Effect of cyclooxygenase 2 on hippocampal neuronal damage induced by aluminum in rats

AIM:To study the relationship between cyclooxygenase 2(COX-2) and the damage of hippocampal neurons by aluminum overload in rats. METHODS:Newborn SD rats(less than 24 h) were used to establish the model of primary culture of hippocampal neurons. The neurons were treated with aluminum at concentration of 200 μmol/L. The techniques of RNA interference(RNAi) and cell transfection were used to study the role of COX-2 in hippocampal neuron. Following RNAi by cell transfection, Western blotting analysis was used to determine the protein expression of COX-2. Cell growth was assayed by the method of MTT. The pathological changes of the neurons were observed by fluorescence labeling. The activity of superoxide dismutase(SOD) and lactate dehydrogenase(LDH), and the content of malondialdehyde(MDA) were detected for evaluating cell damage. RESULTS:COX-2 RNAi by cell transfection significantly decreased the protein expression of COX-2 without changing the neuronal pathomorphology, cell viability, SOD activity and MDA content. However, it obviously improved livability and SOD activity of the hippocampal neurons, which were aluminum-overloaded. Inhibition of COX-2 expression also reduced the leakage of LDH and the content of MDA, and ameliorated the pathological changes in neurons. CONCLUSION:Moderate silence of COX-2 expression not only significantly affects the morphological changes and physiological functions of hippocampal neurons, but also prevents the neurons from aluminum-induced damages.     

番茄匍柄霉叶斑病拮抗细菌的筛选与鉴定

应用平板对峙法从连作多年的番茄根际土样中筛选得到对番茄匍柄霉叶斑病具有较强拮抗活性的细菌菌株ZF161,该菌株对番茄匍柄霉的平板抑制率达到70.51%。离体叶片试验显示,菌株ZF161对番茄匍柄霉叶斑病防治效果达到69.83%。通过菌落形态观察、生理生化特性、Biolog测定和多基因系统发育树综合分析,鉴定菌株ZF161为枯草芽孢杆菌(Bacillussubtilis)。番茄盆栽试验结果表明,菌株ZF161对番茄匍柄霉叶斑病的防治效果达到63.27%。进一步平板对峙抑菌谱试验结果显示,菌株ZF161对其他7种病原真菌也具有较好的抑制效果。上述结果说明,菌株ZF161对番茄匍柄霉叶斑病具有良好的防治效果,且对多种病原真菌也具有抑制作用,具有开发应用的潜力。     

Protective effect of quercetin on L-02 cells by inhibiting DNA damage of INH-induced mitochondrial oxidative stress

AIM: To investigate the role of reactive oxygen species(ROS)-mediated mitochondrial oxidative injury in isonicotinyl hydrazide(INH)-induced DNA damage and the protective effect of quercetin on L-02 cells. METHODS: The injury model of hepatocyte L-02cells in vitro induced by INH was established. The cells were divided into control group, INH group, low-dose quercetin group and high-dose quercetin group. The DNA damage of L-02 cells was evaluated by the comet test. The mitochondrion was prepared, and the level of mitochondrial ROS and the value of mitochondrial membrane potential(ΔΨ_m) were detected by fluorescent probes DCFH-DA and rhodamine 123. The content of MDA was measured by TBA method. The activity of SOD was assessed with the xanthine oxidase method. The protein expression of Bcl-2 and Bax was determined by Western blotting, and the value of Bax/Bcl-2 was calculated. RESULTS: INH induced obvious DNA damage, increased the level of mitochondrial ROS, the content of MDA and the value of Bax/Bcl-2, and markedly reduced the value of ΔΨ_m and the activity of SOD in the L-02 cells. Quercetin attenuated DNA damage, reduced the level of mitochondrial ROS, elevated the value of ΔΨ_m, declined the content of MDA, increased the activity of SOD and decreased the value of Bax/Bcl-2 in the L-02 cells. CONCLUSION: INH induces DNA damage in L-02 cells by generation of mitochondrial oxidative stress. Quercetin has a protective effect on L-02 cells to attenuate the INH-induced DNA damage by inhibiting ROS-mediated mitochondrial oxidative damage.     

