Dose-dependent Manner In RAW264 Research Articles

Megastigmane sesquiterpenoids from whole plants of Viola kunawurensis

Investigation on the chemical constituents of Viola kunawurensis resulted in the isolation of seven undescribed megastigmane sesquiterpenoids including four bicyclic megastigmane glucosides, kunawuronoside A-D, two megastigmane glucosides, kunawuronoside E-F, and a megastigmane, kunawurone A, together with ten known megastigmane sesquiterpenoids. Their structures were established by comprehensive 1D, 2D-NMR and HRESIMS analyses, and their absolute configurations were determined by comparing their calculated ECD data with the experimental ones. Evaluations of the anti-inflammatory activity revealed that kunawuronoside A-D and compounds 14–15 inhibited COX-2 expression with inhibition rates ranging from 36.7% to 58.5%, while the NO production induced by lipopolysaccharide (LPS) was suppressed by the kunawuronoside A-D in a dose-dependent manner in RAW264.7 macrophage cell line.

Silk sericin application increases bone morphogenic protein-2/4 expression via a toll-like receptor-mediated pathway

Bone morphogenic protein-2/4 (BMP-2/4) is an osteoinductive protein that accelerates osteogenesis when administered to bony defects. Sericin is produced by silkworms, and has a biological activity that differs depending on the degumming method used. Our results indicated that the high molecular weight fraction of silk sericin (MW > 30 kDa) obtained via sonication had a more abundant β-sheet structure than the low molecular weight fraction. Administration of the β-sheet structure silk sericin increased BMP-2/4 expression in a dose-dependent manner in RAW264.7 cells and human monocytes. This sericin increased the expression levels of toll-like receptor (TLR)-2, TLR-3, and TLR-4 in RAW264.7 cells. Application of a TLR-2 antibody or TLR pathway blocker decreased BMP-2/4 expression following sericin administration. In the animal model, the bone volume and BMP-2/4 expression were higher in rats treated with a sericin-incorporated gelatin sponge than in rats treated with a gelatin sponge alone or a sponge-incorporated with denatured sericin. In conclusion, sericin with a more abundant β-sheet structure increased BMP-2/4 expression and bone formation better than sericin with a less abundant β-sheet structure.

HSPA12B Secreted by Tumor-Associated Endothelial Cells Might Induce M2 Polarization of Macrophages via Activating PI3K/Akt/mTOR Signaling.

PurposeThe intratumoral microenvironment of head and neck squamous cell carcinoma (HNSC) is highly immunosuppressive. In this study, we explored the potential functional role of HSPA12B secreted by tumor-associated endothelial cells (TECs) in M2 polarization of macrophages.Materials and MethodsBulk-seq data from TCGA-HNSC and single-cell RNA-seq data from GSE103322 (with over 5000 cells from 18 primary HNSC cases) were used for bioinformatic analysis. RAW264.7 cell line was used for in vitro studies.ResultsTECs in HNSC had significantly higher expression and secretion of HSPA12B, compared to normal human umbilical vein endothelial cells (HUVECs). Exogenous HSPA12B treatment increased the expression of M2 macrophage marker CD163 and CD206 on RAW264.7 cells in a dose-dependent manner but had no significant influence on CD86, an M1 macrophage marker. OLR1, a known receptor of HSP70 proteins, was specifically expressed in tumor-associated macrophages (TAMs) in HNSC. OLR1 knockdown significantly impaired HSPA12B uptake by RAW264.7 cells and weakened HSPA12B-induced CD163 and CD206 upregulation. HSPA12B treatment increased the expression of p-PI3K, p-Akt and p-mTOR in a dose-dependent manner in RAW264.7 cells. OLR1 inhibition and LY294002 treatment significantly weakened the effects HSPA12B on activating the PI3K/Akt/mTOR signaling and M2 marker expression.ConclusionBased on these findings, we speculated that aberrantly expressed and secreted HSPA12B by TECs could be taken by macrophages partly via OLR1, leading to subsequent activation of the PI3K/Akt/mTOR signaling pathway and elevated expression of M2 markers. This mechanism shows a novel cross-talk between TECs and TAMs, which contributes to the intratumoral immunosuppressive microenvironment.

