Protective effects of Dragon's Blood on blood coagulation and NO/iNOS level in myocardium and serum of rats in simulated microgravity

To investigate effects of Dragon''s Blood (DB), a traditional Chinese medicine, on blood coagulation and NO/iNOS level in myocardium and serum of rats in simulated microgravity for the first time, Sprague Dawley (SD) rats were randomly divided into six groups:(a) 5-day control group, (b) 5-day model group, (c) 5-day drug group, (d) 21-day control group, (e) 21-day model group, and (f) 21-day drug group. Blood coagulation and NO/iNOS level in myocardium and serum were examined after 5 and 21 days of simulated microgravity respectively. The results showed that blood of tail-suspended rats was in a hypercoagulable state that could not be converted with time extending. Conversely, DB changed these parameters towards normal level and the curative effects became better when tail-suspension lasted till the 21st day. NO concentration of both myocardium and serum for two periods all increased markedly and DB could effectively reduce these increases except that of 21-day myocardium NO. Activity of iNOS increased markedly as early as 5 days and became more serious on the 21st day, while DB showed preventive effect on the 21st day. Western Blot analysis illustrated that the expression of iNOS in the 5-day model group increased significantly over the 5-day control group and the expression in the 5-day drug group dramatically returned to the normal level. The similar trend was observed on the 21-day groups without notable variances. The findings of this study can serve for the further use of Dragon''s Blood in space diseases.     

Cyclic Stretch Induces Inducible Nitric Oxide Synthase and Soluble Guanylate Cyclase in Pulmonary Artery Smooth Muscle Cells

In the pulmonary vasculature, mechanical forces such as cyclic stretch induce changes in vascular signaling, tone and remodeling. Nitric oxide is a potent regulator of soluble guanylate cyclase (sGC), which drives cGMP production, causing vasorelaxation. Pulmonary artery smooth muscle cells (PASMCs) express inducible nitric oxide synthase (iNOS), and while iNOS expression increases during late gestation, little is known about how cyclic stretch impacts this pathway. In this study, PASMC were subjected to cyclic stretch of 20% amplitude and frequency of 1 Hz for 24 h and compared to control cells maintained under static conditions. Cyclic stretch significantly increased cytosolic oxidative stress as compared to static cells (62.9 ± 5.9% vs. 33.3 ± 5.7% maximal oxidation), as measured by the intracellular redox sensor roGFP. Cyclic stretch also increased sGCβ protein expression (2.5 ± 0.9-fold), sGC activity (1.5 ± 0.2-fold) and cGMP levels (1.8 ± 0.2-fold), as well as iNOS mRNA and protein expression (3.0 ± 0.9 and 2.6 ± 0.7-fold, respectively) relative to control cells. An antioxidant, recombinant human superoxide dismutase (rhSOD), significantly decreased stretch-induced cytosolic oxidative stress, but did not block stretch-induced sGC activity. Inhibition of iNOS with 1400 W or an iNOS-specific siRNA inhibited stretch-induced sGC activity by 30% and 68% respectively vs. static controls. In conclusion, cyclic stretch increases sGC expression and activity in an iNOS-dependent manner in PASMC from fetal lambs. The mechanism that produces iNOS and sGC upregulation is not yet known, but we speculate these effects represent an early compensatory mechanism to counteract the effects of stretch-induced oxidative stress. A better understanding of the interplay between these two distinct pathways could provide key insights into future avenues to treat infants with pulmonary hypertension.     

Dietary lutein modulates inducible nitric oxide synthase (iNOS) gene and protein expression in mouse macrophage cells (RAW 264.7)

Lutein is an oxycarotenoid primarily found in dark-green leafy vegetables such as spinach and kale. Other dietary sources which contain moderate amounts of lutein include corn, egg yolks, and fruits like oranges and kiwi. Although a number of in vivo studies have demonstrated the anti-inflammatory effect of lutein, its in vitro anti-inflammatory molecular mechanism of action is unknown. In this study, we have investigated the in vitro anti-inflammatory effect of lutein using LPS-stimulated mouse macrophage cell line (RAW 264.7). The inhibition of LPS-stimulated nitric oxide (NO) was measured and the expression of inducible NO synthase (iNOS) was assessed at the mRNA and protein levels in mouse macrophage cells after treatment with lutein. Lutein decreased the LPS-induced NO production by 50% compared to LPS alone. Real-time PCR analysis showed a 1.9-fold reduction in iNOS expression at the mRNA level. Western blotting revealed that lutein decreased LPS-induced iNOS expression at the protein level by 72.5%. The results of this study suggest the anti-inflammatory properties of lutein demonstrated by the decrease in the expression of iNOS at the mRNA and protein levels in RAW 264.7 mouse macrophage cells.     

