Regulation Of NF-κB Signaling Pathway Research Articles

Ameliorative effects of Magnolia sieboldii buds hexane extract on experimental reflux esophagitis.

Gastroesophageal reflux disease (GERD) is a disease that stomach contents continually refluxing into esophagus causes symptoms and/or complications. The study was working to find natural plant extracts with good effects and small side effects to treat reflux esophagitis (RE). The anti-inflammatory effects of hexane extract of Magnolia sieboldii (MsHE) were conducted on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The ameliorative effects of MsHE on esophageal damage in rats induced by gastric acid reflux was explored in vivo. The results showed that MsHE decreased the production of nitric oxide (NO) and expression levels of iNOS, COX-2 and TNF-α on LPS-stimulated RAW 264.7 cells and MsHE treatment ameliorated the rats' esophageal tissue damage induced by gastric acid and inhibited the increase of inflammatory mediators and pro-inflammatory cytokines by regulating NF-κB signaling pathway. In addition, MsHE protected the function of barrier of epithelial cells against inflammatory conditions through increasing the expression of tight junctions. Furthermore, liquid chromatography-mass spectrometry analysis was used for determine the active ingredients contained in MsHE. The results show that MsHE can alleviate experimental rat RE by regulating NF-κB signaling pathway. In summary, MsHE may be used as a source material of drug candidate for the treatment of RE.

MiR-146a-5p inhibits proliferation and promotes apoptosis of oral squamous cell carcinoma cells by regulating NF-κB signaling pathway.

The aim of this study was to investigate the function and potential mechanism of micro ribonucleic acid (miR)-146a-5p in oral squamous cell carcinoma (OSCC). The expression of miR-146a-5p in OSCC tissues and cell lines was examined by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis. Then, the role of miR-146a-5p in proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. Besides, the proliferation and apoptosis of OSCC cells were detected by the colony formation assay and flow cytometry, respectively. Finally, the regulatory effect of miR-146a-5p/nuclear factor-kappa B subunit 1 (NF-κB1) was determined by Western blotting assay and Luciferase reporter assay system. The expression of miR-146a-5p was markedly upregulated in OSCC cell lines. In addition, the silence of miR-146a-5p inhibited the proliferation and promoted the apoptosis of OSCC cells. According to the results of the Western blotting analysis and Luciferase reporter gene assay, NF-κB1 was identified as a direct target of miR-146a-5p. Moreover, the downregulation of NF-κB1 restored the inhibitory effect of silenced miR-146a-5p on the proliferation of SCC-9 cells. MiR-146a-5p can inhibit the proliferation and accelerate the apoptosis of OSCC cells by directly targeting NF-κB1, and it plays a carcinogenic role in OSCC.

Pien Tze Huang alleviate the joint inflammation in collagen-induced arthritis mice

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis. Pien Tze Huang (PZH) is a Chinese patent medicine with anti-inflammatory and immunomodulatory effects. However, whether PZH could be used in RA therapy is still unknown. Therefore, this study aimed to explore the therapeutic effect and the potential mechanism of PZH on collagen-induced arthritis (CIA) mice.MethodsMale DBA/1J mice were used to establish an animal model of CIA and then treated with different doses of PZH for 4 weeks. The therapeutic effect of PZH on CIA mice was evaluated by arthritis score, pathological staining, and detecting the levels of inflammatory factors in serum and joints. To investigate its possible mechanism, the activity of NF-κB signaling pathway, NLRP3 inflammasome and the level of A20 were detected.ResultsThe results showed that PZH could alleviate the erythema and swelling of hind paws of CIA mice, improve the pathological conditions of joint and decrease the production of IL-1β, IL-6 and IL-17 in serum and joints. Furthermore, PZH could significantly inhibit the activity of NF-κB signaling pathway and NLRP3 inflammasome in the ankle joint of CIA mice compared with the model group. It also increased the level of A20 in the ankle joint of CIA mice.ConclusionThis study indicated that PZH could alleviate the joint inflammation of CIA mice, and the mechanism might be related to the regulation of NF-κB signaling pathway and NLRP3 inflammasome.

