Zaprinast activates MAPKs, NFκB, and Akt and induces the expressions of inflammatory genes in microglia

Previously, the authors reported that zaprinast, an inhibitor of cGMP-selective phosphodiesterases, induced the secretions of TNF-α and IL-1β by microglia and enhanced the induction of iNOS by lipopolysaccharide (LPS). In this study, the signaling mechanism responsible for microglial activation by zaprinast was investigated and the effects of zaprinast and LPS on microglial activation were compared. Zaprinast was found to activate ERK1/2, p38 MAPK, JNK, NFκB, and PI3K/Akt, and subsequently, induce the mRNA expressions of IL-1α, IL-1β, TNF-α, CCL2, CCL4, CXCL1, CXCL2, and CD14. Associations between signaling pathways and gene expressions were examined by treating microglia with signal inhibitors. PDTC inhibited the induction of all the above genes by zaprinast, and SB203580 inhibited all genes except CXCL1. SP600125, PD98059, and LY294002 inhibited the induction of at least CCL2. Microglial activation by zaprinast was then compared with full-blown activation by LPS. The zaprinast-induced phosphorylations of MAPKs and IκB were less prompt than LPS-induced phosphorylations. IκB degradation by LPS was significant at 10min and did not return to normal, whereas zaprinast induced a later, transient degradation. LPS induced the mRNA expressions of IL-1β, TNF-α, IL-6, CCL2, iNOS, and COX-2, and although zaprinast significantly induced the expressions of all except IL-6 and iNOS, these inductions were far less than those induced by LPS. Collectively, zaprinast was found to upregulate microglial activity mainly via NFκB and p38 MAPK signaling and the subsequent expressions of inflammatory genes. Although, zaprinast was found to have obvious effects on microglia, these were weaker than the effects of LPS.     

转录因子D位点结合蛋白对原代大鼠系膜细胞中 免疫炎症因子的调控作用

目的 通过基因表达谱分析,探讨并验证转录因子D位点结合蛋白（DBP）对原代大鼠系膜细胞（RMCs）中 免疫炎症因子的调控作用。方法 从8周龄SD大鼠中分离、培养并鉴定原代RMCs,利用小干扰RNA技术敲低其 DBP表达,进而行RNA-seq测序分析;筛选差异表达基因（DEGs）,并对DEGs行Gene Ontology（GO）注释及KEGG信 号通路分析;采用qPCR验证DBP低表达的RMCs中免疫炎症因子mRNA表达水平;利用质粒转染技术获得DBP过表 达RMCs并采用qPCR检测DBP过表达对免疫炎症因子mRNA表达的影响。结果 成功分离、培养原代RMCs,并获 得DBP低表达的原代RMCs。RNA-seq测序结果显示,与野生原代RMCs相比,DBP低表达的原代RMCs中共有267 个DEGs（上调84个,下调183个）。GO注释及KEGG信号通路分析表明,DEGs主要富集在免疫炎症相关的生物学过程 及信号通路。原代RMCs中,DBP敲低可明显下调免疫炎症因子C-C模体趋化因子配体2（CCL2）、C-X-C模体趋化因 子（CXCL）10、CXCL1、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1、IL-6 mRNA表达,DBP过表达则可明显上调上述免 疫炎症因子mRNA表达。结论 原代RMCs中,DBP可通过调控免疫炎症因子表达参与免疫炎症反应。     

Polarized macrophages treated with nonylphenol differently regulate lipopolysaccharide‐induced sepsis

