植物细胞H_2O_2的产生及其生理作用

过氧化氢（hydrogen peroxide,H2O2）是细胞有氧代谢的产物,激素等发育信号和胁迫刺激都可以诱导细胞内H2O2的产生和积累。H2O2在细胞内浓度的升高和降低以及变化模式可能介导了不同的信号转导途径,并调控相应生长发育、胁迫应答等生物学过程。     

氧化应激对鲤抗氧化状态和免疫功能的影响

为探讨氧化应激对鲤抗氧化状态和免疫功能的影响,本实验以H_2O_2作为活性氧自由基(ROS),将鲤暴露于不同浓度的H_2O_2(0、0.25、0.50和1.00 mmol/L)中,诱导鲤产生氧化应激反应。连续暴露7 d后,采集鲤血液和肝组织,以检测相关生化指标以及基因表达量的变化。结果显示,与空白对照组(0 mmol/L)相比,随着H_2O_2浓度的升高,血清葡萄糖(GLU)、皮质醇(cortisol)和乳酸(LA)含量显著升高;而碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活性仅在1.00 mmol/L H_2O_2处理组中显著高于其他实验组。氧化应激参数显示,与空白对照组(0 mmol/L)相比,0.50和1.00 mmol/L H2O2处理显著降低血清过氧化氢酶(CAT)活性,而提高还原型谷胱甘肽(GSH)、丙二醛(MDA)和总抗氧化能力(T-AOC)水平;在肝脏组织中,1.00 mmol/L H_2O_2处理显著降低了GSH含量,促进了MDA生成。基因表达结果显示,与空白对照组相比,1.00 mmol/L H2O2处理组显著上调了肝脏组织中cyp1a表达,而下调了cyp1b表达;同时0.50和1.00 mmol/L H2O2处理显著上调了hsp70、hsp90、c3、c-lyz和hep的表达。研究表明,氧化应激暴露可诱导鲤产生明显应激反应和脂质过氧化,降低机体抗氧化能力并激发免疫应答反应。     

水杨酸对烟草抗黄瓜花叶病毒的诱导效应

通过外源水杨酸（Salicylic acid,SA）处理烟苗,研究了水杨酸处理的烟草接种黄瓜花叶病毒后,其病情指数及抗病相关酶活性和H2O2含量的变化。结果表明：接种CMV后21d,SA处理烟株的病情指数显著低于对照;SA预处理的烟株在接种CMV前后叶片内的过氧化物酶（POD）活性和过氧化氢（H2O2）含量提高,过氧化氢酶（CAT）活性下降。表明水杨酸具有诱导烟草抗黄瓜花叶病毒的效应。     

Hydrogen sulfide attenuates urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway

AIM:To explore the effect of hydrogen sulfide (H₂S) on urosepsis-induced acute kidney injury. METHODS:New Zealand white rabbits were randomly divided into control group, sham group, model (sepsis) group, NaHS treatment (NaHS) group, and NaHS combined with TAK-242 (a TLR4 inhibitor) treatment (NaHS+TAK-242) group. After treatment for 72 h, HE staining was used to measure the histopathological changes of rabbit kidney. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by automatic biochemical analyzer. The serum levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), procalcitonin (PCT), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The TLR4/MyD88/PI3K signaling pathway-related proteins in the kidney were determined by Western blot. RESULTS:Compared with control group, obvious damage was observed in the kidneys of septic rabbits, but the kidneys were markedly improved by treatment with NaHS. The levels of BUN, SCr, NGAL, KIM-1, PCT, IL-1β, IL-6 and TNF-α in the septic rabbits were higher than those in control group, and decreased significantly in NaHS group and NaHS+TAK-242 group. The protein levels of TLR4, MyD88, p-PI3K and p-Akt in septic rabbit kidneys were higher than those in control group. However, NaHS or NaHS+TAK-242 inhibited the activation of TLR4/MyD88/PI3K signaling pathway in the kidneys of septic rabbits. CONCLUSION:H₂S play a protective effect on the rabbits with urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway to inhibit inflammatory response.     