DIGIRR as a member of the toll/IL-1R family negative regulates NF-κB signaling pathway in miiuy croaker

The double-Ig-IL-1R related molecule (DIGIRR) is a member of the TIR (Toll -Interleukin-1 receptor) superfamily and plays an important role in the immune system, it is also as a negative regulator of the IL-1 signaling pathway. In this study, we identified and characterized the miiuy croaker DIGIRR (mmi-DIGIRR) gene. The results of gene structure analysis indicated that there were differences between the mmi-DIGIRR and mammalian SIGIRR, which there were two immunoglobulin (Ig) domains contained in extracellular region of mmi-DIGIRR. Sequence alignment analysis showed that fish DIGIRR shared some conserved sequences with other vertebrates and the evolution was relatively conservative. In order to further validate the function of mmi-DIGIRR and its expression levels in various tissues of fish, qRT-PCR has been conducted. The results showed DIGIRR has significant expression levels in liver, skin and muscle while expression levels in heart are low. The LPS-induced NF-κB activation was inhibited by overexpression of DIGIRR significantly. In conclusion, the evolution and function of mmi-DIGIRR were comprehensively analyzed in this study, which would provide a theoretical basis for the future research of fish DIGIRR.

Paeoniflorin ameliorates Aβ-stimulated neuroinflammation via regulation of NF-κB signaling pathway and Aβ degradation in C6 glial cells

BACKGROUND/OBJECTIVESAlzheimer's disease is common age-related neurodegenerative condition characterized by amyloid beta (Aβ) accumulation that leads cognitive impairment. In the present study, we investigated the protective effect of paeoniflorin (PF) against Aβ-induced neuroinflammation and the underlying mechanism in C6 glial cells.MATERIALS/METHODSC6 glial cells were treated with PF and Aβ25–35, and cell viability, nitric oxide (NO) production, and pro-inflammatory cytokine release were measured. Furthermore, the mechanism underlying the effect of PF on inflammatory responses and Aβ degradation was determined by Western blot.RESULTSAβ25–35 significantly reduced cell viability, but this reduction was prevented by the pretreatment with PF. In addition, PF significantly inhibited Aβ25–35-induced NO production in C6 glial cells. The secretion of interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha was also significantly reduced by PF. Further mechanistic studies indicated that PF suppressed the production of these pro-inflammatory cytokines by regulating the nuclear factor-kappa B (NF-κB) pathway. The protein levels of inducible NO synthase and cyclooxygenase-2 were downregulated and phosphorylation of NF-κB was blocked by PF. However, PF elevated the protein expression of inhibitor kappa B-alpha and those of Aβ degrading enzymes, insulin degrading enzyme and neprilysin.CONCLUSIONSThese findings indicate that PF exerts protective effects against Aβ-mediated neuroinflammation by inhibiting NF-κB signaling, and these effects were associated with the enhanced activity of Aβ degradation enzymes.

Anti-inflammation and anti-apoptosis effects of growth arrest-specific protein 6 in acute liver injury induced by LPS/D-GalN in mice.

Purpose To investigate the effect of growth arrest-specific protein 6 (Gas6) on acute liver injury in mice and related mechanisms.Methods Thirty C57BL/6 (6-8 weeks old) mice were randomly divided into control, LPS/D-GalN, and LPS/D-GalN+Gas6 groups (10 mice in each group). The LPS/D-GalN group was intraperitoneally administered with LPS (0.25 mg/Kg) and D-GalN (400 mg/Kg) for 5h. The LPS/D-GalN+Gas6 group was intraperitoneally administered with rmGas6 one hour before intraperitoneal application of LPS/D-GalN. All subjects were sacrificed at 5 h for blood and tissue analysis. The expression of protein and mRNA was assessed by western blotting and RT-PCR, respectively.Results Compared with the control group, AST, ALT, IL-1β, TNF-α, IL-6 IL-10, MPO activity were increased in the LPS/D-GalN group. However, they were significantly inhibited by Gas6. Gas6 markedly suppressed the expression of apoptosis-related protein induced by LPS/D-GalN. Moreover, Gas6 attenuated the activation of the NF-κB signaling pathway in acute liver injury induced by LPS/D-GalN.Conclusions Gas6 alleviates acute liver injury in mice through regulating NF-κB signaling pathways. Gas6 can be a potential therapeutic agent in treating LPS/D-GalN-induced acute liver injury in the future.

