有氧运动对慢性阻塞性肺疾病稳定期患者外周血调节性T细胞亚群与心肺运动功能的干预研究

目的:探讨COPD稳定期患者有氧运动前后调节性T细胞(Treg)亚群细胞及心肺运动功能变化情况,研究有氧运动改善COPD稳定期患者临床症状、促进COPD患者康复的机制。方法:选取2016年1月—2017年12月我院住院COPD稳定期患者为研究对象。COPD对照组30例用12周吸氧、化痰、舒张支气管等常规药物治疗,COPD试验组30例用常规治疗同时施加有氧运动干预12周。比较患者治疗前后外周血中的Treg细胞亚群比例、IL-17和TGF-β表达水平与心肺运动功能变化情况。结果:有氧运动干预后,试验组Treg细胞亚群比例较干预前及对照组治疗后升高,且IL-17及TGFβ表达水平降低(P0.05);峰值公斤摄氧量(Peak VO_2/kg)、峰值功率(Peak Power)、峰值CO_2排出量(Peak VCO_2)较干预前及对照组药物治疗后得到一定改善(P0.05)。结论:有氧运动对COPD稳定期患者具有调节免疫功能、减轻炎症反应、改善肺功能和促进肺康复的作用,为临床COPD稳定期患者的有氧运动在免疫学上提供理论依据。     

An Avocado Extract Enriched in Mannoheptulose Prevents the Negative Effects of a High-Fat Diet in Mice

Beginning at 16 weeks of age and continuing for 44 weeks, male C57BL/6J were fed either a control (CON) diet; a high-fat (HF) diet (60% unsaturated); or the HF diet containing an extract of unripe avocados (AvX) enriched in the 7-carbon sugar mannoheptulose (MH), designed to act as a glycolytic inhibitor (HF + MH). Compared to the CON diet, mice on the HF diet exhibited higher body weights; body fat; blood lipids; and leptin with reduced adiponectin levels, insulin sensitivity, VO2max, and falls from a rotarod. Mice on the HF + MH diet were completely protected against these changes in the absence of significant diet effects on food intake. Compared to the CON diet, oxidative stress was also increased by the HF diet indicated by higher levels of total reactive oxygen species, superoxide, and peroxynitrite measured in liver samples by electron paramagnetic resonance spectroscopy, whereas the HF + MH diet attenuated these changes. Compared to the CON, the HF diet increased signaling in the mechanistic target of the rapamycin (mTOR) pathway, and the addition of the MH-enriched AvX to this diet attenuated these changes. Beyond generating further interest in the health benefits of avocados, these results draw further new attention to the effects of this rare sugar, MH, as a botanical intervention for preventing obesity.     

Sod1 integrates oxygen availability to redox regulate NADPH production and the thiol redoxome

Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O₂^•−) by catalyzing its conversion to dioxygen (O₂) and hydrogen peroxide (H₂O₂). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O₂ availability to promote NADPH production. The mechanism involves Sod1-derived H₂O₂ oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O₂ via O₂^•− to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O₂^•− toxicity, which only requires <1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H₂O₂ is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Superoxide dismutases (SODs) serve on the frontline of defense against reactive oxygen species (ROS). SODs, which detoxify O₂^•− by catalyzing its disproportionation into O₂ and hydrogen peroxide (H₂O₂), are unique among antioxidant enzymes in that they also produce a ROS byproduct. While much is known about the necessity of scavenging O₂^•−, it is less clear what the physiological consequences of SOD-derived H₂O₂ are. Paradoxically, increased expression of Cu/Zn SOD (Sod1), which accounts for the majority of SOD activity in cells (1), is associated with reduced cellular H₂O₂ levels (2), suggesting there may be additional unknown mechanisms underlying Sod1 antioxidant activity.The cytotoxicity of O₂^•− stems from its ability to oxidize and inactivate [4Fe-4S] cluster-containing enzymes, which results in defects in metabolic pathways that utilize [4Fe-4S] proteins and Fe toxicity due to its release from damaged Fe/S clusters (3–6). The released Fe can catalyze deleterious redox reactions and, in particular, production of hydroxyl radicals (^•OH) via Haber-Weiss and Fenton reactions, which indiscriminately oxidizes lipids, proteins, and nucleic acids (4, 7). The importance of Sod1 in oxidative stress protection is underscored by reduced proliferation, decreased lifespan, and numerous metabolic defects, including cancer, when SOD1 is deleted in various cell lines and organisms (7–11). It was previously proposed that Sod1 limits steady-state H₂O₂ levels because of its ability to prevent the O₂^•−-mediated oxidation of Fe/S clusters, which results in the concomitant formation of H₂O₂ (2, 12, 13). However, since vanishingly small amounts of Sod1 (<1% of total cellular Sod1) is sufficient to protect cells against O₂^•− toxicity, including oxidative inactivation of Fe/S enzymes (14–16), any changes in Sod1 expression would not be expected to alter H₂O₂ arising from O₂^•− oxidation of Fe/S clusters. How then can Sod1, an enzyme that catalyzes H₂O₂ formation, act to reduce cellular [H₂O₂]?Two previously reported but unexplained metabolic defects in sod1Δ strains of Saccharomyces cerevisiae point to a potential role for Sod1 in regulating the production of NADPH, a key cellular reductant required for reductive biosynthesis and the reduction and regeneration of H₂O₂ scavenging thiol peroxidases (17) and catalases (18, 19). Yeast strains lacking SOD1 exhibit increased glucose consumption (20) and defects in the oxidative phase of the pentose phosphate pathway (oxPPP) (21), the primary source of NADPH. Inhibition of key rate-limiting enzymes in glycolysis—including phosphofructose kinase (22), GAPDH (23, 24), and pyruvate kinase (25, 26)—reduces glucose uptake (27–29) and increases the concentration of glucose-6-phosphate (G6P), a glycolytic intermediate that is also the substrate for the first enzyme in the oxPPP, G6P dehydrogenase (G6PDH), which in turn increases oxPPP flux and NADPH production (30–35). Taking these data together, we surmised that Sod1 negatively regulates a rate-determining enzyme in glycolysis, thereby accounting for the observed metabolic defects in glucose utilization and the oxPPP in sod1Δ cells (20, 21).GAPDH, which catalyzes a rate-determining step in glycolysis (36, 37), is very abundant (38), and contains a H₂O₂-reactive catalytic Cys (k ∼ 10² to 10³ M⁻¹s⁻¹), represents a critical redox regulated node that can toggle flux between glycolysis and the oxPPP (32). As such, we hypothesized that a novel aspect of the antioxidant activity of Sod1 is to oxidatively inactivate GAPDH using Sod1-catalyzed H₂O₂, which would in turn stimulate NADPH production via the oxPPP and enhance cellular peroxide scavenging by thiol peroxidases. This mechanism for Sod1-mediated antioxidant activity would explain a number of prior observations, including the findings that elevated Sod1 expression decreases peroxide levels and loss of SOD1 increases glucose consumption and attenuates oxPPP activity. In addition, more generally, since Sod1-derived H₂O₂ has previously been implicated in the redox regulation of other enzymes, including protein tyrosine phosphatases (39) and casein kinases (15, 16, 40), we also sought to identify proteome-wide redox targets of Sod1.In the present report we provide evidence highlighting an antioxidant function for Sod1-derived H₂O₂ in integrating O₂ availability to control NADPH production to support aerobic growth and metabolism. The mechanism involves the conversion of O₂ to O₂^•− by mitochondrial respiration and an NADPH oxidase, followed by the Sod1-catalyzed conversion of O₂^•− to H₂O₂, which in turn oxidatively inactivates GAPDH. The inhibition of GAPDH serves to reroute metabolism from glycolysis to the oxPPP in order to maintain sufficient NADPH for metabolism in air. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1, suggesting that it provides a privileged pool of peroxides to inactivate GAPDH under physiological conditions. Finally, we revealed a larger network of cysteine-containing proteins that are oxidized in a Sod1-dependent manner using mass spectrometry-based redox proteomics approaches. Altogether, these results highlight a mechanism for O₂ sensing and adaptation, reveal an important but previously unknown antioxidant role of Sod1 that goes beyond O₂^•− scavenging to include the stimulation of aerobic NADPH production, and places Sod1 as a master regulator of proteome-wide thiol oxidation and multiple facets of metabolism.     

