Nuclear factor-kappaB Phosphorylation Research Articles

The Mucilage From the Opuntia ficus-indica (L.) Mill. Cladodes Plays an Anti-Inflammatory Role in the LPS-Stimulated HepG2 Cells: A Combined In Vitro and In Silico Approach.

The effect of a mucilage extracted from Opuntia ficus-indica (L.) Mill (OFI) cladodes was tested in lipopolysaccharide (LPS)-challenged HepG2 hepatocarcinoma cells, through a combined in vitro-in silico approach. The OFI mucilage was characterized by gas chromatography-mass spectrometry and liquid chromatography-high resolution mass spectrometry. In cells treated with OFI (5-10µg/mL) prior to LPS (1 µg/mL, 24h), the gene expression profile of pro-inflammatory mediators, namely tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and cyclo-oxygenase-2, was significantly (p<0.01) reduced if compared to single LPS-challenged cells. The OFI-mediated cytokines reduction was also validated in polystyrene scaffold-grown 3D HepG2 cultures, undergoing treatment with the OFI mucilage (50µg/mL, 24h) and LPS stimulation (50µg/mL, 24h). We further demonstrated that OFI suppresses the LPS-triggered inflammatory response via impairment of the Toll-like receptor 4 (TLR4)/Myeloid differentiation protein-88/Nuclear factor-kappa B (NF-kB) pathway, by interfering with NF-kB phosphorylation at Serine 536. By molecular docking approach, we provided in silico demonstration of the direct molecular interaction between the mucilage monosaccharides and the TLR4 that interferes with the LPS receptor binding and down-stream inflammatory cascade activation. We also demonstrated that OFI cladodes mucilage downregulates the TLR4 pathway, showing an anti-inflammatory potential in HepG2 cells.

Skin collagen peptides of Chinese giant salamander (Andrias davidianus) exert anti-inflammatory effects in LPS-induced RAW264.7 cells via the lincRNA-EPS/NF-κB pathway

ABSTRACT Food-derived collagen peptides have recently been reported to have immunomodulatory functions. The Chinese giant salamander (CGS) has both edible and medicinal values. In this study, we tried to verify the immunoregulatory function of CGS skin collagen peptides (CGSSCPs) on macrophages and elucidate its mechanism. CGSSCPs decreased the secretion of nitric oxide (NO) and downregulated the expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), chemokine (C–C motif) ligand 5 (CCL5) and (C-X-C motif) ligand 10 (CXCL10) in Lipopolysaccharide (LPS)-induced RAW264.7 cells. Moreover, CGSSCPs promoted the expression of lincRNA-EPS, and inhibited the phosphorylation of nuclear factor kappa-B (NF-κB) in LPS-induced RAW264.7 cells. Silencing lincRNA-EPS up-regulated the phosphorylation level of NF-κB and the expression of inflammatory cytokines (IL-6, IL-1β, and TNF-α) and chemokines (CCL5 and CXCL10). These suggest that CGSSCPs may regulate RAW264.7 cells by the lincRNA-EPS/NF-κB pathway, which provides a theoretical basis for the development of new anti-inflammatory drugs.

Acetylcorynoline alleviates acute liver injury via inhibiting TLR4/JNK/NF-ĸB pathway Based on RNA-seq and molecular docking in vivo and in vitro

Acute liver injury is characterized by massive inflammatory cell infiltration, destruction of liver structure and abnormalities in liver function. Acetylcorynoline (AC) is one of the main chemical components of Corydalis bungeana Turcz. which has been shown to have a protective effect against acute liver injury. However, Whether AC is protective against acute liver injury remains unclear. This study aimed to explore the protective mechanism of AC against acute liver injury from in vivo as well as experiments in vitro. In experimental in vivo studies, AC pretreatment reduced the serum levels of ALT and AST and inhibited the expression of inflammatory factors in the liver of LPS/D-GalN-induced mice and alcohol liver disease mice. RNA-sequencing and molecular docking were used to predict that AC exerts its anti-inflammatory effects through the Toll-like receptor signaling pathway. Using RT-qPCR and Western blotting to detect expression levels of key genes and nodal proteins of the Toll-like receptor signaling pathway, AC was found to inhibit the phosphorylation of nuclear factor-kappaB (NF-ĸB) and c-Jun amino-terminal kinase (JNK). This finding was validated in cellular experiments. In conclusion, AC exerts its anti-hepatic injury effect by suppressing inflammation through inhibition of the TLR4/JNK/NF-ĸB pathway.

