miR-125b-5p促进分枝杆菌在宿主细胞和小鼠体内存活的研究

通过观察miR-125b-5p对分枝杆菌在宿主细胞和小鼠体内存活情况的影响,探究其在抗结核免疫过程中的作用。采用不同培养基对分枝杆菌进行培养并计数;以1640培养基加10%胎牛血清培养所有实验用细胞。将终浓度50 nmol/L的miR-125b-5p 模拟物、miR-125b-5p 抑制剂及磷酸盐缓冲液(PBS)对照加入细胞后,在不同时间点收集细胞。用分枝杆菌分别感染宿主细胞(A549、THP-1和RAW264.7)以及C57BL/6小鼠。采用定量聚合酶链反应检测miR-125b-5p的表达量。结果miR-125b-5p在分枝杆菌感染的多种宿主细胞及小鼠中都显著上调表达,其中小鼠肺部的表达量提高了约15倍。分别转染模拟物和抑制剂后,再用分枝杆菌感染细胞,结果发现miR-125b-5p可促进分枝杆菌在宿主细胞内的生长。当miR-125b-5p抑制剂注射到卡介苗(BCG)感染的小鼠体内时,小鼠体内的细菌载量显著降低(P<0.05)。本研究证明miR-125b-5p可调控分枝杆菌在宿主细胞及小鼠体内的生长,在抗结核免疫过程中发挥了重要作用。进一步对其作用机制的深入研究将为临床结核病的治疗提供理论指导。     

TGF-β-induced epithelial-mesenchymal transition of A549 lung adenocarcinoma cells is enhanced by pro-inflammatory cytokines derived from RAW 264.7 macrophage cells

Cancer cells undergo epithelial-mesenchymal transition (EMT) during invasion and metastasis. Although transforming growth factor-β (TGF-β) and pro-inflammatory cytokines have been implicated in EMT, the underlying molecular mechanisms remain to be elucidated. Here, we studied the effects of proinflammatory cytokines derived from the mouse macrophage cell line RAW 264.7 on TGF-β-induced EMT in A549 lung cancer cells. Co-culture and treatment with conditioned medium of RAW 264.7 cells enhanced a subset of TGF-β-induced EMT phenotypes in A549 cells, including changes in cell morphology and induction of mesenchymal marker expression. These effects were increased by the treatment of RAW 264.7 cells with lipopolysaccharide, which also induced the expression of various proinflammatory cytokines, including TNF-α and IL-1β. The effects of conditioned medium of RAW 264.7 cells were partially inhibited by a TNF-α neutralizing antibody. Dehydroxy methyl epoxyquinomicin, a selective inhibitor of NFκB, partially inhibited the enhancement of fibronectin expression by TGF-β, TNF-α, and IL-1β, but not of N-cadherin expression. Effects of other pharmacological inhibitors also suggested complex regulatory mechanisms of the TGF-β-induced EMT phenotype by TNF-α stimulation. These findings provide direct evidence of the effects of RAW 264.7-derived TNF-α on TGF-β-induced EMT in A549 cells, which is transduced in part by NFκB signalling.     

