Efficacy of Sishen Wan on dinitrobenzene sulfonic acid-induced ulcerative colitis and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB signal pathway.

OBJECTIVE: To investigate the therapeutic effect of Sishen Wan (, SSW) on ulcerative colitis (UC) induced by dinitrobenzene sulfonic acid and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB (TLR2/IRAK4/NF-κB) sig-naling pathway in colonic tissue. METHODS: In this study, 120 Sprague-Dawley rats were randomly divided into blank and model groups. The experimental UC model in rats was established by subcutaneous injection of hydrocortisone + senna gavage for 21 d + dinitrobenzene sulfonic acid (DNBS)/ ethanol solution enema. The successful model rats were randomly divided into the model group; mesalazine (0.36 g/kg) group; and high-, medium-, and low- dose SSW (24, 12, and 6 g/kg) groups. The model and blank groups were gavaged with equal volumes of distilled water once a day for 21 d. The general condition of the rats was observed, and the body mass, fecal properties, and occult blood were recorded for calculating the disease activity index (DAI) score. The colonic tissue of the rats was collected, and its general morphology and pathological form were noted for obtaining the colonic mucosal injury index (CMDI) score. Hematoxylin-eosin staining was used to view the pathological changes of the colon tissue in each group, apoptosis of the cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and quantitative real-time polymerase chain reaction was used to measure the expressions of TLR2, myeloid differentiation primary response gene 88 (MyD88), IRAK4, and NF-κB p65 mRNA in the colon tissue. The expressions of TLR2, MyD88, IRAK4, and NF-κB p65 protein were detected using western blotting and immunohistochemistry assay, and the levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the colon tissue were determined using enzyme linked immunosorbent assay. RESULTS: Compared with the blank group, the general condition of the model group was relatively poor. The DAI and CMDI scores of the model group increased significantly (< 0.01), the glands and intestinal mucosa disappeared partially, and several inflammatory cells infiltrated and gathered in the mucosal layer and base layer of the rats in the model group. Furthermore, the cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of rats in the model group increased significantly (< 0.01). The levels of IL-1ß and TNF-α increased significantly in the colon tissue of rats in the model group (< 0.01). After treatment with SSW, compared with the model group, the general condition of the UC rats improved. Moreover, the DAI and CMDI scores of the UC rats decreased significantly (< 0.05), and the pathological changes in the colon tissue of the UC rats tended to be normal. The cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of the UC rats decreased gradually ( < 0.01), and the levels of IL-1ß and TNF-α decreased significantly (< 0.01). CONCLUSION: SSW can improve the general condition and alleviate the intestinal mucosal injury of UC model rats. Additionally, SSW can inhibit the TLR2/IRAK4/ NF-κB signaling pathway, but further studies are required to confirm it.

Inhibition of Inflammatory Responses by Centella asiatica via Suppression of IRAK1-TAK1 in Mouse Macrophages.

Centella asiatica (L.) Urb. (C. asiatica) has been widely treated for inflammation-related diseases in China for thousands of years. While C. asiatica showed relevant effects as traditional medicine, the mechanism of C. asiatica suppressing inflammation has not been thoroughly investigated. Therefore, this study was conducted to reveal the anti-inflammatory mechanism of methanol fraction from C. asiatica (MCA) at the molecular level in murine macrophages. Levels of inflammation-related mediators were observed with treatment of MCA. MCA significantly suppressed nitric oxide production and iNOS expression in RAW 264.7 macrophages. Prostaglandin E2 production was alleviated by MCA via the downregulation of cyclooxygenase-2. MCA treatment also reduced pro-inflammatory tumor necrosis factor-[Formula: see text] and interleukin (IL)-6 levels. LPS/D-GalN-induced acute hepatitis in mouse was alleviated by MCA treatment. In addition, MCA decreased the phosphorylation of inhibitory [Formula: see text]B[Formula: see text] (I[Formula: see text]B[Formula: see text]) at Ser32/36 and thereby blocked I[Formula: see text]B[Formula: see text] degradation. TXY motif phosphorylation in the activation loops of mitogen-activated protein kinases (MAPKs) was also suppressed by MCA treatment. Further investigation revealed that MCA inhibited transforming growth factor-[Formula: see text]-activated kinase 1 (TAK1) phosphorylation and IL-1 receptor-associated kinase (IRAK1) degradation, the upstream kinases activating nuclear factor [Formula: see text]B and MAPKs. Taken together, MCA exhibited anti-inflammatory properties via the downregulation of IRAK1-TAK1 signaling pathways.