Role of ERK and NF-KB pathway in neurally mediated enhanced expression and traffcking of SGLT2 in congestive heart failure

Congestive heart failure (CHF) is characterized by the activation of neurohumoral systems concomitant with avid fluid retention. Recently we showed enhanced expression of sodium-glucose cotransporter 2 (SGLT2) in rats with CHF. However, the exact molecular mechanism linking enhanced sympathetic tone and enhanced expression and translocation of SGLT2 in CHF is not clear. The objective of this study was to investigate the role of enhanced sympathetic tone on the expression and traffcking of SGLT2 via activation of protein kinase A (PKA), extracellular signal-regulated kinases (ERK), and nuclear factor kappa B (NF-KB) in the renal cortex of CHF rats. Male Sprague Dawley rats were subjected to either the left coronary artery ligation for the CHF group or Sham surgery. Four weeks after surgery, expression of protein levels (PKA, ERK and NF-KB) were assessed by Western blotting, whereas the location of SGLT2 expression was determined by immunohistological and immunofluorescent staining in the cortical region of kidneys from Sham and CHF rats. Effect of exogenous norepinephrine (NE) (10μM for 24hrs) on expression and traffcking of SGLT2, phosphorylation of ERK and NF-KB in HK2 cells were examined in an in vitro model. Expression of SGLT2 was significantly enhanced in the renal cortex of rats with CHF versus Sham (0.83±0.06 vs. 0.58±0.02). Also, the translocation of SGLT2 was significantly enhanced towards the membrane in CHF rats (0.92±0.11 vs. 0.33±0.07). Phosphorylation of PKA (0.05±0.01 vs. 0.01±0.01), ERK (0.32±0.02 vs. 0.23±0.02) and NF-KB (0.71±0.14 vs. 0.08±0.06) were significantly enhanced in CHF compared to Sham. NE treatment significantly increased expression of SGLT2 (0.26±0.03 vs. 0.15±0.02), traffcking of SGLT2 (0.45 ± 0.09 vs. 0.15 ± 0.03) towards membrane, phosphorylation of ERK (0.38±0.05 vs. 0.14±0.02) and NF-KB (0.61±0.08 vs. 0.17±0.07) in HK2 cells. Thus, NE treatment enhanced the phosphorylation of ERK, NF-kB as well as traffcking of SGLT2. Immunohistological and immunofluorescent staining demonstrated increased expression of SGLT2 in the kidneys of rats with CHF. Similar results were observed in immunofluorescent staining of human kidney cells treated with NE. In conclusion, these findings indicate that NE activates ERK and NF-KB pathways which leads to enhanced traffcking and overexpression of SGLT2 in CHF. These results provide a molecular link between enhanced sympathetic tone, ERK and NF-KB pathway and the traffcking of SGLT2 to the luminal membrane of renal proximal tubular cells and consequent increase in sodium retention in rats with CHF. This study was funded by NIH R01-DK-129311. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lecithin-complexed oregano essential oil-encapsulated fibrous barriers prevent postoperative adhesions by regulating Nrf2/NF-κB signaling pathways

Postoperative adhesions are prone to a variety of complications, which can adversely affect the quality of life (QOL) of patients. While anti-adhesion scaffolds rapidly degrade, foreign body reactions (FBR), exaggerated inflammatory reactions, and oxidative stress may increase tissue adhesions. This necessitates the development of anti-adhesion scaffolds with multifunctional anti-inflammatory, anti-bacterial, and anti-oxidative characteristics. The objective of this study was to simultaneously leverage natural antimicrobial and anti-oxidative bioactive molecules, such as lecithin (L) and Oregano essential oil (O) to afford poly(lactic-co-glycolic acid) (PLGA)-based electrospun membranes for postoperative anti-adhesion management. A series of assays revealed a beneficial effect of L and O co-loaded membranes (P@LO) to suppress bacterial growth, reduce the proliferation of fibroblasts, and confer anti-oxidative and anti-inflammatory functionalities to membranes than that of the control (P) or only a single cue-loaded groups (P@L and P@O). The underlying molecular mechanism of the anti-inflammatory effect may be an upregulation of the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. An assessment of membranes in an abdominal and a tendon adhesion model showed a significant effect of P@LO membranes to prevent post-operative tissue adhesion. The P@LO membranes were found to up-regulate the phosphorylation of Nrf2 while down-regulate the phosphorylation of nuclear factor kappa-B (NF-κB) P65. This resulted in lower expression of collagen Ⅰ (Col-Ⅰ), collagen Ⅲ (Col-Ⅲ), and alpha-smooth muscle actin (α-SMA) alongside inflammation resolution and less transition of fibroblasts to myofibroblasts. Taken together, these multifunctional scaffolds may have broad implications for postoperative adhesion management.