Pirfenidone prevents and reverses hepatic insulin resistance and steatohepatitis by polarizing M2 macrophages

Nonalcoholic steatohepatitis (NASH) is associated with lipotoxic liver injury, leading to insulin resistance, inflammation, and fibrosis. Despite its increased global incidence, very few promising treatments for NASH are available. Pirfenidone is an antifibrotic agent used to treat pulmonary fibrosis; it suppresses the pulmonary influx of T cells and macrophages. Here, we investigated the effect of pirfenidone in a mouse model of lipotoxicity-induced NASH via a high-cholesterol and high-fat diet. After 12 weeks of feeding, pirfenidone administration attenuated excessive hepatic lipid accumulation and peroxidation by reducing the expression of genes related to lipogenesis and fatty acid synthesis and enhancing the expression of those related to fatty acid oxidation. Flow cytometry indicated that pirfenidone reduced the number of total hepatic macrophages, particularly CD11c+CD206–(M1)-type macrophages, increased the number of CD11c–CD206+(M2)-type macrophages, and subsequently reduced T-cell numbers, which helped improve insulin resistance and steatohepatitis. Moreover, pirfenidone downregulated the lipopolysaccharide (LPS)-induced mRNA expression of M1 marker genes and upregulated IL-4-induced M2 marker genes in a dose-dependent manner in RAW264.7 macrophages. Importantly, pirfenidone reversed insulin resistance, hepatic inflammation, and fibrosis in mice with pre-existing NASH. These findings suggest that pirfenidone is a potential candidate for the treatment of NASH.

4-Hexylresorcinol and silk sericin increase the expression of vascular endothelial growth factor via different pathways

Angiogenesis plays an important role in active inflammation and wound healing. Our results showed that silk sericin and 4-hexylresorcinol (4HR) increased vascular endothelial growth factor (VEGF) expression in a dose-dependent manner in RAW264.7 cells. Unlike 4HR, silk sericin increased the expression of hypoxia inducible factor-1α (HIF-1α) and HIF-2α. Pretreatment with an HIF inhibitor decreased the sericin-induced increase in VEGF expression. However, the HIF inhibitor did not affect the 4HR-induced increase in VEGF expression. An inhibitor of matrix metalloproteinase (MMP) declined the 4HR-induced increase in VEGF expression. Silk sericin increased production of reactive oxygen species (ROS), whereas 4HR decreased ROS. M1 markers were increased by silk sericin treatment, and M2 markers were increased by 4HR treatment. VEGF and angiogenin expression were higher in rats treated with a 4HR-incorporated silk mat than in rats treated with a silk mat alone. In conclusion, silk sericin and 4HR increased VEGF expression in RAW264.7 cells via HIF-mediated and MMP-mediated pathways, respectively. Silk sericin exerted like pro-oxidant effects and 4HR exerted anti-oxidant effects. Rats treated with a 4HR-incorporated silk mat showed higher levels of VEGF and angiogenin than those treated with a silk mat alone.

Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var peltata

ABSTRACTThis work aimed to evaluate the nutritional composition and immunostimulatory activity of an edible green alga Caulerpa racemosa var peltata collected from South China Sea. The results indicated that this alga contained the total carbohydrates (71.67%, DW), crude protein (11.39%, DW), and crude lipid (1.03%, DW), and was rich in taurine (7.71 mg/100 g, DW), essential amino acids (40.07%), monounsaturated fatty acids (25.72%), and polyunsaturated fatty acids (26.33%). Moreover, water extraction of this alga (WEC) increased NO, ROS, TNF-α, and IL-6 in a dose-dependent manner in RAW264.7 macrophage cells. Furthermore, WEC contained the total sugar (34.19 ± 0.46 mg/g), total protein (6.76 ± 0.32 mg/g), total phenolic (1.93 ± 0.04 mg/g), and total flavonoid (4.80 ± 0.10 mg/g). In addition, the MSn analysis of WEC indicated that the presence of 19 compounds. Thus, these findings suggest that this alga may have a potential nutraceutical value.

Chikusetsusaponin V Inhibits LPS-Activated Inflammatory Responses via SIRT1/NF-κB Signaling Pathway in RAW264.7 Cells.

