15d-PGJ2 stimulates HO-1 expression through p38 MAP kinase and Nrf-2 pathway in rat vascular smooth muscle cells

15d-PGJ(2), a potent endogenous ligand for peroxisome proliferators activated receptor-gamma, is a cyclopentenone-type prostaglandin produced by many different types of cells. Pertinent to its effect on vascular smooth muscle cell (VSMC), antiproliferative effects have been most frequently reported. In the present study, we investigated the effect of 15d-PGJ(2) on HO-1 expression that has been reported to inhibit VSMC proliferation. According to our data, 15d-PGJ(2) significantly induced ROS/NO production and HO-1 expression in rVSMCs. We also observed 15d-PGJ(2)-induced translocation of Nrf-2. In addition, ROS scavenger pretreatment suppressed 15d-PGJ(2)-induced HO-1 expression while PPARgamma antagonist did not, suggesting nuclear translocation of Nrf-2 and subsequent HO-1 expression was ROS dependent rather than PPARgamma dependent. Furthermore, an inhibitor of p38 MAPK abolished 15d-PGJ(2)-induced HO-1 expression. These data suggest that 15d-PGJ(2)-induced up-regulation of HO-1 is independent of PPARgamma but dependent of ROS and p38 MAPK pathway. The present study reports for the first time that 15d-PGJ(2) induces HO-1 expression possibly using Nrf-2 pathway as a response to ROS in VSMCs.     

DJ-1保护多巴胺能神经元抵抗过氧化氢氧化应激损伤的研究

目的 探讨DJ-1保护多巴胺能神经元免受过氧化氢(H₂O₂)损伤的机制。 方法 使用神经生长因子(NGF)将大鼠嗜铬细胞瘤细胞(PC12细胞)诱导为多巴胺能神经元模型。H₂O₂处理引起细胞氧化应激损伤模型,CCK-8试剂盒检测细胞活性,DHE染色检测细胞内ROS水平。PI / Hoechst染色检测细胞凋亡,蛋白质免疫印迹法(Western blot)检测DJ-1和TH蛋白的表达。构建DJ-1过表达载体,检测DJ-1对H₂O₂中PC12细胞的保护作用及对细胞内活性氧(ROS)的影响。RT-qPCR检测α-synuclein,p53,Bax,Bcl-2的表达变化。 结果 H₂O₂处理可显着降低PC12细胞的活性,H₂O₂处理24 h以上可引起细胞凋亡。H₂O₂处理下调DJ-1蛋白和TH蛋白的表达,并且在RNA水平上α-突触核蛋白的表达增加。另外 p53, Bax和 caspase-3表达增加, Bcl-2表达减少。DJ-1的过表达可以抑制H₂O₂引起的ROS增加,DJ-1可以维持细胞活性,减少H₂O₂的凋亡。在RNA水平抑制α-突触核蛋白,p53,bax,bcl-2的凋亡和抗凋亡基因表达变化。 结论 DJ-1能够抑制H₂O₂引起的 ROS水平升高,减少α-synuclein积累,抑制 p53,凋亡基因如 bax的表达减弱了多巴胺能神经元中H₂O₂诱导的氧化应激损伤。     

Catalytic activity-independent pathway is involved in phospholipase A2-induced apoptotic death of human leukemia U937 cells via Ca-mediated p38 MAPK activation and mitochondrial depolarization

In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A₂ (PLA₂), the present study is conducted to explore whether PLA₂ induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA₂) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA₂. It was found that BPB–PLA₂-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB–PLA₂, elevation of intracellular Ca²⁺ levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca²⁺ chelator) and nifedipine (L-type Ca²⁺ channel blocker) abrogated Ca²⁺ increase and p38 MAPK activation, and rescued viability of BPB–PLA₂-treated U937 cells. BPB–PLA₂-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA₂ mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA₂ activity, they induced death of U937 cells. The signaling pathway of PLA₂ mutants in inducing cell death was indistinguishable from that of BPB–PLA₂. Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA₂-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca²⁺-mediated p38 MAPK activation.     

