NF-κB Protein Levels Research Articles

Ge-Gen Decoction Exerts an Anti-Primary Dysmenorrhea Effect in Rats by Inactivating the HSP90/NLRP3/NF-κB/COX-2 Pathway.

Although Ge-Gen decoction (GGD) has beneficial effects on primary dysmenorrhea (PD), the underlying mechanisms remain poorly understood. Our previous proteomic data revealed decreased level of heat shockprotein 90 (HSP90) in uterine tissues of rats with PD after GGD treatment. However, the potential role of HSP90 in the anti-PD effect of GGD and the underlying mechanisms remain unexplored. This study investigated the potential role and mechanism of HSP90 in the anti-PD effect of GGD using a PD rat model. Wistar female rats were used to investigate the potential role of HSP90 in the anti-PD effect of GGD. The rat PD model was established by injecting estradiol benzoate and oxytocin. GGD, Terazosin (an agonist of HSP90) or GGD combined with Terazosin were orally administered to the PD rats. The expression levels of protein and cytokines, including HSP90, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX-2) in the uterine tissue of rats in each group were detected by immunohistochemical assay or Western blot. GGD ameliorated the writhing response, suppressed the protein levels of HSP90 and inflammation-associated proteins, including NLRP3, NF-κB, and COX-2 in uterine tissues of rats with PD. Terazosin attenuated the anti-PD effect of GGD and reversed the effects of GGD on the protein levels of NLRP3, NF-κB and COX-2 in uterine tissues. GGD exerts an anti-PD effect and suppresses levels of HSP90 and some inflammation associated proteins in uterine tissues of rats.

Expression of HMGB1-TLR4 in Placentas from Preeclamptic Pregnancies and Its Effect on Proliferation and Invasion of HTR-8/SVneo Cells

Objectives: Placental inflammation possibly underlies preeclampsia (PE) pathogenesis. This study aimed to investigate the expression of the high mobility box group 1 (HMGB1)-toll-like receptor 4 (TLR4) signalling pathway in preeclamptic placentas and determine whether HMGB1 regulates the biological behaviour of trophoblasts in vitro. Design: Placental biopsies were taken from 30 preeclamptic patients and 30 normotensive controls. In vitro experiments were carried out in HTR-8/SVneo human trophoblast cells. Participants/Materials, Setting, Methods: HMGB1, TLR4, and nuclear factor kappa B (NF-κB) mRNA and protein were quantified to compare their expression in human placentas from preeclamptic and normotensive pregnancies. HTR-8/SVneo cells were stimulated with HMGB1 (50–400 μg/L) for 6–48 h, and proliferation and invasion of HTR-8/SVneo cells were measured via Cell Counting Kit-8 and transwell assays. HTR-8/SVneo cells were also transfected with HMGB1 and TLR4 small interfering RNA (siRNA) to investigate the effect of knocking down these proteins. The mRNA and protein expression of TLR4, NF-κB, and matrix metalloproteinase 9 (MMP-9) were determined using quantitative real-time PCR and Western blotting, respectively. Data were analysed with either a t-test or one-way analysis of variance. Results: The mRNA and protein levels of HMGB1, TLR4, and NF-κB were significantly higher in the placentas from preeclamptic pregnancies than from normal pregnancies (p < 0.05). HMGB1 stimulation (at concentrations up to 200 μg/L) of HTR-8/SVneo cells significantly increased invasion and proliferation over time. However, at an HMGB1 stimulation concentration of 400 μg/L, the invasion and proliferation ability of HTR-8/SVneo cells decreased. Compared to controls, mRNA and protein expression levels of TLR4, NF-κB, and MMP-9 increased (mRNA level fold change: 1.460, 1.921, 1.667; protein level fold change: 1.600, 1.750, 2.047) when stimulated with HMGB1 (p < 0.05) but decreased when HMGB1 was knocked down (p < 0.05). TLR4 siRNA transfection combined with HMGB1 stimulation reduced the mRNA (fold change: 0.451) and protein (fold change: 0.289) expression of TLR4 (p < 0.05), while NF-κB and MMP-9 were unaffected (p > 0.05). Limitations: Only one trophoblast cell line was used in this study, and the findings were not confirmed in animal studies. Conclusions: This study explored the pathogenesis of PE from two aspects: inflammation and trophoblast invasion. The overexpression of HMGB1 in placentas from preeclamptic pregnancies suggests this protein may be involved in PE pathogenesis. In vitro, HMGB1 was found to regulate the proliferation and invasion of HTR-8/SVneo cells by activating the TLR4-NF-κB-MMP-9 pathway. These findings have implications for targeting HMGB1 could be a therapeutic strategy for treating PE. In the future, we will further verify this in vivo and in other trophoblast cell lines, further exploring the molecular interactions of the pathway.

