Sustained activation of AMPK ameliorates age-associated vascular endothelial dysfunction via a nitric oxide-independent mechanism

Exercise restores endothelium-dependent dilation (EDD) in old mice by reducing oxidative stress and increasing nitric oxide (NO) bioavailability. Adenosine monophosphate protein kinase (AMPK) activation mimics some effects of exercise. Old (28-30 months) B6D2F1 mice had reduced arterial AMPK expression and superoxide-mediated suppression of EDD vs. young (3-6 months) controls. Pharmacological activation of AMPK by aminoimidazole carboxamide ribonucleotide (AICAR) for 2 weeks increased arterial AMPK and reversed this superoxide-induced impairment of EDD. The improvement in EDD was independent of NO or prostaglandin signaling, suggesting enhanced endothelium-dependent hyperpolarizing factor-related dilation. AMPK activation may represent a novel therapy for treating age-associated vascular dysfunction.     

Nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension

Endothelial dysfunction in the setting of cardiovascular risk factors such as hypercholesterolemia, diabetes mellitus, chronic smoking, as well hypertension, is, at least in part, dependent of the production of reactive oxygen species (ROS) and the subsequent decrease in vascular bioavailability of nitric oxide (NO). ROS-producing enzymes involved in increased oxidative stress within vascular tissue include NADPH oxidase, xanthine oxidase, and mitochondrial superoxide producing enzymes. Superoxide produced by the NADPH oxidase may react with NO, thereby stimulating the production of the NO/superoxide reaction product peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, therefore switching an antiatherosclerotic NO producing enzyme to an enzyme that may accelerate the atherosclerotic process by producing superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and also occurs within the smooth muscle cell layer. Increased superoxide production has important consequences with respect to signaling by the soluble guanylate cyclase and the cGMP-dependent kinase I, which activity and expression is regulated in a redox-sensitive fashion. The present review will summarize current concepts concerning eNOS uncoupling, with special focus on the role of tetrahydrobiopterin in mediating eNOS uncoupling.     

Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo

Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin-1 scaffolding domain (AP-Cav) reduces eNOS-regulated hyperpermeability. We used eNOS-deficient (eNOS−/−) mice and their wild-type control as experimental animals, platelet-activating factor (PAF) at 10⁻⁷ m as the test pro-inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild-type cremaster muscle from a baseline of 2.4 ± 2.2 to a peak net value of 84.4 ± 2.7 units, while the corresponding values in cremaster muscle of eNOS−/− mice were 1.0 ± 0.3 and 15.6 ± 7.7 units ( P < 0.05). Similarly, PAF increased IOI in the mesentery of wild-type mice but much less in the mesentery of eNOS−/− mice. PAF increased IOI to comparable values in the mesenteries of wild-type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP-Cav blocked the microvascular hyperpermeability responses to 10⁻⁷ m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin-1 inhibits eNOS regulation of microvascular permeability in vivo . Our results establish eNOS as an important regulator of microvascular permeability in inflammation.     

Xanthine oxidase does not contribute to impaired peripheral conduit artery endothelium-dependent dilatation with ageing

Vascular oxidative stress is the key mechanism involved in the age-related decline in endothelium-dependent dilatation (EDD). We tested the hypothesis that xanthine oxidase (XO), a major vascular source of reactive oxygen species, contributes to the impairment in EDD with ageing. At baseline, brachial artery flow-mediated dilatation (FMD) was 55% lower in older ( n = 9, 64 ± 2 years, 8M/1F, mean ± s.e.m. ) versus young ( n = 9, 26 ± 1 years, 8M/1F) healthy adults (3.41 ± 0.44 versus 7.53 ± 0.67%, P < 0.001), whereas endothelium-independent dilatation (EID; sublingual nitroglycerin) did not differ between groups. Plasma oxidized low-density lipoprotein (oxi-LDL), a measure of systemic oxidative stress, was greater at baseline in the older subjects (58.3 ± 5.9 versus 46.8 ± 2.4 U l⁻¹, P < 0.05) and inversely correlated with baseline FMD ( r =− 0.54; P < 0.05). Acute administration of allopurinol, a competitive inhibitor of XO, reduced plasma uric acid concentrations similarly in both groups ( P < 0.001), but did not affect FMD, EID, or oxi-LDL in either group. Vascular endothelial protein expression of XO (immunofluorescence) was not different in antecubital venous cells from the young and older subjects (0.56 ± 0.12 versus 0.68 ± 0.19 XO intensity/human umbilical vein endothelial cell intensity, P = 0.49). We conclude that XO does not contribute to oxidative stress-associated reductions in peripheral conduit artery EDD with ageing in humans, possibly due to an absence of age-associated up-regulation of endothelial XO.     