Effect of Silent Information Regulator on the Survival and Osteogenic Differentiation of Inflammatory Bone Marrow Mesenchymal Stem Cells by Regulating NF-κB Signaling Pathway

Osteogenic differentiation of BMSCs is beneficial for osteoarthritis (OA) treatment. Silent information regulator (SIRT1) plays a role in endocrine diseases and aging-related diseases. However, the role of SIRT1 in OA has not yet been elucidated. Rat BMSCs were isolated and divided into control group, inflammation group (BMSCs were cultured with IL-6), SIRT1 group (SIRT1 agonist Resveratrol was added under the action of IL-6) followed by analysis of cell proliferation by MTT assay, Caspase 3 activity, ALP activity, expression of osteogenic genes Runx2 and OC and adipogenic differentiation gene PPARγ2 by Real time PCR, NF-κB expression by western blot and secretion of TNF-α and IL-6 by ELISA. In inflammation group, SIRT1 expression was significantly decreased, cell proliferation was significantly inhibited, and Caspase 3 activity was increased. Meanwhile, ALP activity, Runx2 and OC expression was decreased, PPARγ2 and NF-κB expression was increased, along with elevated TNF-α and IL-6 secretion compared to control (P < 0.05). Resveratrol can significantly promote the expression of SIRT1 in BMSCs of inflammation group, promote cell proliferation, decrease Caspase 3 activity, and increase Runx2 and OC expression. In addition, it decreased PPARγ2 and NF-κB expression and reduced the secretion of TNF-α and IL-6 (P < 0.05). The expression of SIRT1 was decreased in BMSCs under inflammation. SIRT1 overexpression in BMSCs under inflammation inhibits inflammation, promotes proliferation and osteogenic differentiation of BMSCs through regulating NF-κB signaling pathway.

MAST3 modulates the inflammatory response and proliferation of fibroblast-like synoviocytes in rheumatoid arthritis

Via promoting synovitis, pannus growth and cartilage/bone destruction, fibroblast-like synovial cells (FLSs) play a significant role in the pathogenesis of rheumatoid arthritis (RA). In our study, rats were induced with complete freund’s adjuvant (CFA) to be animal models for studying the RA pathogenesis. Microtubule-associated Serine/Threonine-protein kinase 3 (MAST3) has been documented to play a critical role in regulating the immune response of IBD (Inflammatory bowel disease) and involved in the process of cytoskeleton organization, intracellular signal transduction and peptidyl-serine phosphorylation, but its role in the progression of RA remains unknown and is warranted for investigation. So, we tried our best to investigate the mechanism and signaling pathway of MAST3 in RA progression. In the synovial tissue and FLSs of AA rats, we have found that MAST3 was significantly up-regulated than normal. Furthermore, MAST3 overexpression could promote proliferation and inflammatory response of FLSs. In the aspect of mechanism, we discovered that the expression of MAST3 might involve in NF-κB signaling pathway in RA. On the whole, our results suggested that MAST3 might promote the proliferation and inflammation of FLSs by regulating NF-κB signaling pathway.

Icariside II inhibits lipopolysaccharide-induced inflammation and amyloid production in rat astrocytes by regulating IKK/IκB/NF-κB/BACE1 signaling pathway.

β-amyloid (Aβ) is one of the inducing factors of astrocytes activation and neuroinflammation, and it is also a crucial factor for the development of Alzheimer’s disease (AD). Icariside II (ICS II) is an active component isolated from a traditional Chinese herb Epimedium, which has shown to attnuate lipopolysaccharide (LPS)-induced neuroinflammation through regulation of NF-κB signaling pathway. In this study we investigated the effects of ICS II on LPS-induced astrocytes activation and Aβ accumulation. Primary rat astrocytes were pretreated with ICS II (5, 10, and 20 μM) or dexamethasone (DXMS, 1 μM) for 1 h, thereafter, treated with LPS for another 24 h. We found that ICS II pretreatment dose dependently mitigated the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in the astrocytes. Moreover, ICS II not only exerted the inhibitory effect on LPS-induced IκB-α degradation and NF-κB activation, but also decreased the levels of Aβ1–40, Aβ1–42, amyloid precursor protein (APP) and beta secretase 1 (BACE1) in the astrocytes. Interestingly, molecular docking revealed that ICS II might directly bind to BACE1. It is concluded that ICS II has potential value as a new therapeutic agent to treat neuroinflammation-related diseases, such as AD.