Paeoniflorin inhibits activation of the IRAK1-NF-κB signaling pathway in peritoneal macrophages from lupus-prone MRL/lpr mice.

Systemic lupus erythematosus (SLE) is a chronic and multisystemic autoimmune disease. Interleukin-1 receptor-associated kinase 1 (IRAK1) is associated with the susceptibility of SLE in humans and paeoniflorin has recently been reported to exhibit immunosuppressive properties. The aim of this study was to determine the effect of paeoniflorin on lipopolysaccharide (LPS)-triggered macrophage activation and and its role in LPS-induced IRAK1-nuclear factor κB (NF-κB) signaling pathways. Peritoneal macrophages from lupus-prone MRL/lpr mice and ICR mice were isolated, prepared and cultured. Cells were treated with LPS alone or LPS with paeoniflorin, and macrophage proliferation was analyzed using the CCK8 assay. The expression of IRAK1 in cells was analyzed by immunofluorescence staining. The level of gene expression of IRAK1, NF-κB, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was measured by RT-PCR, and TNF-α, IL-6 levels in the cell supernatant were determined by ELISA. The protein expression of IRAK1 and downstream molecules tumor necrosis factor receptor-associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase (IKK), NF-kappa-B inhibitor alpha (IKBα), and NF-κB was detected by Western-blot analysis. Paeoniflorin was found to decrease the phosphorylation of IRAK1 and its downstream proteins induced by LPS and inhibit the expression of TNF-α and IL-6. Taken together, the data obtained indicate that paeoniflorin inhibits LPS-induced cell activation by inhibiting the IRAK1-NF-κB pathway in MRL/lpr mouse macrophages. Therefore, paeoniflorin may be a potential therapy for SLE.

Protective Effects of a Lipid Extract from Hard-Shelled Mussel (Mytilus coruscus) on Intestinal Integrity after Lipopolysaccharide Challenge in Mice.

This study investigated the protective effects of a lipid extract from hard-shelled mussel (HMLE) on intestinal integrity and the underlying mechanisms after a lipopolysaccharide (LPS) challenge in mice by using a 3 × 2 factorial design. Mice received olive oil, fish oil, and HMLE (n = 12 per group) by using gastric gavage for six weeks, respectively. Then half the mice in each group was injected intraperitoneally with LPS and the other half with phosphate buffered saline. Four hours after injection, mice were sacrificed and samples were collected. n-3 PUFAs were significantly enriched in erythrocytes following fish oil and HMLE supplementation. Both fish oil and HMLE improved intestinal morphology by restoring the ileac villus height and barrier function, which is indicated by decreased colonic myeloperoxidase activity and increased diamine oxidase activity as well as enhanced mRNA expression of intestinal tight junction proteins known as occludin and claudin-1 when compared with olive oil. In addition, both fish oil and HMLE increased colon production and the expression of anti-inflammatory cytokine, IL-10, while they inhibited the abnormal production and expression of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 relative to the olive oil. Lastly, in comparison with olive oil, both fish oil and HMLE downregulated the TLR-4 signaling pathway by reducing the expression of two key molecules in this pathway, which are called TLR-4 and MyD88. These results suggest that HMLE had a protective effect on intestinal integrity after the LPS challenge, which was equivalent to that of fish oil. This effect might be associated with the regulation of inflammatory mediators and the inhibition of the TLR-4 signaling pathway.

A novel NF-κB/YY1/microRNA-10a regulatory circuit in fibroblast-like synoviocytes regulates inflammation in rheumatoid arthritis.

