一氧化氮对香烟烟雾提取物诱导大鼠肺泡巨噬细胞核因子κB活化的影响

目的:探讨一氧化氮(NO)对香烟烟雾提取物(CSE)诱导的大鼠肺泡巨噬细胞(AM)中核因子κB(NF—κB)活化的影响及机 制。方法:将大鼠AM与不同浓度的NO前体左旋精氨酸(L-Arg)或iNOS特异性抑制剂N6-(1-亚氨乙基)赖氨酸(L-NIL)及CSE共同培养,用免疫细胞化学染色法检测NF-κB,用Western blot检测I-κB蛋白含量,用Griess法测定培养上清液中NO的水平。结果:CSE可使NF-κB活化细胞的百分率增加,I-κB的水平下降。加入CSE和低浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著高于只加入CSE的AM;而I-κB的水平显著低于只加入CSE的AM。加入CSE和高浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM,而I-κB的水平无显著变化。加入CSE和不同浓度的L-NIL培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM;而I-κB的水平则显著高于只加入CSE的AM,并呈浓度依赖(P<0.01)。结论:内源性NO对香烟烟雾所致NF-κB的活化具有双向调控作用。     

一氧化氮对香烟烟雾提取物诱导大鼠肺泡巨噬细胞核因子кB活化的影响

目的:探讨一氧化氮(NO)对香烟烟雾提取物(CSE)诱导的大鼠肺泡巨噬细胞(AM)中核因子κB(NF—κB)活化的影响及机 制。方法:将大鼠AM与不同浓度的NO前体左旋精氨酸(L-Arg)或iNOS特异性抑制剂N6-(1-亚氨乙基)赖氨酸(L-NIL)及CSE共同培养,用免疫细胞化学染色法检测NF-κB,用Western blot检测I-κB蛋白含量,用Griess法测定培养上清液中NO的水平。结果:CSE可使NF-κB活化细胞的百分率增加,I-κB的水平下降。加入CSE和低浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著高于只加入CSE的AM;而I-κB的水平显著低于只加入CSE的AM。加入CSE和高浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM,而I-κB的水平无显著变化。加入CSE和不同浓度的L-NIL培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM;而I-κB的水平则显著高于只加入CSE的AM,并呈浓度依赖(P<0.01)。结论:内源性NO对香烟烟雾所致NF-κB的活化具有双向调控作用。     

NAC减轻香烟烟雾致COPD大鼠的肺组织损伤北大核心CSCD

目的:分析N-乙酰半胱氨酸(NAC)对香烟烟雾致慢性阻塞性肺疾病(COPD)大鼠肺组织细胞凋亡的保护作用及机制。方法:采用简单随机分组将36只SD大鼠分为对照组、模型组和NAC组,12只/组,造模大鼠采用烟熏联合脂多糖制备大鼠COPD模型。造模后,NAC组灌胃NAC(400 mg/kg),持续灌胃30 d。检测大鼠第0.3秒用力呼气量(FEV_(0.3))、FEV_(0.3)/用力肺活量(FVC)、最大呼气流量(PEF);检测大鼠血清TNF-α、IL-6、IL-8、肺组织MDA、SOD和GSH-Px水平;检测B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、cleaved Caspase-3、雷帕霉素靶蛋白/S6激酶1(mTOR/S6K1)信号通路表达;HE和TUNEL染色观察肺组织损伤。结果:与对照组相比,模型组大鼠FEV_(0.3)、FEV_(0.3)/FVC和PEF水平明显降低(P<0.05),IL-6、IL-8、TNF-α和MDA水平升高(P<0.05),SOD和GSH-Px活性明显降低(P<0.05),细胞凋亡率升高(P<0.05),cleaved Caspase-3、Bcl-2和Bax表达上调(P<0.05);与模型组相比,NAC组大鼠IL-6、IL-8、TNF-α和MDA水平降低(P<0.05),SOD和GSH-Px活性升高(P<0.05),细胞凋亡率降低(P<0.05),cleaved Caspase-3和Bax表达下调(P<0.05),Bcl-2、p-mTOR/mTOR、p-S6K1/S6K1表达上调(P<0.05)。HE染色结果表明,NAC组大鼠肺组织病理变化明显减轻。结论:NAC可抑制香烟烟雾致COPD大鼠的肺组织细胞凋亡,保护肺组织损伤,其作用机制可能与激活mTOR/S6K1信号通路有关。     

