Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorfen

AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS： Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Pudfied Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS： B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION： Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.

Effects of Nitric Oxide on the Germination of Wheat Seeds and Its Reactive Oxygen Species Metabolisms Under Osmotic Stress

Effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on the germination and metabolism of reactive oxygen species were surveyed in wheat (Triticum aestivum L.) seeds. Germination of wheat seeds and even the elongation of radicle and plumule were dramatically promoted by SNP treatments during the germination under osmotic stress. Meanwhile, activities of amylase and EP were enhanced, thus leading to the degradation of storage reserve in seeds. After osmotic stress was removed, higher viability of wheat seeds was also maintained. In addition, the activities of CAT, APX and the content of proline were increased by SNP treatment simultaneously, but activities of LOX were inhibited, and both of which were beneficial for improving the antioxidant capacity during the germination of wheat seeds under osmotic stress. It was also shown that the increase of the activity of amylase induced by SNP in embryoless half-seeds of wheat in the beginning period of germination (6 h) might be indirectly related to GA(3).

Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

Background The vascular endothelial growth factor （VEGF） is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 （ICAM-1） is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells （ECs） is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs. Methods Bovine retinal ECs （BRECs） were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF （100 ng/ml） and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase （eNOS） were detected using Western blotting. Griess reaction was used to detect nitric oxide （NO）. Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C （PKC） altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase （PI3K） or reactive oxygen species （ROS） significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased. Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase

Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase （eNOS）. Methods ＆ Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin （BH4）. Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes： GTP cyclobydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin （BH2） as well as BH4：BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4： eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality （i.e., coupled or uncoupled）, the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Prednisolone inhibits SaOS2 osteosarcoma cell proliferation by activating inducible nitric oxide synthase

AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differentiation were used.SaOS2 show a rather mature phenotype,while U2 OS are negative for almost all osteoblastic markers.The cells were exposed to different concentrations of prednisolone(1-9 μmol/L) with or without antioxidants or the inhibitor of inducible nitric oxide synthase(i NOS) l-N6-(iminoethyl)-lysine-HCl(L-NIL).Cell growth was assessed by counting viable cells.The production of nitric oxide(NO) was measured in the conditioned media by the Griess method.The production of reactive oxygen species was quantified using 2'-7'-dichlorofluorescein diacetate.Western blot with specific antibodies against NOSs was performed on cell extracts.RESULTS:Prednisolone inhibited SaOS2 cell growth in a dose dependent manner.No significant effects were observed in U2OS.The inhibition of SaOS2 growth is not due to oxidative stress,because antioxidants do not rescue cell proliferation.Since high concentrations of NO inhibit bone formation,we also measured NO and found it induced in SaOS2,but not in U2 OS,exposed to prednisolone,because of the upregulation of i NOS as detected by western blot.Therefore,we treated SaOS2 with prednisolone in the presence or in the absence of L-NIL.L-NIL prevented NO release induced by prednisolone at all the concentrations apart from 9 μmol/L.At the same concentrations,we found that L-NIL rescued SaOS2 growth after exposure to prednisolone.In U2 OS cells,prednisolone did not induce NO production nor affected cell growth.All together,these data indicate that a link exists between increased amounts of NO and growth inhibition in response to prednisolone in SaOS2.CONCLUSION:Prednisolone inhibited SaOS2 proliferation by increasing the release of NO through the upregulation of i NOS,while no effect was exerted on U2OS.

Regulation of Nitric Oxide on the Aging Process of Wheat Leaves

When the first fully expanded leaf of wheat ( Triticum aestivum L.) seedlings with two leaves were treated with different concentrations (0.05, 0.10, 0.20 and 0.50 mmol/L) of nitric oxide donor, sodium nitroprusside (SNP), the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were reduced by the lower concentrations (0.05, 0.10 and 0.20 mmol/L), but enhanced by the higher concentration of SNP (0.50 mmol/L). The protective effect of 0.10 mmol/L SNP was the most obvious. Furthermore, the treatment with 0.10 mmol/L SNP on the above seedlings until the fourth leaves were fully expanded attenuated the accumulation of H2O2, superoxide anion radical (O-2(-)) and MDA, also counteracted the degradation of chlorophyll and soluble proteins, especally Rubisco, both leading to the effective delay of aging process in wheat leaves. The effects of different SNP concentrations (0.05, 0.10, 0.20, 0.50, 1.00 and 5.00 mmol/L) also displayed a dual role in an aging experiment of chloroplasts in vitro, one of which, 0.2 mmol/L SNP treatment, protected the membrane structure and attenuated the degradation of Rubisco effectively. Based on the present results, it was inferred that lower concentrations of nitric oxide (NO) might play a role in delaying aging process in wheat leaves, i.e., might attribute to decrease the level of reactive oxygen species (ROS) and the alleviation of further oxidative damage caused by ROS.