Mechanism of Yishen Tonglong Decoction inhibiting TLR4/p38 MAPK/NF-κB signaling pathway against prostate cancer via upregulating miR-145-5p

ObjectiveTo investigate the mechanism of Yishen Tonglong Decoction (益肾通癃汤, YSTLD) inhibiting the toll-like receptor 4/p38 mitogen activated protein kinases/nuclear factor kappa-B (TLR4/p38 MAPK/NF-κB) signaling pathway against prostate cancer by up-regulating miR-145-5p.Methods miRNA microarray technology was used to detect the changes of miRNA expression profile in prostate cancer PC-3 cells treated with YSTLD, and miRNAs with marked differences in miRNA microarray results were screened and validated by real-time polymerase chain reaction (qRT-PCR). Lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, Cell Counting Kit-8 (CCK8) assay, and scratch assay were adopted to detect the effects of miR-145-5p on prostate cancer PC-3 cell proliferation and migration. qRT-PCR and Western blot were employed to detect the effects of miR-145-5p on TLR4/p38 MAPK/NF-κB signaling pathway and the expression levels of apoptosis-related genes caspase3, tumor necrosis factor-α (TNF-α), Bax, and Bcl-2. qRT-PCR and Western blot were used to detect the effects of serum containing YSTLD on miR-145-5p, TLR4/p38 MAPK/NF-κB signaling pathway, and the expression levels of apoptosis-related genes caspase3, TNF-α, Bax, and Bcl-2. ResultsThe expression levels of 35 miRNAs in prostate cancer PC-3 cells treated with YSTLD were significantly different from those in the control group, with miR-145-5p being the most significantly different; qRT-PCR validation revealed that the miR-145-5p levels in prostate cancer PC-3 cells treated with YSTLD were significantly higher than those in the DMSO control group (P < 0.05). After lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, miR-145-5p was found to inhibit the proliferation and migration of prostate cancer PC-3 cells. Overexpression of miR-145-5p up-regulated expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulated expression levels of p38 MAPK, p65 NF-κB, and Bcl-2 mRNA in prostate cancer PC-3 cells (P < 0.05), while up-regulated caspase3 protein expression levels in prostate cancer PC-3 cells and down-regulated expression levels of TLR4, p38 MAPK, and p65 NF-κB protein (P < 0.05). Serum containing YSTLD could up-regulate the expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulate the mRNA expression levels of p38 MAPK, p65 NF-κB, Bcl-2, and TNF receptor-associated factor 1 (TRAF1) in prostate cancer PC-3 cells after intervening prostate cancer PC-3 cells (P < 0.05). Simultaneously, it up-regulated the expression levels of caspase3 protein and down-regulated the protein expression levels of TLR4, p38 MARK, p65 NF-κB, and TRAF1 in prostate cancer PC-3 cells (P < 0.05). ConclusionYSTLD can promote apoptosis of prostate cancer PC-3 cells by up-regulating the expression level of miR-145-5p and inhibiting TLR4/p38 MAPK/NF-κB signaling pathway, which may be an important mechanism of YSTLD against prostate cancer.

The expression levels of NF-κB and IKKβ in epithelial ovarian cancer and their correlation with drug resistance-related genes MDR1, TOPOII, and ERCC1.

To explore the expression levels of nuclear factor kappa B (NF-κB) and inhibitor of nuclear factor kappa B kinase (IKKβ) in epithelial ovarian cancer and the correlation analysis with multi-drug resistance-related genes 1 (MDR1), topoisomerase II (TOPOII) and nucleotide excision repair cross complementary group 1 (ERCC1). Immunohistochemical methods were used to detect the expression levels of NF-κB and IKKβ in epithelial ovarian cancer group (50 cases), ovarian benign tumor group (30 cases), and normal ovary group (10 cases). The expression levels of NF-κB, IKKβ, MDR1, TOPOII and ERCC1 messenger ribonucleic acid (mRNA) and protein were analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. Student's t-test and one-way ANOVA were used for comparison of numerical data. Pearson's chi-squared and Fisher's exact tests were carried out for analysis of non-numerical data. The levels of NF-κB, IKKβ, MDR1 and ERCC1 mRNA and protein were increased (P<0.05), and the expression levels of TOPOII were decreased (P<0.05) in the epithelial ovarian cancer group compared to the normal ovary and benign ovarian tumor groups. The expression of NF-κB and IKKβ in epithelial ovarian cancer was significantly increased in patients with higher tumor stage, lower differentiation and presence of lymph node metastasis and positively correlated with MDR1 expression. NF-κB and IKKβ were negatively correlated with the expression of TOPOII and antagonized each other with TOPOII. The expression of NF-κB and IKKβ was positively correlated with the expression of MDR1, and negatively correlated with the expression of TOPOII. The correlation of NF-κB, IKKβ and resistance related genes, including MDR1, TOPOII, ERCC1, can predict the resistance of chemotherapy individuals to chemotherapy.