脂多糖对大鼠血清及内脏组织一氧化氮产物水平的影响

作者观察了ＬＰＳ对大鼠血清及内脏组织一氧化氮代谢产物ＮＯ２－／ＮＯ３－水平的影响，结果表明于ＬＰＳ后，血清ＮＯ＾－／ＮＯ＾－３水平急剧升高，２４ｈ达峰值，为对照组的２０倍。心，肺组织的ＮＯ＾－２／ＮＯ＾－／３含量也显著增加；但肝脏和肾脏组织ＮＯ＾－２／ＮＯ＾－３仅在短时期内增加。同时血清和内脏组织ｃＧＭＰ水平明显升高。给于ＮＯ合成酶抑制剂Ｌ－ＮＭＭＡ，能降低血清和心，肺组织ＮＯ＾－２／ＮＯ＾－３升     

不同药物对烟雾吸入性损伤大鼠肺保护作用的研究

目的探讨乌司他丁、地塞米松和氨溴索对烟雾吸入性损伤大鼠肺脏损害的保护作用及机制。方法SD大鼠随机分为5组，C组为正常对照组，A组、D组和U组大鼠分别在炯雾致伤后给予氨溴索、地塞米松、乌司他丁腹腔注射，M组大鼠致伤后注射等量生理盐水。各组大鼠在致伤6h后测血清肿瘤坏死因子（TNF-α）、白细胞介素6（IL-6）含量。取肺组织匀浆测超氧化物歧化酶（SOD）、丙二醛（MDA）、一氧化氮（NO）、诱导型一氧化氮合酶（iNOS）含量，右下肺组织病理切片光镜观察。结果光镜下见C组大鼠肺组织正常，M组肺间质出血，肺泡腔内可见大量粒细胞聚集、浸润，肺泡间隔增厚，而U、D组上述病理表现明显减轻。与M组相比，U组TNF-α、IL-6及MDA含量明显降低（P〈0．05），D组上二述指标显著降低（P〈0．01）。与C组比，A组NO、iNOS、SOD浓度明显增高（P〈0．05）。D组和U组iNOS与C组比无统计学差异（P〉0．05）。结论乌司他丁和地塞米松可能通过减少炎性介质和自由基的产生对吸入性损伤大鼠肺脏产生保护作用，氨溴索对吸入性损伤大鼠肺脏保护作用不强。     

香烟烟雾提取物通过PKC途径促进大鼠肺动脉平滑肌细胞bFGF表达

目的： 研究香烟烟雾提取物（CSE）对大鼠肺动脉平滑肌细胞（PASMCs）碱性成纤维细胞生长因子（bFGF）表达的影响及蛋白激酶C（PKC）信号转导途径在其中所起的作用。方法: 组织块法培养正常Wistar大鼠PASMCs。5%CSE作用于PASMCs不同时间及PASMCs先与PKC特异性抑制剂Ro31-8220预孵育，然后再暴露于5%CSE，逆转录-聚合酶链反应（RT-PCR）检测PASMCs bFGF mRNA表达，免疫细胞化学检测bFGF蛋白表达。结果: 对照组PASMCs中有少量bFGF mRNA和蛋白表达（分别为0.093±0.034，0.142±0.013）。暴露于5%CSE后4 h细胞内bFGF mRNA和蛋白表达增加，mRNA于8 h达高峰（0.436±0.103，P＜0.01），蛋白于12 h达高峰（0.237±0.031，P＜0.01），此后逐渐下降，但24 h都仍高于对照组。Ro31-8220预孵育可显著抑制5%CSE诱导的bFGF mRNA和蛋白表达增加。结论: 5%CSE可通过激活PKC途径促进大鼠PASMCs bFGF的表达。     