Effect of LGR4 on synovial cells and inflammatory factors in rats with traumatic osteoarthritis.

Traumatic arthritis is one of the most common diseases in orthopedics. LGR4 is involved in bone formation and bone development. However, the role of LGR4 in synovial cells of rats with traumatic osteoarthritis has not been reported. Sprague Dawley (SD) rats were randomly divided into the control group and model group. The Real Time-Polymerase Chain Reaction (RT-PCR), Western blot, and Enzyme-Linked Immunosorbent Assay (ELISA) were used to analyze the expression of LGR4 in synovial tissue and synovial fluid. Synovial cells were isolated and cultured, followed by transfection of LGR4-pcDNA3.1 plasmid into cells. Cell proliferation was analyzed by MTT and EdU assay, and the Caspase-3 activity was assessed using the Caspase-3 activity kit. The secretion of the inflammatory factors interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was detected by ELISA. NF-κB signaling pathway changes were evaluated by the Western blot. In the model group, LGR4 mRNA expression in synovial tissue was significantly decreased, and the secretion of LGR4 in the synovial fluid was significantly decreased compared with the control group (p<0.05). LGR4 protein expression in the synovial membrane in the model group tissue was reduced. The transfection of LGR4-pcDNA3.1 plasmid into synovial cells promoted the LGR4 expression, inhibited the proliferation of synoviocytes, increased the Caspase-3 activity, the secretion of IL-1, TNF-α, and IL-6, as well as the decreased expression of NF-κB with a statistical significance, compared with the control group (p<0.05). LGR4 expression is reduced in the rat model of traumatic osteoarthritis. The upregulation of LGR4 expression can inhibit the secretion of the inflammatory factors and inhibit the proliferation of the synovial cells by regulating NF-κB signaling pathway, which may alleviate the development of the joint inflammation.

Anti-inflammatory properties of fermented pine (Pinus morrisonicola Hay.) needle on lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells.

This study was aimed to explore the antioxidant and anti-inflammatory activity of various pine needle products (non-fermented, fermented, and commercial) extracted with different solvents (hexane, ethyl acetate, and water) in lipopolysaccharide (LPS) induced RAW 264.7 cells. The phenolic/flavonoid contents of ethyl acetate extract of fermented pine needle (EAE-FPN) is higher than other pine product extracts (hexane/water). The levels of antioxidant indices (TEAC, DPPH) as well as free radical scavenging activity (H2 O2 ) were significantly improved in EAE-FPN than other pine needle product extracts. The levels of ROS and various inflammatory markers (NO, PGE2, TNF-α, and IL-1β/6) were considerably abolished by EAE-FPN in a dose-dependent manner (50-200μg/ml). Moreover, the protein expressions of inducible NO synthase (iNOS), cyclooxygenase 2 (COX-2), and nuclear factor Kappa B (NF-κB) p65 subunit were also markedly downregulated by EAE-FPN. Collectively, EAE-FPN with phenolic/flavonoid content showed excellent antioxidant and anti-inflammatory properties via modulating NF-κB signaling pathway. PRACTICAL APPLICATIONS: Pine needle drink (Pinus morrisonicola Hay) has been used as a functional beverage for many years due to its various biological properties in Asia especially in Taiwan, China, and Korea. Many researchers hinted various biological activity of fermented pine needle product but none of them explored the in-depth mechanism underpinning its antioxidant and anti-inflammatory properties in LPS-induced RAW 264.7 cell model. Hence, the current cell line study was designed to assess the underlying mechanism behind the antioxidant and anti-inflammatory activity of Pine needles extract (both non-fermented and fermented) in LPS-induced RAW 264.7 cells (macrophage). The outcome of this study distinctly showed that EAE-FPN displayed potent antioxidant and anti-inflammatory activities by regulating NF-κB signaling pathway. Therefore, pine needle could be developed into functional drink to abolish the progression of inflammatory responses in various disease condition.