The main etiopathogenesis of rheumatoid arthritis (RA) is overexpressed inflammatory cytokines and tissue injury mediated by persistent NF-κB activation. MicroRNAs widely participate in the regulation of target gene expression and play important roles in various diseases. Here, we explored the mechanisms of microRNAs in RA. We found that microRNA (miR)-10a was downregulated in the fibroblast-like synoviocytes (FLSs) of RA patients compared with osteoarthritis (OA) controls, and this downregulation could be triggered by TNF-α and IL-1ß in an NF-κB-dependent manner through promoting the expression of the YingYang 1 (YY1) transcription factor. Downregulated miR-10a could accelerate IκB degradation and NF-κB activation by targeting IRAK4, TAK1 and BTRC. This miR-10a-mediated NF-κB activation then significantly promoted the production of various inflammatory cytokines, including TNF-α, IL-1ß, IL-6, IL-8, and MCP-1, and matrix metalloproteinase (MMP)-1 and MMP-13. In addition, transfection of a miR-10a inhibitor accelerated the proliferation and migration of FLSs. Collectively, our data demonstrates the existence of a novel NF-κB/YY1/miR-10a/NF-κB regulatory circuit that promotes the excessive secretion of NF-κB-mediated inflammatory cytokines and the proliferation and migration of RA FLSs. Thus, miR-10a acts as a switch to control this regulatory circuit and may serve as a diagnostic and therapeutic target for RA treatment.

Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

Xuebijing (XBJ) is a type of traditional Tibetan medicine, and previous pharmacological studies have shown that the ethanol extract is derived from Chuanxiong, Chishao, Danshen and Honghua. Chuanxiong, Chishao, Danshen and Honghua possesses potent anti-inflammatory activity, and has been used in the treatment of inflammatory infectious diseases. In the present study, we investigated the effects of XBJ on pulmonary permeability and lung injury in cecal ligation and puncture (CLP)-induced sepsis in rats. A CLP sepsis model was established for the control and treatment groups, respectively. Approximately 2 h prior to surgery, an amount of 100 mg/kg XBJ injection was administered to the treatment group. Reverse transcription polymerase chain reaction (PT-PCR) and western blot analysis were used to examine the expression of Toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase 1 (IRAK1), Toll-like receptor 4 (TLR4), nuclear factor-κB65 (NF-κB65) and TNF receptor-associated factor 6 (TRAF6) in lung tissue. ELISA was applied to detect changes of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), interleukin-4 (IL-4) and interleukin-10 (IL-10) levels in bronchoalveolar lavage (BAL) fluid, and intercellular adhesion molecule 1 (ICAM-1) and von wille-brand factor (vWF) in serum. The number of neutrophils, albumin and total cells in the BAL fluid were measured. For histological analysis, hematoxylin and eosin (H&E) stains were evaluated. Lung permeability, the wet/dry weight ratio (W/D) and the lung pathology score were determined following the induction of ALI by CLP for 24 h. The results demonstrated that XBJ upregulated Tollip expression and blocked the activity of IRAK1, TLR4, NF-κß65 and TRAF6. Additionally, the number of neutrophils and total cells were significantly decreased in the XBJ group compared to that in the control group. Lung permeability, the wet/dry weight ratio (W/D) and the lung pathology score were significantly decreased in the XBJ group. The histological results also demonstrated the attenuation effect of XBJ on CLP-induced lung inflammation. The results of the present study indicated that XBJ has a significantly reduced CLP-induced lung permeability by upregulating Tollip expression. The protective effects of XBJ suggest its therapeutic potential in CLP-induced acute lung injury treatment.

Oral free and dipeptide forms of glutamine supplementation attenuate oxidative stress and inflammation induced by endotoxemia.