纳米锰锌铁氧体颗粒对L-02细胞的氧化损伤作用

目的 探讨磁性锰锌铁氧体纳米颗粒(Mn_0.5Zn_0.5Fe₂O₄)对人肝细胞株L-02的毒性作用机制。方法 Mn_0.5Zn_0.5Fe₂O₄ 800 mg·L^-1作用L-02细胞48 h,透射电镜观察细胞形态及超微结构的变化。Mn_0.5Zn_0.5Fe₂O₄ 200, 400和800 mg·L^-1作用48 h后,检测L-02细胞内丙二醛(MDA)的含量、超氧化物歧化酶(SOD)和还原型谷胱甘肽(GSH)的活性;荧光染色观察凋亡细胞形态;流式细胞术检测细胞周期及凋亡;荧光定量PCR仪检测胱天蛋白酶3 mRNA表达。结果 Mn_0.5Zn_0.5Fe₂O₄ 800 mg·L^-1作用48 h后,纳米颗粒进入细胞内,细胞膜发生破损,细胞器消失,染色体异常聚集。与正常对照组比较,Mn_0.5Zn_0.5Fe₂O₄ 200~800 mg·L^-1使细胞内MDA含量逐渐升高,SOD与GSH活性逐渐降低(P<0.05)。Mn_0.5Zn_0.5Fe₂O₄可使细胞周期发生改变,G₀/G₁期细胞百分率有降低的趋势,S期和G₂/M期细胞百分率有升高的趋势。Hoechst33258显示明显的细胞凋亡形态。Mn_0.5Zn_0.5Fe₂O₄可引起L-02细胞发生剂量依赖性的细胞凋亡,Mn_0.5Zn_0.5Fe₂O₄ 800 mg·L^-1作用48 h后,细胞凋亡率达到30.3%,是对照组细胞凋亡率(2.4%)的12.6倍。胱天蛋白酶3 mRNA表达量先增加后降低,但都明显高于正常对照组(P<0.05)。结论 Mn_0.5Zn_0.5Fe₂O₄可破坏细胞膜完整性并进入细胞内,诱导细胞发生氧化应激,改变细胞周期,引发细胞凋亡,产生细胞毒性。     

Involvement of the activation of Nrf2/HO-1, p38 MAPK signaling pathways and endoplasmic reticulum stress in furazolidone induced cytotoxicity and S phase arrest in human hepatocyte L02 cells: modulation of curcumin

Furazolidone (FZD) is extensively used as the antiprotozoal and antibacterial drug in clinic. The previous study has shown that curcumin pretreatment could improve FZD induced cytotoxicity by inhibiting oxidative stress and mitochondrial apoptotic pathway. The current study aimed to investigate the potential roles of endoplasmic reticulum (ER) stress, p38 mitogen-activated protein kinases (p38 MAPK) signaling pathway in curcumin against FZD cytotoxicity by using human hepatocyte L02 cells. The results showed that curcumin could markedly attenuate FZD induced cytotoxicity. Compared with FZD alone group, curcumin pretreatment significantly reduced the expression of phospho (p)-p38, cyclin D1, p-checkpoint kinase 1 (ChK1) and breast cancer associated gene 1 (BRCA1) protein, followed to attenuate S phase arrest. Meanwhile, curcumin pretreatment prevented FZD induced ER stress, evidenced by the inhibition of glucose-regulated protein 78 and DNA damage inducible gene 153/C/EBP-homologous protein (GADD153/CHOP) protein expression. Moreover, compared with the control, FZD exposure activated the protein and mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1), which were further activated by curcumin treatment. These results reveal that curcumin could prevent FZD induced cytotoxicity and S phase arrest, which may involve the activation of Nrf2/HO-1 pathway and the inhibition of p38 MAPK pathway and ER stress.     

Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells

As the applications of industrial nanoparticles are being developed, the concerns on the environmental health are increasing. Cytotoxicities of titanium dioxide nanoparticles of different concentrations (5, 10, 20 and 40mug/ml) were evaluated in this study using a cultured human bronchial epithelial cell line, BEAS-2B. Exposure of the cultured cells to nanoparticles led to cell death, reactive oxygen species (ROS) increase, reduced glutathione (GSH) decrease, and the induction of oxidative stress-related genes such as heme oxygenase-1, thioredoxin reductase, glutathione-S-transferase, catalase, and a hypoxia inducible gene. The ROS increase by titanium dioxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that titanium dioxide nanoparticles exert cytotoxicity by an apoptotic process. Furthermore, the expressions of inflammation-related genes such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), TNF-a, and C-X-C motif ligand 2 (CXCL2) were also elevated. The induction of IL-8 by titanium dioxide nanoparticles was inhibited by the pre-treatment with SB203580 and PD98059, which means that the IL-8 was induced through p38 mitogen-acitvated protein kinase (MAPK) pathway and/or extracellular signal (ERK) pathway. Uptake of the nanoparticles into the cultured cells was observed and titanium dioxide nanoparticles seemed to penetrate into the cytoplasm and locate in the peri-region of the nucleus as aggregated particles, which may induce direct interactions between the particles and cellular molecules, to cause adverse biological responses.     

The aromatic volatile organic compounds toluene, benzene and styrene induce COX-2 and prostaglandins in human lung epithelial cells via oxidative stress and p38 MAPK activation

Toluene, benzene and styrene are volatile organic compounds (VOCs) widely distributed in the environment. Tobacco smoke, traffic exposure and solvents used for paints, rubber and adhesives are known sources for these compounds. The aim of the present study was to investigate whether toluene, benzene and styrene can induce inflammatory reactions in lung cells and to characterize possible underlying mechanisms. A previous study gave evidence that expression of cyclooxygenase-2 (COX-2) is upregulated following exposure to the aromatic VOC chlorobenzene. Here, we investigated the effects of the aromatics toluene, benzene and styrene on human lung cells, with emphasis on COX-2, the rate-limiting enzyme of the prostaglandin pathway. In addition, we studied the potential role of oxidative stress and p38 MAPK activation in the toluene/benzene/styrene-dependent COX-2 induction. Following exposure to the aromatic compounds the expression level of COX-2 increased markedly. In addition, prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α), major products of the COX enzyme, were found to be upregulated in response to toluene, benzene or styrene exposure. Furthermore, we observed an activation of p38 MAPK resulting from aromatic VOC exposure. Treatment of the cells with a specific p38 inhibitor (SB203580) or the antioxidant N-acetylcysteine (NAC) was able to prevent the toluene/benzene/styrene-dependent COX-2 activation, and subsequent increased PGE₂ and PGF_2α secretion. These results suggest that toluene, benzene and styrene induce production and secretion of PGE₂ and PGF_2α in lung epithelial cells via p38 MAPK and COX-2 activation in a redox sensitive manner.     

高效液相色谱-荧光检测法测定沉香中黄曲霉毒素B1、B2、G1、G2

目的建立高效液相色谱–光化学衍生–荧光检测法测定沉香药材中黄曲霉毒素B1、B2、G1、G2。方法采用高效液相色谱法,通过免疫亲和柱提取和净化,荧光检测器检测。Agilent Zorbax Ecilpse Plus C18色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇–水(45∶55);体积流量:0.8 m L/min;柱温:30℃;进样盘温度:4℃;荧光激发波长为360 nm,发射波长为450 nm。结果黄曲霉毒素B1、B2、G1、G2分别在9.3~74.4、3.0~24.0、9.3~74.4、3.5~28.0 pg线性关系良好,r均大于0.998 0;检测限分别为1.86、0.60、1.86、0.70 pg,定量限分别为7.44、2.40、7.44、2.80 pg。平均回收率分别为78%、92%、82%、99%,RSD值分别为4.4%、3.0%、4.3%、2.8%。结论所建立的方法结果准确、重复性、稳定性均良好,可用于沉香药材中黄曲霉毒素的质量控制。     

