MicroRNA-590 Inhibits Lipoprotein Lipase Expression and Prevents Atherosclerosis in apoE Knockout Mice

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE^−/−) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE^−/− mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE^−/− mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion.     

Daxx通过上调caveolin-1的表达介导Ox-LDL诱导巨噬细胞胆固醇蓄积和凋亡

为探讨Daxx对氧化型低密度脂蛋白(oxidized low-density lipoprotein,Ox-LDL)诱导巨噬细胞胆固醇蓄积和凋亡的介导作用及其可能的分子机制,用高效液相色谱法检测细胞内胆固醇含量,油红O染色观察细胞内脂滴的形成情况,流式细胞术和吖啶橙/溴化乙锭(AO/EB)染色法研究Ox-LDL对细胞凋亡的影响,Real time RT-PCR检测细胞内Daxx mRNA的表达水平,Western blot检测caveolin-1蛋白的表达,用特异性siRNA沉默Daxx在RAW264.7 细胞中的表达．Ox-LDL上调Daxx mRNA和caveolin-1的表达、增加细胞内胆固醇含量、促使RAW264.7细胞凋亡,用特异性siRNA干扰Daxx在RAW264.7细胞中的表达能降低caveolin-1的表达、减少细胞内胆固醇含量、以及抑制细胞凋亡．上述结果表明,Daxx对Ox-LDL诱导RAW264.7巨噬细胞胆固醇蓄积和凋亡具有介导作用,这一作用可能与Daxx上调caveolin -1的表达有关．     

NEAT1 contributes to ox-LDL-induced inflammation and oxidative stress in macrophages through inhibiting miR-128

Long noncoding RNAs (lncRNA) have been recognized as significant regulators in the progression of atherosclerosis (AS). Oxidized low-density lipoprotein (ox-LDL) can induce macrophage inflammation and oxidative stress, that serves important roles in AS. However, the exact function of lncRNA NEAT1 and its possible molecular mechanism in AS remain unclear. Here, we concentrated on the roles and molecular mechanisms of NEAT1 in AS development. In our current study, we observed that NEAT1 was elevated by ox-LDL in a dose-dependent and time-dependent manner. RAW264.7 cell survival was greatly enhanced, and cell apoptosis was significantly inhibited by LV-shNEAT1 transfection. In addition, knockdown of NEAT1 in RAW264.7 cells repressed CD36 expression and foam cell formation while NEAT1 overexpression shown an opposite process. Moreover, NEAT1 downregulation inhibited inflammation molecules including IL-6, IL-1β, and TNF-α. Meanwhile, silencing of NEAT1 can also suppress reactive oxygen species (ROS) and malondialdehyde (MDA) levels with an enhancement of superoxide dismutase (SOD) activity in RAW264.7 cells. MicroRNAs are some short RNAs, and they can regulate multiple biological functions in many diseases including AS. Here, we found that miR-128 expression was remarkably decreased in ox-LDL-incubated RAW264.7 cells. Interestingly, miR-128 mimics was able to reverse AS-correlated events induced by overexpression of NEAT1. By using bioinformatics analysis, miR-128 was predicted as a target of NEAT1 and the correlation between them was validated in our study. Taken these together, it was implied that NEAT1 participated in ox-LDL-induced inflammation and oxidative stress in AS development through sponging miR-128.     

miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H(2)O(2)) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H(2)O(2). These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR-21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway.     

High fat diet induced downregulation of microRNA-467b increased lipoprotein lipase in hepatic steatosis

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation and is presently the most common chronic liver disease. However, the mechanisms underlying the development of steatosis remain unclear. MicroRNAs (miRNAs) are small non-coding RNAs that modulate a variety of biological functions. We have investigated the role of miRNA in the development of steatosis. We found that miR-467b expression is significantly downregulated in liver tissues of high-fat diet fed mice and in steatosis-induced hepatocytes. The downregulation of miR-467b resulted in the upregulation of hepatic lipoprotein lipase (LPL), the direct target of miR-467b. Moreover, the interaction between miR-467b and LPL was associated with insulin resistance, a major cause of NAFLD. These results suggest that downregulation of miR-467b is involved in the development of hepatic steatosis by modulating the expression of its target, LPL.     