OBJECTIVE: The aim of the present study was to determine the effects of oral supplementation with L-glutamine plus L-alanine (GLN+ALA), both in the free form and L-alanyl-L-glutamine dipeptide (DIP) in endotoxemic mice. METHODS: B6.129 F2/J mice were subjected to endotoxemia (Escherichia coli lipopolysaccharide [LPS], 5 mg/kg, LPS group) and orally supplemented for 48 h with either L-glutamine (1 g/kg) plus L-alanine (0.61 g/kg) (GLN+ALA-LPS group) or 1.49 g/kg DIP (DIP-LPS group). Plasma glutamine, cytokines, and lymphocyte proliferation were measured. Liver and skeletal muscle glutamine, glutathione (GSH), oxidized GSH (GSSG), tissue lipoperoxidation (TBARS), and nuclear factor (NF)-κB-interleukin-1 receptor-associated kinase 1 (IRAK1)-Myeloid differentiation primary response gene 88 pathway also were determined. RESULTS: Endotoxemia depleted plasma (by 71%), muscle (by 44%), and liver (by 49%) glutamine concentrations (relative to the control group), which were restored in both GLN+ALA-LPS and DIP-LPS groups (P < 0.05). Supplemented groups reestablished GSH content, intracellular redox status (GSSG/GSH ratio), and TBARS concentration in muscle and liver (P < 0.05). T- and B-lymphocyte proliferation increased in supplemented groups compared with controls and LPS group (P < 0.05). Tumor necrosis factor-α, interleukin (IL)-6, IL-1 ß, and IL-10 increased in LPS group but were attenuated by the supplements (P < 0.05). Endotoxemic mice exhibited higher muscle gene expression of components of the NF-κB pathway, with the phosphorylation of IκB kinase-α/ß. These returned to basal levels (relative to the control group) in both GLN+ALA-LPS and DIP-LPS groups (P < 0.05). Higher mRNA of IRAK1 and MyD88 were observed in muscle of LPS group compared with the control and supplemented groups (P < 0.05). CONCLUSION: Oral supplementations with GLN+ALA or DIP are effective in attenuating oxidative stress and the proinflammatory responses induced by endotoxemia in mice.

Brazilin selectively disrupts proximal IL-1 receptor signaling complex formation by targeting an IKK-upstream signaling components.

The ligation of interleukin-1 receptor (IL-1R) or tumor necrosis factor receptor 1 (TNFR1) induces the recruitment of adaptor proteins and their concomitant ubiquitination to the proximal receptor signaling complex, respectively. Such are upstream signaling events of IKK that play essential roles in NF-κB activation. Thus, the discovery of a substance that would modulate the recruitment of key proximal signaling elements at the upstream level of IKK has been impending in this field of study. Here, we propose that brazilin, an active compound of Caesalpinia sappan L. (Leguminosae), is a potent NF-κB inhibitor that selectively disrupts the formation of the upstream IL-1R signaling complex. Analysis of upstream signaling events revealed that brazilin markedly abolished the IL-1ß-induced polyubiquitination of IRAK1 and its interaction with IKK-γ counterpart. Notably, pretreatment of brazilin drastically interfered the recruitment of the receptor-proximal signaling components including IRAK1/4 and TRAF6 onto MyD88 in IL-1R-triggerd NF-κB activation. Interestingly, brazilin did not affect the TNF-induced RIP1 ubiquitination and the recruitment of RIP1 and TRAF2 to TNFR1, suggesting that brazilin is effective in selectively suppressing the proximal signaling complex formation of IL-1R, but not that of TNFR1. Moreover, our findings suggest that such a disruption of IL-1R-proximal complex formation by brazilin is not mediated by affecting the heterodimerization of IL-1R and IL-1RAcP. Taken together, the results suggest that the anti-IKK activity of brazilin is induced by targeting IKK upstream signaling components and subsequently disrupting proximal IL-1 receptor signaling complex formation.

Endocannabinoids affect innate immunity of Muller glia during HIV-1 Tat cytotoxicity.