高效液相色谱-光化学衍生法测定湖南产莲子中黄曲霉毒素G1、G2、B1、B2

目的对不同来源的湖南产莲子中黄曲霉毒素G_1、G_2、B_2、B_1进行高效液相色谱-光化学衍生法测定。方法采用高效液相色谱–光化学衍生法,采用岛津GL Inertsil ODS-3色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇–乙腈–水(35∶13∶52),柱温35℃;光化学衍生器(254 nm)激发波长λ_(ex)=360 nm,发射波长λ_(ex)=450 nm。结果黄曲霉毒素G_1、G_2、B_2、B_1分别在6.7～33.3、11.4～56.8、10.3～51.5、4.1～20.3 pg线性关系良好;平均回收率分别为98.07%、97.72%、96.11%、99.52%,RSD值分别为1.69%、1.40%、2.72%、1.34%(n=6)。9批莲子样品中,有6批未检出黄曲霉毒素,来自于农贸市场农户自存的2批次检出黄曲霉毒素B_1,实验室塑料袋包装储存1年的1批检出黄曲霉毒素B_1、B_2,但质量分数均低于法定标准限量。结论不同来源湖南产莲子中黄曲霉毒素质量分数均低于《中国药典》2020年版规定限量。该法测定结果准确、重复性良好,可为完善莲子安全性控制和质量标准提升提供实验依据。     

岩藻黄素抗H2O2诱导WI-38细胞早衰的作用研究D*

目的：探讨岩藻黄素是否具有抑制H₂O₂引起的WI-38细胞早衰的活性。方法：WI-38细胞在添加岩藻黄素的培养基中培养一定时间后经H₂O₂处理诱导发生早衰,用MTT法检测细胞活力,SA-β-半乳糖苷酶染色法检测细胞内衰老相关的β-半乳糖苷酶活性。 结果：300 μM H₂O₂处理WI-38细胞20 min可成功建立早衰细胞模型。与空白对照组相比,H₂O₂处理组细胞活力明显降低,SA-β-半乳糖苷酶阳性细胞数明显增多。5 μM和10 μM岩藻黄素组细胞活力明显高于H₂O₂处理组,SA-β-半乳糖苷酶阳性细胞数较H₂O₂组明显减少。 结论：岩藻黄素能够抑制由H₂O₂处理引起的细胞早衰,具有一定的抗衰老作用。     

PM10 impairs the antioxidant defense system and exacerbates oxidative stress driven cell death

The aim of this study was to investigate the effect of airborne particulate matter with a mean aerodynamic diameter of ≤10 μm (PM₁₀) on oxidative stress markers and antioxidant enzymatic activity and its relevance in the face of acute oxidative challenge in a human lung epithelial cell line (A549). PM₁₀-induced reactive oxygen species (ROS) generation and oxidative damage with no changes in cellular viability. In addition, PM₁₀ decreased glutathione (GSH) levels (54.9%) and the activity of the antioxidant enzymes superoxide dismutase (65%), catalase (31.2%), glutathione reductase (61.5%) and glutathione-S-transferase (42.39%). Trolox, a scavenger of reactive species, prevented the increase of ROS generation and the decrease in GSH levels but partially prevented PM₁₀-induced oxidative damage. Interestingly, it was unable to avoid the decrease in the activity of antioxidant enzymes. Finally, the survival of the cells previously exposed to PM₁₀ and challenged with hydrogen peroxide was significantly lower. We conclude that the impairment in the antioxidant defense system induced by PM₁₀ weaken ROS detoxification which exacerbates cell death when these cells are exposed to an acute oxidative challenge.     