吸烟大鼠一氧化氮合酶和一氧化氮的变化

目的:观察吸烟对大鼠肺组织iNOS、eNOSmRNA和蛋白表达以及支气管肺泡灌洗液(BALF)中NO的影响, 探讨不同类型的NOS在吸烟所致慢性气道炎症中的作用。方法:选用Wistar大鼠80只随机分为对照组, 被动吸烟组, iNOS抑制剂L-NIL干预组及NOS抑制剂L-NAME干预组。用免疫组化法检测iNOS及eNOS的蛋白表达, 用RT-PCR检测iNOS及eNOSmRNA的表达, 用Griess法测定BALF中的NO^-₂/NO^-₃含量。结果:吸烟大鼠肺组织中iNOSmRNA及其蛋白表达增加, eNOSmRNA及蛋白表达下降, BALF中细胞总数及NO^-₂/NO^-₃显著增加(P＜0.05)。在体实验发现, L-NIL使BALF中细胞总数及NO^-₂/NO^-₃下降(P＜0.05);L-NAME对BALF中细胞总数及NO^-₂/NO^-₃无显著影响(P＞0.05)。结论:吸烟大鼠肺组织iNOSmRNA和蛋白表达增加, eNOSmRNA和蛋白表达减少。活化的iNOS产生大量NO促进炎症发展。     

大鼠肺一氧化氮合酶阳性神经结构

以还原型尼克腕腺嘌呤二核苷酸磷酸辛酰胺脱氢酶组织化学技术研究大鼠肺一氧化氮合酶阳性神经结构。结果表明：（１）：气管，支气管树系统壁内神经节部分神经细胞为一氧化氮合酶阳性神经细胞；（２）部分强阳性神经细胞的突起穿越神经节延向管壁粘膜组织或肺组织；（３）在各级肺动，静脉及肺组织内分布有不同密度的一氧化氮合酶阳性神经纤维。     

香烟烟雾暴露对大鼠肺组织Wnt3a和β-catenin蛋白表达的影响

目的 探讨香烟烟雾暴露对大鼠肺组织Wnt3a和β-catenin蛋白表达的影响.方法 20只雌性SD大鼠,按随机数字表分为对照组和模型组,每组10只.模型组大鼠被动吸入香烟烟雾,每天2次,每次45 min;对照组大鼠不接触香烟烟雾.100d后处死动物,光镜下观察肺组织的病理形态学改变,免疫组化法检测肺组织α-平滑肌肌动蛋白(α-SMA)、Wnt3a和β-catenin蛋白表达,并测定2组大鼠支气管平滑肌厚度.结果 模型组大鼠肺组织呈现慢性炎性反应和肺气肿样改变,小气道壁及平滑肌层增厚明显.α-SMA主要表达于气道和血管的平滑肌层.模型组支气管平滑肌厚度高于对照组[ (3.06±0.62) μm比(1.86±0.43) μm,P＜0.05].Wnt3a和β-catenin主要表达于支气管上皮细胞和肺泡上皮细胞.模型组与对照组支气管上皮细胞Wnt3a蛋白表达分别为74.54±4.14、89.24±3.02;肺泡上皮细胞Wnt3a蛋白表达分别为91.97±2.50、110.46±3.85,组间比较差异均有统计学意义(均P＜0.05).模型组与对照组支气管上皮细胞胞质β-catenin蛋白表达分别为86.97±4.87、103.18±3.77;支气管上皮细胞胞核β-catenin蛋白表达分别为95.75±3.91、116.12±5.76;肺泡上皮细胞胞质β-catenin蛋白表达分别为106.24±4.57、128.81±3.96;肺泡上皮细胞胞核β-catenin蛋白表达分别为125.44±4.89、152.90±4.10,组间比较差异均有统计学意义(均P＜0.05).结论 香烟烟雾可诱导大鼠肺组织Wnt3a和β-catenin蛋白表达上调.     