Combinatorial treatment with Silybum marianum essential oil enhances the therapeutic efficacy of a 5-fluorouracil base therapy for hepatocellular carcinoma.

Silybum marianum seeds contain a family of flavonolignans which can regulate cancer cell proliferation and apoptosis. However, research has rarely focused on the effect of S. marianum essential oil in combination with another anticancer drug. Here, we evaluated the antitumor effect of a combination of 5-fluorouracil (5-FU) and S. marianum essential oils on some pathways in hepatocellular carcinoma (HCC) in vitro and in vivo. Gas chromatography-mass spectrography results indicated there was no significant difference between the components of essential oils isolated from two geographical areas (Khuzestan or Isfahan, Sm-K or Sm-I). Each preparation decreased the viability of H22 cells compared to the control group. S. marianum essential oils alone, and combined with 5-FU, reduced the migration and invasion of H22 cells. Angiogenesis-related proteins were significantly reduced both in vivo and in vitro. Apoptosis and autophagy-related proteins were modulated both in vivo and in vitro. Each treatment decreased phospho-NF-κB (p65) and NF-κB (p65) protein levels. Expression levels of Wnt pathway-related genes were also decreased both in vivo and in vitro. Results revealed that the combination of S. marianum and 5-FU prolonged survival in a mouse model of HCC compared to either treatment alone.

Alpha‐kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway

BackgroundAlpha‐kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1β in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1β and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined.MethodsImmunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1β, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1β and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD.ResultsThe heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68+ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1β in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1β, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA.ConclusionsOur data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

Protein biomarkers for response to XPO1 inhibition in haematologic malignancies.

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision-making in the personalized treatment of cancer patients undergoing treatment with selinexor.

Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction.

Endothelial dysfunction is a critical component of both atherosclerotic plaque formation and saphenous vein graft failure. Crosstalk between the pro-inflammatory TNF-α-NFκB signaling axis and the canonical Wnt/β-catenin signaling pathway potentially plays an important role in regulating endothelial dysfunction, though the exact nature of this is not defined. In this study, cultured endothelial cells were challenged with TNF-α and the potential of a Wnt/β-catenin signaling inhibitor, iCRT-14, in reversing the adverse effects of TNF-α on endothelial physiology was evaluated. Treatment with iCRT-14 lowered nuclear and total NFκB protein levels, as well as expression of NFκB target genes, IL-8 and MCP-1. Inhibition of β-catenin activity with iCRT-14 suppressed TNF-α-induced monocyte adhesion and decreased VCAM-1 protein levels. Treatment with iCRT-14 also restored endothelial barrier function and increased levels of ZO-1 and focal adhesion-associated phospho-paxillin (Tyr118). Interestingly, inhibition of β-catenin with iCRT-14 enhanced platelet adhesion in cultured TNF-α-stimulated endothelial cells and in an ex vivo human saphenous vein model, most likely via elevating levels of membrane-tethered vWF. Wound healing was moderately retarded by iCRT-14; hence, inhibition of Wnt/β-catenin signaling may interfere with re-endothelialisation in grafted saphenous vein conduits. Inhibition of the Wnt/β-catenin signaling pathway with iCRT-14 significantly recovered normal endothelial function by decreasing inflammatory cytokine production, monocyte adhesion and endothelial permeability. However, treatment of cultured endothelial cells with iCRT-14 also exerted a pro-coagulatory and moderate anti-wound healing effect: these factors may affect the suitability of Wnt/β-catenin inhibition as a therapy for atherosclerosis and vein graft failure.

Sparassis latifolia polysaccharides inhibit colon cancer in mice by modulating gut microbiota and metabolism

Sparassis latifolia polysaccharides (SLPs) can regulate inflammatory cytokines. However, little is known about the regulation mechanism of SLPs on colon cancer. In this study, we investigated the mechanism of SLPs on metabolism in mice with colon cancer. The results showed that SLPs could improve the colon morphology and physiological indices, and inhibit the infiltration of immune cells in colon. Moreover, it could improve metabolism disorder of colon cancer via reducing the levels of TNF-α, IL-6, NF-κB, COX-2 and IL-1β mRNA or protein, increasing IκB mRNA or protein expression. In addition, it could comprehensively regulate the colon cancer related metabolism by changing the abundance of key intestinal flora and 35 metabolites including phosphatidylcholine, tryptophan and tetrahydrobiopterin. Some biomarkers associated with colon cancer metabolism were related significantly with the abundance of specific intestinal flora. These findings indicate that SLPs can attenuate metabolism disorder of colon cancer by modulating gut microbiota and metabolites.