In the retina, increased inflammatory response can cause visual impairment during HIV infection in spite of successful anti-retroviral therapy (HAART). The HIV-1 Tat protein is implicated in neurodegeneration by eliciting a cytokine response in cells of the CNS, including glia. The current study investigated whether innate immune response in human retinal Muller glia could be immune-modulated to combat inflammation. Endocannabinoids, N-arachidonoylethanolamide and 2-arachidonoylglycerol are used to alleviate Tat-induced cytotoxicity and rescue retinal cells. The neuroprotective mechanism involved suppression in production of pro-inflammatory and increase of anti-inflammatory cytokines, mainly through the MAPK pathway. The MAPK regulation was primarily by MKP-1. Both endocannabinoids regulated cytokine production by affecting at the transcriptional level the NF-κB complex, including IRAK1BP1 and TAB2. Stability of cytokine mRNA is likely to have been influenced through tristetraprolin. These findings have direct relevance in conditions like immune-recovery uveitis where anti-retroviral therapy has helped immune reconstitution. In such conditions drugs to combat overwhelming inflammatory response would need to supplement HAART. Endocannabinoids and their agonists may be thought of as neurotherapeutic during certain conditions of HIV-1 induced inflammation.

The role of miR-146a in dorsal root ganglia neurons of experimental diabetic peripheral neuropathy.

Sensory neurons mediate diabetic peripheral neuropathy. Using a mouse model of diabetic peripheral neuropathy (BKS.Cg-m+/+Lepr(db)/J (db/db) mice) and cultured dorsal root ganglion (DRG) neurons, the present study showed that hyperglycemia downregulated miR-146a expression and elevated interleukin-1 receptor-activated kinase (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) levels in DRG neurons. In vitro, elevation of miR-146a by miR-146a mimics in DRG neurons increased neuronal survival under high-glucose conditions. Downregulation and elevation of miR-146a in DRG neurons, respectively, were inversely related to IRAK1 and TRAF6 levels. Treatment of diabetic peripheral neuropathy with sildenafil, a phosphodiesterase type 5 inhibitor, augmented miR-146a expression and decreased levels of IRAK1 and TRAF6 in the DRG neurons. In vitro, blockage of miR-146a in DRG neurons abolished the effect of sildenafil on DRG neuron protection and downregulation of IRAK1 and TRAF6 proteins under hyperglycemia. Our data provide the first evidence showing that miR-146a plays an important role in mediating DRG neuron apoptosis under hyperglycemic conditions.

Fish oil alleviates activation of the hypothalamic-pituitary-adrenal axis associated with inhibition of TLR4 and NOD signaling pathways in weaned piglets after a lipopolysaccharide challenge.

Long-chain n-3 (ω-3) polyunsaturated fatty acids exert beneficial effects in neuroendocrine dysfunctions in animal models and clinical trials. However, the mechanism(s) underlying the beneficial effects remains to be elucidated. We hypothesized that dietary treatment with fish oil (FO) could mitigate LPS-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis through inhibition of Toll-like receptor 4 and nucleotide-binding oligomerization domain protein signaling pathways. Twenty-four weaned pigs were used in a 2 × 2 factorial design, and the main factors consisted of diet (5% corn oil vs. 5% FO) and immunological challenge (saline vs. LPS). After 21 d of dietary treatment with 5% corn oil or FO diets, pigs were treated with saline or LPS. Blood samples were collected at 0 (preinjection), 2, and 4 h postinjection, and then pigs were humanely killed by intravenous injection of 40 mg/kg body weight sodium pentobarbital for tissue sample collection. FO led to enrichment of eicosapentaenoic acid and docosahexaenoic acid and total n-3 polyunsaturated fatty acids in hypothalamus, pituitary gland, adrenal gland, spleen, and thymus. FO decreased plasma adrenocorticotrophin and cortisol concentrations as well as mRNA expressions of hypothalamic corticotropin releasing hormone and pituitary proopiomelanocortin. FO also reduced mRNA expression of tumor necrosis factor-α in hypothalamus, adrenal gland, spleen, and thymus, and of cyclooxygenase 2 in hypothalamus. Moreover, FO downregulated the mRNA expressions of Toll-like receptor 4 (TLR4) and its downstream molecules, including cluster differentiation factor 14, myeloid differentiation factor 2, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase 1, tumor necrosis factor-α receptor-associated factor 6, and nuclear factor kappa-light-chain-enhancer of activated B cells p65, and also decreased the mRNA expressions of nucleotide-binding oligomerization domain 1, nucleotide-binding oligomerization domain 2, and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2. These results suggested that FO attenuates the activation of the HPA axis induced by LPS challenge. The beneficial effects of FO on the HPA axis may be associated with decreasing the production of brain or peripheral proinflammatory cytokines through inhibition of TLR4 and nucleotide-binding oligomerization domain protein signaling pathways.