Punicalagin promotes mincle-mediated phagocytosis of macrophages via the NF-κB and MAPK signaling pathways

Punicalagin (PUN) is a polyphenol derived from the pomegranate peel. It has been reported to have many beneficial effects, including anti-inflammatory, anti-oxidant, and anti-proliferation. However, the role of PUN in macrophage phagocytosis is currently unknown. In this study, we found that pre-treatment with PUN significantly enhanced phagocytosis by macrophages in a time- and dose-dependent manner in vitro. Moreover, KEGG enrichment analysis by RNA-sequencing showed that differentially expressed genes following PUN treatment were significantly enriched in phagocyte-related receptors, such as the C-type lectin receptor signaling pathway. Among the C-type lectin receptor family, Mincle (Clec4e) significantly increased at the mRNA and protein level after PUN treatment, as shown by qRT-PCR and western blotting. Small interfering RNA (siRNA) mediated knockdown of Mincle in macrophages resulted in down regulation of phagocytosis. Furthermore, western blotting showed that PUN treatment enhanced the phosphorylation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) in macrophages at the early stage. Mincle-mediated phagocytosis by PUN was inhibited by PDTC (a NF-κB inhibitor) and SB203580 (a p38 MAPK inhibitor). In addition, PUN pre-treatment enhanced phagocytosis by peritoneal and alveolar macrophages in vivo. After intraperitoneal injection of Escherichia coli (E.coli), the bacterial load of peritoneal lavage fluid and peripheral blood in PUN pre-treated mice decreased significantly. Similarly, the number of bacteria in the lung tissue significantly reduced after intranasal administration of Pseudomonas aeruginosa (PAO1). Taken together, our results reveal that PUN enhances bacterial clearance in mice by activating the NF-κB and MAPK pathways and upregulating C-type lectin receptor expression to enhance phagocytosis by macrophages.

Effect of Infliximab on Radiation-Induced Submandibular Gland Dysfunction in Rats.

Inflammatory response is one of the essential parts of various pathogenic mechanisms of radiation-induced salivary dysfunction. The effect of decreasing the levels of inflammatory cytokines on alleviating submandibular gland injuries after irradiation is unclear. This study aimed to explore the effect of the antibody against tumor necrosis factor-alpha, infliximab, on radiation-induced submandibular gland dysfunction in rats. Male Wistar rats received a single 20 Gy dose to the right submandibular gland region or sham irradiated. Meanwhile, the irradiated group was divided into infliximab treatment groups or untreated groups. Animals were euthanized at 1, 6, and 12 weeks postirradiation, and the irradiated submandibular gland was dissected for subsequent detection. Submandibular gland exposure caused obvious pathological changes. The increased levels of inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1β, and interleukin-6, represent an aggravated inflammatory response. The results of the western blot, reverse transcription-quantitative polymerase chain reaction, and immunofluorescence staining showed upregulated levels of claudin-1, claudin-3, and aquaporin 5 and downregulated levels of claudin-4. Moreover, nuclear factor kappa-B phosphorylation levels were also up-regulated. In subsequent experiments, we found that infliximab alleviated inflammatory response, up-regulated tumor necrosis factor-alpha, interleukin-1β, and interleukin-6 levels, and improved claudin-1, claudin-3, claudin-4, and aquaporin 5 expression. Our results indicate that infliximab might improve the para-cellular pathway and trans-cellular pathway destruction by reducing the inflammatory.

Arabinogalactan Alleviates Lipopolysaccharide-Induced Intestinal Epithelial Barrier Damage through Adenosine Monophosphate-Activated Protein Kinase/Silent Information Regulator 1/Nuclear Factor Kappa-B Signaling Pathways in Caco-2 Cells.

