【摘要】 目的 探讨抗氧化应激是否参与参附注射液预处理诱导的肾脏保护作用。 方法 健康成年雄性SD大鼠21只随机分为假手术对照组（Sham组）、肾脏缺血再灌注组（I/R组）和参附注射液组（SF组）；SF组给予参附注射液10 mL/kg腹腔注射，每日1次，连续给药7d。麻醉下行右肾切除后，用无损伤动脉夹钳夹左侧肾蒂60min，再灌注24 h，制备肾缺血再灌注损伤动物模型。比较各组SD大鼠再灌注24 h肾脏组织中超氧化物歧化酶（superonidedismutase，SOD）水平、过氧化氢酶（catalese，CAT）和丙二醛（malonicalaldehyed，MDA）含量。 结果 与Sham组相比，I/R和SF组肾脏组织SOD和CAT显著降低，而MDA明显升高（Plt;0.05）；与I/R组比，参附注射液能明显增加SOD和CAT水平（Plt;0.05），降低MDA含量（Plt;0.05）。 结论 参附注射液预处理可增强缺血再灌注损伤肾脏组织抗氧化应激，其表现为增强SOD和CAT的活力，减少MDA的生成。【Abstract】 Objective To explore the protective effect of Shenfu injection combined with antioxidant system on rats’ kidney after ischemia-reperfusion injury. Methods Twenty-one male Sprague Dawley (SD) rats were randomly divided into 3 groups: sham operation group (Sham group), ischemia-reperfusion group (IR group), and shenfu injection treated group (SF group). The rats were anesthetized with valebarbitone. Bilateral kidneys were exposed through midline incision. The right kidney underwent the nephrectomy and left renal pedicels were occluded for 60 minutes with a traumatic mini-clamp and then unclamped for 24 hours. Animals in SF group received Shenfu injection (10 mL/kg) through intraperitoneal injection every day for 7 days. About 24 hours after reperfusion, superoxide dismutase (SOD), CAT and malonical aldehyde (MDA) were measured. Results The levels of MDA were lower in SF group than those in IR group (Plt;0.05). The level of SOD and CAT in SF group increased more significantly than which did in IR group (Plt;0.05). Conclusion Our finding suggests that antioxidant system in SF group works more efficiently than IR group to overcome oxidative stress in renal ischemia-reperfusion injury.
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 observe the effects of hydrogen peroxide on the expression of transforming growth factorβ1(TGF-β1) and Smad3 protein in A549 cells. Methods A549 cells were cultured with different concentrations of hydrogen peroxide. MTT assay was used to determine the cell growth and survival rates. The level of TGF-β1 and p-Smad3 protein were detected by western blotting. Results It was observed that hydrogen peroxide significantly inhibit proliferation of A549 cells. When the concentration of hydrogen peroxide was 1.0 mmol/L, the inhibition ratio reaches 46.34%, and the level of TGF-β1 and p-Smad3 protein were increased in a time-dependence manner and reached a peak after 24 h, then decreased a little but also remained at high level. Conclusions In the early oxidative damage, A549 cells express high level of TGF-β1 and p-Smad3 protein. It may be relevant to tissue repair and remodeling after lung injury.
Objective To observe the effects of cigarette smoke extract ( CSE) on the proliferation and secretion of hydrogen peroxide ( H2O2 ) in human lung fibroblasts ( HLFs) induced by transforming growth factor-β1 ( TGF-β1 ) . Methods Cultured HLFs were divided into a normal group and a model group induced by TGF-β1 ( 5 ng/mL) , then intervened with CSE at different concentrations ( 0% , 2. 5% , 5% ,10% , respectively) . Brdu ELISA assay was used to detect cell proliferation. H2O2 release from cultured cells was assayed using a fluorimetric method. Cellular localization of H2O2 and expression of α-SMA were performed using a fluorescent-labeling strategy. Results TGF-β1 stimulated group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In TGF-β1 stimulated group, 2. 5% and 5% CSE promoted cell proliferation ( P lt; 0. 01 or 0. 05) , while 10% CSE inhibited cell proliferation ( P lt; 0. 01) . In the normal group, both low and high concentration of CSE inhibited cell proliferation ( P lt; 0. 01 or P lt; 0. 05) , and the inhibition effect was dose-dependent. HLF induced by TGF-β1 generated low constitutive levels of extracellular H2O2 that was markedly enhanced by CSE treatment ( P lt; 0. 01) . Pretreatment with DPI, an inhibitor of NADPH oxidase, abolished secretion of H2O2 . Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast. Conclusions Low concentration of CSE can promote myofibroblast proliferation, and markedly increase extracellular secretion of H2O2 . CSE possibly take part in the development and progress of idiopathic pulmonary fibrosis by increasing oxidative stress.
Objective To study the influence of the immersed time by hydrogen dioxide on the characteristics of bovine cancellous bone granules in various periods. Methods Ten 24-month-old Qinchuan bovine, male or female, weighing 150-170 g, were selected. Cancellous bone granules from metaphysic of bovine long bone were cut into cubes of 5 mm × 5 mm ×5 mm and immersed by 8.8 mol/L hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72 hours separately. Determination of ash, scanning electron microscope, X-ray energy spectrum and micro CT were used to investigate the changes of composition, structure and qual ity of bone. Results With the immersed time increasing, the contents of organics in the bone cancellous were reduced gradually, and obviously decreased during the periods of 0 to 24 hours and 60 to 72 hours (P lt; 0.05). The contents of calcium and phosphorus decreased gradually, they could not be detected almost after 60 days (P lt; 0.05). Bone mineral density and bone mineral content were decreased obviously after 60 hours (P lt; 0.05). The bone trabecula became sl immer and trabecular spacing became larger. Conclusion Hydrogen dioxide can be used to remove the antigen in xenogeneic bone; however as the time increasing (more than 60 hours) the composition and structure will be damaged. Thus it is important to control the immersed time for maintaining the biological characteristics of xenogeneic bone substitute as well as el iminating antigen by hydrogen dioxide.