Anti-inflammatory effect of augmented nitric oxide production in chronic lung infection

Chronic infection of the lungs with Pseudomonas aeruginosa complicates many long-term lung diseases including cystic fibrosis, bronchiectasis, chronic obstructive lung disease, and mechanical ventilation. In acute inflammatory lung diseases, increased nitric oxide synthase (NOS-2) expression leads to excess nitric oxide (NO) production, resulting in the production of reactive nitrogen intermediates, which contribute to tissue damage. In contrast, the contribution of NO to pulmonary damage in chronic Pseudomonas infection of the lung has not been directly examined and is unclear. Although NOS-2 expression is increased in this condition, NO production is not abnormally elevated. It was hypothesized that chronic infection of the airways does not cause increased NO production but, in contrast, leads to inappropriately low NO concentrations that are pro-inflammatory. A rodent model of chronic airway infection was used to examine the effects on lung damage of augmenting or inhibiting NO production after airway infection with P. aeruginosa was well established. Three days post-infection, L-arginine, which augments NO production, or L-NAME, an inhibitor of NO production, was administered in drinking water. Lung damage was assessed 12 days later. L-arginine treatment reduced tissue damage, inhibited neutrophil recruitment, and reduced the pro-inflammatory cytokine interleukin (IL)-1beta. Treatment with L-NAME caused loss of alveolar walls, greater vascular damage, and increased levels of the pro-inflammatory cytokine IL-6. Thus, in chronic airway infection, inhibition of NO production worsened lung damage, whereas augmenting NO ameliorated this damage. This is the first demonstration that augmenting endogenous NO production in chronic infective lung disease caused by P. aeruginosa is anti-inflammatory. Given that infection with this organism complicates many chronic lung diseases, most notoriously cystic fibrosis, these findings have important clinical implications.     

No effect of metabolic acidosis on nitric oxide production in hypoxic and hyperoxic lung regions in pigs

Aim: In the severely ill intensive care patients metabolic acidosis and hypoxia often co‐exist. We studied the effects of metabolic acidosis on nitric oxide synthase (NOS) dependent and NOS independent nitric oxide (NO) production in hypoxic and hyperoxic lung (HL) regions in a pig model. Methods: Eighteen healthy anaesthetized pigs were separately ventilated with hypoxic gas to the left lower lobe (LLL) and hyperoxic gas to the rest of the lung. Six pigs received HCl infusion (HCl group), six pigs received the non‐specific NOS inhibitor N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME) and HCl infusions (l ‐NAME + HCl group) and six pigs received buffered Ringer’s solution (control group). NO concentration in exhaled air (ENO), NOS activity in lung tissue, and regional pulmonary blood flow were measured. Results: Metabolic acidosis, induced by infusion of HCl, decreased the relative perfusion to the hypoxic LLL from 7 (3) [mean (SD)] to 3 (1) % in the HCl group (P < 0.01), and from 4 (1) to 1 (1) % in the l ‐NAME + HCl group (P < 0.05), without any measurable significant changes in ENO from hypoxic or HL regions There were no significant differences between the HCl and control groups for Ca²⁺‐dependent (cNOS) or Ca²⁺‐independent NOS (iNOS) activity in hypoxic or HL regions. Conclusions: Metabolic acidosis augmented the hypoxic pulmonary vasoconstriction, without any changes in pulmonary NOS dependent or NOS independent NO production. When acidosis was induced during ongoing NOS blockade, the perfusion of hypoxic lung regions was almost abolished, indicating acidosis‐induced pulmonary vasoconstriction was not NO dependent.     

束缚应激对大鼠血小板-氧化氮释放的影响

目的 观察急性应激对大鼠血小板一氧化氮(NO)释放的影响及其机制.方法 大鼠浸水-束缚应激(WRS)2、4和8 h,以胃溃疡指数(UI)作为应激损伤的标识,采用Greiss法测定血小板孵育液中亚硝酸盐(NO2-)含量;同位素法测其一氧化氮合酶(NOS)活性和L精氨酸(L-Arg)转运量.结果 WRS 2 h血小板L-Arg转运量、NOS活性和孵育液NO2-含量较对照组显著增加,但随应激时间延长,其呈下降趋势,应激8 h时均显著低于对照组,胃溃疡逐渐加重.结论 WRS应激早期阶段可上调血小板L-Arg/NO通路,促进血小板NO生成;长时间应激下调L-Arg/NO通路,减少NO释放.     