Knockdown of TRIM28 inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.

Down-regulation of microRNA-98 promoted apoptosis of TNF-α stimulated human fibroblast-like synoviocytes via up-regulating IL-10

In this study, we constructed a tumor necrosis factor α (TNF-α)-induced synovial cell inflammatory model using human synoviocytes (HS) cell line to explore the function of miR-98 in rheumatoid arthritis (RA). miR-98 mimics or miR-98 inhibitor were transfected into HS cells to up-regulate or down-regulate the expression of miR-98. The proliferation and apoptosis of HS cells were determined using CCK8 assay and flow cytometry, respectively. TargetScan website was utilized to predict the targets of miR-98. Luciferase assay was carried out to verify that IL-10 is a target of miR-98. Western blot was performed to analyze the expression of IL-10, apoptosis-related and NF-κB signaling pathway-related proteins. Our results demonstrated that the expression of miR-98 was up-regulated in HS cells stimulated by TNF-α. Down-regulation of miR-98 by inhibitor in TNF-α-stimulated HS cells dramatically inhibited cell proliferation and promoted cell apoptosis compared with the miR-98 inhibitor NC group. The protein expression of Bcl-2 was declined while the levels of Bax and Bim were increased by miR-98 inhibitor in TNF-α-stimulated HS cells. IL-10 was predicted and verified as a target of miR-98. qRT-PCR and western blot results revealed that the level of IL-10 was negatively regulated by miR-98. Finally, we identified that down-regulation of miR-98 reduced the expression level of p-p65 and p-IκBα in TNF-α-stimulated HS cells. In summary, our present study demonstrated that down-regulation of miR-98 inhibited the proliferation and promoted the apoptosis of TNF-α-stimulated HS partly by targeting IL-10 and regulating NF-κB signaling pathway, insinuating miR-98 as a candidate biomarker in RA.

Effect of ginkgo biloba L. extract on apoptosis and NF-κB signaling pathways in the spleen tissue of mice with radiation damage

Objective To investigate the influence of Ginkgo biloba L. extract (EGB) on the expression of apoptosis-related protein and NF-κB signaling pathway in the spleen tissue of mice with radiation damage. Methods The mice were divided randomly into normal control group (NC), irradiation control group (IC), low dose EGB group (IC+EGBL), medium dose EGB group (IC+EGBM) and high dose EGB group (IC+EGBH) according to the random number table, with 12 rats in each group. The mice in the low, middle and high dose EGB groups were given EGB respectively by 5, 10, 20 mg/kg, and the normal and irradiation control group were given saline by intraperitoneal injection once daily for 14 days. On the 15th day, the mice in all groups were uniformly irradiated with 4.0 Gy γ-rays for one time except normal control group. After 24 hours, Bcl-2, Bax and caspase-3 protein expression were measured by immunohistochemical method. The IKKβ expression was detected by qRT-PCR method, and the content of NF-κB p65 and IKKα in serum was detected by Elisa method in spleen tissue. Results Comparing with IC group, the expression of Bax (54.31 ± 1.59, 42.04 ± 1.56, 32.08 ± 2.43 vs. 68.68 ± 3.12) and caspase-3 protein (55.73 ± 2.61, 45.81 ± 2.59, 36.78 ± 2.23 vs. 72.18 ± 1.84) in IC+EGBL, IC+EGBM and IC+EGBH group were significantly decreased (P<0.05), the expression of Bcl-2 protein (30.33 ± 1.28, 39.80 ± 2.86, 44.42 ± 3.64 vs. 22.80 ± 2.01) in IC+EGBL, IC+EGBM and IC+EGBH group significantly increased (P<0.05), the expression of IKKβ mRNA (1.43 ± 0.06, 1.31 ± 0.06, 1.17 ± 0.09 vs. 1.64 ± 0.10) and the level of NF-κB p65 (129.38 ± 8.41 pg/ml, 111.28 ± 9.09 pg/ml, 95.41 ± 6.88 pg/ml vs. 145.64 ± 6.29 pg/ml) and IKKα (160.10 ± 8.94 pg/ml, 144.00 ± 8.36 pg/ml, 108.84 ± 13.74 pg/ml vs. 176.38 ± 8.54 pg/ml) in IC+EGBL, IC+EGBM and IC+EGBH group significantly decreased (P<0.05). Conclusions The EGB can reduce the expression of apoptotic protein Bax and caspase-3 in spleen cells induced by radiation, elevate the expression level of apoptotic protein Bcl-2, and inhibit the damage caused by radiation by regulating NF-κB signaling pathway. Key words: Ginkgo folium; Extracts; Radiation protection; Apoptosis; NF-kappa B; Mice