Intestinal epithelial barrier (IEB) damage is an important aspect in inflammatory bowel disease (IBD). The objective of this study was to explore the protective effects and mechanisms of arabinogalactan (AG) on lipopolysaccharide (LPS)-stimulated IEB dysfunction. The results show that AG (1, 2, and 5 mg/mL) mitigated 100 μg/mL LPS-stimulated IEB dysfunction through increasing transepithelial electrical resistance (TEER), reducing fluorescein isothiocyanate (FITC)-dextran (4 kDa) flux, and up-regulating the protein and mRNA expression of tight junction (TJ) proteins (Claudin-1, Zonula occludens-1 (ZO-1) and Occludin). In addition, AG ameliorated LPS-stimulated IEB dysfunction by reducing interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β levels, decreasing the reactive oxygen species (ROS) level, increasing superoxide dismutase (SOD) activity, increasing the glutathione (GSH) level, and decreasing the levels of malondialdehyde (MDA) and intracellular calcium ([Ca2+]i). Furthermore, 2 mg/mL AG up-regulated the expression of silent information regulator 1 (SIRT1), the phosphorylated adenosine monophosphate-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and inhibited the phosphorylation of nuclear factor kappa-B (NF-κB) and the inhibitor of NF-κBα (IκBα). Therefore, AG could maintain IEB integrity by activating AMPK/SIRT1 and inhibiting the NF-κB signaling pathway. In conclusion, AG can regulate the AMPK/SIRT1/NF-κB signaling pathway to reduce inflammation and oxidative stress, thus alleviating LPS-stimulated IEB damage.

Downregulation of XBP1s aggravates lipopolysaccharide-induced inflammation by promoting NF-κB and NLRP3 pathways’ activation in goat endometrial epithelial cells

After delivery, bacterial contamination and uterine tissue degeneration in animals can lead to the development of uterine diseases, such as endometritis, accompanied by endoplasmic reticulum stress (ERS). Increasing evidence suggests that spliced X-box binding protein 1 (XBP1s), a critical component of ERS, is involved in several pathological processes in various organisms. However, the specific molecular mechanisms by which XBP1s mediates the inflammatory response in goat endometrial epithelial cells (gEECs) remain largely unknown. In the present study, XBP1s protein was induced into the nucleus in the lipopolysaccharide (LPS, 5 μg/mL)-induced inflammatory response of gEECs. Lipopolysaccharide-induced expression and nucleation of XBP1s were reduced by the inhibition of Toll-like receptor 4 (TLR4) using TAK-242 (1 μM; a TLR4 inhibitor). Expression and nucleation of XBP1s were similarly reduced when the activity of inositol-requiring enzyme 1α (IRE1α) was inhibited using 4μ8C (10 μM; an IRE1α inhibitor). In addition, inhibition of IRE1a increased IL-1β, TNF-α, and IL-8 levels and secretion of IL-6 induced by LPS. Notably, phosphorylation of nuclear factor kappa-B (NF-κB) P65 protein and expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) were similarly increased. Furthermore, knockdown of XBP1s in gEECs consistently promoted NF-κB P65 protein phosphorylation, NLRP3 protein expression, and inflammatory cytokine secretion. In summary, the current results suggest that in the LPS-induced inflammatory response in gEECs, LPS generates intracellular signaling cascades in gEECs via TLR4, which may promote XBP1s protein expression and nucleation by activating IRE1a. However, downregulation of XBP1s expression exacerbates inflammation by promoting activation of the NF-κB and NLRP3 inflammatory vesicle pathways. These results will potentially contribute to the treatment and prevention of endometritis in ruminants.

Geniposide protected against cerebral ischemic injury through the anti-inflammatory effect via the NF-κB signaling pathway.

Accumulated evidence indicates that geniposide exhibits neuroprotective effects in ischemic stroke. However, the potential targets of geniposide remain unclear. We explore the potential targets of geniposide in ischemic stroke. Adult male C57BL/6 mice were subjected to the middle cerebral artery occlusion (MCAO) model. Mice were randomly divided into five groups: Sham, MCAO, and geniposide-treated (i.p. twice daily for 3 days before MCAO) at doses of 25, 75, or 150 mg/kg. We first examined the neuroprotective effects of geniposide. Then, we further explored via biological information analysis and verified the underlying mechanism in vivo and in vitro. In the current study, geniposide had no toxicity at concentrations of up to 150 mg/kg. Compared with the MCAO group, the 150 mg/kg group of geniposide significantly (P < 0.05) improved neurological deficits, brain edema (79.00 ± 0.57% vs 82.28 ± 0.53%), and infarct volume (45.10 ± 0.24% vs 54.73 ± 2.87%) at 24 h after MCAO. Biological information analysis showed that the protective effect was closely related to the inflammatory response. Geniposide suppressed interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) expression in the brain homogenate, as measured by enzyme-linked immunosorbent assay (ELISA). Geniposide upregulated A20 and downregulated TNF receptor-associated factor-6 and nuclear factor kappa-B phosphorylation in the MCAO model and lipopolysaccharide-treated BV2 cells at 100 μM. Geniposide exhibited a neuroprotective effect via attenuating inflammatory response, as indicated by biological information analysis, in vivo and in vitro experiments, which may provide a potential direction for the application of geniposide in the treatment of ischemic stroke.