Endothelial dysfunction in cirrhosis: Role of inflammation and oxidative stress

This review describes the recent developments in the pathobiology of endothelial dysfunction(ED) in the context of cirrhosis with portal hypertension and defines novel strategies and potential targets for therapy. ED has prognostic implications by predicting unfavourable early hepatic events and mortality in patients with portal hypertension and advanced liver diseases. EDcharacterised by an impaired bioactivity of nitric oxide(NO) within the hepatic circulation and is mainly due to decreased bioavailability of NO and accelerated degradation of NO with reactive oxygen species. Furthermore, elevated inflammatory markers also inhibit NO synthesis and causes ED in cirrhotic liver. Therefore, improvement of NO availability in the hepatic circulation can be beneficial for the improvement of endothelial dysfunction and associated portal hypertension in patients with cirrhosis. Furthermore, therapeutic agents that are identified in increasing NO bioavailability through improvement of hepatic endothelial nitric oxide synthase(e NOS) activity and reduction in hepatic asymmetric dimethylarginine, an endogenous modulator of e NOS and a key mediator of elevated intrahepatic vascular tone in cirrhosis would be interesting therapeutic approaches in patients with endothelial dysfunction and portal hypertension in advanced liver diseases.

Danlou Fang(丹蒌方)reduces microvascular obstruction through the endothelial/inducible nitric oxide synthase pathway in a rat model

OBJECTIVE:To investigate the efficacy of the Danlou Fang(DL)Traditional Chinese Medicine formula on microvascular obstruction(no-reflow)through the endothelial/inducible nitric oxide synthase(eNOS/iNOS)pathway in a rat model.METHODS:Sprague-Dawley rats were subjected to 60 min of coronary artery occlusion(or sham procedure)followed by 2 h of reperfusion and were then divided into treatment groups:sham,model,DL(500 mg/kg),DL(500 mg/kg)+eNOS inhibitor L-nitroarginine(L-NNA;7.5 mg/kg),and sodium nitroprusside(SNP;0.5 mg/kg).There were 16 per group.Areas of no-reflow were determined by thioflavin S staining of heart tissue.Cardiac function was assessed by echocardiography.Myocardial enzymes and antioxidants in serum were measured and analyzed.The relative protein expression levels of eNOS and iNOS were determined by western blotting.RESULTS:DL had a myocardial protective effect on myocardial reperfusion and reduced the area of no-reflow.The serum levels of creatine kinase(CK),myocardial CK isoenzyme CK-MB,and lactate dehydrogenase were significantly lower in the DL group than in the model(P<0.05).DL treatment also decreased the serum content of malondialdehyde and reactive oxygen species(ROS),increased the activity of superoxide dismutase and nitric oxide,and promoted eNOS expression(P<0.05)while lowering iNOS expression.CONCLUSION:DL reduced the area of no-reflow and had a myocardial protective effect that may be associated with the eNOS/iNOS pathway.

Resveratrol Reverses the Impaired Vasodilation Observed in 2K-1C Hypertension through Endothelial Function Improvement