β-glucan protects against necrotizing enterocolitis in mice by inhibiting intestinal inflammation, improving the gut barrier, and modulating gut microbiota

BackgroundNecrotizing enterocolitis (NEC) is a devastating gastrointestinal disease with high morbidity and mortality, affecting preterm infants especially those with very low and extremely low birth weight. β-glucan has manifested multiple biological effects including anti-inflammatory, regulation of gut microbiota, and immunomodulatory activities. This study aimed to investigate the effects of β-glucan on NEC.MethodsNeonatal C57BL/6 mice were randomly divided into three groups: Control group, NEC group and β-glucan group. Newborn 3-day-old mice were gavaged with either 1 mg/ml β-glucan or phosphate buffer saline at 0.03 ml/g for 7 consecutive days before NEC induction and a NEC model was established with hypoxia combined with cold exposure and formula feeding. All the pups were killed after 72-h modeling. Hematoxylin–eosin staining was performed to assess the pathological injury to the intestines. The mRNA expression levels of inflammatory factors in intestinal tissues were determined using quantitative real-time PCR. The protein levels of TLR4, NF-κB and tight junction proteins in intestinal tissues were evaluated using western blotting and immunohistochemistry. 16S rRNA sequencing was performed to determine the structure of the gut microbiota.Resultsβ-glucan administration ameliorated intestinal injury of NEC mice; reduced the intestinal expression of TLR4, NF-κB, IL-1β, IL-6, and TNF-α; increased the intestinal expression of IL-10; and improved the expression of ZO-1, Occludin and Claudin-1 within the intestinal barrier. Pre-treatment with β-glucan also increased the proportion of Actinobacteria, Clostridium butyricum, Lactobacillus johnsonii, Lactobacillus murinus, and Lachnospiraceae bacterium mt14 and reduced the proportion of Klebsiella oxytoca g Klebsiella in the NEC model.Conclusionβ-glucan intervention prevents against NEC in neonatal mice, possibly by suppressing the TLR4-NF-κB signaling pathway, improving intestinal barrier function, and partially regulating intestinal microbiota.

Azilsartan Modulates HMGB1/NF-κB/p38/ERK1/2/JNK and Apoptosis Pathways during Renal Ischemia Reperfusion Injury

Renal ischemia/reperfusion (IR) injury is characterized by an unexpected impairment of blood flow to the kidney. Azilsartan is an angiotensin receptor blocker that is approved for the management of hypertension. The present study aimed to investigate, on molecular basics, the nephroprotective activity of azilsartan on renal IR injury in rats. Rats were assigned into four groups: (1) Sham group, (2) Azilsartan group, (3) IR group, and (4) IR/Azilsartan-treated group. Histological examination and renal function were evaluated. Levels of KIM-1, HMGB1, caspase 3, GPX, SOD, NF-κB, and p53 proteins were investigated using ELISA. mRNA levels of IL-1β, IL6, IL10, TNF-α, NF-κB, p53, and bax were assessed by qRT-PCR. Expression of p38, JNK, and ERK1/2 proteins was investigated by Western blotting. IR injury resulted in tissue damage, elevation of creatinine, BUN, KIM-1, HMGB1, caspase 3, NF-κB, and p53 levels, decreasing GPX and SOD activities, and up-regulation of NF-κB, IL-1β, IL6, TNF-α, p53, and bax genes. Furthermore, it up-regulated the expression of phosphorylated/total ratio of p38, ERK1/2, and JNK proteins. Interestingly, treatment of the injured rats with azilsartan significantly alleviated IR injury-induced histopathological and biochemical changes. It reduced the creatinine, BUN, KIM-1, HMGB1, caspase-3, NF-κB, and p53 levels, elevated GPX and SOD activities, down-regulated the expression of NF-κB, IL-1β, IL6, TNF-α, p53, and bax genes, and up-regulated IL10 gene expression. Furthermore, it decreased the phosphorylated/total ratio of p38, ERK1/2, and JNK proteins. Azilsartan exhibited nephroprotective activity in IR-injured rats via its antioxidant effect, suppression of inflammation, attenuation of apoptosis, and inhibition of HMGB1/NF-κB/p38/ERK1/2/JNK signaling pathway.

MiR-124-Antagonist-Loaded Liposomal Nanoparticles Negatively Regulate the Toll-Like Receptor (TLR)-Signaling Pathway in Alveolar Epithelial Cells in Pulmonary Tuberculosis