Protective effects of Stephania pierrei tuber-derived oxocrebanine against LPS-induced acute lung injury in mice.

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) have high mortality rates. Though corticosteroids are commonly used for the treatment of these conditions, their efficacy has not been conclusively demonstrated and their use can induce various adverse reactions. Hence, the application of corticosteroids as therapeutic modalities for ALI/ARDS is limited. Meanwhile, the aporphine alkaloid oxocrebanine isolated from Stephania pierrei tubers has demonstrated anti-inflammatory efficacy in murine/human macrophage cell lines stimulated by lipopolysaccharide (LPS). Accordingly, the primary objectives of the present study are to investigate the anti-inflammatory effects of oxocrebanine on LPS-induced murine alveolar epithelial (MLE-12) cells and its efficacy against LPS-induced murine ALI. Results show that oxocrebanine downregulates the abundance of interleukin (IL)-1beta, IL-6, and inducible nitric oxide synthase, as well as the phosphorylation of nuclear factor-kappaB (NF-κB), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), p38, protein kinase B (Akt), and glycogen synthase kinase-3beta signalling proteins in LPS-inducedMLE-12 cells. Moreover, in a murine ALI model, oxocrebanine lowers lung injury scores and lung wet/dry weight ratios while reducing inflammatory cell infiltration. It also suppresses LPS-induced tumour necrosis factor-alpha and IL-6 in the bronchoalveolar lavage fluid and plasma. Moreover, oxocrebanine downregulates NF-κB, SAPK/JNK, p38, and Akt phosphorylation in the lung tissues of LPS-treated mice. Taken together, the foregoing results show that oxocrebanine provides significant protection against LPS-induced ALI in mice primarily by suppressing various inflammatory signalling pathways in alveolar epithelial cells and lung tissues. Hence, oxocrebanine might prove effective as an anti-inflammatory agent for the treatment of lung inflammation.

Melatonin-Driven NLRP3 Inflammation Inhibition Via Regulation of NF-κB Nucleocytoplasmic Transport: Implications for Postoperative Cognitive Dysfunction.

The aseptic inflammatory response of the central nervous system is one of the important causes of neurodegenerative diseases in individuals and is also recognized in postoperative cognitive dysfunction (POCD). Inflammasome is thought to be closely related to brain homeostasis. However, there are few drugs targeting the inflammasome to suppress inflammation in clinical practice. Here, we showed that the neuroinflammatory response mediated by the NLRP3 (NLR family, pyrin domain containing 3) inflammasome was involved in the pathological process of POCD. Melatonin protected mice from nerve damage by inhibiting activation of the NLRP3-caspase-1-interleukin 1 beta (IL-β) pathway and thus reduced the secretion of IL-1β inflammatory factors in microglia. Further research found that melatonin has a potential binding effect with NLRP3 protein, and at the same time could reduce the phosphorylation of nuclear factor kappa-B (NF-κB) and inhibit its nuclear translocation. The underlying mechanism was that melatonin inhibited the expression of acetylation of histone H3 and melatonin attenuated the binding of NF-κb to the NLRP3 promoter region 1-200bp, where there are two potential binding target sites of NF-κb and NLRP3, namely the sequences 5'-GGGAACCCCC-3' and 5'-GGAAATCCA -3'. Therefore, we confirmed a novel mechanism of action of melatonin in the prevention and treatment of POCD.

Effects of Avenanthramide on the Small Intestinal Damage through Hsp70-NF-κB Signaling in an Ovalbumin-Induced Food Allergy Model.