miR-181a Regulates Inflammation Responses in Monocytes and Macrophages

miR-181a has been presumed to target the 3′-untranslated regions (3′-UTR) of IL1a based on software predictions. miR-181a and IL1a have opposite expression levels in monocytes and macrophages in the inflammatory state. This led us to suspect that mir-181a has an important function in regulating inflammatory response by targeting IL1a. Fluorescence reporter assays showed that miR-181a effectively binds to the 3′-UTR of IL1a. The anti-inflammatory functions of miR-181a were investigated in lipopolysaccharides (LPS)-induced Raw264.7 and phorbol 12-myristate 13-acetate (PMA)/LPS-induced THP-1 cells. We found that miR-181a mimics significantly lowered IL1a expression levels in these cells and, interestingly, miR-181a inhibitors reversed this decrease. In addition, miR-181a mimics significantly inhibited increase in the levels of inflammatory factors (IL1b, IL6, and TNFa) in these cells. Furthermore, miR-181a mimics and inhibitors decreased and increased, respectively, production of reactive oxygen species in PMA/LPS-induced THP-1 cells. These results indicate that miR-181a regulates inflammatory responses by directly targeting the 3′-UTR of IL1a and down-regulating IL1a levels. Interestingly, we found that miR-181a inhibited production of inflammatory factors even in IL1a-induced THP-1 cells, suggesting that the anti-inflammatory effects of miR-181a possibly involves other targets in addition to IL1a. Thus, we provide the first evidence for anti-inflammatory effects of miR-181a mediated at least in part by down-regulating IL1a.     

Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

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-34a inhibits lipopolysaccharide-induced inflammatory response through targeting Notch1 in murine macrophages

Inflammatory responses are complex events occurring when the host immune system fights against invading pathogens, which are double-edged swords requiring appropriate control. MicroRNAs (miRNAs), emerging as a new layer of gene-regulation mechanism, have been reported to have crucial effects on inflammation. In the current study, we identified miR-34a, previously known for its potent tumor suppressive role, to be a novel inflammation regulator. We found that the expression of miR-34a was downregulated in macrophages after lipopolysaccharide (LPS) stimulation. MiR-34a mimics decreased, while the inhibition of miR-34a increased, the expression of inflammatory cytokines tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) in LPS treated RAW264.7 cells. Bioinformatics predictions revealed a potential binding site of miR-34a in 3' untranslated region (UTR) of Notch1 and it was further confirmed by luciferase assay. Moreover, both the mRNA and protein level of Notch1 were downregulated by miR-34a in RAW264.7. Subsequently, knockdown of Notch1 with either genetic or pharmacological inhibition exhibited similar effects as miR-34a mimics on LPS-induced macrophage inflammatory response. Furthermore, the NF-κB activation induced by LPS was also significantly suppressed by miR-34a. These results together identify, for the first time, miR-34a as a negative regulator in LPS-induced inflammation at least partially by targeting Notch1. Besides extending the knowledge of miR-34a from tumor suppressor to inflammation regulator, this study also provides an implication that compounds which can enhance miR-34a expression or miR-34a itself may hold a promise in anti-inflammatory drugs development.     

miR-135a inhibition protects A549 cells from LPS-induced apoptosis by targeting Bcl-2

Acute lung injury (ALI) is a severe clinical condition with high morbidity and mortality. Apoptosis is a key pathologic feature of ALI, and Bcl-2 plays an important role during the pathogenesis of ALI via the regulation of apoptosis. However, the regulation of Bcl-2 during ALI, particularly through microRNAs, remains unclear. We hypothesize that certain miRNAs may play deleterious or protective roles in ALI via the regulation of Bcl-2. The LPS stimulation of A549 cells was used to mimic ALI in vitro. First, we confirmed that Bcl-2 is involved in LPS-induced apoptosis in A549 cells. Then, bioinformatic analyses and quantitative real-time polymerase chain reaction assays were performed to screen for miRNAs targeting Bcl-2. We observed that miR-135a was markedly increased in LPS-challenged A549 cells. miR-135a inhibition markedly restored Bcl-2 expression and protected A549 cells from LPS-induced apoptosis. Furthermore, bioinformatic analysis and luciferase activity assays were conducted to confirm that miR-135a binds directly to the 3′-untranslated region of Bcl-2 and suppresses its expression. Interestingly, the inhibition of miR-135a did not attenuate apoptosis under LPS-treated conditions when Bcl-2 was knocked down. Therefore, we suggest that miR-135a regulation of LPS-induced apoptosis in A549 cells may depend in part on the regulation of Bcl-2. The miR-135a/Bcl-2 signaling pathway may be a novel therapeutic target for the prevention of ALI.     