Baicalin alleviated APEC-induced acute lung injury in chicken by inhibiting NF-κB pathway activation

Bacterial pneumonia is a leading cause of death in the animal husbandry. Acute lung injury (ALI), most often seen as a part of systemic inflammatory process, characterized by progressive hypoxemia, edema, and neutrophil accumulation in the lung. Baicalin has been reported to inhibit inflammatory response, but its role in ALI remains unknown. The purpose of our study was to determine the protective effect and possible mechanism of baicalin against avian pathogenic Escherichia coli (APEC)-induced ALI in chicken. Chickens were conditioned with baicalin 1 week before intratracheally instilled with APEC. Then, chickens were sacrificed by CO2 inhalation 12 h later and the lung tissues were collected for examining histopathological changes, wet/dry (W/D) ratio, myeloperoxidase (MPO) activity, levels of pro-inflammatory cytokines and activation of NF-κB signaling pathway. The results showed that pre-treatment of chickens with baicalin significantly alleviated the death rate, histopathological changes in lung tissues. The W/D ratio, MPO activity and production of cytokines, such as IL-1β, TNF-α, IL-6 of lung tissues were also decreased following treatment with baicalin. Furthermore, the mechanism responsible for these effects was attributed to the inhibitory effect of baicalin on nuclear factor-κB (NF-κB) signaling activation. These data thus support the application of baicalin as a potential medicine for the treatment of E. coli-induced ALI by regulating NF-κB signaling pathway.

Molecular mechanisms of interleukin-38 inhibiting inflammatory bowel disease in children by regulating nuclear factor-κB and signal transduction and activator of transcription 3 pathway

Objective To explore the role of interleukin (IL)-38 in inhibiting inflammatory bowel disease (IBD) in children and to investigate the potential molecular mechanisms. Methods From January 2014 to October 2017, 67 patients with ulcerative colitis (UC) and 115 patients with Crohn′s disease (CD) admitted to Wuhan Children′s Hospital were recruited, and 40 individuals with normal endoscopic findings were selected as control. Serum levels of IL-38 of IBD patients and healthy control were determined by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining (IHC) was used to detect the expression level of IL-38, nuclear factor κB (NF-κB), phosphorylated signal transduction and activator of transcription 3 (p-STAT3), C-reaction protein (CRP) and erythrocyte sedimentation rate (ESR) in the intestinal mucosa of IBD patients and healthy controls. The extent of disease, therapeutic agents and disease activity scores (Mayo score system for UC patients, Crohn′s disease activity index (CDAI) for CD patients) were evaluated. IL-38-C57BL/6 transgenic mice model was established, and dextran sulfate sodium was used to induce IBD mice model. The intestinal inflammation levels were compared between the wild type IBD mice and IL-38 transgenic IBD mice. The levels of IL-38, NF-κB and p-STAT3 in intestinal mucosa of mice of different groups were determined by IHC. The ratio of CD4+ IL-17+ T helper (Th) 17 cells in peripheral blood of mice of different groups was detected by flow cytometry. Independent sample t test, chi square test and Pearson correlation were performed for statistical analysis. Results The results of ELISA showed that the serum levels of IL-38 of UC and CD patients were (6.1±1.9) ng/L and (9.8±2.1) ng/L, respectively, which both were lower than that of healthy controls ((16.4±2.7) ng/L), and the differences were statistically significant (t=23.107 and 15.853, both P<0.05). The results of IHC indicated that the levels of IL-38 in the intestinal mucosal tissues of UC and CD patients were 0.04±0.01 and 0.03±0.01, respectively, which were both lower than that of healthy controls (0.18±0.02), and the differences were statistically significant (t=48.186 and 69.443, both P<0.05). The levels of NF-κB and p-STAT3 of UC and CD patients were 0.150±0.030, 0.160±0.040 and 0.130±0.030, 0.110±0.010, which were all higher than those of healthy controls (0.020±0.003 and 0.010±0.002), and the differences were statistically significant (tUC=27.273 and 23.078, tCD=23.657 and 62.684; all P<0.05). The number of patients with disease at active phase, CRP level, ESR and disease activity scores of UC and CD patients with low IL-38 expression were all significantly higher than those of patients with high IL-38 expression (χUC2=11.552, tUC=7.118, 8.991 and 7.086; χCD2=5.675, tCD=9.559, 9.358 and 11.268; all P<0.05). The results of Pearson correlation analysis demonstrated that the level of IL-38 in the intestinal mucosal tissue of UC patients was negatively correlated with CRP, ESR and Mayo scores (r=-0.291, -0.672 and -0.639; all P<0.05). And the level of IL-38 in the intestinal mucosal tissue of CD patients was negatively correlated with CRP, ESR and CDAI (r=-0.559, -0.471 and -0.353; all P<0.05). The IHC results showed that the levels of NF-κB and p-STAT3 of IL-38 transgenic IBD mice were lower than those of wild type IBD mice (0.14±0.02 vs. 0.32±0.06, 0.12±0.02 vs. 0.44±0.07), and the differences were statistically significant (t=6.971 and 10.767, both P<0.05). The results of flow cytometry showed that the ratio of CD4+ IL-17+ Th17 cells in the peripheral blood of IL-38 transgenic IBD mice was lower than that of wild type IBD mice (0.030±0.006 vs. 0.280±0.050), and the difference was statistically significant (t=12.160, P<0.05). Conclusions The expression level of IL-38 significantly decreases in the intestinal mucosal tissues of IBD patients, while the level of NF-κB and p-STAT3 significantly increases. IL-38 may inhibit IBD by regulating NF-κB and p-STAT3 signaling pathway to alleviate intestinal immune reaction. Key words: Inflammatory bowel diseases; NF-kappa B; interleukin-38; STAT3