葡萄籽原花青素体外抗血小板聚集的机制研究

目的: 研究葡萄籽原花青素(PC)体外抗血小板聚集的可能机制。方法: 应用血小板聚集仪研究PC、二亚苯基碘鎓(DPI,非特异性NADPH氧化抑制剂)和夹竹桃麻素(apocynin,特异性NADPH氧化酶抑制剂)对胶原诱导的健康志愿者血小板最大聚集率的影响。用化学发光仪检测PC对血小板NADPH氧化酶活性、一氧化氮(NO)含量和超氧阴离子(O₂^-·)水平的影响。用流式细胞术观察PC对血小板活化标志物PAC-1和CD62P表达率的影响。结果: PC(100 μmol/L)、apocynin(10 μmol/L)和DPI(100 μmol/L )均可显著抑制胶原蛋白诱导的血小板最大聚集率(P<0.01)。胶原蛋白激活的血小板NO含量显著降低,O₂^-·含量显著增加,100 μmol/L PC可使二者明显恢复(P<0.05)。添加了100 μmol/L PC的样本中,血小板NADPH氧化酶活性受到明显的抑制(P<0.01),血小板PAC-1 和CD62P表达率也显著降低(P<0.05)。结论: 葡萄籽原花青素可能通过抑制NADPH氧化酶的活性,进而影响血小板NO和O₂^-·含量,在一定程度上阻断血小板活化过程,最终达到抗血小板聚集的作用。     

载脂蛋白A-I模拟肽D4F通过抑制caspase-12减轻氧化低密度脂蛋白诱导的巨噬细胞凋亡

目的:探讨载脂蛋白A-I(apolipoprotein A-I,Apo A-I)模拟肽D4F对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的巨噬细胞凋亡和内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键分子caspase-12的影响,并阐明其可能的分子机制。方法:体外培养RAW264.7巨噬细胞,给予12.5、25和50 mg/L D4F、5 mmol/L ERS抑制剂4-苯丁酸(4-phenylbutyric acid,PBA)或5μmol/L二亚苯基碘鎓(diphenyleneiodonium,DPI)预处理1 h后,再加入100 mg/L ox-LDL或4 mg/L ERS诱导剂衣霉素(tunicamycin,TM)继续培养24 h。MTT法检测细胞活力;TUNEL法检测细胞凋亡情况;试剂盒测定细胞内丙二醛(malondialdehyde,MDA)和活性氧(reactive oxygen species,ROS)水平,以及超氧化物歧化酶(superoxide dismutase,SOD)和烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶活性;Western blot法检测caspase-12的表达变化。结果:与ERS抑制剂PBA相似,D4F可抑制ox-LDL或TM所致的巨噬细胞活力降低和凋亡,且呈浓度依赖性(P0.05)。与氧化应激抑制剂DPI相似,D4F显著抑制ox-LDL诱导的氧化应激反应,表现为ROS和MDA生成减少(P0.01)、SOD活性增加以及NADPH氧化酶活性降低(P0.05);与PBA和DPI相似,D4F可减轻ox-LDL诱导的巨噬细胞caspase-12活化,且呈浓度依赖性(P0.05);另外,D4F还可抑制TM诱导的caspase-12活化(P0.05)。结论:D4F能够抑制ox-LDL诱导的巨噬细胞凋亡,其机制至少部分是通过减轻氧化应激继而抑制caspase-12活化实现的。     

Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS-/-) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS-/- femurs showed bone volume and bone formation rate was reduced by up to 45% (P: < 0.01) and 52% (P: < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P: < 0.05) and spinal bone mineral densities (P: < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS-/- mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17ss-estradiol and had an attenuated chemotactic response to transforming growth factor-beta. In conclusion, eNOS is involved in the postnatal regulation of bone mass and lack of eNOS gene results in reduced bone formation and volume and this is related to impaired osteoblast function.     

