非诺贝特改善脂多糖诱导的急性肺损伤及其机制研究

摘要：目的　探究非诺贝特（Fen）对急性肺损伤（ALI）的作用及机制。方法　（1）体内实验。30只C57BL/6雄性小鼠采用随机数字表法分成6组：正常组,模型组（LPS组）,阳性药地塞米松（DXMS）组,Fen低、中、高剂量（20、40、80 mg/kg）组。分组给药12 h后,收集肺泡灌洗液（BALF）,处死小鼠,获取肺组织,记录肺组织湿/干质量比;酶联免疫吸附试验（ELISA）检测肺组织肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6和IL-1β含量;BCA比色法和瑞氏-吉姆萨染色检测BALF中总蛋白含量和免疫细胞数目;苏木精-伊红（HE）染色观察肺组织病变并进行病理评分。Western blot检测肺组织B淋巴细胞瘤-2蛋白（Bcl-2）、Bcl-2相关X蛋白（Bax）及c-Jun氨基末端激酶（JNK）、磷酸化JNK（p-JNK）表达。（2）体外实验。实验设Control、LPS（10 mg/L）、LPS+Fen（5 μmol/L）、LPS+Fen（10 μmol/L）、LPS+Fen（20 μmol/L）组。A549细胞经LPS（10 mg/L）处理后分别加入5、10、20 μmol/L的Fen处理12 h,分别用DCFH-DA、Annexin V-FITC/PI法和Western blot检测Fen对A549细胞ROS、凋亡和凋亡相关蛋白Bcl-2、Bax及p-JNK影响。另选取LPS（10 mg/L）+Fen（20 μmol/L）处理后添加H2O2（100 μmol/L）,观察细胞ROS、凋亡率和凋亡相关蛋白Bcl-2、Bax表达变化。此外,选取LPS（10 mg/L）+Fen（20 μmol/L）处理后添加JNK激动剂Anisomycin（3 μmol/L）,观察Bcl-2、Bax表达变化。结果　（1）与正常组相比,模型组小鼠肺组织湿/干质量比,肺组织损伤评分,TNF-α、IL-6、IL-1β含量,肺泡灌洗液总蛋白含量,免疫细胞数目,p-JNK和Bax表达均出现显著升高,Bcl-2表达显著降低。与模型组相比,Fen低、中、高剂量组上述指标变化均被逆转。（2）Fen能显著减少LPS诱导的A549细胞ROS含量,降低凋亡率,抑制p-JNK和Bax表达并促进Bcl-2表达。H2O2可逆转Fen引起的上述效应。Anisomycin也能抑制Fen引起的Bcl-2上调和Bax蛋白下调。结论　Fen可通过ROS/JNK信号抑制细胞凋亡,从而减轻ALI。     

Standardized myrtol attenuates lipopolysaccharide induced acute lung injury in mice

Context: Standardized myrtol, an essential oil containing primarily cineole, limonene and α-pinene, has been used for treating nasosinusitis, bronchitis and chronic obstructive pulmonary disease (COPD).

Objective: To investigate the effects of standardized myrtol in a model of acute lung injury (ALI) induced by lipopolysaccharides (LPS).

Materials and methods: Male BALB/c mice were treated with standardized myrtol for 1.5?h prior to exposure of atomized LPS. Six hours after LPS challenge, lung injury was determined by the neutrophil recruitment, cytokine levels and total protein concentration in the bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in the lung tissue. Additionally, pathological changes and NF-κB activation in the lung were examined by haematoxylin and eosin staining and western blot, respectively.