Poria cocos polysaccharides attenuated ox-LDL-induced inflammation and oxidative stress via ERK activated Nrf2/HO-1 signaling pathway and inhibited foam cell formation in VSMCs

Oxidative stress, inflammation, and foam cell formation in vascular smooth muscle cells (VSMCs) are considered to play crucial roles in the pathogenesis of atherosclerosis. Poria cocos polysaccharides (PCP) has been shown to possess anti-inflammatory, antitumor and anti-oxidative properties. In this study we explored the effects of PCP on ox-LDL-induced inflammation, oxidative stress and foam cell formation in VSMCs. PCP significantly attenuated ox-LDL-induced oxidative stress, as evidenced by the decreased reactive oxygen species (ROS) and MDA levels, and the increased SOD activity in VSMCs. PCP suppressed the induction effect of ox-LDL on inflammatory cytokines and inflammatory mediators. PCP also substantially inhibited VSMCs foam cell formation and intracellular lipids accumulation. Mechanistically, PCP suppressed ox-LDL-induced up-regulation of LOX-1, which is responsible for ox-LDL uptake. Western blotting suggested that PCP activated ERK1/2 signaling pathway, increased Nrf2 translocated from cytoplasm to nucleus and heme oxygenase-1 (HO-1) expression. Up-regulation of PCP on Nrf2/HO-1 signaling was reversed by pretreatment with ERK inhibitor PD98059, indicating the involvement of ERK in PCP activation of Nrf2/HO-1 signaling. In conclusion, these results demonstrated that PCP exerted its protection against oxidative stress and inflammation via the ERK/Nrf2/HO-1 signaling pathway and that PCP may be a promising candidate for the therapy of atherosclerosis.     

Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells

CMS-9, a phospholipase A₂ (PLA₂) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca²⁺ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca²⁺ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca²⁺ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA₂ activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca²⁺/ROS-evoked p38 MAPK and JNK activation.     

人参皂苷Rg1对H2O2诱导的HaCaT细胞氧化损伤保护作用研究

目的 观察人参皂苷Rg₁对H₂O₂诱导的HaCaT细胞氧化损伤保护作用,并探讨其机制。方法 体外培养HaCaT细胞, H₂O₂诱导细胞建立氧化应激损伤模型,分为空白组、H₂O₂损伤组、人参皂苷Rg₁保护组。细胞增殖与毒性检测试剂盒(CCK-8)检测细胞存活率,Hochest染色法检测细胞凋亡情况,活性氧检测试剂盒测定细胞活性氧(ROS)水平,Western blot检测细胞中caspase-3、caspase-6、caspase-8、GAPDH蛋白表达。结果 H₂O₂诱导HaCaT细胞半数抑制浓度为100 μg?mL^-1;与H₂O₂损伤组比较,5、10和15mg?L^-1人参皂苷Rg₁预处理后,HaCaT细胞存活率明显升高(P<0.05),细胞核皱缩损伤状态明显改善,细胞凋亡数量显著减少。同时,人参皂苷Rg₁预处理可显著降低HaCaT细胞ROS水平,下调凋亡相关标志蛋白-活化型caspase-3、caspase-6、caspase-8蛋白表达水平。结论 人参皂苷Rg₁对H₂O₂诱导的HaCaT细胞氧化应激损伤具有一定的保护作用,其机制可能与增强细胞清除自由基能力及抑制凋亡相关。     

Role of hMLH1 in sterigmatocystin-induced G2 phase arrest in human esophageal epithelial Het-1A cells in vitro

Sterigmatocystin (ST), a common environmental contaminant found across the world, is generally recognized as a potential carcinogen, mutagen and teratogen. Our previous epidemiological studies suggested that ST exposure might be a risk factor for esophageal cancer. However, the direct effects of ST on human esophageal epithelial cells are currently unknown. In the present study, we examined the effect of treating a human esophageal epithelial cell line (Het-1A) with ST on DNA damage, DNA repair mechanisms, and cell cycle distribution. We found that ST treatment could induce DNA damage and lead to a G₂ phase arrest, associated with a marked up-regulation of G₂/M regulatory proteins, including Cyclin B1, Cdc2/p-Cdc2, and Cdc25C/p-Cdc25C. Additionally, we found that the expression of two mismatch repair (MMR) proteins, hMLH1 and hMSH2, was up-regulated at both the mRNA and protein levels after ST treatment, suggesting that ST could induce the MMR system in Het-1A cells. Interestingly, ST-induced G₂ phase arrest was mediated by hMLH1 up-regulation, but was independent of hMSH2. Treatment with hMLH1-siRNA prevented the up-regulation of Cyclin B1, Cdc2/p-Cdc2 and Cdc25C/p-Cdc25C in ST-treated cells, thereby inhibiting the subsequent G₂ phase arrest of Het-1A cells. Moreover, we found that hMLH1 may act as a direct sensor of ST-mediated DNA damage. In conclusion, the study demonstrated that ST caused DNA damage and triggered G₂ phase arrest in Het-1A cells, and hMLH1 participated in the ST-induced G₂ phase arrest by up-regulating G₂/M regulatory proteins.     