The mechanism of neuronal injury and repair after focal cerebral ischemia

AIM:To explore the mechanism of neuronal injury and repair by investigating the expression of caspase-3 and apurinic/apyrimidinic endonuclease (APE/Ref-1) after focal cerebral ischemia. METHODS:A model of middle cerebral artery occlusion in rats was performed. The expression of caspase-3P₂₀ and APE/Ref-1 was examined by immunohistochemistry staining, TUNEL was applied to detected DNA damage, and double labeling with TUNEL and APE/Ref-1 was used to determine the relationship between APE/Ref-1 and DNA damage. RESULTS:The active subunit P₂₀ of caspase-3 was predominantly expressed within ischemic penumbra. The peak time of caspase-3P₂₀ positive cells preceded the appearance of TUNEL. With aggravation of cerebral ischemia, APE/Ref-1 immunoreactive cells in penumbra were significantly decreased. CONCLUSION:The activation of caspase enzymatic cascade following cerebral ischemia leads to degradation in DNA, meanwhile, decrease in DNA repair molecules or the failure of DNA repair may deteriorate the course.     

Effects of soybean isflavones on mitochondrial damage and neuronal apoptosis induced by cerebral ischemia/reperfusion

AIM: To study the effects of soybean isoflavones on mitochondrial ultrastructure, neuronal apoptosis and expression of cytochrome C, caspase-9 and caspase-3 in the rats with cerebral ischemia/reperfusion.METHODS: Adult healthy SD rats (n=60) were randomly divided into 3 groups: sham group, ischemia/reperfusion injury (I/R) group and soybean isoflavone (SI) pretreatment group. Soybean isoflavones (120 mg·kg-1·d-1) were fed by gastric lavage for 21 d. The global ischemia/reperfusion model of the rats was established by blocking 3 vessels, and then reperfused for 1 h after 1 h of ischemia. The morphological change of the cerebral cortex cells was observed under light microscope. The mitochondrial ultrastructure of the cerebral cortex cells was determined by transmission electron microscope. The apoptotic rate of the cerebral cortex cells was detected by flow cytometry. The expression of cytochrome C, caspase-9 and caspase-3 in the cerebral cortex cells was determined by semi-quantitative RT-PCR and immunohistochemical techniques.RESULTS: Disintegration of mitochondria membrane and disappearance of the mitochondrial cristae were seen in I/R group. Compared with I/R group, the change of ultrastructure of mitochondria was significantly improved by soybean isoflavone pretreatment, and the neuronal apoptotic rate was also significantly decreased (P<0.01). The mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in I/R group were obviously higher than those in sham group (P<0.01). Compared with I/R group, the mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in SI group were significantly decreased (P<0.01).CONCLUSION: Soybean isoflavones attenuate cerebral ischemia/reperfusion injury by stabilizing the structure of mitochondria, preventing cytochrome C release to the cytoplasm, inhibiting the activation of caspase-9 and caspase-3 and decreasing cell apoptosis．     

The adaptive response of low dose sodium nitrite pretreatment on cultured CHL cells to DNA damage caused by high dose sodium nitrite