Porphyromonas gingivalis promotes the motility of esophageal squamous cell carcinoma by activating NF-κB signaling pathway

Esophageal carcinoma, with a increasing incidence, is one of the most aggressive carcinomas in gastrointestinal tract. Epidemiologic studies demonstrate an association of oral pathogens with multiple diseases, including rheumatoid arthritis, cardiovascular diseases, diabetes, and gastrointestinal malignancies. Nevertheless, a causal relationship between oral pathogens and esophageal squamous cell carcinoma (ESCC) has not been elucidated. Here, we found that Porphyromonas was significantly enriched in the saliva of patients with ESCC, compared with that in normal human. In vitro studies showed that Porphyromonas gingivalis (P. gingivalis) promoted the proliferation and motility of ESCC cells, as evidenced by up regulated expression of key molecules implicated in NF-κB signaling pathway. These findings, for the first time, demonstrated a role of oral pathogens in inducing ESCC tumorigenesis and metastasis, which might involve regulation of NF-κB signaling pathway.

Cellular mechanism of Tβ4 intervention in liver fibrosis by regulating NF-κB signaling pathway.

To investigate the inhibitory effect of thymosin-β4 (Tβ4) on the activation of the human hepatic stellate cell line (HSC-LX2) induced by interleukin (IL)-1β. There were 5 groups in this study, i.e., blank control group, negative control group (SI-NC, empty plasmid), model group (20 ng/ml of IL-1β), siRNA-Tβ4 knockdown group (IL-1β and si-Tβ4) and Tβ4 treatment group (IL-1β and 1000 ng/ml of Tβ4). Cell proliferation rate was measured using the Cell Counting Kit-8 (CCK-8) method. The cell cycle change and percentage of apoptotic cells were determined by Propidium Iodide (PI) DNA staining and Annexin V-fluorescein isothiocyanate (FITC) double staining. Cellular nucleic acid levels of p-IKB and nuclear factor-kappa B (NF-κB)/p65 proteins were measured by fluorescent quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Double immunofluorescence staining and Western blot were used to detect nuclear translocation of NF-κB and p65 and levels of cytoplasmic p-IKB protein and nuclear p65 protein. Due to the G0/G1 phase arrest, the number of cells in the Tβ4 treatment group increased, compared with the model group and the siRNA-Tβ4 knockdown group (p<0.01). In the same between-group comparison, apoptotic rate in the Tβ4 treatment group increased significantly (p<0.05). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were markedly higher in the model group and the siRNA-Tβ4 knockdown group than in the blank control group (p<0.01). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were remarkably lower in the Tβ4 treatment group than in the siRNA-Tβ4 knockdown group (p<0.01). The expression levels of NF-κB/p65 and NF-κB/p50 were significantly lower in the Tβ4 treatment group. The expression levels of cytoplasmic p-IKB and nuclear NF-κB/p65 were lower in the Tβ4 treatment group than in the model group (p<0.01). Tβ4 significantly inhibited IL-1β-induced HSC-LX2 cell proliferation. The mechanism may involve decreased activation of the NF-κB pathway, decreased expression of p-IKB and nuclear translocation of p65. Therefore, Tβ4 had the effect of reversing liver fibrosis.