Inhibition of calcium-independent phospholipase A2 activates p38 MAPK signaling pathways during cytostasis in prostate cancer cells

The p38 mitogen-activated protein kinase (MAPK) signaling pathways activated during cytostasis induced by Ca²⁺-independent phospholipase A₂ (iPLA₂) inhibition in prostate cancer cells were investigated. iPLA₂ inhibition using siRNA, or the selective inhibitor bromoenol lactone (BEL) and it's enantiomers, decreased growth in LNCaP (p53 positive) and PC-3 (p53 negative) human prostate cancer cells. Decreased cell growth correlated to time- and concentration-dependent activation of the mitogen-activated protein kinase p38 in both cell lines. Inhibition of cytosolic iPLA₂β using S-BEL, induced significantly higher levels of P-p53, p53, p21 and P-p38 expression than inhibition of microsomal iPLA₂γ using R-BEL. Inhibition of p38 using SB202190 or SB203580 inhibited BEL-induced increases in P-p53 (ser15), p53 and p21, and altered the number of cells in G1 in LNCaP cells, and S-phase in PC-3 cells. BEL treatment also induced reactive species in PC-3 and LNCaP cells, which was partially reversed by pretreatment with N-acetyl-cysteine (NAC). NAC subsequently inhibited BEL-induced activation of p38 and p53 in LNCaP cells. In addition, treatment of cells with NAC partially reversed the effect of BEL on cell growth and preserved cell morphology. Collectively, these data demonstrate the novel findings that iPLA₂ inhibition activates p38 by inducing reactive species, and further suggest that this signaling kinase is involved in p53 activation, cell cycle arrest and cytostasis.     

Effects of inhaled nano-TiO2 aerosols showing two distinct agglomeration states on rat lungs

Nano-aerosols composed of large agglomerates (LA) (>100 nm) are more likely to promote pulmonary clearance via macrophages phagocytosis. Small agglomerates (SA) (<100 nm) seem to escape this first defense mechanism and are more likely to interact directly with biological material. These different mechanisms can influence pulmonary toxicity. This hypothesis was evaluated by comparing the relative pulmonary toxicity induced by aerosolized nano-TiO₂ showing two different agglomeration states: SA (<100 nm) and LA (>100 nm) at mass concentrations of 2 or 7 mg/m³. Groups of Fisher 344 male rats were nose-only exposed for 6 h. The median number aerodynamic diameters were 30 and 185 nm at 2 mg/m³, and 31 and 194 nm at 7 mg/m³. We found in rat's bronchoalveolar lavage fluids (BALF) a significant 2.1-fold increase in the number of neutrophils (p < 0.05) in the group exposed to the 7 mg/m³ LA nano-aerosol suggesting a mild inflammatory response. Rats exposed to the 7 mg/m³ SA nano-aerosol showed a 1.8-fold increase in LDH activity and 8-isoprostane concentration in BALF, providing evidence for cytotoxic and oxidative stress effects. Our results indicate that biological responses to nanoparticles (NP) might depend on the dimension and concentration of NP agglomerates.     

4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B₄ (LTB₄) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT₁ (cysLT₁) receptor antagonist, REV-5901 as well as a BLT₁ receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB₄ and cysLT (LTC₄ and LTD₄) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB₄ and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.