 μmol/L DFO作用于HL-60细胞24 h，caspase-3酶活性升高明显，与对照组相比，有统计学意义（Plt;0.001）；相关分析结果显示，HL-60细胞LIP的改变与caspase-3活性变化呈负相关系（r=-0.887，Plt;0.05）。 结论 DFO诱导白血病细胞凋亡的作用可能与螯合细胞内铁，降低细胞LIP，激活caspase-3，最终实施细胞凋亡密切相关。【Abstract】 Objective To observe the changes of caspase-3 activity during apoptosis of HL-60 cells induced by an iron deferoxamine (DFO). Methods Exponentially growing HL-60 cells (1×106/mL) were used in this experiment from July 2003 to December 2003. The study groups were divided as follows: DFO group, iron+DFO group and control group. The viability was detected by typanblue, apoptosis was assessed by morphological study and flow cytometry (FCM) assay, and the caspase-3 activity was detected by melorimetry. The intracellular label iron pool (LIP) was measured with a fluorimetric assay using the metalsensitive probe calcein-AM. Results ①When HL-60 cells were incubated with different concentrations of DFO, viability assay was lower than that in the control group at the 12th, 24th and 48th hour (Plt;0.05). ② The cells incubated with different concentrations of DFO showed dose-time dependence and was much higher than that in the control group (Plt;0.01). ③The caspase-3 activity was significantly higher in the apoptotic cells than that in the control cells. Conclusions The apoptosis of HL-60 cells induced by DFO may be correlated with the decrease of cellular LIP and activity of caspase-3.
目的:探讨白藜芦醇对人脑胶质瘤U251细胞的凋亡相关因子caspase-3的作用。方法:应用RT-PCR方法和Western-blot分别检测凋亡因子caspase-3的RNA和蛋白水平的表达。结果:白藜芦醇能明显促进U251细胞的凋亡相关因子caspase-3的表达,存在剂量依赖与时间依赖性。结论:白藜芦醇能通过caspase－3途径诱导U251细胞的凋亡。
ObjectiveTo investigate the expression of caspase-3 and Toll-like receptor 4 (TLR4) in the incised rat skin healing process and its relationship with the wound time and to provide an experimental evidence for the prediction of injury time. MethodsAfter the rat incised wound model was established, hematoxylin-eosin dyeing technology and immunohistochemical staining technique were used to observe the expression of caspase-3 and TLR4. Then Image Pro Plus Image analysis software and SPSS statistical analysis software were used to deal with the experimental results. ResultsCaspase-3- and TLR4-positive cells were detected in epidermis, hair follicle and sebaceous gland cells in the control skin. The expression of caspase-3 and TLR4 of the ante mortem groups were significantly different compared with the control group except the 0 h group (P<0.05). Caspase-3- and TLR4-positive cells were detected in neutrophils around the hair follicle half an hour later. Caspase-3- and TLR4-positive cell rate increased with the infiltration of inflammatory cells. Caspase-3- and TLR4-positive cell rate reached the maximum on the 3 rd day, and then it began to decrease, and they were mainly expressed in fibroblasts and mononuclear macrophages. Caspase-3- and TLR4-positive cells were mainly expressed in fibroblasts on the 10th day. There was no significant differences between the postmortem injury groups and the normal control groups (P>0.05). ConclusionCaspase-3- and TLR4-positive cell rate is time dependent and stable in 25℃ temperature environment which makes it possible to determine the time of injury.