Nonylphenol (NP) as well‐known “endocrine disrupter” influences sexual and reproductive development. Here, we investigated the effect of NP on M1‐/M2‐type macrophages and their role in lipopolysaccharide (LPS)‐induced sepsis. Polarized macrophages of M1‐ and M2‐types were obtained by the treatment with LPS and interleukin‐4 (IL‐4) to bone marrow‐derived macrophages (BMDM), respectively. Coincubation of M1‐macrophages with NP decreased COX‐2, iNOS, IL‐6, and TNF‐α expression but no changes were detected in the production of nitric oxide (NO). Survival probability of LPS‐induced sepsis mice was enhanced by the injection of NP‐treated BMDM as compared to the injection of NP‐untreated control BMDM. In the meanwhile, the expression of arginase 1(Arg1), a marker for M2‐polarized macrophages was increased by the stimulation with LPS in BMDM. Arg1 expression was also enhanced by the treatment with IL‐4 in BMDM, which was reduced by the coincubation with NP. Survival probability of LPS‐induced sepsis mice was decreased by the injection of BMDM treated with IL‐4 and NP as compared to the injection of IL‐4‐treated BMDM. It suggests that NP might inhibit macrophage function and the polarization to M2‐macrophages. Taken together, data demonstrate that NP could differently affect immune responses of polarized macrophages resulted in the modulation of LPS‐induced sepsis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2081–2089, 2016.     

Effect of lipopolysaccharide on the xenobiotic-induced expression and activity of hepatic cytochrome P450 in mice

Infection-associated inflammation can alter the expression levels and functions of cytochrome P450s (CYPs). Cyp gene expression is regulated by the activation of several nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). These receptors can be activated by xenobiotics, including medicines. Here, to study the xenobiotic-induced fluctuations in CYP during inflammation, we examined the effect of lipopolysaccharide (LPS) treatment on the level of mRNAs encoding hepatic CYPs induced by xenobiotic-activated nuclear receptors, in mice. Both the mRNA induction of Cyp genes and the metabolic activities of CYP proteins were examined. LPS treatment caused a significant decrease in the induced expression of the mRNAs for Cyp3a11, 2c29, 2c55, and 1a2, but not for Cyp2b10. To assess the CYP enzymatic activities, CYP3A-mediated testosterone 6β-hydroxylation and the intrinsic clearance (CL(int)) of nifedipine in liver microsomes were measured in mice treated with the xenobiotic pregnenolone-16alpha-carbonitrile (PCN) with or without LPS administration. Both assays revealed that the CYP3A activity, which was induced by PCN, declined significantly after LPS treatment, and this decline correlated with the Cyp3a11 mRNA level. In addition, we found that the mRNAs for interleukin (IL)-1β and tumor necrosis factor (TNF) α were increased after treatment with LPS plus xenobiotics. Our findings demonstrated that LPS treatment reduces the PXR- and AhR-mediated, and possibly CAR-mediated Cyp gene expression and further suggest that these decreases are dependent on inflammatory cytokines in the liver.     

前列腺素E1抑制血管平滑肌细胞增殖的作用

目的 从细胞周期蛋白和凋亡相关基因探讨前列腺素E₁(PGE₁)对血管平滑肌细胞(VSMC)增殖的调控作用。方法 采用培养的新生牛主动脉VSMC，以胎牛血清及白介素1(IL-1)分别作为促增殖剂，测定不同浓度PGE₁对VSMC增殖、细胞内周期蛋白d₁ mRNA表达、p53及Bcl-2基因表达和培养液内NO含量的影响。结果 PGE₁浓度依赖性抑制胎牛血清促VSMC增殖作用；显著减少细胞周期蛋白d₁ mRNA含量；浓度依赖性诱导IL 1孵育下VSMC产生大量NO, 同时抑制VSMC增殖；并促进p53基因表达，下调bcl-2基因，诱导细胞凋亡。结论 PGE₁具有抑制VSMC增殖的作用，其机制可能与其阻滞VSMC生长有关；在炎性因子存在情况下，PGE₁可诱导VSMC内大量产生NO，抑制VSMC增殖，并加速VSMC凋亡。     