The autophagy enhancer spermidine reverses arterial aging

Arterial aging, characterized by stiffening of large elastic arteries and the development of arterial endothelial dysfunction, increases cardiovascular disease (CVD) risk. We tested the hypothesis that spermidine, a nutrient associated with the anti-aging process autophagy, would improve arterial aging. Aortic pulse wave velocity (aPWV), a measure of arterial stiffness, was ～20% greater in old (O, 28 months) compared with young C57BL6 mice (Y, 4 months, P < 0.05). Arterial endothelium-dependent dilation (EDD), a measure of endothelial function, was ～25% lower in O (P < 0.05 vs. Y) due to reduced nitric oxide (NO) bioavailability. These impairments were associated with greater arterial oxidative stress (nitrotyrosine), superoxide production, and protein cross-linking (advanced glycation end-products, AGEs) in O (all P < 0.05). Spermidine supplementation normalized aPWV, restored NO-mediated EDD and reduced nitrotyrosine, superoxide, AGEs and collagen in O. These effects of spermidine were associated with enhanced arterial expression of autophagy markers, and in vitro experiments demonstrated that vascular protection by spermidine was autophagy-dependent. Our results indicate that spermidine exerts a potent anti-aging influence on arteries by increasing NO bioavailability, reducing oxidative stress, modifying structural factors and enhancing autophagy. Spermidine may be a promising nutraceutical treatment for arterial aging and prevention of age-associated CVD.     

Interaction between nitric oxide and oxygen radicals in regulation of tubuloglomerular feedback

NADPH oxidase, nitric oxide synthase (NOS) and cyclooxygenase are oxidases that are expressed in the juxtaglomerular apparatus (JGA) or blood vessels and can generate oxygen radicals (O-2) during partial reduction of molecular oxygen. O-2 interacts rapidly and irreversibly with nitric oxide (NO) to yield peroxynitrite (ONOO-), thereby restricting the half-life, diffusion distance and bioactivity of NO in tissues. NO generated by a neuronal (n) NOS isoform that is heavily expressed in macula densa (MD) cells, is generated during NaCl reabsorption at the MD and blunts the expression of the tubuloglomerular feedback (TGF) response. Therefore, we tested the hypothesis that O-2 formed in the JGA of the normal rat limits NO signalling. Tempol is a membrane-permeable superoxide dismutase (SOD) mimetic. Maximal TGF responses were assessed from the fall in proximal stop flow pressure during orthograde perfusion of artificial tubular fluid (ATF) into the loop of Henle. Microperfusion of tempol (10-4 M) into the efferent arteriole (EA) of Wistar-Kyoto rats blunted maximal TGF response (8. 2 +/- 0.4 vs. 6.4 +/- 0.4 mmHg; n=8; P < 0.05). Graded doses of the NO donor compound, S-nitroso-acetylpenicillamine (SNAP; 10-7-10-4 M) microperfused into the lumen of the MD produces graded buffering of TGF. During EA microperfusion of tempol, responses to luminal SNAP at 10-6 M and greater were enhanced significantly (P < 0.05 or <0. 01). In conclusion, O-2 generated in the JGA can be metabolized by a membrane-permeable SOD mimetic. O-2 enhances the basal TGF response and limits NO signalling from the macula densa. Therefore, O-2 and NO interact in the JGA to modulate the TGF response.     

