外源物质诱导对甜瓜枯萎病抗性和防御酶活性的影响

以甜瓜抗病品系MR-1、感病品系M1-15为试材,以甜瓜枯萎菌为供试菌,采用营养钵栽培的方法,研究了水杨酸、茉莉酸甲酯和Ca~(2+)诱导对甜瓜幼苗枯萎病抗性的影响。结果表明:3种外源物质对甜瓜苗期枯萎病抗性的诱导效果不同,其中水杨酸和茉莉酸甲酯效果最好。1.0mmol·L~(-1)水杨酸诱导处理对甜瓜枯萎病的相对防效可达50.7%和45.7%;1.2mmol·L~(-1)茉莉酸甲酯诱导处理对甜瓜枯萎病的相对防效可达66.7%和40.3%,显著高于对照和Ca~(2+)处理;水杨酸、茉莉酸甲酯处理后,甜瓜植株叶片相关防御酶系多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)活性呈上升趋势。     

BTH诱导的一种黄瓜叶片胞间隙病程相关蛋白的纯化及鉴定

 对苯并噻二唑(BTH) 处理和接种霜霉病菌后的黄瓜幼苗叶片胞间隙液中病程相关蛋白的诱导积累及纯化鉴定进行了研究。结果表明, BTH处理和接种霜霉病菌均可诱导黄瓜叶片胞间隙液产生分子量为27 kD的新蛋白, 利用SDS-PAGE结合电洗脱的方法纯化了该蛋白。通过基体辅助激光解析电离飞行时间质谱(MALDI-TOFMS) 分析, 将所得的肽指纹图谱( PMF) 在NCBInr蛋白质数据库中比对, 发现该蛋白是一种酸性几丁质酶。酶活性测定及Western blotting分析进一步证实了上述结果。     

茉莉酸甲酯诱导辣椒抗青枯病与活性氧代谢的关系

为探究茉莉酸甲酯(Methyl Jasmonate,Me JA)诱导辣椒抗青枯病效应与活性氧代谢相关酶的关系,以辣椒易感青枯病品种‘粤红1号’和抗青枯病品种‘辛香8号’幼苗为试验材料,在0.1mmol·L-1 Me JA喷雾处理后12、24、48、72和96 h接种青枯劳尔氏菌(Ralstonia solanacearum),对其进行青枯病病情指数与活性氧代谢相关酶——超氧化酶歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)、过氧化物酶(Peroxidase,POD)和抗坏血酸过氧化物酶(Ascorbate Peroxidase,APX)活性以及丙二醛(Malondialdehyde,MDA)含量测定及相互关系的分析。结果表明:‘粤红1号’和‘辛香8号’的病情指数随喷Me JA处理时间的推移表现为先降后升,但都低于对照;而诱导效果则相反,Me JA处理对两个辣椒品种幼苗抗性的最适诱导时间均为接种前48 h。接种后0~96 h,Me JA喷雾处理的辣椒幼苗叶片SOD、CAT、POD和APX酶活性均显示先升后降的趋势,且明显高于对照,接种后24~48 h达到最高,而MDA含量则明显低于对照。因此,Me JA可诱导辣椒幼苗抗青枯病,其实现途径可能与提高活性氧代谢相关酶活性和缓解膜脂过氧化有关。     

嫁接对网纹甜瓜果实发育、糖含量及蔗糖代谢相关酶活性的影响

 试验研究了嫁接对日光温室栽培的网纹甜瓜果实发育、糖含量及蔗糖代谢相关酶活性的影响。结果表明: 嫁接加快了果实的膨大速度, 但对中果肉厚度影响不大; 降低了网纹甜瓜中果肉内的果糖、葡萄糖含量; 改变了网纹甜瓜蔗糖积累模式, 嫁接网纹甜瓜中果肉内蔗糖积累时间较自根提前了7 d左右,蔗糖含量高于自根; 但嫁接降低了网纹甜瓜中果肉的总糖含量及酸性转化酶(AI) 和中性转化酶(NI) 活性, 提高了蔗糖磷酸合成酶( SPS) 和蔗糖合成酶( SS) 活性。     