Nobiletin reduces LPL-mediated lipid accumulation and pro-inflammatory cytokine secretion through upregulation of miR-590 expression

Nobiletin has protective effects on cardiovascular diseases, but the mechanism is not clear. In this study, we examined whether nobiletin affects the expression of miR-590/LPL and its relative effects on lipid accumulation and pro-inflammatory cytokine secretion in human THP-1 macrophages. RT-qPCR analysis showed that nobiletin increased the expression of miR-590. Western blot analysis showed that nobiletin-suppressed LPL expression was enhanced by miR-590 mimic and abrogated by miR-590 inhibitor. Oil Red O staining and high-performance liquid chromatography assays showed that nobiletin attenuated lipid accumulation in macrophages. Treatment with nobiletin and miR-590 mimic decreased cellular lipid accumulation, whereas treatment with miR-590 inhibitor increased cellular lipid accumulation. ELISA illustrated that nobiletin alleviated pro-inflammatory cytokine secretion in macrophages as measured by, which was reduced by miR-590 mimic and increased by miR-590 inhibitor. In conclusion, nobiletin may alleviate lipid accumulation and secretion of pro-in?ammatory cytokines by enhancing the inhibitory effect of miR-590 on LPL expression, suggesting a promising strategy for potential drug development for atherosclerosis.     

Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.     

Anti-Inflammatory Activity of S. Marianum and N. Sativa Extracts on Macrophages

Background:Nigella sativa (N. sativa) and Silybum marianum (S. marianum) are used to regulate macrophage polarization in lipopolysaccharide-induced RAW 264.7 cells and thioglycollate-elicited peritoneal inflammation.Methods:Cytotoxicity assays and acute toxicity tests were performed to investigate the safe dose and toxicity of the prepared extracts. Also, nitric oxide production was determined by Griess assay on RAW264.7 and peritoneal macrophage supernatants. After RNA extraction from macrophages, real-time PCR was performed to measure the relative gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β, and IL-10. Finally, regulatory T cells (Treg cells) were counted by flow cytometry.Results:S. marianum methanolic extract (SME), N. sativa ethanolic extract (NEE), and their mixture (SME+NEE) decreased NO levels significantly in RAW264.7 and peritoneal murine macrophages. N. sativa ethanolic extract significantly increased IL-10 gene expression and significantly decreased IL-6 and TNF-α expression in RAW264.7 cells. In mixture-treated peritoneal macrophages, IL-10 and TGF-β expression were significantly increased, while IL-6 and TNF-α were significantly decreased. Also, the percentage of Treg cells was significantly greater in the mixture-treated cells than in controls.Conclusion:These results suggest that an SME and NEE mixture has anti-inflammatory and immunomodulatory activities and may be useful in the treatment of diseases of immunopathologic origin characterized by macrophage hyperactivation.Key Words: Cytokine, Inflammation, Nigella sativa, Nitric oxide, Silybum marianum     

Oxidized lipoprotein induces the macrophage ascorbate transporter (SVCT2): Protection by intracellular ascorbate against oxidant stress and apoptosis

To assess whether ascorbic acid decreases the cytotoxicity of oxidized human low density lipoprotein (oxLDL) in cells involved in atherosclerosis, its interaction with oxLDL was studied in murine RAW264.7 macrophages. Macrophages took up ascorbate to millimolar intracellular concentrations and retained it with little loss over 18 h in culture. Culture of the macrophages with oxLDL enhanced ascorbate uptake. This was associated with increased expression of the ascorbate transporter (SVCT2), which was prevented by ascorbate and by inhibiting the NF-κB pathway. Culture of RAW264.7 macrophages with oxLDL increased intracellular dihydrofluorescein oxidation and lipid peroxidation, both of which were decreased by intracellular ascorbate. Ascorbate also protected the cells against oxLDL-induced cytotoxicity and apoptosis, but it did not affect macrophage accumulation of lipid from oxLDL or oxLDL-induced increases in macrophage cytokine secretion. These results suggest that ascorbate protects macrophages against oxLDL-induced oxidant stress and subsequent apoptotic death without impairing their function.     