miR-124 is intensively expressed in the alveolar epithelial cells of pulmonary tuberculosis. This study focused on exploring the negative regulation of miR-124-antagonist-loaded liposomal nanoparticles on the Toll-like receptor (TLR)-signal transduction pathway in the alveolar epithelial cells from pulmonary tuberculosis, aiming to provide theoretical evidence for the treatment of pulmonary tuberculosis. The purchased alveolar epithelial cells were grouped into Blank group, Empty-vector group, Bacillus Calmette-Guerin (BCG) group, Nanoparticle+MiR-124 Antagonist group, MiR-124 Antagonist group, and MiR-124 Agonist group. The liposomal nanoparticles were identified. The following aspects were investigated: mRNA level of miR-124, mRNA and protein levels of Myeloid differentiation factor 88 (MyD 88), Toll-like receptor the 6 (TLR 6) and their downstream molecules Nuclear Factor-κB (NF-κB) and Tumor necrosis factor TNF receptor-associated factor 6 (TRAF 6) secretion level of cytokines (NF-κB, IL-8, IL-1α, TNF-α and IL-6), as well as the regulatory link between miR-124-antagonists with TLR6 and MyD88. The liposomal nanoparticles were uniform in size, with an average particle size of (35.25±10.58) nm and an average Zeta potential of (−48.55±10.27) mV. The miR-124 level was the strongest in the MiR-124 Agonist group, while being the lowest in the Blank group. The miR-124 level was relatively higher in the BCG group and Empty-vector group, while being significantly reduced in the Nanoparticle+MiR-124 Antagonist group, which was higher than the Blank group. The miR-124 level in the MiR-124 Antagonist group was higher than that in the Nanoparticle+MiR-124 Antagonist group (P &lt;0.05). The mRNA and protein levels of MyD88, TLR6, NF-κB and TRAF6 were the highest in the MiR-124 Agonist group, while being the lowest in the Blank group. The transcription and translation levels of TRAF6, TLR6, NF-κB and MyD88 were relatively higher in the BCG group and Empty-vector group, while being significantly reduced in the Nanoparticle+ MiR-124 Antagonist group, which were higher than in the Blank group. The transcription and translation levels of TRAF6, TLR6, NF-κB and MyD88 were in the MiR-124 Antagonist group were higher than that in the Nanoparticle+MiR-124 Antagonist group (P &lt;0.05). The secretion levels of inflammatory factors (NF-κB, IL-8, IL-1α, TNF-α and IL-6) were the highest in the MiR-124 Agonist group, while being the lowest in the Blank group. The levels of these inflammatory factors were relatively higher in the BCG group and Empty-vector group, while being significantly reduced in the Nanoparticle+MiR-124 Antagonist group, which were elevated compared to that in the Blank group. The secretion quantities of these inflammatory factors in the MiR-124 Antagonist group were higher than that in the Nanoparticle+MiR-124 Antagonist group (P &lt;0.05).Dual luciferase experiments indicated that miR-124-antagonists may retard TLR6 and MyD88 to affect the immune response of pulmonary alveolar epithelial cells in pulmonary tuberculosis. The fluorescence intensity of mutant plasmid was significantly stronger than that of wild-type plasmid (P &lt; 0.05). In the alveolar epithelial cells from pulmonary tuberculosis, the miR-124-antagonistloaded liposomal nanoparticles can significantly reduce the expression of TLR6 and MyD88, and their downstream molecules (NF-κB and TRAF6), leading to the reduced secretion of the inflammatory factors. As a result, the inflammatory response of lung tissue was alleviated, while the immune function was restored. This regulation was achieved by the miR-124-antagonist-loaded liposomal nanoparticles via negatively regulating the TLR6/MyD88 pathways.

TSAb Modulates Microglia M1 Polarization via Activation of the TSHR-Mediated NF-κB Signaling Pathway

Introduction: Thyrotropin receptor-stimulating antibody (TSAb) is a pathogenic antibody in the serum of patients with Graves’ disease. The binding of TSAb to thyroid-stimulating hormone receptor (TSHR) in non-thyroid tissue may be associated with the occurrence and development of Graves’ disease-related complications. However, only few studies have been conducted on the effects of TSAb on the brain, and the pathogenesis of acute hyperthyroidism myopathy (ATM) is unclear. Therefore, this study aimed to explore the effect of TSAb on the polarization of BV-2 cells in the brain and its possible mechanism and provide a basic experimental basis for ATM. Methods: BV-2 cells were treated with different concentrations of TSAb. The relative survival rate of BV-2 cells was determined using the CCK-8 assay; the migration ability of BV-2 cells was detected using the Transwell migration assay; and the expression levels of M1/M2 polarization markers (CD86, inducible nitric oxide synthase [iNOS], CD206, and arginase 1 [Arg-1]), TSHR, tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-κB) protein in BV-2 cells were measured using WB. Results: Compared with the negative control group, the proliferative activity of BV-2 cells was significantly increased in the 20, 50, and 100 ng/mL TSAb groups, and the migration ability of BV-2 cells was significantly enhanced in the 50 and 100 ng/mL TSAb groups. The expression levels of M1 polarization markers (CD86 and iNOS), TSHR, TNF-α, and NF-κB protein in BV-2 cells treated with 50 and 100 ng/mL TSAb for 24 h were significantly upregulated, whereas those of M2 polarization markers (CD206 and Arg-1) significantly decreased. Conclusions: TSAb can induce abnormal activation of microglia, polarize to the M1 phenotype, and promote the inflammatory cascade reaction, in which TSHR plays a key role in NF-κB activation and proinflammatory cytokine release.