INA诱导的香蕉果实抗病性与早期活性氧积累的关系

 研究2,6–二氯异烟酸（INA）对采后香蕉果实抗病性的诱导作用和早期果皮活性氧含量、抗病相关酶活性及基因表达量的变化。结果表明：香蕉果实经0.5 g · L-1 INA处理后0、3、6、12、24 h接种炭疽病菌孢子,贮藏8 d后,INA处理果实的病斑直径比对照果实的明显减小;INA处理果实的H2O2含量和NADPH氧化酶活性分别在6 h和3 ~ 12 h明显高于对照;过氧化氢酶（CAT）活性均明显低于对照,抗坏血酸氧化酶（APX）活性稍低于对照,CAT和APX基因的表达也受到一定的抑制;内切几丁质酶（CHI）活性在0、3、6、12 h高于对照,外切CHI活性在24 h内逐步增加,而对照保持不变;CHI1和CHI2基因的表达在前6 h略高于对照。综上所述,INA诱导采后香蕉果实抗病性增加与其早期活性氧水平及抗病相关酶活性增加有密切关系,由于活性氧合成酶活性增加、清除酶活性减少而产生的高水平活性氧可能作为信号分子启动了抗病应答反应。     

香蕉枯萎病菌4号生理小种毒素解毒剂的筛选及对香蕉防御酶活性的影响

从15种候选解毒剂中筛选出对香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)4号生理小种(简称Foc 4)毒素解毒效果最好的2种解毒剂——硫酸锌和井冈霉素。在2 g.L-1浓度下,硫酸锌和井冈霉素对Foc 4毒素的钝化率分别为76.67%和71.33%,而在5 g.L-1浓度下,它们的钝化率分别为90.33%和80.00%。探索了毒素—硫酸锌和毒素—井冈霉素处理体系在2 g.L-1和5 g.L-1浓度下对香蕉苗5种防御酶(PAL、POD、PPO、SOD和CAT)活性的影响。结果表明,在这个处理体系中,香蕉苗PAL和PPO酶活高峰出现的时间(PAL 36 h、PPO 36 h和48 h)较毒素单独处理(PAL 48 h、PPO 60 h)早;SOD和CAT酶活高峰出现的时间与对照基本一致;而POD酶活高峰出现的时间较复杂。总体上,这个处理体系的酶活高于毒素单独处理和无菌水对照。"毒素—硫酸锌处理体系"在PAL、SOD整体酶活上高于"毒素—井冈霉素处理体系",而后者在其他3种酶活上普遍高于前者。     

Erianin induces apoptosis of human lung cancer A549 cells via ROS/p38 MAPK pathway

AIM:To investigate the effect of erianin on the viability and apoptosis of human lung cancer A549 cells and its possible mechanism. METHODS:A549 cells and BEAS-2B cells were cultured in vitro and treated with erianin at 10, 20, 40, 80 and 160 nmol/L for 48 h. The cell viability was measured by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. The activity of superoxide dismutase (SOD) was detected by WST-8 method, and the content of malondialdehyde (MDA) was detected by barbituric acid method. The protein levels of nuclear factor E2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), p38 MAPK, p-p38 MAPK, caspase-3 and cleaved caspase-3 were determined by Western blot.RESULTS:Erianin remarkably reduced the viability of A549 cells in a dose-dependent manner (P<0.05) with IC₅₀ at 52.64 nmol/L. Erianin also induced apoptosis (P<0.05), increased ROS level and MDA content (P<0.05), diminished SOD activity (P<0.05), and down-regulated the protein levels of Nrf2, NQO1 and HO-1 (P<0.05), in a dose-dependent manner. Meanwhile, erianin up-regulated the levels of p-p38 MAPK and cleaved caspase-3 (P<0.05), and these effects were inhibited by N-acetyl-L-cysteine and SB203580 (P<0.05).CONCLUSION:Erianin may induce apoptosis of human lung cancer A549 cells most likely via inhibiting SOD activity and down-regulating the protein levels of Nrf2, NQO1 and HO-1, thus resulting in an increase in ROS and activation of p38 MAPK.     

Hydrogen sulfide suppresses the increase in reactive oxygen species in neurons induced by angiotensin II via ERK1/2 signal pathway

AIM: To investigate the effect of hydrogen sulfide (H₂S) on the reactive oxygen species (ROS) level in medullary neurons induced by angiotensin II (Ang II). METHODS: Primarily cultured rat medullary neurons were divided into 5 groups as follows: control group, Ang II group, sodium hydrosulfide(NaHS) group, NaHS with Ang II group, and PD98059 (an inhibitor of p-ERK1/2) with Ang II group. ROS production was measured with dihydroethidium (DHE) staining. The expression of p-ERK1/2 and ERK1/2 was determined by Western blotting. The activity of neurons was detected by CCK-8 assay. RESULTS: Ang II at concentration of 100 nmol/L significantly increased ROS level in the neurons, but the effect was inhibited by NaHS at concentrations of 50~200 μmol/L, while NaHS alone had no influence on the ROS level in neurons. Additionally, PD98059 also depressed the ROS level in neurons induced by Ang II. Furthermore, the enhanced expression of p-ERK1/2 in the neurons induced by Ang II was significantly reduced by NaHS. CONCLUSION: H₂S remarkably inhibits the ROS level in the neurons induced by Ang II via activation of MAPK signal pathways, especially p-ERK1/2, indicating that H₂S rescues neurons from oxidative stress through declining the enhanced expression of p-ERK1/2.     