Effects of hydrogen sulfide on impaired wound healing in ob/ob mice

AIM: To investigate the role of hydrogen sulfide(H₂S) on impaired wound healing in ob/ob mice and the underlying mechanism.METHODS: The ob/ob mice were randomly divided into 3 groups, including vehicle, insulin and NaHS for treatment. C57BL/6 mice were treated with vehicle as control. Full-thickness punch biopsy wounds were created on the mice. Firstly, H₂S concentrations in the skins and granulation tissues were measured. The mRNA expression of cystathionine γ-lyase(CSE) was detected by RT-qPCR. The protein expression of CSE and MMP-9 were determined by Western blot. The neutrophil and monocyte/macrophage infiltration was analyzed by immunohistochemistry me-thod. The levels of tumor necrosis factor(TNF)-α and interleukin(IL)-6 were measured by ELISA.Collagen formation was measured by Masson staining.RESULTS: The H₂S levels in the skin and granulation were significantly decreased in ob/ob mice and increased in the NaHS-treated mice(P<0.05). CSE expression at mRNA and protein levels was significantly decreased in ob/ob mice compared with the control mice(P<0.05). The wound healing period was significantly shorter in NaHS group than that in vehicle-treated ob/ob mice group(P<0.05), in which the insulin group had no difference with vehicle ob/ob mice group. The neutrophil and monocyte/macrophage infiltration, and TNF-α and IL-6 levels were significantly increased in ob/ob groups, but were decreased in NaHS group(P<0.01 or P<0.05). Meanwhile, NaHS increased collagen formation in the granulation tissues of ob/ob mice.CONCLUSION: H₂S/CSE down-regulation contributes to impaired wound healing in diabetes, which is alleviated by exogenous H₂S possibly through anti-inflammation.     

Neuroprotective effect of H2S by inhibiting autophagy after restoration of spontaneous circulation in rats with cardiac arrest

AIM: To investigate the neuroprotective effect of hydrogen sulfide (H₂S) after cardiopulmonary resuscitation in rats with cardiac arrest (CA), and to explore the effects of H₂S on neuron autophagy. METHODS: The CA model was established through asphyxia. Male Wistar rats were randomly divided into sham group, model group and NaHS group. The levels of beclin-1 and LC3 II/I were measured by Western blot at 2 h, 4 h, 12 h and 24 h after the restoration of spontaneous circulation (ROSC). At 12 h after ROSC, the formation of autophagic vacuole with LC3 dots was determined by immunohistochemical (IHC) method. The phenomenon of neuron autophagy was observed under transmission electron microscope. The numbers of apoptotic neurons were counted by TUNEL staining at 72 h after ROSC. The neurolo-gic deficit score (NDS) was used to evaluate the neurologic function after ROSC. RESULTS: The level of beclin-1 was gradually increased in model group, but it was increased and then gradually recovered in NaHS group (P < 0.05). The conversion of LC3 II in the cerebral cortex was the same as beclin-1. The results of IHC showed that LC3-positive nuclei in model group were more than those in NaHS group (P < 0.05). The number of autophagic vacuole in model group was more than that in NaHS group (P < 0.05). The number of the TUNEL-positive cells in model group was more than that in NaHS group (P<0.05). The NDS of the animals in NaHS group after ROSC was lower than that in model group(P < 0.05). CONCLUSION: H₂S inhibits neuronal autophagy, decreases apoptosis and improves neurologic function in CA rats after ROSC.     

肉鸡Trx1和Trx2蛋白的表达纯化及其抗氧化应激效果评价

试验旨在对肉鸡Trx1和Trx2蛋白进行表达和纯化,并评价其抗氧化特性。将原核表达载体GgTrx1-pET28a(+)和GgTrx2-nsp-pET28a(+)转入大肠埃希菌中进行蛋白表达;应用镍柱亲和层析的方法对该融合蛋白进行纯化;采用胰岛素还原法对重组肉鸡Trx1和Trx2蛋白(GgTrx1和GgTrx2)进行活性鉴定;通过细胞体外试验分析比较GgTrx1和GgTrx2对大鼠肝细胞BRL-3A氧化应激保护作用的影响。结果显示,试验成功获得两种重组蛋白:GgTrx1和GgTrx2,最适诱导条件分别为37℃、0.4mmol/L IPTG诱导4h和37℃、1.0mmol/L IPTG诱导4h;纯化的重组蛋白GgTrx1和GgTrx2纯度可达到90%,浓度分别达到4.0和5.0 mg/mL。重组蛋白GgTrx1和GgTrx2均具有较高的还原胰岛素二硫键的能力,并呈现出浓度效应。体外细胞试验发现,重组蛋白GgTrx1和GgTrx2均能显著降低过氧化氢诱导的BRL-3A膜脂过氧化,保护抗氧化酶SOD和CAT活性,且重组蛋白GgTrx2效果更明显。本试验结果表明,重组蛋白GgTrx1和GgTrx2均具有生物学活性和良好的抗氧化应激特性,且线粒体型Trx2为临床治疗氧化应激性疾病提供了更多可能。     

银杏内酯对活性氧自由基和过氧化氢清除作用的研究

研究了银杏叶中内酯类提取物对超氧阴离子自由基、羟自由基和过氧化氢的清除作用。为了进一步分析内酯类在细胞内的清除作用,在反应体系中加入了大肠杆菌提取物以部分模拟细胞环境,结果表明,内酯类提取物对自由基和过氧化氢的清除作用不显著。在清除过程中,内酯类对黄酮类自由基清除的协同作用比较复杂。但可以肯定的是大肠杆菌提取物对自由基和过氧化氢的清除没有影响或影响较小。