虫草素抑制脂多糖诱导的小胶质细胞活化及神经保护作用

目的：研究虫草素抑制脂多糖（lipopolysaccharides,LPS）诱导小胶质细胞激活及其神经保护作用。方法：培养原代小鼠小胶质细胞,以LPS激活小胶质细胞使NO大量释放,同时给予不同浓度的虫草素（0.1～10 μmol/L）处理,四甲基偶氮唑蓝（3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT）法检测细胞存活率,Griess法测定小胶质细胞NO释放,逆转录聚合酶链式反应法检测细胞内诱导型一氧化氮合酶（inducible nitric oxidesynthase,iNOS）的mRNA表达。以硝普钠作为NO供体,以1,1-二苯基苦基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基作为自由基的供体,分别考察虫草素对NO和DPPH自由基的直接清除作用。分别以H2O2和以LPS激活的小胶质细胞条件培养液损伤小鼠PC12神经元细胞,MTT法考察虫草素对PC12细胞损伤的保护作用。此外,以羟胺法测定超氧化物歧化酶（superoxide dismutase,SOD）活性。结果：虫草素能够显著抑制LPS激活的原代小鼠小胶质细胞NO产生及iNOS mRNA表达,同时不影响细胞的存活率;虫草素具有显著的NO清除和DPPH自由基清除作用;虫草素本身对PC12小鼠神经元细胞的生长没有显著影响,但能够显著抑制H2O2或活化小胶质细胞的条件培养液引起的PC12细胞损伤。此外,虫草素可显著提高H2O2损伤的PC12细胞中SOD活性。结论：虫草素可通过抑制iNOS转录和直接清除NO而抑制LPS激活小胶质细胞的NO产生;虫草素还可通过抑制小胶质细胞激活而对PC12神经元细胞产生保护作用,也可通过提高SOD活性而保护H2O2损伤的PC12细胞。因此,虫草素可能对与小胶质细胞激活相关的神经退行性疾病具有潜在的防治作用。     

β-Casomorphin-7 cause decreasing in oxidative stress and inhibiting NF-κB-iNOS-NO signal pathway in pancreas of diabetes rats

β-Casomorphin-7 (β-CM-7) is a milk biological active peptide. The present study is aimed to investigate the protective effects of β-CM-7, against oxidative stress in pancreas of streptozotocin-induced diabetic rats by assaying malondialdehyde (MDA), nitric oxide (NO) level, the activity of enzymatic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), and NF-κB, inducible nitric oxide synthase (iNOS) gene expression. A significant increase in the level of oxidative stress was observed in pancreas of the diabetic rats when compared to control rats. After 15 d oral administration of β-CM-7 (7.5 × 10(-8) mol/d), the pancreas MDA level was markedly reduced. Oral administration of β-CM-7 to diabetic rats showed an obviously increase in the activity of catalase in pancreas, oral administration of β-CM-7 to the diabetic group of rats also showed a reduction of NF-κB and iNOS gene expression in pancreas. The elevated pancreas NO level was markedly reduced by the oral administration of β-CM-7. Thus, the results of the present study suggest that β-CM-7 may cause protective effects such as pronounced decreasing in oxidative stress and inhibiting NF-κB-iNOS-NO signal pathway in pancreas of diabetes rats.     

Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD₈₀ dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan–Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG_1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb’s efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.     

烷基双胍硫酸盐的合成及iNOS抑制活性的测定

二腈二胺在60℃下与硫酸铜反应1 5h得配合物(Ⅰ);然后分2次加入烷基胺(间隔时间为1h)于70℃(对Ⅱa、Ⅱb)或80℃(对Ⅱc～Ⅱe)下反应10h合成了5种烷基双胍硫酸盐(Ⅱa～Ⅱe),Ⅱa、Ⅱb、Ⅱc、Ⅱd、Ⅱe的收率分别为83 75%、84 21%、88 45%、87 62%、86 51%,以元素分析、IR、NMR、UV对化合物Ⅱa～Ⅱe的结构进行了确认。对化合物Ⅱa～Ⅱe及配合物(Ⅰ)的诱生型一氧化氮合酶(iNOS)抑制活性进行了测定,结果表明,化合物Ⅱb及配合物(Ⅰ)有iNOS抑制活性,其中配合物(Ⅰ)的活性强于对照物氨基胍。     

Terpenoids from the Octocoral Sinularia gaweli

Two eudesmane sesquiterpenoids, verticillatol (1) and 5α-acetoxy-4(14)-eudesmene-1β-ol (2) and two cembrane diterpenoids, (–)-leptodiol acetate (3) and sinulacembranolide A (4) were isolated from the octocoral Sinularia gaweli and compounds 2–4 are new isolates. The structures of new terpenoids 2–4 were elucidated by spectroscopic methods and by comparison the spectral data with those of known analogues. Terpenoid 4 was found to inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 marcophage cells.