Glutamate Metabolism Shapes Osteoclast Differentiation: New Potential Therapeutic Target to Block Osteoclastic Bone Resorption in Multiple Myeloma Patients

Multiple Myeloma (MM) cells are characterized by an impaired Glutamine (Gln) metabolism and Gln-addiction feature. As a result of this, MM patients have lower levels of Gln and higher levels of its main metabolite Glutamate (Glu) in the bone marrow (BM) microenvironment as compared with Smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS) patients. Moreover, we have recently shown that MM-dependent Gln-exhausted microenvironment impairs osteoblast differentiation. Based on this evidence, we have hypothesized that alterations of Gln/Glu axis may have a significant impact on the activation of osteoclasts (OCLs) and in the development of osteolytic lesions. We purified BM CD14+ monocytes from a cohort of 30 patients with monoclonal gammopathies (5 MGUS, 12 SMM and 13 newly diagnosed MM (ND-MM)). Cells have been incubated in osteoclastogenic medium in the presence or absence of Glu. The samples were subsequently analyzed by real-time PCR, Western blot and TRAP staining after 3 or 14 days. Amino acid uptake and amino acid content have been assessed under the same conditions. Glu uptake inhibitors have been tested throughout osteoclastogenesis. Our data showed that the presence of Glu significantly increases the number of OCLs generated from MGUS/SMM CD14+ compared with cells differentiated in the absence of the amino acid (p<0.01). In these patients, Glu increases the expression of RANK as well as NF-κB and NFATc1 protein level pointing to a stimulatory effect on osteoclastogenic signaling pathway. On the contrary, CD14+ obtained from MM patients have a lower response to Glu probably as a consequence of the high Glu microenvironment imposed by MM cells. Consistently, MM CD14+ cells are prone to generate more OCLs also in the absence of Glu compared with CD14+ cells obtained from premalignant samples (median number of OCLs: MGUS/SMM 33, MM 123, p=0.02). The analysis of intracellular amino acids (Gln, Glu, and α-ketoglutarate (2-OG)) indicated that Glu content significantly increases after 8 days of differentiation while decreasing after 14 days pointing to a fast accumulation of Glu upon differentiation. The content of both Gln and 2-OG do not vary appreciably throughout the differentiation. These data suggest that OCLs differentiation is associated with an increased uptake of extracellular Glu. Consistently, the activity of the sodium-dependent Glu transporters EAATs, increases already after 3 days of incubation in differentiating medium without Glu, with a further increase in the presence of the amino acid. Expression analysis revealed that, among EAATs, EAAT1 (encoded by SLC1A3 gene) is responsible for the uptake of extracellular Glu upon differentiation both in the presence and in the absence of the amino acid. The expression of other Glu transporters do not change during differentiation. Selective inhibitors of EAAT1, UCPH101 and TFB-TBOA completely block the formation of multinucleated OCLs in the presence of Glu (p<0.01). In parallel to EAAT1 induction, Glu treatment increases the expression of Calcineurin, the serine/threonine phosphatase activated by increased intracellular calcium levels indicating the possible modulation of calcium signaling by Glu. In conclusion, our results indicate that high BM Glu level sustains OCLs formation by stimulating of NF-κB/NFATc1/calcineurin pathway. EAAT1 transporter is responsible for the uptake of extracellular Glu during osteoclastogenesis while its inhibition markedly blocks OCL formation. Therefore, targeting glutamate metabolism could be a therapeutic strategy to block OCL formation in MM patients.

Treponema pallidum FlaA2 inducing the release of pro-inflammatory cytokines is mediated via TLR2 in keratinocytes

BackgroundSyphilis, caused by Treponema pallidum (T. pallidum), is a multi-organ, multiple systems, multi-stage sexually transmitted diseases with various clinical manifestations, among of which pathological lesions of skin and mucosa are the typical clinical manifestations of syphilis. However, the immunopathogenesis of this process is poorly understood. T. pallidum flagellin FlaA2, as a part of the important organelle responsible for the causative agent's motility, may contributes to the host skin inflammatory response. ObjectivesTo determine the mechanisms of T. pallidum FlaA2 stimulating the expression of pro-inflammatory cytokines in human keratinocytes. MethodsRecombinant FlaA2 protein was performed to stimulate human keratinocytes. The mRNA transcription levels and protein expression levels of IL-6 and IL-8 were detected by qRT-PCR and ELISA, respectively. Western blot was used to detect the total protein and phosphorylation levels of ERK, p38, JNK and NF-κB, respectively. The intracellular location of NF-κB p65 was detected by immunofluorescence staining. ResultsRecombinant FlaA2 could considerably induced the expression of pro-inflammation cytokines IL-6 and IL-8 in HaCaT cells, and FlaA2-induced IL-6 and IL-8 secretion could be decreased by inhibiting TLR2 using pZERO-hTLR2. Further investigation showed that FlaA2 could activate the phosphorylation of ERK, p38 and IκBα and FlaA2-stimulated secretion of IL-6, IL-8 were attenuated by ERK, p38 and NF-κB inhibitors in HaCaT cells. Moreover, FlaA2 activates the ERK, p38 and NF-κB pathways through TLR2 signaling pathway in HaCaT cells. ConclusionsFrom the findings above, these results confirm that T. pallidum FlaA2 activates ERK, p38 and NF-κB signaling pathway through TLR2 pathway to induce the production of IL-6 and IL-8, which could contribute to enhance the understanding of the skin inflammatory response induced by the pathogen in syphilis patients.