Obtusifolin isolated from the seeds of Cassia obtusifolia regulates the gene expression and production of MUC5AC mucin in airway epithelial cells via affecting NF-κB pathway.

We investigated whether obtusin, obtusifolin, and cassiaside isolated from the seeds of Cassia obtusifolia inhibit the gene expression and production of airway mucin 5AC (MUC5AC). Confluent NCI-H292 cells were pretreated with obtusin, obtusifolin, or cassiaside for 30min and then stimulated with epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA), or tumor necrosis factor-α (TNF-α) for 24hr. The MUC5AC mucin gene expression was measured by reverse transcription-polymerase chain reaction. Production of MUC5AC mucin protein was measured by enzyme-linked immunosorbent assay. To elucidate the action mechanism of obtusifolin, effect of obtusifolin on PMA-induced nuclear factor kappa B (NF-κB) signaling pathway was investigated by western blot analysis. Obtusin, obtusifolin, or cassiaside inhibited the expression of MUC5AC mucin gene and the production of MUC5AC mucin protein, induced by EGF, PMA, or TNF-α. Obtusifolin inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase, and thus phosphorylation and degradation of inhibitory kappa B alpha. Obtusifolin inhibited PMA-induced nuclear translocation of NF-κB p65. These results suggest that obtusifolin can regulate the production and gene expression of mucin by acting on airway epithelial cells through regulation of NF-κB signaling pathway.

MiR-155 affects osteosarcoma cell proliferation and invasion through regulating NF-κB signaling pathway.

Osteosarcoma can form tumor osteoid tissue and bone tissue directly or indirectly through cartilage stage. It mainly occurs in children and adolescents with high mortality. MicroRNA (miRNA) possesses tissue sensitivity as tumor biomarker and plays a promoting or inhibitory role in tumor pathogenesis as oncogene or tumor suppressor gene. It was found that miR-155 was abnormally expressed in tumor and could be treated as a biomarker for cancer progression. However, miR-155 expression in osteosarcoma and related mechanism still remains unclear. This study aimed to explore the role of miR-155 in osteosarcoma occurrence and development. Osteosarcoma cell line MG-63 was cultured in vitro and transfected by miR-155 mimics or inhibitor. MiR-155 expression was examined by Real-time PCR (RT-PCR). Cell proliferation was evaluated by MTT assay. Caspase 3 activity was determined by caspase 3 activity detection kit. Cell invasion was measured by transwell assay. B-cell lymphoma-2 (Bcl-2) and nuclear factor κB (NF-κB) protein expressions were assessed by Western blot. MiR-155 mimics significantly up-regulated miR-155 expression, promoted MG-63 cell proliferation and invasion, inhibited caspase 3 activity, and up-regulated expressions of NF-κB and Bcl-2 compared with control group (p < 0.05). However, miR-155 inhibitor significantly inhibited MG-63 cell proliferation and invasion, enhanced caspase 3 activity, and reduced expressions of NF-κB and Bcl-2 compared with control group (p < 0.05). MiR-155 affected osteosarcoma cell proliferation and apoptosis through regulating NF-κB signaling pathway, indicating it might be a new biomarker for osteosarcoma diagnosis and treatment.