Dietary supplementation with geranylgeraniol suppresses lipopolysaccharide-induced inflammation via inhibition of nuclear factor-κB activation in rats.

PURPOSE: The isoprenoid geranylgeraniol (GGOH) inhibits nuclear factor-kappa B (NF-κB) activation in the liver, yet the mechanism remains unclear. We investigated the modulation and inhibition of lipopolysaccharide (LPS)-induced NF-κB signaling in the liver of rats fed a GGOH-supplemented diet. METHODS: Rats were fed a diet supplemented with or without GGOH for 10 days. Rats were then intraperitoneally injected with 0.5 mg/kg LPS or vehicle (sterilized saline) and fasted for 18 h. Plasma levels of the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, and the liver damage indicators alanine and aspartate aminotransferases (ALT and AST) were assessed. Liver mRNA and proteins were assayed for changes in NF-κB target genes and signal transduction genes. RESULTS: Rats fed a high-dose, GGOH-supplemented diet showed significantly lower levels of plasma inflammatory cytokines and ALT and AST activities. In the liver, GGOH significantly suppressed NF-κB activation and mRNA expression of its pro-inflammatory target genes. Furthermore, GGOH supplementation substantially suppressed mRNA expression of signal transducer genes upstream of the IκB kinase complex. Western blotting of liver extracts further demonstrated the substantial decrease in total IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6), leading to lower signal transduction and inhibition of NF-κB after LPS. CONCLUSION: A 10-day, high-dose, GGOH-supplemented diet was sufficient to inhibit LPS-induced inflammation and activation of NF-κB in rat livers. GGOH significantly modulated NF-κB signaling molecules, inhibiting its signal transduction and activation in the liver, thus protecting against liver damage.

Kalopanaxsaponin B inhibits LPS-induced inflammation by inhibiting IRAK1 Kinase.

The stem bark of Kalopanax pictus Nakai (KP, family Araliaceae), of which main constituent is kalopanaxsaponin B, has been used for inflammation in Chinese traditional medicine. We isolated kalopanaxsaponin B from KP and investigated its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated peritoneal macrophages and on LPS-stimulated systemic inflammation in male ICR mice. Kalopanaxsaponin B inhibited the expression of TNF-α, IL-1ß, iNOS and COX-2 in LPS-stimulated peritoneal macrophages. Kalopanaxsaponin B also inhibited the activation of IRAK1, IKK-ß, NF-κB and MAP kinases (ERK, JNK, p-38). Treatment with LPS in the presence of kalopanaxsaponin B inhibited LPS-induced IRAK1 degradation and phosphorylation. Kalopanaxsaponin B inhibited IRAK1 kinase binding activity. However, kalopanaxsaponin B did not inhibit the NF-κB activation in active IKK-ß-transfected macrophages. Kalopanaxsaponin B did not inhibit the binding of LPS on toll-like receptor-4 of the macrophages. Kalopanaxsaponin B inhibited LPS-induced systemic inflammation in mice. Based on these findings, kalopanaxsaponin B ameliorates LPS-induced systemic inflammation by inhibiting IRAK1 kinase.

Standardized curcuminoid extract (Curcuma longa l.) decreases gene expression related to inflammation and interacts with associated microRNAs in human umbilical vein endothelial cells (HUVEC).