It is becoming increasingly accepted that macrophage activation play a crucial role in many diseases associated with chronic inflammation, such as metabolic disease, including atherosclerosis, obesity, and diabetes. Recent studies have indicated that the regulation of inflammation and energy metabolism are connected together through an antagonistic crosstalk between NF-κB and SIRT1 signaling pathways. In order to investigate anti-inflammatory drugs, we investigated whether SIRT1 is implicated in the lipopolysaccharide (LPS)-stimulated NF-κB signaling pathway in macrophage RAW264.7 cells when pretreated with chikusetsusaponin V (CsV). Griess assay and enzyme-linked immunosorbent assay were used to determine the effect of CsV on LPS-induced inflammatory cytokine secretion. Western blot was used to assess the effect of CsV on LPS-induced SIRT1 and Ac-NF-κB p65 expression. Results showed that CsV suppressed LPS-induced inflammatory cytokine NO, TNF-α, and IL-1β production in RAW264.7 cells. In addition, downregulation of SIRT1 and upregulation of Ac-NF-κB p65 induced by LPS were abolished by CsV in a dose-dependent manner in RAW264.7 cells. Meanwhile, inflammatory cytokines were regulated with CsV through SIRT1-Ac-NF-κB p65 signaling pathway. Therefore, our results demonstrate that CsV exerts an anti-inflammatory effect partly through SIRT1/NF-κB signaling pathways and SIRT1 may be a new target for anti-inflammation therapies.

Momordica charantia Inhibits Inflammatory Responses in Murine Macrophages via Suppression of TAK1.

Momordica charantia known as bitter melon is a representative medicinal plant reported to exhibit numerous pharmacological activities such as antibacterial, antidiabetic, anti-inflammatory, anti-oxidant, antitumor, and hypoglycemic actions. Although this plant has high ethnopharmacological value for treating inflammatory diseases, the molecular mechanisms by which it inhibits the inflammatory response are not fully understood. In this study, we aim to identify the anti-inflammatory mechanism of this plant. To this end, we studied the effects of its methanol extract (Mc-ME) on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Specifically, we evaluated nitric oxide (NO) production, mRNA expression of inflammatory genes, luciferase reporter gene activity, and putative molecular targets. Mc-ME blocked NO production in a dose-dependent manner in RAW264.7 cells; importantly, no cytotoxicity was observed. Moreover, the mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were decreased by Mc-ME treatment in a dose-dependent manner. Luciferase assays and nuclear lysate immunoblotting analyses strongly indicated that Mc-ME decreases the levels of p65 [a nuclear factor (NF)-[Formula: see text]B subunit] and c-Fos [an activator protein (AP)-1 subunit]. Whole lysate immunoblotting assays, luciferase assays, and overexpression experiments suggested that transforming growth factor [Formula: see text]-activated kinase 1 (TAK1) is targeted by Mc-ME, thereby suppressing NF-[Formula: see text]B and AP-1 activity via downregulation of extracellular signal-regulated kinases (ERKs) and AKT. These results strongly suggest that Mc-ME exerts its anti-inflammatory activity by reducing the action of TAK1, which also affects the activation of NF-[Formula: see text]B and AP-1.

β-elemene attenuates macrophage activation and proinflammatory factor production via crosstalk with Wnt/β-catenin signaling pathway