AIM: To study if low dose NaNO₂ can induce the adaptive response of cultured Chinese hamster lung cells（CHL cells）to DNA damage. METHODS: Single cell gel electrophoresis technique was used to detect the DNA damage in CHL cells exposed to NaNO₂ at different concentrations. CHL cells were pretreated with NaNO₂ of concentrations of 0.01 mg/L, 0.1 mg/L and 1 mg/L respectively. And the adaptive response to the toxicity of 1g/L NaNO₂ was observed. The activity of polyADP- ribose polymerase (PARP-1) of CHL cells was inhibited with 3-aminobenzamide(3AB) before or after pretreated with low dose of NaNO₂. And the changes of the adaptive response were observed. RESULTS: The rate of tailing cells was 7.87% when the cells were exposed to 1 g/L NaNO₂ without pretreatment with low dose NaNO₂. An extremely remarkable statistics significance (P＜0.01) was observed when compared the difference to control group. NaNO₂ of 0.01 mg/L and 0.1 mg/L could induce the adaptive response of cultured CHL cells to DNA damage caused by 1 g/L NaNO₂. The rate of tailing cells was 3.55% and 1.06％ respectively, which was much lower than that of no-pretreatment group(P＜0.05；P＜0.01). But the rate of tailing cells was 6.09% when the cells were exposed to 1 g/L NaNO₂ with pretreatment of 1 mg /L NaNO₂, which had no significant difference compared with the rate of tailing cells in control group (P＞0.05). The adaptive response could be blocked when the activity of PARP-1 was inhibited with 3AB before the low dose pretreatment, but could not be blocked when the activity of PARP-1 was inhibited after low dose NaNO₂ pretreatment 6 h. CONCLUSION: NaNO₂ of concentration that equals to or lowers than 0.1 mg/L can induce the adaptive response of cultured CHL cells to DNA damage caused by high dose NaNO₂ through PARP-1 activation. And the dose of NaNO2 that can induce adaptive response might not cause the DNA damage.     

Effect of MCPH1 on irradiation induced DNA damage in esophageal cancer cells

AIM: To discover the effect of MCPH1 on the DNA damage induced by ionizing radiation in esophageal cancer cells. METHODS: ECA109 cancer cells were radiated at dose of 8 Gy. The nuclear foci of relevant factors were detected 1 h after irradiation in the ECA109 cells after silence of MDC 1 gene. A cell line was established that was stable low expression of MCPH 1 . The nuclear foci induced by ionizing radiation after silence of MCPH 1 were determined. RESULTS: The MCPH1 gene silenced ECA109 cell line was successfully constructed. A strong relationship between MDC1, MCPH1 and γ-H2AX was observed 1 h after 8 Gy irradiation. Silence of MDC 1 did not affect the nuclear foci formation of γ-H2AX and MCPH1. The nuclear foci of MDC1 but not γ-H2AX significantly reduced after silencing of MCPH 1 . CONCLUSION: MCPH1 is located in the downstream of H2AX and upstream formation of MDC1, and regulates the nuclear foci formation of MDC1 during DNA damage response.     

CUDC-907 induces DNA damage and autophagy in glioma cells

AIM:To investigate the effect of CUDC-907, a dual histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K) inhibitor, on the DNA damage, cell cycle distribution and autophagy in human glioma U251 cells. METHODS:U251 cells were treated with CUDC-907 of different concentrations, and the cell viability was detected by MTT assay. The quantitative γ-H2AX foci were determined by laser scanning confocal microscopy. The cell cycle distribution of U251 cells was examined by flow cytometry. The protein expression was determined by Western blot analysis. RESULTS:CUDC-907 inhibited the cell viability and the phosphorylation of Akt and p70 ribosomal protein S6 kinase (p70s6K) in the U251 cells (P<0.05). In CUDC-907-treated cells, the number of γ-H2AX foci and protein expression of γ-H2AX were increased significantly (P<0.05). CUDC-907 also induced cell arrest in the G₂/M phase by up-regulating the expression of p21, and inhibiting the protein level of cyclin B1 and the phosphorylation of cell division cycle protein 2 (Cdc2). In addition, CUDC-907 triggered cell autophagy, and inhibition of autophagy increased CUDC-907-induced DNA damage of U251 cells. CONCLUSION:CUDC-907 significantly inhibits PI3K/Akt signaling pathway, induces DNA damage and arrests cell cycle in G₂/M phase. Blockage of autophagy promotes CUDC-907-induced DNA damage of U251 cells.     