Cl-amidine attenuates lipopolysaccharide-induced inflammation in human gingival fibroblasts via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.

Cl-amidine has been reported to have anti-inflammatory properties in a variety of diseases. However, the role of Cl-amidine in periodontal disease remains unclear. Here, the purpose of this study was to investigate the effect of Cl-amidine on lipopolysaccharide (LPS)-induced inflammation in human gingival fibroblasts (HGFs). The cytotoxic effect of Cl-amidine was measured with the Cell Counting Kit-8 (CCK-8) assay and Annexin V-FITC/PI staining. The protein levels of IL-6 and IL-8 in culture supernatants were measured with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines, TLR4 and MyD88 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression patterns of IL-6, TNF-ɑ, and IL-1β in HGFs were tested with western blot. The levels of NF-κB, MAPK, and Nrf2 pathway-related proteins were detected by western blot. Immunofluorescence (IF) staining was used to examine the nuclear translocation of NF-κB p65. Moreover, a rat gingivitis model was established to further clarify the role of Cl-amidine. Our results showed that Cl-amidine suppressed LPS-induced gingival inflammation both in vitro and in vivo. Mechanistically, Cl-amidine inhibited LPS-induced MyD88 expression, NF-κB activation, and JNK phosphorylation. Additionally, Cl-amidine upregulated Nrf2 and Ho-1 expression both with and without LPS stimulation but did not alter ROS levels or Keap1 expression. Overall, our data suggest that Cl-amidine acts as an inhibitor of LPS-induced gingival inflammation via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.

Neuroprotective effects of etanercept on diabetic retinopathy via regulation of the TNF-α/NF-κB signaling pathway

Purpose: To study the influence of etanercept on diabetic retinopathy in rats via tumor necrosis factor alpha (TNF-α)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. &#x0D; Methods: Thirty-six Sprague-Dawley (SD) rats were randomly divided into normal, model and etanercept groups. The expression of Caspase-3 was determined by immunohistochemistry, while the relative protein and mRNA expression levels of TNF-α and NF-κB were determined by Western blotting and quantitative polymerase chain reaction, respectively. Besides, the contents of TNF-α and interleukin-1 beta (IL-1β) were evaluated using enzyme-linked immunosorbent assay (ELISA), while cell apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). &#x0D; Results: Immunohistochemical studies showed that the mean optical density of tissues positive for caspase-3 in both the model and etanercept groups were significantly higher than in the normal group (p &lt; 0.05), while the mean optical density in the etanercept group was significantly lower than that in the model group (p &lt; 0.05). The protein expression levels of TNF-α and NF-κB in the etanercept group were significantly lower than those in the model group (p &lt; 0.05). Furthermore, mRNA expressions of TNF-α and NF-κB declined in the etanercept group (p &lt; 0.05); in addition, TNF-α, and IL-1β levels in the etanercept group were lower than in the model group (p &lt; 0.05). Cell apoptosis in the etanercept group was also lower than in the model group. &#x0D; Conclusion: Etanercept suppresses TNF-α/NF-κB signaling pathway thereby repressing inflammation and cell apoptosis in diabetic retinopathy rats. Therefore, etenercept’s neuroprotective effect may potentially be useful in developing a suitable therapy for diabetic neuropathy.