Results: In LPS-challenged mice, standardized myrtol at a dose of 1200?mg/kg significantly inhibited the neutrophile counts (from 820.97?±?142.44 to 280.42?±?65.45, 10³/mL), protein concentration (from 0.331?±?0.02 to 0.183?±?0.01, mg/mL) and inflammatory cytokines level (TNF-α: from 6072.70?±?748.40 to 2317.70?±?500.14, ng/mL; IL-6: from 1184.85?±?143.58 to 509.57?±?133.03, ng/mL) in BALF. Standardized myrtol also attenuated LPS-induced MPO activity (from 0.82?±?0.04 to 0.48?±?0.06, U/g) and pathological changes (lung injury score: from 11.67?±?0.33 to 7.83?±?0.79) in the lung. Further study demonstrated that standardized myrtol prevented LPS-induced NF-κB activation in lung tissues.

Discussion and conclusion: Together, these data suggest that standardized myrtol has the potential to protect against LPS-induced airway inflammation in a model of ALI.     

硫化氢对大鼠内毒素性急性肺损伤的影响

目的观察硫化氢(H2S)对脂多糖(LPS)所致大鼠内毒素性急性肺损伤(ALI)的影响,探讨其对肺脏的作用机制。方法♂SD大鼠共64只,随机分为8组,每组8只:Ⅰ:3h空白对照组;Ⅱ:LPS3h组;Ⅲ:6h空白对照组;Ⅳ:LPS6h组;Ⅴ:LPS+NaHS低剂量组;Ⅵ:LPS+NaHS中剂量组;Ⅶ:LPS+NaHS高剂量组;Ⅷ:LPS+PPG组。LPS3h和LPS6h组给予LPS,其相应空白对照组给予生理盐水,分别在3h或6h时处死大鼠;LPS+NaHS低、中、高剂量组和LPS+PPG组分别在给LPS3h时腹腔注射低、中、高剂量氢硫化钠(NaHS)或炔丙基甘氨酸(PPG),再观察3h后处死大鼠。光、电镜下观察肺组织形态学改变,检测肺系数、肺湿/干重比(W/D)、血浆中H2S含量、肺组织胱硫醚-γ-裂解酶(CSE)活性,以及肺组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性的变化。结果LPS3h和LPS6h组与相应的空白对照组比较,光、电镜下肺组织明显受损,超微结构明显改变,肺系数、肺湿/干重比和肺组织MDA含量升高,血浆中H2S的含量、肺组织CSE以及SOD、GSH-Px活性降低。LPS+NaHS低、中、高剂量组与LPS6h组比较,光、电镜下肺组织受损情况均有不同程度改善,肺系数、肺湿/干重比和肺组织MDA含量降低,血浆H2S含量、肺组织CSE以及SOD、GSH-Px活性升高。LPS+PPG组与LPS6h组比较,光、电镜下肺组织损伤无改善,肺系数、肺湿/干重比和肺组织MDA含量升高,血浆中H2S的含量、肺组织CSE和SOD活性降低,肺组织GSH-Px活性无明显变化。结论CSE/H2S体系可能参与内毒素性ALI的病理生理过程,外源性补充H2S可减轻内毒素性ALI。     

Protective effect of taraxasterol on acute lung injury induced by lipopolysaccharide in mice

Taraxasterol, a pentacyclic-triterpene isolated from Taraxacum officinale, has been reported to have potent anti-inflammatory properties. However, the effect of taraxasterol on lipopolysaccharide (LPS)-induced mice acute lung injury has not been investigated. The aims of this study were to investigate whether taraxasterol could ameliorate the inflammation response in LPS-induced acute lung injury and to clarify the possible mechanism. Male BALB/c mice were pretreated with taraxasterol 1 h before intranasal instillation of LPS. 7 h after LPS administration, the myeloperoxidase (MPO) in lung tissues, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) in the BALF were measured by ELISA. The extent of phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 were determined by western blotting. The results showed that taraxasterol attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), lung wet/dry ratio, and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in a dose-dependent manner. Additionally, western blotting results showed that taraxasterol inhibited the phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 caused by LPS. Our data suggest that anti-inflammatory effects of taraxasterol against the LPS-induced ALI may be due to its ability of inhibition of the NF-κB and MAPK signaling pathways.     