Objective To investigate the effect of advanced glycation end products (AGEs) on the catalase activity and the levels of malondialdehyde in cultured bovine retinal capillary pericytes (BRPs), and to investigate the relationship between oxidative stress and diabetic retinopathy. Methods Cultured BRPs were exposed to AGEs (0, 8, 32, 125, 500, 2 000 μg/ml) for four days. Activity and the levels of catalase and malondialdehyde in cultured BRPs were examined by spectrophotometry. Results AGEs decreased the catalase activity, whereas increased the levels of malondialdehyde of cultured BRPs in a dose-dependent manner (r=-0.714, r=0.748, P＜0.01).There were significant differences between BRPs cultured in 32 μg/ml AGEs and in control group (P＜0.01), while no significant differences between BRPs cultured in non-glycated bovine serum albumin and absence of bovine serum albumin were found. Conclusion Oxidative stress may be one of the reasons why the pericyte disappears in diabetic retinopathy. (Chin J Ocul Fundus Dis, 2002, 18: 143-145)
With the oxidative damage model established in rat myocardial cells by hydrogen peroxide (H2O2), the expression of myocardin and nuclear factor erythroid 2-related factor 2 (Nrf2) during oxidative damage and effect of myocardin on Nrf2 were preliminarily explored. The expression of the target gene was increased or decreased by transfection of plasmid DNA or shRNA, respectively. Cell proliferation was detected by sulforhodamine B (SRB) assay. The expression of myocardin mRNA and Nrf2 mRNA was detected by Real-time PCR, and their protein levels were detected by Western blot. The results showed that oxidative damage was induced by H2O2 with an optimized incubation condition of 200 μmol/L H2O2 for 24 hours. H2O2 inhibited expression of myocardin in mRNA and protein levels, and increased expression of Nrf2 in mRNA and protein levels. The overexpression of myocardin or the knockdown of Nrf2 significantly decreased cell viability compared with the control group, while the knockdown of myocardin or the overexpression of Nrf2 significantly increased cell viability. The overexpression of myocardin significantly down-regulated the expression of Nrf2 in mRNA and protein levels, while the knockdown of myocardin dramatically up-regulated the expression of Nrf2. Thus, it is deduced that myocardin may inhibit cell proliferation and Nrf2 may promote cell proliferation. Oxidative damage induced by H2O2 in rat myocardial cell might activate Nrf2-related signaling pathway through down-regulation of myocardin.
Objective To observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro. Methods RPE cells were cultured and divided into a normal group, normal+H2O2 group, Vec+H2O2group, PSF+H2O2 group according to the experimental design. Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells, then RPE cells were exposed to H2O2. The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay. The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit. Meanwhile, intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method. Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining, and effectively reduce dead cells number shown by Live/Dead staining. After H2O2 stimulation, the survival rate, apoptosis rate and ROS production level in PSF overexpression group were 0.68±0.12, 0.44±0.08 and 18 616±3 382.54 respectively, showing significant difference in comparison with the vector plasmid group and normal group (P＜0.05). Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells.
ObjectiveTo observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).MethodsHuman retinal Müller cells cultured in vitro were divided into normal control group, model group (H2O2 group) and experimental group (H2O2+NBP group). The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μmol/L H2O2 for 2 h. Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 μmol/L NBP. The normal control group was a conventional cultured cells. Müller cells were identified by immunofluorescence staining. Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes. MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention. Hoechst33258 staining was used to observe the apoptosis. LIVE/DEAD ® cell activity/cytotoxicity kit was used to detect cell viability. Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. One-way ANOVA combined with Dunnett statistical method were used for data analysis.ResultsHE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group. MTT assay showed that after 24 h and 48 h of NBP intervention, the differences in cell viability between the normal control group and the H2O2 group, the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96, 3.658, 47.58, 20.33; P＜0.001, 0.022). The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced, and the blue fluorescence of the remaining cells was uniform. The LIVE/DEAD ® cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly, and the number of viable cells with green fluorescence decreased significantly. In the H2O2+NBP group, the number of viable cells with green fluorescence increased, and the number of dead cells with red fluorescence decreased. The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced; the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.ConclusionNBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.
ObjectiveTo investigate the disinfection effect of dry-fogging hydrogen peroxide (DFHP) on ambulance inner surfaces.MethodsThis study was carried out using simulated field test and field test from October to December 2018. In the simulated field test, the carriers with Geobacillus stearothemopilus (ATCC12980) spores were placed in 6 places in the ambulance, and disinfected for 60 minutes with DFHP of 0.38–0.72 g/m3. The carriers were cultured for up to 7 days to observe whether the bacteria were eliminated. Before and after the DFHP disinfection, the microbial sampling of the surface in the ambulance was carried out, and the colonies were counted after the cultivation.ResultsThe eliminating rate of the bacteria carriers on the uncovered surface was 100% (20/20), and that of the covered surface was 10% (1/10). The pass rate of microbial sampling was 100% (26/26).ConclusionsThe DFHP had a significant decontamination effect on the ambulance inner uncovered surfaces. The DFHP equipment is automated and their disinfecting quality is consistent, therefore it is suitable for the disinfection of ambulance inner surfaces. But the limitation of disinfection effect on covered surfaces should be avoided.