脑啡肽对培养新生大鼠海马胶质细胞白介素1α基因表达的影响

用逆转录－聚合酶链反应技术研究了脑啡肽对培养的新生大鼠海马胶质细胞ＩＬ－１α基因表达的影响．结果发现，脂多糖（１０ｍｇ·Ｌ－１）能诱导海马胶质细胞ＩＬ－１α基因表达，当预先用甲硫脑啡肽（０．１ｎｍｏｌ·Ｌ－１）或亮脑啡肽（０．１ｎｍｏｌ·Ｌ－１）处理海马胶质细胞，则脂多糖诱导的海马胶质细胞ＩＬ－１α基因表达受到抑制，且脑啡肽的作用可被阿片受体阻断剂纳洛酮所阻断．结果提示脑啡肽对脂多糖诱导大鼠海马胶质细胞ＩＬ－１α基因表达的抑制性影响可能是通过海马胶质细胞或神经细胞上的阿片受体介导的     

Concentration‐dependent cytokine responses of silica nanoparticles and role of ROS in human lung epithelial cells

Reactive oxygen species (ROS) is regarded as a critical denominator in nanoparticle toxicology and inflammation. Previously, we have shown that silica nanoparticles sized 50 nm (Si50) induce release of CXCL8 and IL‐6 from BEAS‐2B cells, via mechanisms involving NFκB, p38 MAP kinase and TGF‐α‐activated EGF receptor. In the present study, the role of ROS‐mediated mechanisms in the concentration‐dependent Si50 induction of CXCL8 and IL‐6 responses was examined. Si50 (200 µg/mL) induced a time‐dependent ROS formation and a postponed increase in expression of haem oxygenase (HO‐1) mRNA and protein. Pre‐treatment with the ROS inhibitors N‐acetyl cysteine (NAC) and diphenyleneiodonium (DPI) partially attenuated CXCL8 and IL‐6 responses to 200 µg/mL, but not to 100 µg/mL Si50. The release of TGF‐α induced by Si50 (200 µg/mL) was significantly reduced by NAC, but not by DPI nor siRNA against NADPH oxidase DUOX‐1 (siDUOX‐1). Furthermore, siDUOX‐1 reduced Si50‐induced CXCL8, but not IL‐6. Both p38 and p65 phosphorylations were inhibited by siDUOX‐1, but for NAC only p65 phosphorylation reached a significant reduction. Neither NAC nor DPI reduced Si50‐induced CXCL8 and IL‐6 gene expressions. In conclusion, Si50‐induced CXCL8 and IL‐6 involved both ROS‐dependent and ROS‐independent mechanisms. Notably, the role of ROS seemed restricted to effects of higher concentrations of Si50 and not mediated via the gene expression.     

The effect of trimethylamine N-oxide on Helicobacter pylori-induced changes of immunoinflammatory genes expression in gastric epithelial cells

Colonization of Helicobacter pylori (H. pylori) induces immune and inflammatory response in gastric mucosa. Trimethylamine N-oxide (TMAO), from diet and metabolite through the action of gut microbiota, has been linked to inflammatory diseases. To investigate the effects of TMAO and H. pylori infection on gene expression in gastric epithelial cells, Human gene chip Affymetrix HTA 2.0 was used in this study. 1312 genes were identified as differentially expressed genes in GES-1 cells with H. pylori and TMAO co-treatment compared to the control. GO and KEGG analyses indicated that the functions of these differentially expressed genes were related closely with immune inflammation. GO-network showed that Toll-like receptor signaling pathway was the most important biological processes and 49 up-regulated genes related to immune inflammation were obtained. The synergistic effects of H. pylori and TMAO enhanced the genes expression of IL-6, CXCL1, CXCL2, FOS and C3 related to immune inflammation in comparison with those of non-infected control cells, H. pylori-infected cells, and TMAO-stimulated cells. RT-PCR verified the expression levels of IL-6, CXCL1. Additionally, expression levels of 2053 genes were altered and 52 immunoinflammatory genes were upregulated in comparison with H. pylori-infected cells. This study suggested that TMAO altered the expression levels of immunoinflammatory genes induced by H. pylori infection, and the synergistic effects of H. pylori and TMAO provided novel insights into the development of chronic gastritis, gastric ulcer and gastric cancer.     