Effects of inhaled nanoparticles on acute lung injury induced by lipopolysaccharide in mice

We have previously shown that intratracheal instillation of carbon nanoparticles exacerbates lung inflammation related to bacterial endotoxin (lipopolysaccharide, LPS) and subsequent systemic inflammation with coagulatory disturbance in mice [Inoue, K., Takano, H., Yanagisawa, R., Hirano, S., Sakurai, M., Shimada, A., Yoshikawa, T., 2006b. Effects of airway exposure to nanoparticles on lung inflammation induced by bacterial endotoxin in mice. Environ. Health Perspect. 114, 1325-1330]. The present study was performed to determine whether inhalation of diesel engine-derived nanoparticles also exacerbates the model. ICR mice were exposed for 5h to clean air or diesel engine-derived nanoparticles at a concentration of 15, 36, or 169 microg/m(3) after intratracheal challenge with 125 microg/kg of LPS or vehicle, and were sacrificed for evaluation 24h after the intratracheal challenge. Nanoparticles alone did not induce lung inflammation. Nanoparticle inhalation increased LPS-elicited inflammatory cell recruitment into the bronchoalveolar lavage fluid and lung parenchyma as compared with clean air inhalation in a concentration-dependent manner. Lung homogenates derived from the nanoparticle+LPS groups tended to have increased tumor necrosis factor-alpha level and chemotaxis activity for polymorphonuclear leukocytes as compared to those from the LPS group or the corresponding nanoparticle groups. Nanoparticle inhalation did not significantly increase lung expression of proinflammatory cytokines or facilitate systemic inflammation and coagulatory disturbance. Isolated alveolar macrophages (AMs) from nanoparticle-exposed mice showed greater production of interleukin-1beta and keratinocyte chemoattractant stimulated with ex vivo LPS challenge than those from clean air-exposed mice, although the differences did not reach statistical significance. These results suggest that acute exposure to diesel nanoparticles exacerbates lung inflammation induced by LPS.     

Preventive effect of Imperatorin on acute lung injury induced by lipopolysaccharide in mice

Imperatorin, a linear furanocoumarin, has many pharmacological effects such as antibacterial, anti-inflammatory and antiviral effects. The purpose of this study was to investigate the effect of Imperatorin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. BALB/c mice were pretreated with Imperatorin 1 h before LPS challenge. We found that the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) were decreased significantly, and the level of interleukin-10 (IL-10) was up-regulated 8 h after Imperatorin treatment. Pretreatment with Imperatorin (15 or 30 mg/kg) decreased lung wet-to-dry weight (W/D) ratio, the number of inflammatory cells and myeloperoxidase (MPO) activities. Additionally, Imperatorin attenuated lung histopathological changes and significantly inhibited the phosphorylation of IκB, JNK, ERK and p38/MAPK. These findings demonstrate that Imperatorin protects against LPS-induced ALI in mice.     

白藜芦醇对脂多糖诱导小鼠急性肺损伤的保护作用

目的研究白藜芦醇对脂多糖(LPS)致小鼠急性肺损伤(ALI)的保护作用,探讨其可能的作用机制。方法以小鼠气道滴注LPS制备急性肺损伤模型,检测气道吸气阻力(Ri)、气道呼气阻力(Re)和动态肺顺应性(Cdyn)的变化,测定支气管肺泡灌洗液(BALF)中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的含量,检测肺湿/干比值和毛细血管通透性,观察组织病理学变化。结果白藜芦醇能明显抑制Ri、Re增长和Cdyn降低,降低BALF中IL-1βI、L-6、TNF-α的含量,降低肺湿/干比值和渗透性,减轻肺组织病理学的损伤。结论白藜芦醇对LPS诱导的ALI具有保护作用,作用机制可能与抑制炎症因子的合成与释放有关。     