Defects in activation of nitric oxide synthases occur during delayed angiogenesis in aging

Angiogenesis, the formation of new vessels from pre-existing vasculature, is impaired in aging. This is due, in part, to a lack of regulatory molecules such as nitric oxide (NO). We wished to test the hypothesis that there are deficits in the pathways that mediate NO production during angiogenesis (as defined by fibrovascular invasion into a polyvinyl alcohol (PVA) sponge implant), in aged mice in comparison to young mice. Sponges were implanted subcutaneously in young (6-8 months old, n=11) and aged (23-25 months old, n=13) mice and sampled at 14 and 19 days. Sections from the implants were stained with antibodies against vascular endothelial growth factor receptor 2 (VEGFR-2), Akt, phosphorylated Akt (p-Akt), endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), inducible NOS (iNOS), and 3-nitrotyrosine (3-NT, a marker for nitrosylated proteins). Expression of VEGFR-2 was similar in the sponges of young and aged mice. Moreover, there were no significant differences in levels of Akt or its phosphorylated form in sponges from young and aged mice at 14 and 19 d. In marked contrast, levels of eNOS, p-eNOS and iNOS were significantly decreased in sponges from aged mice relative to young mice (p<0.02 for eNOS, p-eNOS and <0.01 for iNOS between young and aged mice). Concomitantly, there was diminished expression of 3-NT in the sponges from aged mice (p<0.05). Our data indicate that defects in the activation of nitric oxide synthases result in decreased NO production in aged tissues relative to young tissues. We propose that the subsequent lack of NO contributes to impaired angiogenesis in aging.     

Redox regulation of the afferent arteriole and tubuloglomerular feedback

Oxidative stress implies an increased production of reactive oxygen species (ROS) or a decreased capacity to metabolize them. Superoxide anion (O) can bioinactivate nitric oxide (NO). Therefore, many effects of ROS are manifest as NO deficiency. The afferent arteriole and macula densa cell both contain a full complement of components of nicotine adenine dinucleotide phosphate (NADPH) oxidase that generates O. Nitric oxide synthase (NOS) type 1 or neuronal NOS (nNOS) is expressed in the macula densa and NOS type II or endothelial NOS (eNOS) in the afferent arteriole. Whole animal studies in models of hypertension and oxidative stress demonstrate that metabolism of O by a superoxide dismutase (SOD) mimetic can reduce renal vascular resistance. In vivo studies of single nephron function and in vitro studies with the double-perfused juxtaglomerular apparatus preparation have shown extensive interaction between O and NO in macula densa to regulate afferent arteriolar tone mediated by the tubuloglomerular feedback response. In vitro studies of rabbits isolated, perfused afferent arterioles have shown a similar interaction in this vessel. These data indicate important roles for O in the macula densa and afferent arterioles to enhance preglomerular resistance in animal models of oxidative stress. As an increase in afferent arteriolar resistance can precede hypertension, oxidative stress could be important in determining the long-term blood pressure and thereby contribute to hypertension.     

Dynamic exercise attenuates spontaneous baroreceptor reflex sensitivity in conscious rats

In humans, it has been reported that dynamic exercise attenuates spontaneous baroreceptor reflex sensitivity (sBRS), which is an index of the gain of the baroreceptor-cardiac reflex. We demonstrated previously that endogenously produced NO from endothelial nitric oxide synthase (eNOS) within the nucleus tractus solitarii (NTS), the central terminal site of baroreceptor afferents, depressed sBRS. In this study, we investigated whether eNOS activity within the NTS plays any role in down-modulating the sBRS during dynamic exercise. In conscious Wistar rats arterial pressure and heart rate (HR) were monitored continuously and chronically using radiotelemetry before and during wheel cage running at 6 m min(-1) for 10 min. sBRS was determined by a time-series method. During dynamic exercise systolic blood pressure (SBP) and HR were significantly increased (SBP: 138 +/- 2 vs. 125 +/- 2 mmHg, P < 0.001; HR: 447 +/- 6 vs. 362 +/- 8 beats min(-1), P < 0.001) while sBRS was significantly decreased (0.53 +/- 0.03 vs. 1.08 +/- 0.08 ms mmHg(-1), P < 0.001). In sino-aortic denervated rats the change in SBP in response to dynamic exercise was significantly larger than that in baroreceptor-intact rats (denervated: 21.6 +/- 2.5 mmHg; intact: 12.0 +/- 2.8 mmHg, P < 0.05). In contrast, denervation made no difference to the change in HR. Although disabling eNOS activity in the NTS by adenoviral-directed expression of a dominant negative mutant form of eNOS increased resting sBRS (1.48 +/- 0.20 vs. 1.09 +/- 0.15 ms mmHg(-1), P < 0.05), the absolute level reached during dynamic exercise was identical to control. These results demonstrate that during dynamic exercise (i) the sBRS decreases around the operating point of the baroreceptor-cardiac reflex function curve in normotensive rats, (ii) the baroreceptor reflex operates to limit the rise in arterial pressure, and (iii) the attenuation of sBRS is not mediated by changes in eNOS activity within the NTS.     