Blocking autophagy magnifies MK-2206-induced DNA damage in SGC-7901 cells

AIM: To investigate the effect of MK-2206, an inhibitor of protein kinase B(Akt), on the DNA damage of SGC-7901 cells.METHODS: SGC-7901 cells were treated with different concentrations of MK-2206, and phosphorylated histone H2AX(γ-H2AX) foci formation was detected by immunofluorescence staining. Western blot analysis was used to exam the levels of DNA damage-related protein. The expression of LC3-Ⅱ was determined to evaluate the change of autophagy.RESULTS: MK-2206 treatment increased the formation of γ-H2AX foci and histone H2AX phosphorylation in the SGC-7901 cells. The levels of DNA damage response protein were also increased. In addition, MK-2206-treated SGC-7901 cells increased the expression of LC3-II, a hallmark of autophagy. Inhibition of autophagy significantly enhanced MK-2206-mediated histone H2AX phosphorylation.CONCLUSION: MK-2206 induces DNA damage and autophagy in SGC-7901 cells. Blocking autophagy potentiates the response of MK-2206-induced DNA damage.     

Effects of cigarette smoke extract on DNA damage and cell stress in human bronchi smooth muscle cells

AIM: To investigate DNA damage and cell stress (heat shock protein 70 expression) in human bronchi smooth muscle cells by cigarette smoke extract (CSE) in vitro. METHODS: 30 mL smog was dissolved in 1 mL culture medium as stock solution of CSE. Human bronchi smooth muscle cells were cultured 3 hours with 1∶16, 1∶10, 1∶8, 1∶6 and 1∶4 of CSE. The DNA damage and HSP70 expression were determined by single cell gel assay (comet assay) and Western blot, respectively. RESULTS: Associated with rising CSE concentration, DNA damage aggravated. Compared with the untreated group, except 1∶16 of CSE, the level of DNA damage was significantly different (P＜0.05). The level of HSP70 expression rised in 1∶16 and 1∶10 of CSE, but it gradually decreased in others. There was significantly different in the fifth and sixth group with negative control (P＜0.05). CONCLUSION: CSE results in DNA damage and decreasing of HSP70 expression, which may be related with chronic obstructive pulmonary diseases.     

GDC-0032 inhibits cell viability and induces DNA damage in U251 human glioma cells

AIM: To investigate the effect of phosphatidylinostiol 3-kinase (PI3K) inhibitor GDC-0032 on cell viability, cell cycle and DNA damage in human glioma U251 cells. METHODS: The cell viability was analyzed by MTT assay. The cell cycle distribution of U251 cells was examined by flow cytometry. The protein expression was examined by Western blot. The expression and localization of γ-H2AX were determined by laser-scanning confocal microscopy. RESULTS: GDC-0032 inhibited the cell viability in a dose-dependent manner. U251 cells showed G₁ phase arrest accompanied with upregulation of p27 protein after exposure to GDC-0032, while the expression of cell division cycle protein 2 (Cdc2) was inhibited. GDC-0032 treatment increased the formation of γ-H2AX foci and histone H2AX phosphorylation in the U251 cells. In addition, GDC-0032 upregulated the phosphorylation levels of mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), in the glioma cells, while the expression of survivin was inhibited. CONCLUSION: GDC-0032 inhibits U251 cell growth by inhibition of cell viability and cell cycle progression. GDC-0032 also induces DNA damage of U251 cells. The anticancer activity of GDC-0032 is associated with increasing the activity of ERK and JNK and downregulating the expression of survivin.     

黄瓜老化种子基因组DNA的损伤与修复的RAPD研究

应用RAPD技术对人工老化和PEG处理的黄瓜种子进行扩增多态性DNA分析.建立了黄瓜种子基因组DNA的损伤及其修复的RAPD指纹图谱.在92条引物中有22条可应用于老化种子的研究.16条可应用于PEG处理种子的研究,并从中找出了6条引物S190、S300、S359、S475、S1140、S2144用来比较黄瓜老化种子及其修复的RAPD扩增带型的变化.结果发现,人工老化处理的黄瓜种子的DNA带减少甚至消失.而PEG渗调修复后的黄瓜老化种子DNA带重新出现或者与对照基本相同,从而建立了黄瓜老化种子基因组DNA的损伤与修复的RAPD指纹图谱.     

Mechanism of systemic inflammatory response syndrome induced by EC DNA

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-α and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-α and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-JB was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-α and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-α peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-α and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NFκB. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, andinduces serum TNF- αand IL- 6 level to increase in rats and ANA- 1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF- κB may be its importantmolecular mechanism, but other pathway probably exists to play an important role.     