腺苷酸环化酶抑制剂和激动剂在脂多糖诱导的急性肺损伤中的作用

目的探讨腺苷酸环化酶抑制剂(SQ22536)和激动剂(Forskolin)在脂多糖(LPS)诱导的急性肺损伤(acute lung injury,ALI)中的作用。方法 ICR小鼠随机分为生理盐水组(N组)、模型组(L组)、地塞米松组(D组)、SQ22536组(SQ组)和Forskolin组(F组),气道内滴入LPS制备ALI模型。6 h后观察肺组织病理改变,测定肺泡灌洗液(BALF)中白细胞、中性粒细胞和白蛋白含量,检测肺组织中髓过氧化物酶(MPO)、肿瘤坏死因子-α(TNF-α)、白介素-1β(IL~(-1)β)、白介素-6(IL-6)和c AMP含量。结果 Forskolin可明显改善肺组织病理变化,降低BALF中白细胞、中性粒细胞和蛋白含量,MPO活性和TNF-α含量明显降低,c AMP含量升高;SQ22536与M组相比差异均无显著性。结论 Forskolin对ALI的保护机制可能与升高c AMP水平、抑制中性粒细胞黏附和趋化及降低TNF-α等相关炎症因子含量有关。     

Protective effects of apocynin nitrone on acute lung injury induced by lipopolysaccharide in rats

Acute lung injury (ALI) is a major cause of mortality and morbidity worldwide. In a previous study we reported the synthesis of a series of apocynin derivatives. Although the anti-inflammatory activity of them contributes to these cytoprotective effects. However, the mechanisms and effects of improving LPS-induced ALI in vivo remain unknown, so the purpose of our investigation was designed to reveal the effect of apocynin derivatives on LPS-induced acute lung injury in rats. The present study showed that apocynin derivatives reduces overall protein levels and tumor necrosis factor α (TNF-α) level, inhibits the activation of NADPH oxidase, and increases the levels of superoxide dismutase (SOD). Especially, compound 11 significantly reduces pulmonary vascular permeability, white blood cell content and protein expressions of p67^phox and p47^phox. These results suggest that compound 11 can ameliorate ALI induced by LPS through inhibition of NADPH oxidase.     

蛹虫草提取物对内毒素引起小鼠急性肺损伤的保护作用

目的 :观察蛹虫草提取物对内毒素引起小鼠急性肺损伤的保护作用。方法 :建立内毒素急性损伤小鼠肺病理模型 ,造模 4和 8h后 ,用蛹虫草提取物治疗 ,造模 2 4h后 ,对血细胞和肺灌洗液中细胞进行计数并测定肺灌洗液中蛋白酶的活性和蛋白的含量。结果 :蛹虫草提取物对血液成分没有明显影响 ,但可以使肺灌洗液中白细胞、粒细胞的含量有所降低 ,而淋巴细胞和单核细胞的数量有所增加 ,蛋白酶活性降低。结论 :蛹虫草提取物可调节机体组织的免疫功能 ,可以缓解由于内毒素或类似物质造成的肺部炎症 ,具有一定的肺保护作用。     