A food allergy is caused by an abnormal immune reaction and can induce serious intestinal inflammation and tissue damage. Currently, the avoidance of food allergens is still the most effective way to prevent or reduce allergic symptoms, so the development of new strategies to treat allergies is important. Avenanthramide (AVA) is a bioactive polyphenol derived from oats with a wide range of biological activities; however, it is still not clear whether or how AVA alleviates intestinal damage under allergic situations. The aim of this study was to explore the effect of AVA on the small intestinal damage in an ovalbumin (OVA)-induced food allergy model and its mechanism. In experiment 1, 10 mg/kg bw and 20 mg/kg bw doses of AVA both decreased the serum levels of OVA-specific IgE, histamine, and prostaglandin D induced by OVA. The AVA administration relieved inflammation indicated by the lower serum concentrations of pro-inflammatory cytokines including interleukin-1β, IL-6, and tumor necrosis factor-α. The levels of tight junction proteins including Claudin-1, ZO-1, and Occludin in the jejunum were elevated after AVA administration, accompanied by the improved intestinal morphology. Furthermore, AVA elevated the protein expression of heat shock protein 70 (Hsp70) and inhibited the phosphorylation of nuclear factor kappa-B (NF-κB), thus the apoptozole, which a Hsp70 inhibitor, was applied in experiment 2 to assess the contribution of Hsp70-NF-κB signaling to the effects of AVA. In the experiment 2, the inhibition of Hsp70 signaling treatment abolished the beneficial effects of AVA on the small intestinal damage and other allergic symptoms in mice challenged with OVA. Taken together, our results indicated that AVA exerted an intestinal protection role in the OVA-induced allergy, the mechanism of which was partly mediated by the Hsp70-NF-κB signaling.

Glucagon induces the hepatic expression of inflammatory markers in vitro and in vivo.

Glucagon exerts multiple hepatic actions, including stimulation of glycogenolysis/gluconeogenesis. The liver plays a crucial role in chronic inflammation by synthesizing proinflammatory molecules, which are thought to contribute to insulin resistance and hyperglycaemia. Whether glucagon affects hepatic expression of proinflammatory cytokines and acute-phase reactants is unknown. Herein, we report a positive relationship between fasting glucagon levels and circulating interleukin (IL)-1β (r=0.252, p=.042), IL-6 (r=0.230, p=.026), fibrinogen (r=0.193, p=.031), complement component 3 (r=0.227, p=.024) and high sensitivity C-reactive protein (r=0.230, p=.012) in individuals without diabetes. In CD1 mice, 4-week continuous treatment with glucagon induced a significant increase in circulating IL-1β (p=.02), and IL-6 (p=.001), which was countered by the contingent administration of the glucagon receptor antagonist, GRA-II. Consistent with these results, we detected a significant increase in the hepatic activation of inflammatory pathways, such as expression of NLRP3 (p < .02), and the phosphorylation of nuclear factor kappaB (NF-κB; p < .02) and STAT3 (p < .01). In HepG2 cells, we found that glucagon dose-dependently stimulated the expression of IL-1β (p < .002), IL-6 (p < .002), fibrinogen (p < .01), complement component 3 (p < .01) and C-reactive protein (p < .01), stimulated the activation of NLRP3 inflammasome (p < .01) and caspase-1 (p < .05), induced the phosphorylation of TRAF2 (p < .01), NF-κB (p < .01) and STAT3 (p < .01). Preincubating cells with GRA-II inhibited the ability of glucagon to induce an inflammatory response. Using HepaRG cells, we confirmed the dose-dependent ability of glucagon to stimulate the expression of NLRP3, the phosphorylation of NF-κB and STAT3, in the absence of GRA-II. These results suggest that glucagon has proinflammatory effects that may participate in the pathogenesis of hyperglycaemia and unfavourable cardiometabolic risk profile.

Interleukin-33 regulates the endoplasmic reticulum stress of human myometrium via an influx of calcium during initiation of labor.

Inflammation is currently recognized as one of the major causes of premature delivery. As a member of the interleukin-1β (IL-1β) family, interleukin-33 (IL-33) has been shown to be involved in normal pregnancy as well as a variety of pregnancy-related disorder. This study aims to investigate the potential function of IL-33 in uterine smooth muscle cells during labor. Myometrium samples from term pregnant (≥37 weeks gestation) women were either frozen or cells were isolated and cultured. Immunohistochemistry and western blotting were used to assess the distribution of IL-33. Cultured cells were incubated with lipopolysaccharide (LPS) to mimic inflammation as well as in the presence of 4μ8C (IRE1 inhibitor III) to block endoplasmic reticulum (ER) stress and BAPTA-AM, a calcium chelator. LPS reduced the expression of nuclear IL-33 in a time-limited manner and induced ER stress. However, knockdown of IL-33 increased LPS-induced calcium concentration, ER stress and phosphorylation of nuclear factor kappa-B (NF-κB), and P38 mitogen-activated protein kinase (P38 MAPK). In addition, siRNA IL-33 further stimulates LPS enhanced cyclooxygenase-2 (COX-2) expression via NF-κB and p38 pathways. IL-33 expression was decreased in the nucleus with the onset of labor. LPS-induced ER stress and increased expression of the labor-associated gene, COX-2, as well as IL-6 and IL-8 in cultured myometrial cells. IL-33 also increased COX-2 expression, but after it was knocked down, the stimulating effect of LPS on calcium was enhanced. 4μ8C also inhibited the expression of COX-2 markedly. The expression of calcium channels on the membrane and intracellular free calcium ion were both increased which was accompanied by phosphorylated NF-κB and p38. These data suggest that IL-33 may be involved in the initiation of labor by leading to stress of the ER via an influx of calcium ions in human uterine smooth muscle cells. This study was supported by grants from the National Natural Science Foundation of China (No. 81300507).