β-elemene, extracted from Rhizoma zedoariae, has been widely used as a traditional medicine for its antitumor activity against a broad range of cancers. However, the effect of β-elemene in inflammation disorders has yet to be determined. The present study was designed to investigate the anti-inflammatory effects and potential molecular mechanisms of β-elemene in lipopolysaccharide (LPS)-induced murine macrophage cells RAW264.7. We found that the production of pro-inflammatory mediators, including interleukin-6(IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), induced by LPS was significantly suppressed by β-elemene in a dose-dependent manner in RAW264.7 macrophage cell line. Also, β-elemene inhibited LPS-induced nitric oxide synthase (iNOS) and interleukin-10 (IL-10) expression by RAW264.7, which was related to the down-regulation of Wnt/β-catenin signaling pathway. Importantly, this study demonstrates that β-catenin was significantly inhibited by β-elemene, which appeared to be largely responsible for the down-regulation of Wnt/β-catenin signaling pathway. Accordingly, the deletion of β-catenin in primary macrophages reversed β-catenin-elicited inhibition of immune response. Furthermore, β-catenin expression and Wnt/β-catenin signaling pathway induced by LPS in RAW264.7 was also significantly inhibited by α-humulene, one isomeric sesquiterpene of β-elemene. α-humulene was also found to significantly inhibit LPS-induced production of proinflammatory cytokines. However, α-humulene showed more cytotoxic ability than β-elemene. Collectively, our data illustrated that β-elemene exerted a potent inhibitory effect on pro-inflammatory meditator and cytokines production via the inactivation of β-catenin, and also demonstrated the protective functions of β-elemene in endotoxin-induced inflammation. β-elemene may serve as potential nontoxic modulatory agents for the prevention and treatment of inflammatory diseases.

Bioactivity of bovine lung hydrolysates prepared using papain, pepsin, and Alcalase

Bovine lung tissue was hydrolyzed using three different proteases, papain, pepsin, or Alcalase, to generate hydrolysates. Hydrolysates showed little antioxidant activity in noncellular or cellular assays. The anti-inflammatory activity of hydrolysates was assessed in RAW264.7 macrophages and Jurkat T cells. Treatment with the Alcalase hydrolysate significantly decreased the production of the pro-inflammatory cytokines interleukin (IL)-6 and IL-1β in a dose dependent manner in RAW264.7 cells. Nitric oxide production also significantly decreased following treatment with this hydrolysate. The papain hydrolysate decreased IL-6, IL-1β, and NO production in RAW264.7 cells and IL-2 production in Jurkat T cells. However, the decrease was likely due to cytotoxicity of this hydrolysate toward these cell lines. The pepsin hydrolysate had no effect on cytokine production in RAW264.7 cells and only slightly decreased IL-2 production in Jurkat T cells. Practical applications Meat production generates large amounts of protein rich co-products, which are often discarded as waste or sold as low value animal feeds. The enzymatic hydrolysis of these protein co-products may produce hydrolysates that are capable of reducing oxidation or inflammation. Novel hydrolysates were generated from the hydrolysis of bovine lung and results from this study indicate that the hydrolysis of bovine lung using the commercially available protease Alcalase may have potential as an anti-inflammatory agent.

Bergapten prevents lipopolysaccharide-induced inflammation in RAW264.7 cells through suppressing JAK/STAT activation and ROS production and increases the survival rate of mice after LPS challenge

Bergapten (BG) is a cumarine-derivate compound in many medicinal plants. Here, in vitro and in vivo experimental results indicated that BG possesses anti-inflammatory properties, Based on this, we further investigated the precise molecular mechanisms of BG in LPS-stimulated inflammation response. Studies revealed that BG inhibited LPS-stimulated productions of TNF-α, IL-1β, IL-6, PGE2 and NO as well as the expression of iNOS and COX-2, and at the same time, it increased LPS-induced release of IL-10 in a dose-dependent manner in RAW264.7 cells. Mechanistically, BG suppressed the activations of JAK/STAT, but not that of MAPKs and NF-κB. In addition, BG, as an antioxidant, prevented the accumulation of ROS, which further exerted its anti-inflammatory function. In vivo researches revealed that BG decreased LPS-induced mortality in mice. In conclusions, BG may be a potential candidate for inflammation therapy via inhibiting JAK/STAT activation and ROS production in RAW264.7 cells.

Atorvastatin increases lipopolysaccharide-induced expression of tumour necrosis factor-α-induced protein 8-like 2 in RAW264.7 cells.