The anti-inflammatory effects of curcuminoids have been extensively investigated. However, few studies investigate the mechanistic involvement of microRNAs (miRNAs) in their activity. The objective of this study was to examine the protective effects of standardized curcuminoid extract (SCE) in vascular inflammation of human umbilical vein endothelial cells (HUVEC) and the potential involvement of miRNA-126 and miRNA-146a. Escherichia coli lipopolysacharides (LPS) were used to induce inflammation. LPS-challenge increased gene-expression of toll-like receptor-4 (TLR-4) and downstream genes IL-1 receptor-associated kinase 1 (IRAK-1) and tumor necrosis factor receptor-associated factor 6 (TRAF-6) up to 2.58-, 2.39-, and 3.73-fold, respectively, relative to DMSO-treated controls that were not challenged with LPS. LPS up-regulated TLR-4, IRAK-1, and TRAF-6 in SCE pretreated cells (5 mg L(-1)), only up to 0.69-, 1.28-, and 1.15-fold, respectively. miRNA-146a can be up-regulated by transcription nuclear factor kappa B (NF-κB) and acts as a negative feedback loop regulator involving IRAK-1 and TRAF-6 downregulation. In this study, the down-regulation of NF-κB was accompanied by reduced miRNA-146a expression. LPS-challenge induced mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) up to 5.65- and 10.65-fold, respectively. SCE prevented this effect and increases of up to only 2.92- and 5.26-fold of DMSO-treated controls not challenged with LPS were observed. miRNA-126 regulates endothelial expression of VCAM-1, but was not inversely correlated to the expression of its target gene VCAM-1 upon SCE treatment; therefore, miRNA-126 does not appear to be involved in the down-regulation of VCAM-1. Overall, curcuminoids are confirmed to have anti-inflammatory properties in HUVEC; however, neither miRNA-146a nor miRNA-126 seem to be involved in the SCE-induced down-regulation of the NF-κB-target genes IRAK-1, TRAF-6, and VCAM-1.

Low-dose endotoxin induces inflammation by selectively removing nuclear receptors and activating CCAAT/enhancer-binding protein δ.

Subclinical levels of circulating endotoxin are associated with the pathogenesis of diverse human inflammatory diseases, by mildly inducing the expression of proinflammatory mediators. In this study, we examined the molecular mechanism responsible for the effect of low-dose LPS in macrophages. In contrast to high-dose LPS, which activates NF-κB and induces the robust expression of proinflammatory mediators, we observed that low-dose LPS failed to activate NF-κB. Instead, it selectively activated C/EBPδ and removed nuclear repressors, including peroxisome proliferator-activated receptor α and retinoic acid receptor α, enabling a mild and leaky expression of proinflammatory mediators. The effect of low-dose LPS required IRAK-1, which interacts with and acts upstream of IκB kinase ε to contribute to LPS-mediated induction of C/EBPδ and proinflammatory mediators. Additionally, mice fed a high-fat diet acquired elevated levels of endotoxin and proinflammatory mediators in an IRAK-1-dependent fashion. Taken together, these data reveal a distinct pathway preferentially used by low-dose endotoxin in initiating low-grade inflammation.

Dramatic change in function and expression pattern of a gene duplicated by polyploidy created a paternal effect gene in the Brassicaceae.

