Content Of P65 Research Articles

Jiawei Shengjiangsan's Effect on Renal Injury in Diabetic Nephropathy Mice is Investigated via the PI3K/Akt/NF-κB Signaling Pathway.

This study aimed to investigate the intervention mechanism of Jiawei Shengjiangsan (JWSJS) on kidney injury in diabetic nephropathy mice. Thirty 8-week-old db/db mice were randomly divided into five groups: model group, Perindopril group, and JWSJS low-, medium-, and high-dose groups (n=6 per group) based on body weight. Additionally, a blank control group was established consisting of 6 db/m mice aged 8 weeks. The blank and model groups received daily intragastric administration of 7g/kg/d pure water. The remaining groups were assigned to JWSJS low (3.5g/kg/d), medium (7g/kg/d), high (14g/kg/d) dosage groups, and perindopril positive control group (0.48mg/kg/d) for 12 weeks. Post-experiment, serum creatinine (SCr) and blood urea nitrogen (BUN) were analyzed using an automatic biochemical analyzer. Enzyme-linked immunosorbent assay (ELISA) measured 24-hour urinary albumin, neutrophil gelatinase-associated lipocalin (NGAL), TNF-α, IL-1β, VCAM-1, MCP-1, and HbA1c. Western blot assessed the protein expressions of p-PI3K, p-Akt, and p-NF-κB p65, while pathological kidney changes were observed. Compared to the blank group, the model group exhibited increased SCr, BUN, 24-hour urinary albumin, serum NGAL, TNF-α, IL-1β, VCAM-1, MCP-1, HbA1c, p-PI3K, and p-Akt, alongside increased p-NF-κB p65 expression, indicating significant kidney pathology. After treatment, the JWSJS group showed decreased SCr, BUN, 24-hour urinary microalbumin, NGAL, HbA1c, TNF-α, IL-1β, VCAM-1, MCP-1 levels, increased p-PI3K and p-Akt expression (P<0.05), and reduced p-NF-κB p65 content (P<0.05). Histopathological analysis revealed that JWSJS ameliorated renal tubular epithelial cell damage, glomerular capillary and basement membrane injuries, and facilitated the repair of damaged podocytes in diabetic nephropathy mice. JWSJS demonstrated efficacy in reducing renal inflammation in diabetic nephropathy mice, with its mechanism likely associated with the inhibition of the PI3K/Akt/NF-κB signaling pathway.

MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY

Abstract Background and Aims Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4). Conclusion DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.

Alogliptin Attenuates Lipopolysaccharide-Induced Neuroinflammation in Mice Through Modulation of TLR4/MYD88/NF-κB and miRNA-155/SOCS-1 Signaling Pathways

BackgroundEndotoxin-induced neuroinflammation plays a crucial role in the pathogenesis and progression of various neurodegenerative diseases. A growing body of evidence supports that incretin-acting drugs possess various neuroprotective effects that can improve learning and memory impairments in Alzheimer’s disease models. Thus, the present study aimed to investigate whether alogliptin, a dipeptidyl peptidase-4 inhibitor, has neuroprotective effects against lipopolysaccharide (LPS)-induced neuroinflammation and cognitive impairment in mice as well as the potential mechanisms underlying these effects.MethodsMice were treated with alogliptin (20 mg/kg/d; p.o.) for 14 days, starting 1 day prior to intracerebroventricular LPS injection (8 μg/μL in 3 μL).ResultsAlogliptin treatment alleviated LPS-induced cognitive impairment as assessed by Morris water maze and novel object recognition tests. Moreover, alogliptin reversed LPS-induced increases in toll-like receptor 4 and myeloid differentiation primary response 88 protein expression, nuclear factor-κB p65 content, and microRNA‐155 gene expression. It also rescued LPS-induced decreases in suppressor of cytokine signaling gene expression, cyclic adenosine monophosphate (cAMP) content, and phosphorylated cAMP response element binding protein expression in the brain.ConclusionThe present study sheds light on the potential neuroprotective effects of alogliptin against intracerebroventricular LPS-induced neuroinflammation and its associated memory impairment via inhibition of toll-like receptor 4/ myeloid differentiation primary response 88/ nuclear factor-κB signaling, modulation of microRNA-155/suppressor of cytokine signaling-1 expression, and enhancement of cAMP/phosphorylated cAMP response element binding protein signaling.