Background: The production of endothelial-derived factors induces either vasoconstriction or vasodilation;nitric oxide (NO) is the most distinguished relaxing factor. Endothelial dysfunction is associated with hypertension. The partial loss in the NO-promoted vasodilation is due to its decreased bioavailability and/or to an activity reduction of endothelium NO synthase (eNOS). Reactive oxygen species (ROS), present in oxidative stress, seize NO and diminish its bioavailability. Transresveratrol (RESV) has been proved to increase NO and eNOS levels. Thus, RESV could be capable of improving NO dependent vascular relaxation on aortic rings isolated from treated 2K-1C animals through ROS damage reduction. Aim: Evaluate the effects of RESV treatment on the relaxation of aortic rings isolated from treated 2K-1C rats while focusing on the effects of the treatment on systolic blood pressure. Methods: Male Wistar rats (180 g) were grouped: two 2K-1C and two Sham groups, one of each was treated with RESV (20 mg/kg, gavage) dissolved in Tween 80 and one of each was treated with water plus Tween 80 (control) for six weeks. The rats had their systolic blood pressure (SBP) measured before and after the treatments. Vascular reactivity studies were conducted in order to observe and compare acetylcholine (ACh)-induced relaxations in the presence and absence of the NOS inhibitor L-NAME (10-4 mol/L). Results: SBP for 2K-1C was significantly reduced in the treated group (179.13 ± 4.90 mmHg, n = 23) when compared to the untreated group (196.66 ± 6.06 mmHg, n = 15, p < 0.01). The maximum relaxation of aortic rings isolated from the 2K-1C treated group showed a higher efficacy (116.63% ± 1.72%, n = 12) than that from the untreated group (85.97% ± 0.69%, n = 6, p < 0.001);L-NAME exposure was responsible for a significant decrease in each group’s maximum relaxation efficacy. Conclusions: SBP reduction observed after RESV treatment in rat renal hypertension could be due to the reestablishment of vascular relaxation depend of NO.

硝化应激在肺动脉高压中的研究进展

肺动脉高压(pulmonary hypertension,PH)是一种进行性发展的肺血管疾病,病理基础包括内皮功能障碍、平滑肌细胞异常增生、炎症浸润以及肺纤维化。PH的发生机制尚不完全清楚,但硝化应激已经证实在PH中发挥了重要作用。该文综述了活性氮(reactive nitrogen species,RNS)的种类及肺循环中RNS的来源,以及由此引发的硝化应激在PH发生发展中的作用,以期为靶向抗硝化治疗的临床应用提供参考依据。

NADPH oxidase： recent evidence for its role in erectile dysfunction

Important roles for reactive oxygen species （ROS） in physiology and pathophysiology have been increasingly recognized. Under normal conditions, ROS serve as signaling molecules in the regulation of cellular functions. However, enhanced ROS production as a result of the activation of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase contributes significantly to the pathogeneses of vascular diseases. Although it has become evident that increased ROS is associated with erectile dysfunction （ED）, the sources of ROS in the penis remain largely unknown. In recent years, emergent evidence suggests the possible role of NADPH oxidase in inducing ED. In this review, we examine the relationship between ROS and ED in different disease models and discuss the current evidence basis for NADPH oxidase-derive＇d ROS in ED.

Screening of Antioxidant Potential of Selected Barks of Indian Medicinal Plants by Multiple in vitro Assays

Objective To evaluate the antioxidant potential in herbal extract barks of five therapeutically important medicinal plants native to India, i.e. Crataeva nurvala Buch.-Ham., Buchanania lanzan Spreng., Aegle marmelos Corr., Dalbergia sissoo Roxb. ex DC., and Cedrela toona Roxb. Methods Standardized aqueous alcoholic extracts from the selected barks having different target radicals, such as superoxide radical, nitric oxide, ABTS radical, and peroxidative decomposition of phospholipids, were prepared and screened by multiple in vitro assays. These extracts were also tested for total phenolic and tannin content and correlated with antioxidant capacity. Results Total phenolic and tannin contents were found to be the highest in C. nurvala （195 GAE mg/g and 218.3 mg/g CE）. SOD mimetic activity was found to be the highest in Crataeva nurvula, although all barks showed activity more than IO3 units/mg extract. Lipid peroxidation inhibitory potential was found to be the highest in Crataeva nurvala （83.4% inhibition of MDA formation/10 μg extract）, and also showed a comparatively high NO quenching capacity （45.5% per 10 μg extract）. The highest NO quenching potential was found in Aegle marmelos （47.3% per 10 μg extract）. Cedrela toona showed the lowest LPO inhibitory potential and NO quenching capacity （50.5% and 30.5%, respectively）. Buchanania lanzan, a medicinal plant extensively used for inflammatory disorders and Dalbergia sissoo also showed 72.5% and 69.1% LPO inhibitory potential/10 μg extract. Trolox equivalent antioxidant capacity ranged from 0.24 to 0.39 mmol/L TEAC/mg extract, indicating that all the barks tested had ABTS^＋ radical quenching capacity. Conclusion Bark of Crataeva nurvula has the highest antioxidant capacity and a positive correlation between antioxidant activity and their plendic content was found.