运动及运动强度对荷瘤小鼠移植肿瘤生长的影响

目的:通过不同运动强度的游泳运动,探讨运动及运动强度对小鼠接种肝癌H22和黑色素瘤B16-F10移植瘤生长的影响及机制。方法:将小鼠随机分为正常对照组、荷瘤对照组、持续有氧组、持续力竭组、荷瘤后有氧组、荷瘤后力竭组6组:其中正常对照组不荷瘤,不采取其他实验措施;所有荷瘤组小鼠均在实验开始1周后接种肿瘤细胞:ICR小鼠接种小鼠肝癌细胞H22,C57BL/6小鼠接种小鼠黑色素瘤细胞B16-F10;荷瘤对照组小鼠接瘤建模前后不采取其他实验措施;持续有氧组小鼠每天进行有氧游泳训练,时间从30min逐渐延长至60min,荷瘤后有氧组小鼠从接瘤以后开始进行上述有氧训练;持续力竭组小鼠进行每天负重游泳至力竭的训练,荷瘤后力竭组小鼠从接瘤以后开始进行上述力竭训练。所有组小鼠21d后安乐死,测定其体重、瘤重、胸腺重、脾脏重,计算胸腺指数、脾指数,并检测小鼠脾淋巴细胞增殖能力和脾NK细胞杀伤能力。结果:荷瘤后有氧组ICR小鼠瘤重明显降低(P0.05),C57BL/6小鼠瘤重没有明显变化,荷瘤后有氧组ICR和C57BL/6小鼠脾指数升高(P0.001,P0.01),荷瘤后有氧组ICR小鼠脾淋巴细胞增殖能力和脾NK细胞杀伤能力均明显提高(P0.01,P0.001),而且ICR小鼠荷瘤后有氧组胸腺指数显著升高(P0.05),荷瘤后有氧组C57BL/6小鼠胸腺指数没有明显变化;持续力竭组C57BL/6小鼠瘤重显著增加(P0.05),ICR小鼠瘤重没有明显变化,持续力竭组C57BL/6小鼠脾淋巴细胞增殖能力和脾NK细胞杀伤能力均明显下降(P0.05,P0.001),而持续力竭组ICR小鼠脾淋巴细胞增殖能力虽有下降趋势,但与脾NK细胞杀伤能力一样,与荷瘤对照组相比无显著性差异,胸腺指数却显著升高(P0.01)。结论:在实验时间范围内,运动及运动强度对小鼠肿瘤的生长有明显影响,瘤重的变化与运动强度对小鼠脾脏和胸腺功能的影响相关。     

Wortmannin阻断PI3K/AKT途径对食管癌缺氧诱导因子-1α及糖酵解的影响

目的 探讨人食管癌细胞TE1、TE13中应用wortmannin阻断PI3K/AKT通路对缺氧诱导因子(HIF)-1α的抑制效果及对糖酵解相关基因表达的影响,分析PI3K/AKT-HIF-1α途径对食管癌细胞糖酵解通路之间的关系.方法 wortmannin(2μmol/L)预处理食管癌细胞TE1、TE13后常氧和缺氧培养,分为①常氧组(N);②缺氧组(H);③常氧处理组(N+W);④缺氧处理组(H+W).采用Western印迹检测细胞中HIF-1α蛋白及己糖激酶(HK)-Ⅱ、葡萄糖载体蛋白(GLUT)-1、乳酸脱氢酶(LDH )-A等糖酵解相关基因蛋白的表达;实时定量PCR检测HIF-1α及HK-Ⅱ、GLUT-1、LDH-A等糖酵解相关基因mRNA的表达;分光光度法测定胞液中LDH、HK-Ⅱ活性和培养上清液中乳酸浓度.结果 常氧状态下,在TE1细胞中存在HIF-1α蛋白的表达,wortmannin(2 μmol/L)能抑制HIF-1α蛋白表达,12 h后抑制效应最明显,故选取12 h为后续实验的缺氧时间.TE1、TE13细胞经wortmannin预处理后HIF-1α、HK-Ⅱ、GLUT-1、LDHA蛋白表达较未加药细胞明显减弱(P＜0.05);HIF-1α、HK-ⅡmRNA表达较未加药细胞明显减弱(P＜0.05).常氧和缺氧条件下加用wortmannin的TE1,TE13组食管癌细胞胞液LDH、HK-Ⅱ活性均较未加药细胞组明显减弱(P＜0.05),未加药细胞缺氧后酶活性增强(P＜0.05).常氧和缺氧条件下加用wortmannin组较未加药组细胞上清液乳酸浓度明显减低(P＜0.05),加wortmannin组细胞缺氧后表达增强(P＜0.05).结论 常氧及缺氧条件下,wortmannin能通过抑制食管癌细胞HIF-1α和糖酵解相关基因的表达导致乳酸水平降低,表明PI3K/AKT- HIF-1α途径与食管癌细胞糖酵解通路密切相关.