ROS mediates regulation of intracellular Ca2+ induced by angiotensin II in primarily cultured medullary neurons

AIM: To investigate the role of reactive oxygen species (ROS) in the regulation of intracellular Ca²⁺ induced by angiotensin II (Ang II) in the primarily cultured medullary neurons. METHODS: Primarily cultured medullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study. The identification of medullary neurons was assessed by double-labeling immunofluorescence. To explore the role of ROS, mainly the superoxide (O₂^·), the O₂^·generation was measured using the fluorogenic probe dihydroethidium (DHE). To determine intracellular free calcium concentration ([Ca²⁺]_i), the neurons were loaded with the Ca²⁺-specific dye Fura-2/AM. The cell viability after adding Ang II was also examined using CCK-8 assay. RESULTS: Most of the cultured cells were medullary neurons, more than 80% of which were glutamate positive neurons. Ang II (5 μmol/L) increased the level of ROS within 10 min in the medullary neurons. Ang II at 5 μmol/L induced a significant[Ca²⁺]_i increase in the medullary neurons, and the effect of Ang II occurred rapidly and reached a peak within 20 min after administration. The level of[Ca²⁺]_i started to decline after washout. The Ca²⁺ elevation induced by Ang II was significantly decreased by apocynin or TEMPOL. No significant difference in the cell viability between control group and 5 μmol/L Ang II treatment group was observed. CONCLUSION: ROS is involved in the regulation of[Ca²⁺]_i induced by Ang II in the primarily cultured medullary neurons, suggesting a potential intracellular signaling mechanism involved in the Ang II-mediated oxidant regulation of central neural control of blood pressure.     

Smad pathway participates the process of proliferation of vascular smooth muscle cells induced by extracellular signal regulated kinase pathway

AIM: To investigate whether Smad pathway participates the process of extracellular signal regulated kinase (ERK) induced the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Human umbilical artery smooth muscle cells (hUASMCs) were divided into four groups: control group, PDGF (platelet derived growth factor) group, ERK blocking agent group and PDGF+ERK blocking agent group. MTT assay was used to detect the proliferation of hUASMCs (A value). Immunohistochemical technique was used to detect the expression of PCNA, phosphorylated ERK (p-ERK) and phosphorylated Smad2/3 (p-Smad2/3) protein in hUASMCs. The expression of Smad2/3 mRNA in hUASMCs was detected by RT-PCR. RESULTS: The proliferation of hUASMCs and the expression of PCNA, p-ERK and p-Smad2/3 proteins in hUASMCs in PDGF group were increased obviously than those in other groups (P<0.01). No difference in the expression of Smad2/3 mRNA in hUASMCs among groups was observed. CONCLUSION: Smad pathway participates the process of ERK pathway that induces the proliferation of hUASMCs at the level of protein.     

Over-expression of SIRT1 gene inhibits high glucose-stimulated H9c2 cardiomyocyte apoptosis and ROS level through PI3K/AKT signaling pathway

AIM: To investigate the effects of silent information regulator 1 (SIRT1) over-expression on the apoptosis and the level of reactive oxygen species (ROS) in high glucose-induced H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were transfected with empty plasmid (pcDNA3.1-NC) and SIRT1 over-expression plasmid (pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3K and AKT protein levels were down-regulated (P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3K and AKT protein levels (P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3K/AKT signaling pathway.     

Astragaloside IV attenuates apoptosis of H9c2 cardiomyocytes induced by angiotensin II