一氧化氮参与调控油菜素内酯增强高山离子芥悬浮细胞抗寒性

为探究油菜素内酯(BRs)诱导提高植物的抗寒性是否受一氧化氮(NO)信号分子调控,以高山离子芥(Chorispora bungeana)悬浮细胞为材料,分别用24-表油菜素内酯(EBR)、NO供体SNP、NO清除剂PTIO、一氧化氮合成酶(NOS)抑制剂L-NAME、EBR+PTIO以及EBR+L-NAME进行处理,分析在低温胁迫下以上处理对细胞抗寒性、活性氧(ROS)水平以及抗氧化防御系统的影响。结果表明:(1)外源EBR处理可以缓解低温胁迫对悬浮细胞活力的抑制以及离子渗漏和膜脂过氧化程度的加剧,从而增强细胞对低温的抗逆能力。SNP处理对上述生理指标的影响与EBR类似。(2)在低温胁迫下,与EBR处理细胞相比,EBR+PTIO以及EBR+L-NAME处理会导致悬浮细胞活力下降,离子渗漏和膜脂过氧化程度显著升高,表明阻断NO信号会降低EBR诱导提高的抗寒性。(3)与仅受低温胁迫的细胞相比,外源EBR处理导致细胞NO含量和NOS活性进一步升高,而EBR诱导的NO积累可以被PTIO或L-NAME所抑制。(4)EBR、SNP处理均可以明显抑制低温胁迫下离子芥悬浮细胞过氧化氢(H_(2)O_(2))含量、超氧阴离子(O_(2)·^(-))产生速率和羟自由基(OH-)含量的升高,并显著增强抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)、过氧化物酶(POD)和超氧化物歧化酶(SOD)的活性以及增加抗坏血酸(AsA)和还原性谷胱甘肽(GSH)的含量,从而缓解低温造成的氧化损伤,而PTIO、L-NAME会不同程度抑制低温胁迫下EBR的这些保护功能。综上所述,EBR通过激活离子芥悬浮细胞的NOS活性来促进细胞内源NO积累,从而提高其抗寒性。EBR处理可以抑制低温胁迫下ROS过量积累并增强细胞的抗氧化防御能力,这两个过程都受NO信号分子调控,从而缓解低温造成的氧化损伤,防止膜脂过氧化程度加剧,提高细胞对低温胁迫的抗逆性。因此,低温胁迫下,高山离子芥悬浮细胞NOS来源的NO是EBR信号转导的下游信号分子。

α-常春藤皂苷抗肿瘤活性研究进展

α-常春藤皂苷(α-hederin)是一种单桥糖三萜皂苷,存在于常春藤叶片,具有广泛的药理活性。其中,人们发现α-常春藤皂苷最诱人的功能是其极高的抗肿瘤活性。目前,α-常春藤皂苷被发现的抗肿瘤机制包括自噬激活、一氧化氮调控、上皮间质转化阻滞、Hippo-Yap信号通路激活和促进活性氧积累等,且不仅于此。在这些细胞效应中,活性氧是α-常春藤皂苷发挥功能的核心桥梁。有足够的证据表明,α-常春藤皂苷诱导的肿瘤细胞凋亡、糖代谢水平下调、氧化应激发生和铁死亡敏感性上调都是由ROS所介导的;同时,也有理由相信,ROS还可能参与了α-常春藤皂苷诱导的Hippo信号通路激活和自噬启动。近年来,有一些研究对α-常春藤皂苷进行了改良,包括靶向递送的设计,生物利用度的调整和溶血活性的规避等。这给α-常春藤皂苷的开发和应用增加了更多的可能性。目前,关于α-常春藤皂苷的抗肿瘤药理研究仍然在广泛地开展,其丰富的生物学功能和极高的生物学活性使之成为一种十分具有前景的化合物。以近些年α-常春藤皂苷在抗肿瘤药理学上的成果作一综述,以期为该先导化合物的后续研发和临床应用提供理论参考依据,并提供新的思路。