Effects of curcumin on liver injury induced by chronic alcohol addiction

Objective: To investigate the intervention effects of curcumin (Curc) on liver injury induced by chronic alcohol addiction in mice. Methods: Thirty Balb/c mice were randomly divided into normal control group (Control), model group (Model), low-dose Curc group (5 mg/kg, Curc-L), medium dose Curc group (10 mg/kg, Curc-M) and high-dose Curc group (15 mg/kg, Curc-H), with 6 mice in each group. The chronic alcohol addiction liver injury model was prepared with 20% liquor. The mice in control group were given 2 ml of normal saline every day. The mice in model group were given 5 ml/kg of 20% liquor every day, and the mice in Curc treatment group were treated with Curc at the doses of 5, 10, 15 mg/kg in 2 ml saline every day for 35 days. The weight of liver was measured and the health status of mice was observed. Serum ALT, AST, ALP and liver TG, TC, HDL-C, LDL-C, MDA, SOD, GSH-Px and NO were measured. The pathological changes of liver tissues stained with hematoxylin and eosin were observed. Results: Compared with the control group, the liver mass and serum levels of ALT, AST, ALP, MDA, NO, TC, TG, HDL-C, LDL-C in the model group were increased significantly (P＜0.05, P＜0.01), the activities of SOD and GSH-Px were decreased significantly (P＜0.05, P＜0.01), the liver cells were vacuolated and infiltrated with inflammatory cells, and the expression levels of NF-κB and MAPK protein in liver tissues were increased significantly (P＜0.01). Compared with the model group, the levels of ALT, AST, ALP, MDA, NO, TC, TG, HDL-C, LDL-C in Curc group were decreased significantly nd the activities of SOD and GSH-Px were increased significantly (P＜0.05, P＜0.01). Conclusion: Curc can effectively reduce liver tissue damage by regulating NF-κB/MAPK signal pathway.

Sini Decoction inhibits TLR4/NF-κB signaling pathway to improve airway remodeling of allergic asthmatic mice

This study aims to explore the effect of Sini Decoction on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway in the mice with allergic asthma(AA). Forty-eight SPF-grade BALB/c mice were randomly assigned into a blank control group, a model group, a dexamethasone group, and high-, medium-, and low-dose Sini Decoction groups, with 8 mice in each group. The sensitization solution made of ovalbumin and aluminum hydroxide powder was injected intraperitoneally in other groups except the blank control group which was injected with an equal volume of normal saline. The solution(or normal saline) was injected three times in total with an interval of 7 days. At the same time of sensitization, external cold stimulation and ice water were administered in a 4 ℃ climate box for 20 min every day. After modeling, the mice in each group were administrated with corresponding drugs by gavage for 3 weeks. At the end of administration, pentobarbital sodium(30 mg·kg~(-1)) was used for anesthesia, and then the samples were collected for the determination of various indexes. The phenol red test was conducted to evaluate tracheal excretion function. The histopathological changes of lung tissue were observed via hematoxylin-eosin(HE) staining. Masson staining was employed to reveal the deposition of blue collagen fibers around bronchi in lung tissue and the area occupied by blue collagen fibers was calculated. Immunofluorescence method was used to measure the expression of bronchial type Ⅰ collagen(Col-Ⅰ) and α-smooth muscle actin(α-SMA). The protein and mRNA levels of TLR4, NF-κB, cysteinyl aspartate specific proteinase-1(caspase-1), and interleukin-13(IL-13) were determined by Western blot and real-time fluorescence quantitative polymerase chain reaction(real-time PCR), respectively. Compared with the model group, Sini Decoction significantly increased the phenol red excretion from trachea, lowered the lung inflammation score, reduced subepithelial collagen deposition, and decreased Col-Ⅰ and α-SMA levels. Furthermore, the decoction down-regulated the protein and mRNA levels of TLR4, NF-κB, caspase-1, and IL-13 in mouse lung tissue. In conclusion, Sini Decoction can improve air remodeling by inhibiting the TLR4/NF-κB signaling pathway.

Genistein attenuates neuroinflammation and oxidative stress and improves cognitive impairment in a rat model of sepsis-associated encephalopathy: potential role of the Nrf2 signaling pathway.

Oxidative stress and inflammation seem to be the main factors responsible for cognitive impairment in sepsis. Genistein (GEN) is claimed to exert many beneficial effects on health, however, its possible effects on brain sepsis remains unclear. Here, we assess the influence and underling mechanisms of GEN on cognitive impairments in cecal ligation and puncture (CLP)-induced septic model. Rats were randomly divided into Sham, Sham + GEN, CLP, CLP + GEN gropus. Rats were treated with GEN (15mg/kg at 0 and 12h after CLP, i.p). Twenty-four hours after CLP, protein levels of cytokines, NF-kB and Nrf2, myeloperoxidase (MPO) activity, oxidative damage to lipids and proteins, the activities of antioxidant enzymes and the expression of Nrf2-target genes were evaluated in the hippocampus. At 10 days after sepsis induction, behavioral tests were conducted to evaluate cognitive impairment. The results indicate that GEN can enhance survival percentage and improve cognitive function. Genistein administration significantly reduced TNF-α and IL-1β levels, MPO activity and protein level of NF-kB in the hippocampus of septic rats. Genistein also decreased the levels of oxidative stress parameters (MDA and protein carbonyls) and elevated the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in septic rats. Furthermore, nuclear Nrf2 and the expression of HO-1 and NQO-1 were also elevated by GEN treatment. These findings suggest that GEN improves cognition impairment in septic rats via decreasing inflammatory responses and oxidative stress, and activation of the Nrf2 pathway.