Kaempferol 3-O-(2G-glucosylrutinoside)-7-O-glucoside isolated from the flowers of Hosta plantaginea exerts anti-inflammatory activity via suppression of NF-κB, MAPKs and Akt pathways in RAW 264.7 cells

Kaempferol 3-O-(2G-glucosylrutinoside)-7-O-glucoside (KGG) has isolated from Hosta plantaginea flowers and possessed an inhibitory effect on cyclooxygenase 2 (COX-2), could be effective in inhibiting inflammation. However, the anti-inflammatory activity and mechanism of KGG remain unknown. In this study, for the first time, the anti-inflammatory effect of KGG and its potential molecular mechanisms were explored in cells. KGG had no cytotoxicity at concentrations of 1.25, 2.5, 5, 10, 20, and 40 μM by Cell Counting kit-8 assay in RAW 264.7 cells. Besides, KGG concentration-dependently (1.25, 2.5, and 5 μM) inhibited secretions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Western blot showed that the phosphorylation of nuclear factor kappa-B (NF-κB) p65, inhibitor of NF-κB (IκB), p38 MAPK, c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (Erk), and protein kinase B (Akt), together with inducible nitric oxide synthase (iNOS) and COX-2 were significantly attenuated by KGG (1.25, 2.5, and 5 μM) in a concentration-dependent relationship. Meanwhile, KGG remarkably enhanced the protein expression of IκB. Taken together, KGG may be one of bioactive phytochemicals from H. plantaginea flowers, and be an anti-inflammatory agent via inhibiting NF-κB, mitogen-activated protein kinases (MAPKs), and Akt signaling pathways.

Taxifolin ameliorates lipopolysaccharide-induced intestinal epithelial barrier dysfunction via attenuating NF-kappa B/MLCK pathway in a Caco-2 cell monolayer model

Intestinal epithelial barrier dysfunction can cause several intestinal diseases. Flavonoids have been shown to be beneficial to the intestinal epithelial barrier function. However, the effects of taxifolin (TAX), a naturally occurring flavonoid, on the intestinal epithelial barrier function are unclear. Thus, the aims of this study were to investigate the protective effect and potential mechanism of TAX against lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction in a Caco-2 cell monolayer model. Our results showed that TAX increased the transepithelial electrical resistance (TEER) and decreased the fluorescein isothiocyanate (FITC)-dextran (4 kDa) flux in the damaged intestinal epithelial barrier. Meanwhile, TAX inhibited an LPS-induced decrease in mRNA and protein expression of tight junction (TJ) proteins (claudin-1, zonula occludens [ZO]-1, and occludin), and ameliorating the continuous distribution pattern disrupted of TJs. These results suggested that TAX ameliorated intestinal epithelial barrier dysfunction. Regarding the underlying mechanism, TAX reduced the LPS-induced secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in Caco-2 cell monolayers. In addition, TAX suppressed the phosphorylation of nuclear factor kappa-B (NF-κB), inhibitor protein of NF-κBα (IκBα), and myosin light chain (MLC), and downregulated the expression of myosin light chain kinase (MLCK) in LPS-treated Caco-2 cells. In summary, TAX can maintain TJ proteins by inhibiting the NF-κB/MLCK pathway and pro-inflammatory factor secretion to ameliorate LPS-induced intestinal epithelial barrier dysfunction. Thus, TAX is a promising candidate agent for use in functional food to ameliorate intestinal barrier dysfunction.

VX-765 ameliorates inflammation and extracellular matrix accumulation by inhibiting the NOX1/ROS/NF-κB pathway in diabetic nephropathy.