The effects of diethyldithiocarbamate,a SOD inhibitor,on endothelial function in sedentary and exercised db/db mice

The study was designed to evaluate the effects of diethyldithiocarbamate (DDC), an superoxide dismutase (SOD) blocker on endothelial function in db/db mice. The db/db and wild-type (WT) mice were randomly divided into low intensity exercise, moderate intensity exercise and control (sedentary) groups. Mice were exercised daily, 5 days per week. After 6 weeks, ring segments of aortae were mounted in wire myograph and acetylcholine (ACh) concentration response curves were recorded in absence or presence of DDC and DDC plus SOD.Results showed that ACh concentration response curve was similar in WT and WT exercised groups. Incubation of aorta rings of WT mice with DDC suppressed the maximum ACh response (p < 0.05). Subsequent incubation with SOD restored vasodilatory response in WT mice. The vasodilatation to ACh was significantly reduced in sedentary db/db mice compare to WT (p < 0.05) and incubation with DDC did not further decrease this response, however, addition of SOD restored the vasodilatation to ACh to that observed in WT mice. Mild and moderate exercised db/db mice had ACh response similar to that in WT mice. Incubation with DDC incubation reduced ACh induced vasodilatation and addition of SOD restored this response.Our results support the conclusion that SOD mimetics can be used to improve superoxide-mediated endothelial dysfunction in diabetic db/db mice.     

脂联素对大鼠缺血再灌注心肌缝隙连接蛋白43表达的影响

目的: 观察脂联素(APN)对大鼠缺血再灌注心肌缝隙连接蛋白43(Cx43)表达的影响,进一步探讨其抗心律失常的可能机制。方法: 将48只雄性Wistar大鼠随机分成:(1)假手术 (SM)组;(2)缺血再灌注(I/R)组:结扎冠状动脉左前降支,30 min后松开结扎线,再灌注120 min;(3)脂联素+缺血再灌注组1(I/R+APN1):先阻断血流30 min,于再灌注120 min开始时给予3.5 μg/kg APN;(4)脂联素+缺血再灌注组2(I/R+APN2):缺血前10 min给予3.5 μg/kg APN,余同I/R组。观察各组心律失常的发生情况;应用RT-PCR观察各组心室肌细胞 Cx43 基因表达;用免疫组织化学方法观察Cx43分布的变化;应用硫代巴比妥酸(TBA)法和黄嘌呤氧化酶法测各组动物血清中丙二醛(MDA)的含量和超氧化物歧化酶(SOD)的活性;用RT-PCR及Western blotting法分析内皮型一氧化氮合酶(eNOS)mRNA及蛋白的表达,用电镜观察各组心室肌超微结构的改变。结果: (1)I/R组与SM组比较,心律失常评分和血清MDA含量显著增高(P<0.01),SOD活性降低(P<0.01);心室肌Cx43表达明显减少,差异有统计学意义(P<0.01),Cx43分布紊乱,失去正常的规律性;心肌细胞超微结构有明显损伤;心室肌eNOS mRNA及蛋白的表达明显降低,差异有统计学意义(P<0.01)。(2)无论缺血前还是缺血后使用脂联素处理,与I/R组相比,心律失常评分显著降低(P<0.01);Cx43及eNOS表达增高(P<0.01),Cx43分布紊乱的程度减轻;心肌细胞超微结构损伤明显改善。结论: APN可能通过氧化应激调节Cx43的功能,从而发挥抗缺血/再灌注心律失常的作用。     