New gene formation by polyploidy has been an ongoing process during the evolution of various eukaryotes that has contributed greatly to the large number of genes in their genomes. After duplication, some genes that are retained can acquire new functions or expression patterns, or subdivide their functions or expression patterns between duplicates. Here, we show that SHORT SUSPENSOR (SSP) and Brassinosteroid Kinase 1 (BSK1) are paralogs duplicated by a polyploidy event that occurred in the Brassicaceae family about 23 Ma. SSP is involved in paternal control of zygote elongation in Arabidopsis thaliana by transcription in the sperm cells of pollen and then translation in the zygote, whereas BSK1 is involved in brassinosteroid signal transduction. Comparative analysis of expression in 63 different organs and developmental stages revealed that BSK1 and SSP have opposite expression patterns in pollen compared with all other parts of the plant. We determined that BSK1 retains the ancestral expression pattern and function. Thus, SSP has diverged in function after duplication from a component of the brassinosteroid signaling pathway to a paternal regulator of the timing of zygote elongation. The ancestral function of SSP was lost by deletions in the kinase domain. Our sequence rate analysis revealed that SSP but not BSK1 has experienced a greatly accelerated rate of amino acid sequence changes and relaxation of purifying selection. In addition, SSP has been duplicated to create a new gene (SSP-like1) with a completely different expression pattern, a shorter coding sequence that has lost a critical functional domain, and a greatly accelerated rate of amino acid sequence evolution along with evidence for positive selection, together indicative of neofunctionalization. This study illustrates two dramatic examples of neofunctionalization following gene duplication by complete changes in expression pattern and function. In addition, our findings indicate that paternal control of zygote elongation by SSP is an evolutionarily recent innovation in the Brassicaceae family.

[Effect of Dureping injection on TIR signal pathway on Ana-1 cells].

OBJECTIVE: To investigate the influence of Dureping injection to the murinal celiac macrophage Ana-1 on TIR signal pathway. METHOD: Ana-1 cell line was infected by influenza virus FM1 strain and treated with the Dureping injection in different concentrations (10.1 mg x L(-1) group) for 12 h and 24 h. Then we collected the cells, extracted mRNA and measured the expressions of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 respectively by RT-PCR. RESULT: Dureping injection down-regulated the expression of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 mRNA in Ana-1 cell line infected by influenza virus, in a dose-dependent manner significantly. CONCLUSION: Dureping injection has an obvious effect against influenza virus FM1 strain by regulating the TIR signal pathway.

miR-146a suppresses invasion of pancreatic cancer cells.

The aggressive course of pancreatic cancer is believed to reflect its unusually invasive and metastatic nature, which is associated with epidermal growth factor receptor (EGFR) overexpression and NF-kappaB activation. MicroRNAs (miRNA) have been implicated in the regulation of various pathobiological processes in cancer, including metastasis in pancreatic cancer and in other human malignancies. In this study, we report lower expression of miR-146a in pancreatic cancer cells compared with normal human pancreatic duct epithelial cells. Reexpression of miR-146a inhibited the invasive capacity of pancreatic cancer cells with concomitant downregulation of EGFR and the NF-kappaB regulatory kinase interleukin 1 receptor-associated kinase 1 (IRAK-1). Cellular mechanism studies revealed crosstalk between EGFR, IRAK-1, IkappaBalpha, NF-kappaB, and MTA-2, a transcription factor that regulates metastasis. Treatment of pancreatic cancer cells with the natural products 3,3'-diinodolylmethane (DIM) or isoflavone, which increased miR-146a expression, caused a downregulation of EGFR, MTA-2, IRAK-1, and NF-kappaB, resulting in an inhibition of pancreatic cancer cell invasion. Our findings reveal DIM and isoflavone as nontoxic activators of a miRNA that can block pancreatic cancer cell invasion and metastasis, offering starting points to design novel anticancer agents.

Paternal control of embryonic patterning in Arabidopsis thaliana.

The YODA (YDA) mitogen-activated protein kinase pathway promotes elongation of the Arabidopsis zygote and development of its basal daughter cell into the extra-embryonic suspensor. Here, we show that the interleukin-1 receptor-associated kinase (IRAK)/Pelle-like kinase gene SHORT SUSPENSOR (SSP) regulates this pathway through a previously unknown parent-of-origin effect. SSP transcripts are produced in mature pollen but do not appear to be translated. Instead, they are delivered via the sperm cells to the zygote and the endosperm, where SSP protein transiently accumulates. Ectopic expression of SSP protein in the leaf epidermis is sufficient to activate YDA-dependent signaling. We propose that SSP protein produced from paternal transcripts upon fertilization triggers zygotic YDA activity, providing an essential temporal cue for the regulation of the asymmetric first division.