Stereospecific induction of nuclear factor-kappaB activation by isochamaejasmin

The root of Stellera chamaejasme L. is a traditional Chinese herb termed Rui Xiang Lang Du and has been used to treat solid tumors, tuberculosis and psoriasis. Exactly how S. chamaejasme L. regulates cellular responses remains unclear. We examined four biflavonoids isolated from S. chamaejasme L., including isochamaejasmin, two of its stereo-isomers and a methyl derivative, in functional assays originally designed to screen ligands for the G protein-coupled formyl peptide receptor-like 1 (FPRL1). Isochamaejasmin was found to induce the expression of a nuclear factor (NF)-kappaB-directed reporter gene in transfected HeLa cells with an EC50 of 3.23 microM, independently of FPRL1. The isochamaejasmin-stimulated NF-kappaB reporter activity was accompanied by nuclear translocation of NF-kappaB proteins and was blocked by a dominant-negative construct of IkappaBalpha. Isochamaejasmin also induced time-dependent phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 and p38, and a novel protein kinase C (PKCdelta). Likewise, inhibition of these kinases with the respective pharmacological inhibitors significantly reduced the isochamaejasmin-stimulated NF-kappaB activation. It is noteworthy that the two stereoisomers and the methyl derivative did not induce detectable activation of NF-kappaB and were more cytotoxic than isochamaejasmin, which could partially rescue cycloheximide-induced apoptosis. Inhibition of NF-kappaB activation reversed the anti-apoptotic effect of isochamaejasmin. These results provide the first evidence for a potential mechanism of action by S. chamaejasme L., and indicate that structurally similar compounds derived from S. chamaejasme L. may have different pharmacological properties.     

Protective effect of raloxifene on lipopolysaccharide and acid- induced acute lung injury in rats

Aim： To evaluate the protective effect of oral raloxifene on acute lung injury. Methods： Thirty adult, male Sprague-Dawley rats each weighing 180-210 g were used and divided into 3 groups： the raloxifene-lipopolysaccharide （LPS）-HCI group （n=10）, the LPS-raloxifene-HCl group （n=10）, and the placebo group （n=10）. All the rats were injected intraperitoneally （ip） with 5 mg/kg LPS, and raloxifene （30 mg/kg） was orally administered 1 h before and 14 h after LPS injection into the raloxifene-LPS-HCl and the LPS-raloxifene-HCl groups, respectively; the placebo group received nothing. Sixteen hours after LPS injection, all the animals were anesthetized and the femoral artery was cannulated. All the rats received a direct intratracheal （IT） injection ofHCl （pH 1.2; 0.5 mL/kg）. The mean arterial pressure （MAP） and blood gas concentrations were measured. Fifteen rats （5 in each group, respectively） underwent a micro positron emission tomography （microPET） scan of the thorax 4 h after HCI instillation. The wet/dry （W/D） weight ratio determination and histopathological examination were also performed. Results： The rats in the LPS-raloxifene-HCl group had a lower [^18F]fluorodeoxyglucose uptake compared with the rats in the placebo group （4.67±1.33 vs 9.01±1.58, respectively, P〈0.01）. The rats in the LPS-raloxifene-HCl group also had a lower histological lung injury score （8.20± 1.23 vs 12.6±0.97, respectively, P〈0.01） and W/D weight ratio （5.335±0.198 vs 5.886±0.257, respectively, P〈0.01） compared to the placebo group. The rats in this group also showed better pulmonary gas exchange and more stable mean arterial pressure （MAP） compared to the placebo group. Conclusion： Raloxifene provides a significant protective effect on acute lung injury in rats induced first by LPS ip injection and then by HCI IT instillation.     

Effect of 15-deoxy-delta 12,14-prostaglandin J2 on acute lung injury induced by lipopolysaccharide in mice

15-Deoxy-delta(12,14)-prostaglandin J(2) (15d-prostaglandin J(2)) has received attention for its anti-inflammatory properties. The present study investigated the efficacy of 15d-prostaglandin J(2) on acute lung injury induced by lipopolysaccharide in mice. ICR mice were administered with 15d-prostaglandin J(2) (10 microg/kg, 100 microg/kg, or 1 mg/kg) before intratracheal challenge with lipopolysaccharide (125 microg/kg). Treatment with 15d-prostaglandin J(2) did not ameliorate rather enhanced at a dose of 1 mg/kg the neutrophilic lung inflammation and pulmonary edema by lipopolysaccharide. The enhancement was concomitant with the increased lung expression of interleukin-1 beta, macrophage inflammatory protein-1 alpha, and macrophage chemoattractant protein-1. 15d-prostaglandin J(2) increased the nuclear protein expression of peroxisome proliferator-activated receptor (PPAR)-gamma and inhibited the nuclear localization of nuclear factor-kappa B related to lipopolysaccharide. 15d-prostaglandin J(2) increased the phosphorylation of c-Jun in the presence or absence of lipopolysaccharide. Our data suggest that 15d-prostaglandin J(2) may not be useful but potentially harmful for the therapeutic option of acute lung injury.     