持续低温引起菠萝蜜田间寒害症状调查及抗寒性分析

为选育出抗寒性能较强的菠萝蜜优株(系),对经历持续低温天气后的8个菠萝蜜优株(系)进行田间寒害调查,比较优株的抗寒表现并进行寒害分级,同时测定与抗寒性能有关的几个生理指标并分析其与寒害等级的相关性。结果表明,持续低温会造成菠萝蜜优株(系)寒害,其寒害程度与品种(系)抗寒性有关;8个菠萝蜜优株中,一级寒害4个,二级寒害3个,三级寒害1个。叶片电导率与菠萝蜜寒害等级呈显著正相关;可溶性糖含量与菠萝蜜寒害等级呈显著负相关;维生素C含量与菠萝蜜寒害等级无相关性。研究得出抗寒性能较强的菠萝蜜优株(系)为优8、优6、优5、优3,叶片电导率和可溶性糖含量可作为评价菠萝蜜新品种(系)抗寒能力选育的参考。     

Role of polymerase β in DNA metabolism and genomic instability

The major role of DNA polymerase β was thought to be limited in its involvement in short patch base excision repair by removing 5’-deoxyribose phosphate and base insertion. However, the recent researches indicate that polymerase β might take part in a wide spectrum of DNA metabolism reactions, including long patch base excision repair, DNA replication, recombination, meiosis and transleisional DNA synthesis. Because of its wide and important cellular function, an inappropriate intracellular polymerase β level might be associated with genomic instability. Down-regulation or mutation of polymerase β is mutagenic due to deficient in DNA repair, while overexpression of this error-prone β polymerase might perturb the normal function of other accurate polymerases and cause genomic instability as well.     

APE/Ref-1 protein and ischemia/reperfusion injury of neurons in the brain

Cerebral ischemia and the aftermath of reperfusion form a hypoxic/hyperoxic sequence of events that can trigger DNA damage in neurons of central nervous system. Neuronal apoptosis will happen without immediate DNA repair. APE/Ref-1 is a multifunctional protein involoved in DNA base excision repair pathway and in redox reguiation of DNA-binding activity of AP-1 family members, which may play an important role in protection of postischemic neuronal damage.     

Advance in function of deubiquitination enzyme BRCC36

The protein of BRCC36 is a kind of enzyme specifically hydrolyzing K63-linked poly-ubiquitin chain and widely found in a variety of eukaryotic cells. BRCC36 recognizes diverse substrate proteins, and participates in various kinds of pathophysiological responses such as DNA damage repair, cell signal transduction and cell cycle control. It plays an important role in the process of cancer, angiogenesis and cardiac injury. This review discusses the progress in the investigation on BRCC36 protein to provide the necessary information for searching new therapeutic targets of many diseases.     

大白菜脱春化效果及脱春化前后DNA 甲基化分析

针对春大白菜极易经历低温春化而先期抽薹的现象,以大白菜一代杂种阳春为试材,在光照培养箱条件下进行绿体脱春化、种子脱春化及在温室条件下进行绿体脱春化研究。结果表明：高温可以使大白菜脱春化;随着高温处理时间的延长,脱春化效果逐渐增强;种子脱春化效果比绿体脱春化效果好。基因组DNA 甲基化水平检测结果表明：春化时大白菜植株发生去甲基化,脱春化处理可诱导其重新甲基化。     

Effect of Xinmailing Solution on neuronal cell injury induced by glutamate

AIM: To detect the effects of Xinmailing Solution and MK-801on injury of neuronal cell induced by glutamate. METHODS: Cultured neuronal cell injuried by glutamate was prepared and the content of malondialdehyde and nitrite in cell supernatant was measured. Morphology changes were also observed with discrepancy microscope at the same time. RESULTS: Xinmailing Solution and MK-801attenuated cell injury induced by glutamate,and inhibited increase in malondialdehyde and nitrite in cell supernatant. CONCLUSION: Xinmailing Solution had a protective effect on neuronal cell at cell level.     

Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.