AIM: To investigate the protective effect of astragaloside IV (ASIV) on angiotensin II (Ang II)- induced apoptosis of H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were treated with different concentrations of Ang II and ASIV. The effects of Ang II and ASIV on the viability of H9c2 cells was measured by CCK-8 assay. The optimum concentration of Ang II was 1 μmol/L and the concentrations of ASIV were 25, 50 and 100 μmol/L. The H9c2 cells was divided into 6 groups:control group, ASIV group, Ang II group, Ang II+ASIV (25 μmol/L) group, Ang II+ASIV 50 (μmol/L) group and Ang II+ASIV (100 μmol/L) group. The morphological changes of the H9c2 cells were observed under inverted phase-contrast microscope. Apoptosis was detected by TUNEL assay. The generation of reactive oxygen species (ROS) was detected by DCFH-DA staining. The protein expression of Bax, Bcl-2, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. H9c2 cardiomyocytes were transfected with negative control shRNA (NC) or Nrf2-shRNA (shRNA), and the cells were divided into 8 groups:NC+control group, NC+AngⅡgroup, NC+ASIV group, NC+AngⅡ+ASIV group, shRNA+control group, shRNA+AngⅡgroup, shRNA+ASIV group and shRNA+AngⅡ+ASIV group. ROS level was detected by ROS detection kit. The protein expression of Nrf2 and HO-1 was determined by Western blot. RESULTS: Ang II decreased the viability of H9c2 cells in a concentration-dependent manner (P<0.05). ASIV reversed the effect of Ang II on the viability of H9c2 cells in a concentration-dependent manner (P<0.05). Compared with control group, the apoptotic rate, the level of ROS and the protein expression of Bax in Ang II group were increased significantly, while the protein expression of Bcl-2, Nrf2 and HO-1 was decreased significantly (P<0.05). Compared with Ang II group, ASIV reversed the increase in apoptotic rate of H9c2 cells induced by Ang II in a concentration-dependent manner, reduced ROS level, down-regulated the protein expression of Bax and up-regulated the protein expression of Bcl-2, Nrf2 and HO-1 (P<0.05). After shRNA transfection, the effects of ASIV decreasing ROS production induced by Ang II and up-regulating the expression of Nrf2 and HO-1 were eliminated. CONCLUSION: ASIV protects H9c2 cardiomyocytes from apoptosis induced by Ang II, which may be related to reducing ROS generation and mediating the Nrf2/HO-1 signaling pathway.     

草莓白化相关病毒中国分离物全基因组分析

草莓白化相关病毒(strawberrypallidosis-associatedvirus,SPa V)属于长线形病毒科(Closteroviridae)毛形病毒属(Crinivirus),可引起草莓病害,2017年在中国首次报道。采用高通量测序、RACE和RT-PCR技术获得了SPa V中国分离物(FJ)的基因组全长。该病毒含有两条正单链基因组RNA1和RNA2。RNA1全长8 048 nt,5′和3′非编码区序列分别为264和197 nt,含有3个开放阅读框(ORF),分别编码ORF 1a/1b融合蛋白和p9蛋白。RNA2全长7 977 nt,5′和3′非编码区序列分别为248和186 nt,含有8个开放阅读框(ORF),分别编码HSP70h、CPh、CP、CPm、p7、p6、p9和p28等8个蛋白。RNA1和RNA2与美国M1分离物分别具有98.5%和99.0%的核苷酸一致性;系统发育分析结果表明,SPa V中国分离物(FJ)单独处在一个分支。对SPa V来源的小RNA的分析表明,来源于SPa V的小RAN长度以21和22 nt为主。     

Salvianolic acid B reduces apoptosis of bone marrow stem cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolic acid B (Sal B) on apoptosis of rat bone mesenchymal stem cells(BMSCs) induced by hydrogen peroxide(H₂O₂). METHODS: BMSCs were incubated with Sal B at the concentration of 1, 10 or 100 μmol/L while treated with lethal concentration of H₂O₂ (500 μmol/L). The effect of Sal B at different concentrations on the viability of BMSCs was detected by MTT. Flow cytometry were used to determine the protective role of Sal B in apoptosis of BMSCs. The changes of chromatin distribution in BMSCs were observed by Hoechst 33342 staining. The expression of p-ERK1/2 was detected by Western blotting. RESULTS: Sal B protected the BMSCs against H₂O₂ as the cell viability was increased from (53.60±4.21)% to (85.33±9.08)% or (75.78±6.28)% in a dose-dependent manner. After exposed to H₂O₂, about 50%-65% BMSCs displayed apoptotic morphology. Treatment with Sal B at the concentrations of 10 and 100 μmol/L reduced the cytotoxic effect of H₂O₂ on BMSCs to about 32% and 47%, respectively. The results of flow cytometric analysis confirmed the cytoprotective effect of Sal B. This protective effect was concomitant with significant reduction of ROS generation. Moreover, H₂O₂ time-dependently induced a pronounced increase in ERK1/2 phosphorylation,which was effectively inhibited by Sal B.CONCLUSION: Sal B protects BMSCs against H₂O₂-induced apoptosis. Sal B may exert its protective effect on BMSCs by triggering intracellular anti-apoptosis mechanism as well as reducing the oxidative stress.