This study explores the potential role of a highly selective caspase-1 inhibitor, VX-765, on extracellular matrix (ECM) accumulation and inflammation in diabetic nephropathy (DN) and the underlying mechanisms. DN rats, induced via high-fat diet/streptozotocin, were used to assess the effects of VX-765. Parallel experiments were carried out on rat mesangial cell line HBZY-1 exposed to high glucose (HG) to reveal the molecular mechanism of VX-765 in preventing DN. Survival analysis, biochemical parameters and renal oxidative stress of rats were observed, and Western blotting and immunofluorescence were evaluated. In vitro, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX)1 silencing by RNA interference and quantitative real-time PCR (qPCR) assays were conducted in HBZY-1 cells exposed to HG levels. In vivo, VX-765 significantly reduced the increase in urine albumin excretion and ECM accumulation. The phosphorylation of nuclear factor kappa-B (NF-κB) and the expression of pro-inflammatory cytokines IL-1β, IL-6 and tumor necrosis factor (TNF)-α were significantly down-regulated. Furthermore, the generation of reactive oxygen species (ROS), phosphorylation of NF-κB and the expression of the NOX1 gene or protein were significantly decreased in HBZY-1 with VX-765 (5 μM) treatment in vitro. Our results demonstrated that VX-765 exerts favourable effects on DN via the simultaneous alleviation of systemic metabolic syndrome and down-regulating the renal NOX1/ROS/NF-κB pathway, suggesting that it has therapeutic potential for DN.

MiR-200c-3p inhibits LPS-induced M1 polarization of BV2 cells by targeting RIP2

BackgroundMicroglia are important immune cells, which can be induced by lipopolysaccharide (LPS) into M1 phenotype that express pro-inflammatory cytokines. Some studies have shown that microRNAs play critical roles in microglial activation.ObjectiveThis study was designed to investigate the role of miR-200c-3p in regulating inflammatory responses of LPS-treated BV2 cells.MethodsThe expression of miR-200c-3p in BV2 cells was detected by real-time PCR. Receptor-interacting protein 2 (RIP2) was predicted as a target gene of miR-200c-3p. Their relationship was verified by dual-luciferase reporter assay. The function of miR-200c-3p and RIP2 in microglial polarization and NF-κB signaling was further evaluated.ResultsLPS treatment reduced miR-200c-3p expression in a dose-dependent and time-dependent manner in BV2 cells. LPS treatment increased the expression of M1 phenotype markers inducible nitric oxide synthase (iNOS) and major histocompatibility complex class (MHC)-II, promoted the release of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and enhanced the nuclear translocation and phosphorylation of nuclear factor-kappaB (NF-κB) p65. Reversely, miR-200c-3p mimics down-regulated the levels of these inflammatory factors. Furthermore, RIP2 was identified to be a direct target of miR-200c-3p. RIP2 knockdown had a similar effect to miR-200c-3p mimics. Overexpression of RIP2 eliminated the inhibitory effect of miR-200c-3p on LPS-induced M1 polarization and NF-κB activation in BV2 cells.ConclusionsMiR-200c-3p mimics suppressed LPS-induced microglial M1 polarization and NF-κB activation by targeting RIP2. MiR-200c-3p/RIP2 might be a potential therapeutic target for the treatment of neuroinflammation-associated diseases.

Arsenic (III) induces oxidative stress and inflammation in the gills of common carp, which is ameliorated by zinc (II)

Arsenic (As) is widely present in the environment in form of arsenite (AsIII) and arsenate (AsV). Oxidative stress and inflammation are believed to be the dominant mechanisms of AsIII toxicity in vivo and in vitro. The aim of this study was to investigate whether zinc (Zn2+) alleviates exogenous gill toxicity in carp induced by AsIII and to gain insight into the underlying mechanisms. Exposure of carp to 2.83 mg As2O3/L for 30 days reduced superoxide dismutase activity by 4.0%, catalase by 41.0% and glutathione by 19.8%, while the concentration of malondialdehyde was increased by 16.4% compared to the control group, indicating oxidative stress. After the exposure of carp to AsIII the expression of inflammatory markers, such as interleukin-6, interleukin-8, tumor necrosis factor α and inducible nitric oxide synthase in gill tissue were significantly increased. In addition, the phosphorylation of nuclear factor kappa-B (NF-κB) was increased by 225%. 1 mg ZnCl2/L can relieve the toxicity of AsIII based on histopathology, antioxidase activity, qRT-PCR and western results. Zn2+ attenuated AsIII-induced gill toxicity that suppressed intracellular oxidative stress and NF-κB pathway by an upregulation of metallothionein. Therefore, the toxic effect of AsIII on the gill cells of carp was reduced. This study provides a theoretical basis for exploring the alleviation of the toxic effects of metalloids on organisms by heavy metals and the biological assessment of the effects.