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.     

Anti-inflammatory effect of roasted licorice extracts on lipopolysaccharide-induced inflammatory responses in murine macrophages

Licorice, the roots of Glycyrrhiza inflata, is used by practitioners of alternative medicine to treat individuals with gastric or duodenal ulcers, bronchitis, cough, arthritis, adrenal insufficiency, and allergies. We investigated the anti-inflammatory properties of 4 licorice extracts: extracts of roasted licorice obtained by ethanol (rLE) or water extraction (rLW) and extracts of raw licorice obtained by ethanol (LE) or water extraction (LW). rLE demonstrated strong anti-inflammatory activity through its ability to reduce nitric oxide and prostaglandin E(2) production in the LPS-stimulated mouse macrophage cell, RAW264.7. It also inhibited the production of pro-inflammatory cytokines and CD14 expression on the LPS-stimulated RAW264.7 cells. Further study indicated that LPS-induced degradation and phosphorylation of Ikappa-Balpha, along with DNA-binding of NF-kappaB, was significantly inhibited by rLE exposure in RAW264.7 cells. In the murine model, we found that in vivo exposure to rLE-induced an increase in the survival rate, reduced plasma levels of TNF-alpha and IL-6, and increased IL-10 production in LPS-treated mice. Collectively, these data suggest that the use of rLE may be a useful therapeutic approach to various inflammatory diseases.     

TLR-4/microRNA-125a/NF-κB signaling modulates the immune response to Mycobacterium tuberculosis infection

Tuberculosis (TB), caused by Mycobacterium tuberculosis, could lead to kinds of clinical disorders and remains a leading global health problem, resulting in great morbidity and mortality worldwide. Previous studies have firmly demonstrated that M. tuberculosis (M.tb) has evolved to utilize different mechanisms to evade or attenuate the host immune response, such as regulation of immune-related genes by modulation of miRNAs of host or bacteria. However, the knowledge of functions of miRNAs during M.tb infection remains limited. Here, we reported that a host microRNA, miR-125a, was significantly up-regulated by M.tb infection in both RAW264.7 and THP-1cells, in a TLR4 signaling-dependent manner. Subsequently, our results demonstrated that miR-125a was a negative regulator of NF-kB pathway by directly targeting TRAF6, resulting in the suppression of cytokines, attenuation of immune response and promotion of M.tb survival. Taken together, our findings provide a novel detailed molecular mechanism in which miR-125a was enhanced to inhibit inflammatory cytokines secretion and attenuate the immune response during M.tb infection in RAW264.7 and THP-1 cells, and suggest an intrinsic a promising anti-M.tb therapeutic target.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H₂O₂-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.     

MicroRNA-467b targets LPL gene in RAW 264.7 macrophages and attenuates lipid accumulation and proinflammatory cytokine secretion

LPL (lipoprotein lipase) is a rate-limiting enzyme involved in the hydrolysis of triglycerides. Previous studies have shown that microRNA (miR)-467b regulates hepatic LPL expression and plays a role in the progression of steatosis or abnormal lipid retention in obese mice. Macrophage-derived LPL has been shown to promote atherosclerosis. However, if miR-476b influences macrophage LPL expression and the subsequent effects are unknown. Here, we utilized oxLDL-treatment RAW 264.7 macrophages that were transfected with miR-467b mimics or inhibitors to investigate the potential roles of macrophage miR-476b. We found that miR-467b significantly decreased lipid accumulation and IL-6, IL-1β, TNF-α and MCP-1 secretions. Furthermore, our studies suggested an additional explanation for the regulatory mechanism of miR-467b on its functional target, LPL in RAW 264.7 macrophages. Thus, our findings indicate that miR-467b may regulate lipid accumulation and proinflammatory cytokine secretion in oxLDL-stimulated RAW 264.7 macrophages by targeting the LPL gene.     

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.