长链非编码RNA-MEG3在人心脏微血管内皮细胞缺氧损伤中的作用

目的研究长链非编码RNA-MEG3在人心脏微血管内皮细胞(HCMEC)缺氧损伤中的作用。方法将HCMEC分为对照组、缺氧/复氧(H/R)组及缺氧/复氧联合MEG3敲减(H/R+siMEG3)组。荧光定量PCR检测MEG3及炎性因子的表达情况[白介素1β(IL-β)、IL-6、IL-8、IL-10、肿瘤坏死因子(TNF-α)];CCK-8检测HCMEC细胞的增殖活性;流式细胞术分析细胞凋亡情况;Western blotting检测PI3K/AKT/eNOS信号通路表达变化,ELISA检测一氧化氮(NO)、超氧化物歧化酶(SOD)、血管内皮生长因子(VEGF)和活性氧(ROS)表达水平。结果利用qPCR检测对照组及H/R组细胞MEG3表达,结果发现:H/R组MEG3相对表达倍数[(6.87±0.239)倍]较对照组MEG3表达倍数(1.00±0.026)倍显著升高(n=3,t=42.32,P<0.0001),提示MEG3可能在心脏微血管内皮细胞IRI中起到重要作用。H/R组细胞与对照组比较,细胞增殖活性显著减弱(P<0.01);而H/R+siMEG3组与H/R组比较,细胞增殖活性进步受到抑制(P<0.01)。H/R组较对照组PI3K、AKT及eNOS磷酸化水平显著减低,而H/R+siMEG3组细胞较H/R组磷酸化水平更低(P<0.05)。H/R组与对照组比较,NO、SOD及VEGF显著减低,而ROS水平升高(P<0.05);而H/R+siMEG3组与H/R组比较,NO、SOD及VEGF水平进步下降,ROS升高显著。利用qPCR检测各处理组细胞相关炎症基因表达,结果发现:H/R组较对照组基因表达显著升高(P<0.05);H/R+SiMEG3组较H/R组基因表达进一步升高(P<0.01)。结论长链非编码RNA-MEG3在心脏微血管内皮细胞H/R损伤过程中起到重要保护作用,靶向提升MEG3水平有望成为减缓心脏微血管IRI的潜在治疗靶点。

一氧化氮供体型药物对牙龈卟啉单胞菌体外抗菌活性的影响

目的研究一氧化氮(nitric oxide,NO)供体型药物对牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)体外抗菌活性的影响。方法采用菌落计数法、总NO检测试剂盒、活性氧检测试剂盒、钙离子(Ca^(2+))荧光探针Fluo-4 AM检测不同浓度的NO(10、30、50 mmol/L)对P.g的体外抗菌性能,以及对P.g胞内活性氧(reactive oxygen species,ROS)水平和Ca^(2+)浓度的影响。结果NO对P.g有显著的体外抗菌活性,并且其抗菌活性与NO药物浓度和作用时间具有明显的正相关性(P<0.05)。NO处理后显著提高了P.g胞内ROS水平和Ca^(2+)浓度(P<0.05)。结论NO能够有效抑制P.g的活性和生长能力,并引起P.g胞内ROS和Ca^(2+)代谢紊乱。NO供体型药物对种植体周围炎具有潜在的治疗作用和临床应用价值。

Ferrostatin-1 protects HT-22 cells from oxidative toxicity

Ferroptosis is a type of programmed cell death dependent on iron.It is different from other forms of cell death such as apoptosis,classic necrosis and autophagy.Ferroptosis is involved in many neurodegenerative diseases.The role of ferroptosis in glutamate-induced neuronal toxicity is not fully understood.To test its toxicity,glutamate(1.25–20 mM)was applied to HT-22 cells for 12 to 48 hours.The optimal experimental conditions occurred at 12 hours after incubation with 5 mM glutamate.Cells were cultured with 3–12μM ferrostatin-1,an inhibitor of ferroptosis,for 12 hours before exposure to glutamate.The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Autophagy was determined by monodansylcadaverine staining and apoptosis by caspase 3 activity.Damage to cell structures was observed under light and by transmission electron microscopy.The release of lactate dehydrogenase was detected by the commercial kit.Reactive oxygen species were measured by flow cytometry.Glutathione peroxidase activity,superoxide dismutase activity and malondialdehyde level were detected by the appropriate commercial kit.Prostaglandin peroxidase synthase 2 and glutathione peroxidase 4 gene expression was detected by real-time quantitative polymerase chain reaction.Glutathione peroxidase 4 and nuclear factor erythroid-derived-like 2 protein expression was detected by western blot analysis.Results showed that ferrostatin-1 can significantly counter the effects of glutamate on HT-22 cells,improving the survival rate,reducing the release of lactate dehydrogenase and reducing the damage to mitochondrial ultrastructure.However,it did not affect the caspase-3 expression and monodansylcadaverine-positive staining in glutamate-injured HT-22 cells.Ferrostatin-1 reduced the levels of reactive oxygen species and malondialdehyde and enhanced superoxide dismutase activity.It decreased gene expression of prostaglandin peroxidase synthase 2 and increased gene expression of glutathione peroxidase 4 and protein expressions of glutathione peroxidase 4 and nuclear factor(erythroid-derived)-like 2 in glutamate-injured HT-22 cells.Treatment of cultured cells with the apoptosis inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone(2–8μM),autophagy inhibitor 3-methyladenine(100–400μM)or necrosis inhibitor necrostatin-1(10–40μM)had no effect on glutamate induced cell damage.However,the iron chelator deferoxamine mesylate salt inhibited glutamate induced cell death.Thus,the results suggested that ferroptosis is caused by glutamate-induced toxicity and that ferrostatin-1 protects HT-22 cells from glutamate-induced oxidative toxicity by inhibiting the oxidative stress.