NO在脂联素抑制高脂血症大鼠血小板聚集中的作用

目的: 探讨一氧化氮(NO)信号转导通路在脂联素抑制高脂血症血小板聚集机制中的作用。方法: 采用成年大鼠饲以高脂饲料14周,分离其血小板并以重组脂联素(rAPN)孵育。采用免疫荧光、Western blotting等方法观察检测血小板聚集、NO含量、超氧化物含量、内皮型一氧化氮合酶(eNOS)/诱导型一氧化氮合酶(iNOS)的表达和抗氧化物活性。结果: 采用rAPN处理能抑制高脂血症诱导的血小板聚集(P<0.05),并导致血小板NO的生成显著减少。同时,在高脂血症血小板中,采用rAPN处理还能显著减少超氧化物的生成(降低62%, P<0.05) 并增强其抗氧化能力(增加38%, P<0.05)。此外,高脂血症诱导的eNOS磷酸化的降低和iNOS表达的增加在rAPN处理后被显著逆转(P<0.05, P<0.01)。结论: 脂联素是一种抑制高脂血症血小板聚集的脂肪细胞因子,其机制与减少超氧化物水平、增加抗氧化物活性和阻断iNOS的表达有关。     

Mechanisms regulating superoxide generation in experimental models of phenylketonuria: an essential role of NADPH oxidase

This study was designed to investigate whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a superoxide-producing enzyme, could be involved in phenylketonuria (PKU)-associated oxidative stress. A Pah^enu2-BTBR PKU mouse model, and an in vitro cell culture model of PKU mimicking high phenylalanine insults in PKU, were employed for this study. The concentration of phenylalanine in mouse cerebral cortex was determined by liquid chromatography-tandem mass spectrometry. Superoxide production was displayed with dihydroethidium staining. NADPH oxidase expression level was measured by real-time RT-PCR, Western blotting and immunofluorescence. NADPH oxidase activity was measured by the colorimetric method. The phenylalanine concentrations in cerebral cortices of PKU mice were significantly higher than those in wild-type control mice. Similar results concerning superoxide production and NADPH oxidase protein expression and activity, were also found in this brain region. In addition, it was found that cerebral cortical neurons subjected to an in vitro high phenylalanine insult, displayed increased superoxide production accompanied by increases of NADPH oxidase protein expression and activity. Pretreatment with the inhibitor of this oxidase (diphenylene iodonium or apocynin) prevented this superoxide-increasing effect. Collectively, these findings provide evidence that NADPH oxidase might be a key enzyme involved in enhanced superoxide production in PKU and suggest that it may be a potential therapeutic target in neuroprotective strategies against phenylalanine-evoked oxidative brain injury in PKU.     

葡萄多酚对雨蛙肽诱导的急性胰腺炎胰腺组织的保护作用

 目的:研究葡萄多酚对雨蛙肽诱导的小鼠急性胰腺炎（AP）的保护作用。方法: 2月龄ICR雌鼠共21只,随机分为正常对照组（NC组）、AP模型组（AP组）和药物处理AP组,每组7只。造模前,药物处理AP组小鼠连续7 d胃内灌注葡萄多酚（1.5 g/kg）水溶液,NC组和AP组小鼠则以同法给予生理盐水作为对照。第7天开始造模。AP组以及药物处理AP组小鼠予以腹腔注射雨蛙肽(50 μg/kg)造模,每小时1次,共注射7次;NC组小鼠同法注射等量生理盐水。于24 h处死小鼠取其胰腺及肺组织。测量各组胰腺组织的相对重量、病理形态学改变、巨噬细胞的浸润情况及炎症因子、氧化应激分子的表达水平;检测肺脏组织的髓过氧化物酶（MPO）活性以考察远隔器官的炎症。 结果: 与AP组小鼠相比,药物处理AP组小鼠胰腺组织的水肿、炎症以及空泡的评分明显降低（P<0.05）,但坏死和病理总评分没有明显变化;巨噬细胞的浸润明显减少;肿瘤坏死因子α(TNF-α)和单核细胞趋化蛋白1(MCP-1)的表达水平也明显降低(P<0.05);超氧化物歧化酶1（SOD-1)、SOD-2和NADPH氧化酶2（NOX-2）的表达水平也明显降低（P<0.05, P<0.05, P<0.01）。此外,药物处理AP组小鼠肺组织的MPO活性显著降低（P<0.01）,提示中性粒细胞的浸润减少。结论: 葡萄多酚对雨蛙肽诱导的小鼠急性胰腺炎胰腺组织具有明显保护作用,机制与下调炎症因子和氧化应激分子的表达有关。     