NLRP3 inflammasome activation involved in LPS and coal tar pitch extract‐induced malignant transformation of human bronchial epithelial cells

Inflammatory microenvironment has been found as a new characteristic of cancer; however, the mechanisms of inflammation‐related lung cancer remain unclear. To explore the role of NLRP3 inflammsome activation in inflammation‐related lung carcinogenesis, a cell model was set up. Human bronchial epithelial cells (BEAS‐2B) were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 hours, and then treated with 2.4 μg/mL coal tar pitch extract (CTPE) for 24 hours, after removal of LPS and CTPE, the cells were numbered passage 1 and were passaged and treated in this way until passage 30, which was called LPS + CTPE group. DMSO and Saline were used as vehicle controls. Malignant transformation of cells in passage 30 was evaluated by morphological change, platelet clone formation assay, and tumor formation in nude mice. The mRNA levels of NLRP3 and IL‐1β were detected by real time‐PCR. The combination of NLRP3 and caspase‐1 were determined using immunofluorescence and confocal. The protein expression of NLRP3, cleaved caspase‐1(p10), and cleaved IL‐1β was detected using Western blot. It was shown that CTPE, LPS + CTPE‐stimulated BEAS‐2B cells of passage 30 changed a lot morphologically. The clone formation rates, the rates of positive cells of NLRP3 and caspase‐1 combination, the mRNA levels of NLRP3 and IL‐1β, the protein expression of NLRP3, cleaved caspase‐1(p10) and cleaved IL‐1β of cells exposed with CTPE and LPS + CTPE at passage 30 were significantly increased compared to vehicle controls. Furthermore, the ability of tumor formation in nude mice, the rates of clone formation and positive cells, mRNA and protein levels of NLRP3 inflammasome activation‐related factors in LPS + CTPE‐induced cells were all higher than those in cells stimulated with CTPE alone. In conclusion, the cell model of inflammation‐related lung cancer is set up successfully, and NLRP3 inflammasome activation may be involved in the malignant transformation of BEAS‐2B cells which induced by CTPE alone or LPS combined with CTPE.     

Deoxynivalenol-induced cytotoxicity,cytokines and related genes in unstimulated or lipopolysaccharide stimulated primary porcine macrophages

The cytotoxicity of deoxynivalenol (DON) as well as the induction of cytokines and related genes was investigated in porcine pulmonary alveolar macrophages (PAM) in absence or presence of lipopolysaccharides (LPS). IC₂₀ values were 1.1, 0.4 and 1.0 μM DON in the MTT, neutral red and alamar blue assay, respectively, and did not differ significantly in the presence of LPS. The mRNA expression of tumour necrosis factor (TNF)-α peaked at 3 h, whereas LPS and DON showed synergistic effects resulting in an approximately 20-fold increase at 500 nM DON as compared to untreated controls. The supernatant concentrations of TNF-α showed similar synergistic effects. The expression of interleukin (IL)-1β was significantly induced by DON (except for 12 h) and LPS. An induction of the mRNA expression of IL-6 by DON was evident only at 3 h, whereas the supernatant concentrations of LPS stimulated PAM incubated with 500 nM DON were significantly decreased at most time points. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression did not seem to contribute to the effects of DON in porcine macrophages. The results of the present investigation suggest a contribution of cytokines, especially TNF-α and IL-1β, induced by DON in porcine macrophages to the effects observed in vivo.     

Benzo[a]pyrene modulation of acute immunologic responses in red Sea bream pretreated with lipopolysaccharide

The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[a]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM‐hepc) gene and the activities of some immune‐associated parameters in the lipopolysaccharide (LPS)‐challenged red sea bream (Pagrus major). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major. However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 μg L^?1) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM‐hepc mRNA expression at 3, 6, and 12‐h post‐LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM‐hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM‐hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS‐challenged P. major; conversely BaP exposure could enhance the mRNA expression of PM‐hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune‐associated parameters. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 517–525, 2014.