Immunostimulatory effect by aqueous extract of <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.     

广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7免疫调节作用受体TLR4和TLR2的影响

确定广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7的免疫调节作用受体,探索广叶绣球菌β-D-葡聚糖的免疫调节机制。采用MTT法测定不同浓度广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7增殖活力的影响,筛选出促进巨噬细胞增殖能力最强的浓度。用筛选出的β-D-葡聚糖浓度作用巨噬细胞RAW264.7;TLR4抗体和TLR2抗体分别作用巨噬细胞RAW264.7 1h,再用含有β-D-葡聚糖的细胞培养液培养。收集细胞培养上清和细胞,检测细胞培养上清中NO、IL-6、TNF-α、IFN-β的生成量;提取细胞内总RNA,采用RT-PCR测定巨噬细胞TLR4 mRNA表达量;提取巨噬细胞总蛋白,采用蛋白免疫印迹western blot测定TLR4的蛋白表达。广叶绣球菌β-D-葡聚糖能够促进巨噬细胞RAW264.7增殖,增加NO、IL-6、TNF-α、IFN-β的生成量,提高TLR4 mRNA表达和蛋白表达,差异极显著（P<0.01）。TLR4抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量明显下降,差异极显著（P<0.01）。TLR2抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量下降,但差异不显著。广叶绣球菌β-D-葡聚糖可以通过细胞表面受体TLR4激活信号转导通路,增强下游细胞因子的释放,从而调节巨噬细胞RAW264.7的免疫功能。TLR2可能不是广叶绣球菌β-D-葡聚糖的免疫受体。     

Histone deacetylase inhibition by sodium valproate regulates polarization of macrophage subsets

Recent studies suggest that change of macrophage phenotype (M1/M2) is associated with autoimmune diseases. Sodium valproate (VPA) is a class I histone deacetylase (HDAC) inhibitor, which has immunomodulatory function in graft-versus-host disease. However, its impact on macrophage polarization has not been defined. We evaluated the effects of VPA on both mouse macrophage cell line RAW264.7 and primary mouse bone marrow macrophages (BMMs). Exposure to VPA significantly repressed the production of interleukin 12 (IL-12), and tumor necrosis factor α by lipopolysaccharide (LPS)-induced macrophage activation, in contrast, promoted IL-10 expression. VPA also affected the costimulatory molecule expression on LPS-stimulated RAW264.7 and BMMs (downregulation of CD40 and CD80, and upregulation of CD86). Specifically, VPA inhibited macrophage-mediated T helper 1 (Th1) effector but enhanced Th2 effector cell activation. Together, our preclinical study demonstrates that VPA significantly affects the phenotype and function of macrophage, indicating an important role of HDAC activity in immune regulation and inflammation. It also provides a rationale to evaluate VPA activity for the treatment of macrophage dysfunction-associated diseases.     

黄芪甲苷对马兜铃酸诱导的巨噬细胞M1型极化的作用及机制研究

目的:探讨黄芪甲苷对马兜铃酸诱导的RAW264.7细胞向M1型极化的影响,并初步探索其可能的作用机制.方法:分别采用马兜铃酸和脂多糖(LPS)刺激RAW264.7细胞24h,伴或不伴黄芪甲苷进行药物干预处理.采用细胞计数检测试剂盒-8(CCK8)检测细胞活性变化,流式细胞仪检测巨噬细胞分型,酶联免疫吸附试验(ELISA...     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded RNA and viral infection