RAW264.7 cells are one of the major sources of productive inflammatory biomediators, including tumour necrosis factor-α (TNF-α) and interleukin (IL)-6. TNF-α-induced protein 8-like 2 (TIPE2) is an essential negative regulator of Toll-like and T-cell receptors, and the selective expression in the immune system prevents hyper-responsiveness and maintains immune homeostasis. The aim of the present study was to investigate whether atorvastatin upregulates the expression of TIPE2 and further regulates the inflammatory response and oxidation emergency response in RAW264.7 cells. RAW264.7 cells were incubated in Dulbecco’s modified Eagle’s medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. Following incubation, the medium was collected and the levels of TNF-α and IL-6 were measured using an enzyme-linked immunosorbent assay. The cells were harvested, and the mRNA and protein expression levels of TIPE2, macrophage migration inhibitory factor (MIF), IκB and nuclear factor (NF-κB)-κB were analysed using quantitative polymerase chain reaction and western blotting analysis, respectively, the expression of NOS, COX-2 and HO-1 protein were essayed by western blotting analysis, NO and ROS activities were determined. The results revealed that LPS increased the mRNA and protein expression levels of TIPE2, MIF and NF-κB, as well as the production of IL-6 and TNF-α, in a dose and time dependent manner in RAW264.7 cells. Meanwhile, LPS enhanced the expression of NOS and COX-2 protein, blocked HO-1 protein expression, increased NO and PGE2 production and ROS activity in a dose dependent manner in RAW264.7 cells. Atorvastatin significantly increased LPS induced expression of TIPE2, downregulated the expression of NOS, COX-2, MIF and NF-κB and the production of PGE2, NO, IL-6 and TNF-α in a time and dose dependent manner, and increased HO-1 protein expression, reduced ROS production in a dose dependent manner. The observations indicated that atorvastatin upregulated LPS induced expression of TIPE2 and consequently inhibited MIF, NF-κB, NOS and COX-2 expression and the production of NO, PGE2, TNF-α and IL-6, increased HO-1 expression, and inhibited ROS activity in cultured RAW264.7 cells.

JAK/STAT Pathway Plays a Critical Role in the Proinflammatory Gene Expression and Apoptosis of RAW264.7 Cells Induced by Trichothecenes as DON and T-2 Toxin

Deoxynivalenol (DON) and T-2 toxin commonly affect cells of the immune system and cause inflammation and apoptosis. Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is highly associated with inflammatory process and apoptosis and is worth investigating its role when cells were exposed to trichothecenes. The results showed that DON and T-2 upregulated the messenger RNA (mRNA) expressions of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, JAK1-2, STAT1-3, and suppressors of cytokine signaling members and activated the tyrosine phosphorylation of STAT1 and STAT3 with a dose-dependent manner in RAW264.7 cells. AG490 and Stattic, the specific inhibitors of JAK/STAT pathway, blocked the STAT1 and STAT3 tyrosine phosphorylation and decreased the gene expressions of proinflammatory cytokines induced by trichothecenes. Interestingly, the time when the mRNA levels of STAT1 and STAT3 were significantly upregulated was at 12 h, which was much later than the time when mitogen-activated protein kinase was activated, indicating that STATs might be the downstream targets of the trichothecenes. With the intervention of AG490 and Stattic, DON and T-2 toxin induced apoptosis in a strengthened way, with the loss of mitochondrial membrane potential and the decrease ratios of the B-cell leukemia/lymphoma 2 (Bcl-2)/bcl-2-associated X (Bax) and B-cell lymphoma-extra large (Bcl-xL)/Bax. After exposing to DON and T-2 toxin, cells exhibited G2/M and G0/G1 phase arrest, respectively. The increased mRNA expressions of STAT target genes p21 and cyclin D1 for DON and the increases in p21 mRNA and the decreases in cyclin D1 for T-2 toxin were observed. These results demonstrated for the first time that the activation of JAK/STAT might be a critical mediator to induce the inflammatory response and apoptosis in macrophage in response to trichothecenes.

Ethanol Extract of Elaeocarpus petiolatus Inhibits Lipopolysaccharide-Induced Inflammation in Macrophage Cells

Elaeocarpus petiolatus is known to exert active oxygen scavenging, anti-aging, and whitening actions. However, the biological effects of E. petiolatus on inflammation and the underlying mechanisms are yet to be established. In the present study, we investigated the anti-inflammatory effects of the ethanol extract from E. petiolatus (EPE) bark in murine Raw264.7 macrophages stimulated with lipopolysaccharide (LPS). EPE inhibited the production of PGE(2), TNF-α, and IL-1β in a dose-dependent manner in Raw264.7 cells stimulated with LPS. The decrease in PGE(2) production was correlated with reduced COX-2 expression. Furthermore, EPE suppressed the phosphorylation of extracellular signal-related kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 as well as translocation of the NF-κB p65 subunit from the cytosol to nucleus. Our results suggest that EPE exerts anti-inflammatory activity through inhibition of inflammatory mediators, such as PGE(2), TNF-α, and IL-1β, and downregulation of COX-2 via suppression of NF-κB translocation and phosphorylation of ERK, JNK, and p38 in LPS-stimulated Raw264.7 cells.

ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts

The binding of lipopolysaccharides (LPS) to macrophages results in inflammatory responses. In extreme cases it can lead to endotoxic shock, often resulting in death. A broad range of antioxidants, including tocopherols, can reduce LPS activity in vitro and in vivo. To elucidate the underlying mechanisms of their action, we investigated the effect of the sodium salt of γ-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinylglycine (ESeroS-GS), a novel α-tocopherol derivative, on LPS-induced inflammation in vitro and in vivo. ESeroS-GS reduced the transcription of TNF-α, IL-1β, IL-6 and iNOS genes in a dose-dependent manner in RAW264.7 macrophages, and inhibited the release of these inflammatory factors. In addition, ESeroS-GS inhibited LPS-induced mortality in a mouse sepsis model. Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that ESeroS-GS down-regulated the transcriptional activity of NF-κB. By analyzing the partitioning of CD14 and Toll-like receptor 4 (TLR-4) in cell membrane microdomains, we found that ESeroS-GS attenuates the binding of LPS to RAW264.7 cells via interfering with the relocation of CD14 and TLR-4 to lipid rafts, blocking the activation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and inhibiting the consequent phosphorylation of TAK1 and IKKα/β, which together account for the suppression of NF-κB activation. Taken together, our data suggest that ESeroS-GS can modulate LPS signaling in macrophages by impairing TLR-4 complex assembly via a lipid raft dependent mechanism.

Preparation, characterization, and antioxidant properties of gallic acid- grafted-chitosans

Gallic acid- grafted-chitosans (GA- g-chitosans) with four different grafting ratios were prepared by a free radical-induced grafting reaction in order to improve antioxidant and water-solubility. To verify the synthesis of GA- g-chitosans, 1H NMR and thin layer chromatography were employed, and the results revealed that GA was grafted onto the chitosan. The antioxidant properties of the GA- g-chitosans were evaluated using several in vitro models. GA- g-chitosan (I), which has the highest GA content, showed 92.26% scavenging activity against 2,2-diphenyl-1-picrylhydrazyl and 93.15% hydrogen peroxide scavenging activity at 50 μg/mL. GA- g-chitosan (I) was also showed higher reducing power compared to others. All GA- g-chitosans showed improved antioxidant capacities compared to plain chitosan treated in the same conditions without gallic acid grafting. Furthermore, the GA- g-chitosans also exhibited good cytocompatibility and effectively inhibited the formation of intracellular reactive oxygen species (ROS) in time- and dose-dependent manner in RAW264.7 macrophages.

Regulated expression of endothelial lipase in atherosclerosis

Endothelial lipase (EL) is a major determinant of HDL metabolism and associated with the development of atherosclerosis, however the regulated expression of EL in atherosclerosis is unclear. In this study, we investigated EL expression in rat atherosclerosis and explored the potential mechanisms regulating EL expression by employing LPS on Raw264.7 cells in vitro. Rat atherosclerosis model was established fed on high-cholesterol diet (HCD) combined with vitamin D(2) (VD). Western blotting and immunochemistry staining revealed that EL expression was increased in the aorta, especially the atherosclerotic lesions in HCD rats. LPS increased EL expression in a time and dose dependent manner in Raw264.7 cells and NFkappaB inhibitor, PDTC attenuated the effects of LPS on EL. EMSA revealed that LPS stimulated NFkappaB binding to EL promoter. In summary, EL was upregulated in rat atherosclerosis and LPS stimulates EL expression in vitro through NFkappaB activation.

Expression of PTEN and Akt phosphorylation in lipopolysaccharide‐treated NIH3T3 cells

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 microg/ml, 0-6h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.