Advanced glycation end products:Key mediator and therapeutic target of cardiovascular complications in diabetes

The incidence of type 2 diabetes mellitus is growing in epidemic proportions and has become one of the most critical public health concerns.Cardiovascular complications associated with diabetes are the leading cause of morbidity and mortality.The cardiovascular diseases that accompany diabetes include angina,myocardial infarction,stroke,peripheral artery disease,and congestive heart failure.Among the various risk factors generated secondary to hyperglycemic situations,advanced glycation end products(AGEs)are one of the important targets for future diagnosis and prevention of diabetes.In the last decade,AGEs have drawn a lot of attention due to their involvement in diabetic pathophysiology.AGEs can be derived exogenously and endogenously through various pathways.These are a nonhomogeneous,chemically diverse group of compounds formed nonenzymatically by condensation between carbonyl groups of reducing sugars and free amino groups of protein,lipids,and nucleic acid.AGEs mediate their pathological effects at the cellular and extracellular levels by multiple pathways.At the cellular level,they activate signaling cascades via the receptor for AGEs and initiate a complex series of intracellular signaling resulting in reactive oxygen species generation,inflammation,cellular proliferation,and fibrosis that may possibly exacerbate the damaging effects on cardiac functions in diabetics.AGEs also cause covalent modifications and cross-linking of serum and extracellular matrix proteins;altering their structure,stability,and functions.Early diagnosis of diabetes may prevent its progression to complications and decrease its associated comorbidities.In the present review,we recapitulate the role of AGEs as a crucial mediator of hyperglycemia-mediated detrimental effects in diabetes-associated complications.Furthermore,this review presents an overview of future perspectives for new therapeutic interventions to ameliorate cardiovascular complications in diabetes.

Nitric Oxide Signaling in Plant Responses to Abiotic Stresses

Nitric oxide （NO） plays important roles in diverse physiological processes in plants. NO can provoke both beneficial and harmful effects, which depend on the concentration and location of NO in plant cells. This review is focused on NO synthesis and the functions of NO in plant responses to abiotic environmental stresses. Abiotic stresses mostly induce NO production in plants. NO alleviates the harmfulness of reactive oxygen species, and reacts with other target molecules, and regulates the expression of stress responsive genes under various stress conditions.

Nitric Oxide： A Multitasked Signaling Gas in Plants

Nitric oxide （NO） is a gaseous reactive oxygen species （ROS） that has evolved as a signaling hormone in many physiological processes in animals. In plants it has been demonstrated to be a crucial regulator of development, acting as a signaling molecule present at each step of the plant life cycle. NO has also been implicated as a signal in biotic and abiotic responses of plants to the environment. Remarkably, despite this plethora of effects and functional relationships, the fundamental knowledge of NO production, sensing, and transduction in plants remains largely unknown or inadequately characterized. In this review we cover the current understanding of NO production, perception, and action in different physiological sce- narios. We especially address the issues of enzymatic and chemical generation of NO in plants, NO sensing and downstream signaling, namely the putative cGMP and Ca^2＋ pathways, ion-channel activity modulation, gene expression regulation, and the interface with other ROS, which can have a profound effect on both NO accumulation and function. We also focus on the importance of NO in cell-cell communication during developmental processes and sexual reproduction, namely in pollen tube guidance and embryo sac fertilization, pathogen defense, and responses to abiotic stress.