急性肾损伤诱导急性肺损伤的研究进展

急性肾损伤是一种ICU患者中常见的疾病,致死率较高,当合并肺损伤时,致死率显著提高,可达到80%。急性肾损伤可导致全身体液灌注量增加,血浆渗透压升高进而引起肺水肿和急性呼吸衰竭。同时,炎症反应,氧化应激,细胞凋亡和可溶性调节因子代谢异常等也可能参与急性肾损伤诱导的肺损伤。对急性肾损伤诱导肺损伤的临床认识和可能发病机制的研究,将有助于临床疾病的治疗和死亡率的降低,同时也将有助于对其它肾脏疾病发病机制的认识。     

Shikonin exerts anti-inflammatory effects in a murine model of lipopolysaccharide-induced acute lung injury by inhibiting the nuclear factor-kappaB signaling pathway

Shikonin, an analog of naphthoquinone pigments isolated from the root of Lithospermum erythrorhyzon, was recently reported to exert beneficial anti-inflammatory effects both in vivo and in vitro. The present study aimed to investigate the potential therapeutic effect of shikonin in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Dexamethasone was used as a positive control to evaluate the anti-inflammatory effect of shikonin in the study. Pretreatment with shikonin (intraperitoneal injection) significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, shikonin significantly reduced the concentrations of TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid induced by LPS. Compared with the LPS group, lung histopathologic changes were less pronounced in the shikonin-pretreated mice. Additionally, Western blotting results showed that shikonin efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that shikonin exerts anti-inflammatory properties in LPS-mediated ALI, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. Shikonin may be a potential agent for the prophylaxis of ALI.     

补体旁路激活致小鼠急性肺损伤的炎症病理机制研究

目的研究补体旁路激活所导致的小鼠肺部急性炎症的发生发展及相关指标的变化,为药物筛选及干预研究提供理想的小鼠肺部急性炎症病理模型。方法 SPF级昆明小鼠尾静脉注射眼镜蛇毒因子(CVF)激活血清补体旁路途径,根据注射后取样时间不同,分为15 min、30 min、1 h、2 h、6 h组,同时平行设置PBS对照组。取肺组织测定肺系数、肺含水量,并行病理切片检查,肺组织匀浆测定髓过氧化物酶(MPO)活性;制备支气管肺泡灌洗液(BALF)和血清,测定BALF中的细胞数和蛋白含量,采用ELISA法分别测定BALF和血清中的IL-6、TNF-α、P-selectin和ICAM-1含量。结果小鼠尾静脉注射CVF后可致肺部炎性细胞明显浸润,MPO活性明显上调,BALF中细胞总数和蛋白浓度明显增加。BALF和血清中的IL-6、TNF-α、P-selectin水平及血清中ICAM-1的含量均明显升高,其中,BALF中P-selectin含量在30 min时间点出现1个小高峰,IL-6和TNF-α在1 h时间点出现1个高峰,在2 h时间点均无进一步上升,但在6 h时间点各指标均又明显升高;血清中IL-6和TNF-α含量在1 h时间点达到峰值,随后浓度降低,P-selectin和ICAM-1水平随着时间的延长持续上升。而肺系数、肺含水量及BALF中ICAM-1的含量与PBS组相比无明显变化。结论补体旁路激活可导致小鼠肺部急性炎症的发生,以30 min至1 h的炎症反应最为明显,该实验可以为药物筛选及干预研究提供理想的动物肺部急性炎症病理模型。