In this study the regulation of macrophage expression of cyclooxygenase-2 (COX-2) in response to dsRNA and virus infection was examined. Treatment of RAW 264.7 macrophages with dsRNA results in COX-2 mRNA accumulation and protein expression and the production of PGE(2). Similar to dsRNA, encephalomyocarditis virus (EMCV) infection of RAW 264.7 cells stimulates COX-2 expression and PGE(2) accumulation. The dsRNA-dependent protein kinase (PKR), which has been shown to participate in the regulation of gene expression in response to dsRNA and virus infection, does not appear to participate in the regulation of COX-2 expression by macrophages. Expression of dominant negative mutants of PKR in RAW 264.7 cells fails to attenuate dsRNA- and EMCV-induced COX-2 expression or PGE(2) production. Furthermore, dsRNA and EMCV stimulate COX-2 expression and PGE(2) accumulation to similar levels in macrophages isolated from wild-type and PKR-deficient mice. Recently, a novel PKR-independent role for the calcium-independent phospholipase A(2) (iPLA(2)) in the regulation of inducible NO synthase expression by macrophages in response to virus infection has been identified. The selective iPLA(2) suicide substrate inhibitor bromoenol lactone prevents dsRNA- and EMCV-stimulated inducible NO synthase expression; however, bromoenol lactone does not attenuate dsRNA- or EMCV-induced COX-2 expression by macrophages. In contrast, inhibition of NF-kappaB activation prevents dsRNA-stimulated COX-2 expression and PGE(2) accumulation by macrophages. These findings indicate that virus infection and treatment with dsRNA stimulate COX-2 expression by a mechanism that requires the activation of NF-kappaB and that is independent of PKR or iPLA(2) activation.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.     

microRNA-17-5p Modulates Bacille Calmette-Guerin Growth in RAW264.7 Cells by Targeting ULK1

To explore the potential roles of miRNAs in controlling the survival of mycobacteria in macrophages, miR-17-5p in the regulation of Bacillus Calmette-Guérin(BCG)growth in the macrophage RAW264.7 cells was interrogated. Our results reveal that an infection of BCG shows a time-dependent up-regulation of miR-17-5p in RAW264.7 cells in early phase; importantly, excessive expression of miR-17-5p in these cells exhibits an increased propagation of intracellular BCG. Mechanistically, the Unc-51 like autophagy activating kinase 1 (ULK1), an initial molecular of autophagy are identified as novel target of miR-17-5p, the miR-17-5p is capable of targeting down-regulating the expression of ULK1 protein. In addition, an overexpression of miR-17-5p in RAW264.7 cells is correlated with repression of ULK1 and the autophagosome related proteins LC3I/II. These results imply that miR-17-5p may be able to arrest the maturation of mycobacterial phagosomes in part by targeting ULK1, subsequently reduces the ability of host cells to kill intracellular BCG.     

miR-140-3p suppresses the proliferation and migration of macrophages

Macrophages benefit myelin debris removal, blood vessel formation, and Schwann cell activation following peripheral nerve injury. Identifying factors that modulate macrophage phenotype may advantage the repair and regeneration of injured peripheral nerves. microRNAs (miRNAs) are important regulators of many physiological and pathological processes, including peripheral nerve regeneration. Herein, we investigated the regulatory roles of miR-140-3p, a miRNA that was differentially expressed in injured rat sciatic nerves, in macrophage RAW264.7 cells. Observations from EdU proliferation assay demonstrated that elevated miR-140-3p decreased the proliferation rates of RAW264.7 cells while suppressed miR-140-3p increased the proliferation rates of RAW264.7 cells. Transwell-based migration assay showed that up-regulated and down-regulated miR-140-3p led to elevated and reduced migration abilities, respectively. However, the abundances of numerous phenotypic markers of M1 and M2 macrophages were not significantly altered by miR-140-3p mimic or inhibitor transfection. Bioinformatic analysis and miR-140-3p-induced gene suppression examination suggested that Smad3 might be the target gene of miR-140-3p. These findings illuminate the inhibitory effects of miR-140-3p on the proliferation and migration of macrophages and contribute to the cognition of the essential roles of miRNAs during peripheral nerve regeneration.