Oxidation, glycoxidation, lipoxidation, nitration, and responses to oxidative stress in the cerebral cortex in Creutzfeldt-Jakob disease

Gel electrophoresis and Western blotting of frontal cortex homogenates have been carried out in sporadic Creutzfeldt–Jakob disease (CJD) cases and age-matched controls to gain understanding of the expression of glycation-end products (AGEs). N-Carboxymethyl-lysine (CML) and N-carboxyethyl-lysine (CEL) were used as markers of glycoxidation; 4-hydroxynonenal (4-HNE) and malondialdehyde-lysine (MDAL) as markers of lipoxidation; and nitrotyrosine (N-tyr) and neuronal, endothelial and inducible nitric oxide synthase (nNOS, eNos and iNos) as markers of protein nitration and as sources of NO production, respectively. Age receptor (RAGE) and Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) expression levels were also examined. The results showed a significant increase in the expression levels of AGE (p < 0.05), CEL (p < 0.001), RAGE (p < 0.05), HNE-modified proteins (p < 0.01), nNOS, iNOS and eNOS (p < 0.01 and p < 0.05, respectively), N-tyr (p < 0.05), and SOD1 (p < 0.05) and SOD2 (p < 0.05). No relationship was observed between PrP genotype, PrP type, PrP burden, and expression levels of oxidative stress markers. The present findings demonstrate oxidative, glycoxidative, lipoxidative and nitrative protein damage, accompanied by increased oxidative responses, in the cerebral cortex in sporadic CJD. These results provide support for the concept that oxidative stress may have important implications in the pathogenesis of prion diseases.     

从氧自由基、一氧化氮探讨四逆汤抗急性失血性休克的肝脏机制

目的:从氧自由基、一氧化氮探讨四逆汤抗急性失血性休克的肝脏机制。方法:复制急性失血性休克大鼠模型, 分为假手术对照组;单纯休克模型组;休克+生理盐水复苏组;休克+四逆注射液复苏组。四逆注射液(浓度1000g生药/L), 剂量0.1mL/200g大鼠。用生理盐水或四逆注射液治疗3h后处死动物并取组织。测定各组肝脏超氧化物歧化酶(SOD)活性、丙二醛(MDA)水平, 一氧化氮(NO)水平。采用免疫组化染色法, 观察诱导型一氧化氮合酶(iNOS)在肝细胞中的变化特点。RT-PCR观测肝细胞iNOS和内皮源性一氧化氮合酶(eNOS)基因表达的变化。结果:模型组在休克1h后SOD活性明显低于对照组(P＜0.01)、MDA水平明显高于对照组(P＜0.01)。四逆汤组复苏3h后肝组织SOD明显高于生理盐水组(P＜0.01)、MDA低于生理盐水组(P＜0.01)、NO水平明显高于生理盐水组(P＜0.01)。生理盐水组iNOS在肝细胞中染色阳性单位明显高于四逆汤组(P＜0.05)。生理盐水组促进iNOSmRNA的表达。四逆汤组eNOSmRNA的表达增强。结论:四逆汤通过清除氧自由基, 升高NO, 改善肝组织微循环, 减少诱导型iNOS表达的各种因素, 理论上减轻了NO与氧自由基生成的ONOO的细胞毒作用和血管的低反应性, 并对肝脏起到保护作用。