哮喘模型大鼠肺组织一氧化氮合酶的分布

研究一氧化氮 (NO)在哮喘大鼠肺组织中的作用。采用组化法观察一氧化氮合酶 (NOS)在大鼠哮喘模型肺组织中的分布 ,应用免疫组化法观察大鼠哮喘模型气道mIL 2R+ 细胞变化。结果显示 ,哮喘大鼠肺NADPH染色呈强阳性 ,并波及肺泡膈。肺组织中NOS含量明显高于对照组 [哮喘组 (37 44± 0 77)pmol/mg,对照组 (8 73± 0 79)pmol/mg],气道炎性细胞增多 ,特别是mIL 2R+ 细胞 [哮喘组mIL 2R+ 细胞为 (2 3 8± 7 9)个 ,对照组为 0个 ],而NOS抑制剂DMA组气道炎性细胞少 ,NADPH呈阴性。提示NO是哮喘大鼠的炎性效应分子。     

Anti-inflammatory effect of Mentha longifolia in lipopolysaccharide-stimulated macrophages: Reduction of nitric oxide production through inhibition of inducible nitric oxide synthase

Abstract

Mentha longifolia is an aromatic plant used in flavoring and preserving foods and as an anti-inflammatory folk medicine remedy. The present study assessed the effects of M. longifolia extracts, including essential oil and crude methanol extract and its fractions (ethyl acetate, butanol and hexane), on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in lipopolysaccharide (LPS)-stimulated J774A.1 cells using real-time polymerase chain reaction (PCR). The cytotoxic effects of the extracts on the cells were examined and non-cytotoxic concentrations (<0.2?mg/ml) were used to examine their effects on NO production and iNOS mRNA expression. Only the hexane fraction that contained high levels of phenolic and flavonoid compounds at concentrations from 0.05–0.20?mg/ml significantly reduced NO production in LPS-stimulated cells (p?<?0.001). Real-time PCR analysis indicated the ability of this fraction at the same concentrations to significantly decrease iNOS as well as TNFα mRNA expression in the cells (p?<?0.001). All extracts were able to scavenge NO radicals in a concentration-dependent manner. At concentrations greater than 0.2?mg/ml, total radicals were 100% scavenged. In conclusion, M. longifolia possibly reduces NO secretion in macrophages by scavenging NO and inhibiting iNOS mRNA expression, and also decreases TNFα pro-inflammatory cytokine expression, thus showing its usefulness in the inflammatory disease process.     

人内皮型一氧化氮合酶基因在小鼠肺内的表达

目的 观察人内皮型一氧化氮合酶(heNOS)重组腺病毒感染小鼠肺组织后heNOS基因在小鼠肺组织内的表达.方法 采用反转录PCR(RT-PCR)法克隆heNOS基因,利用体外连接法构建复制缺陷型heNOS重组腺病毒表达载体;免疫组织化学分析heNOS在小鼠肺组织内的表达情况.结果 (1)序列分析表明,克隆所得heNOS基因片段含有完整开放阅读框架,与GenBank中heNOS DNA序列(*163729)同源性达99.93%.(2)该基因在转染细胞中能表达具有生物活性功能的NOS蛋白.(3)体外连接法成功构建出复制缺陷型heNOS重组腺病毒转移载体.(4)滴入heNOS重组腺病毒后,小鼠肺血管内皮细胞、平滑肌细胞、细支气管上皮细胞内heNOS阳性表达明显增加.结论 本实验成功构建的复制缺陷型heNOS重组腺病毒转移载体,能经呼吸道转移至肺组织,并在小鼠肺血管、支气管和肺泡上皮细胞表达所需的目的 蛋白heNOS.