Anti-inflammatory activity of the lipophilic metabolites from Scolymus hispanicus L

The golden thistle, Scolymus hispanicus L. (Asteraceae), is a prickly perennial herb that is widely grown in Mediterranean and in Southeastern Europe. The aim of current study was to isolate and identify the constituents of S. hispanicus L. chloroform fraction, and assess their anti-inflammatory activity in-vitro. Chemical investigation led to isolation of one new guaianolide sesquiterpene; namely iso-japonicolactone (4), three tritepenes namely lupeol acetate (1), lupeol (2), and oleanolic acid (5), and the sterol stigmasterol (3). The isolated compounds demonstrated free radical scavenging activity against DPPH with compound 4 being able to scavenge more than 90% of DPPH radical at 100 µM. Treatment of the peripheral blood mononuclear cells (PBMC) stimulated with phytohaemagglutinin (PHA) with the isolated compounds resulted in a decrease in nuclear factor kappa (NFκB) p65 content in a dose-dependent manner. Compound 4 was the most efficient in decreasing NFκB p65 at the lowest concentration (1 µM), followed by compound 5. In addition, cell treatment with isolated compounds resulted in dose-related reduction of interleukins (IL-6 and IL-1β), and tumor necrosis factor (TNF-α). To the best of our knowledge this is the first report for the isolation of iso-japonicolactone and the detailed study of its anti-inflammatory activity. The potent anti-inflammatory compounds in this study could be considered as a nucleus for developing anti-inflammatory drugs through further biological studies on the molecular level.

Anti-inflammatory activity of Kleinia odora

IntroductionKleinia odora is a wild plant growing in Saudi Arabia and consumed by both animals and people. The plant is rich in triterpenes that are known for their anti-inflammatory activity. In this study, the major triterpenes from this plant were isolated, identified and assessed for their anti-inflammatory activity. MethodsDifferent chromatographic and spectroscopic techniques were used to isolate and identify the major triterpenes isolated from the aerial parts of K. odora. Anti-inflammatory activities of isolated compounds were assessed according to radicle scavenging activity and their impact on NFκB. The assessment of anti-inflammatory activity was performed by measuring supernatant concentrations of inflammatory cytokines released from human Peripheral Blood Mononuclear Cell (PBMCs) previously fuelled by phytohaemagglutinin (PHA). ResultsChemical investigation of K. odora lead to isolation of lupeol acetate (1), lupenone (2), friedelin (3), lupeol (4), epilupeol (5) and oleanolic acid (6).Compounds 1 and 2 showed the best activity as a free radical scavenger in DPPH assay. Moreover, they showed the highest potency as anti-inflammatory through decreasing NFκB p65 content, TNF-α IL-6, and IL-1β levels in PHA-stimulated (PBMCs). ConclusionOptimum anti-inflammatory effects have been shown at 100 μM but at lower concentrations only lupane nucleus with acetate or ketone at position 3 showed the optimum activity as in compounds 1 and 2, respectively. It could be concluded that maximum anti-inflammatory activity is obtained from lupine skeleton with acetate or ketone at position C-3.

PO-035 LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitor

IntroductionThe transcriptional factor NF-κB, composed by five subunits (RelA/p65, c-Rel, RelB, p50, p52), is largely involved in many facets of cellular physiology such as innate and adaptive immunity as well as inflammation. In addition, NF-kB play a central role in cancer cell survival and chemoresistance partly by its implication in cross-talks with redox-regulating proteins. Ferritin is the major iron storage protein; it is composed by a variable assembly of Heavy (FHC) and Light (FLC) subunits. FHC, in particular, has been widely demonstrated to be devoted in iron uptake and release thus controlling the redox homeostasis.Material and methodsK562 erythroleukemia cells were stably silenced for FHC by using the shRNA method. Then FHC reconstitution was achieved by transient transfection of a FHC specific expression vector. ROS were determined by incubating cells with the redox-sensitive probe 2‘−7‘-DCF. NAC was used to inhibit ROS production. MTT assay was performed to analyse cell viability. Increasing concentrations of Doxorubicin, ranging from 0 to 5 µM, were used to treat K562 cells.Results and discussionsThe results of this study highlighted that FHC amounts negatively affect NF-kB activation in K562 cells. FHC silencing was accompanied by an increased expression of the nuclear NF-kB subunit p65, FHC rescue determined nuclear p65 decrease. FHC silencing is responsible for intracellular ROS production and ROS are implicated in NF-kB pathway. To elucidate the relationship between ROS amount and nuclear p65 content, we determined ROS amounts in our in vitro model and evaluated p65 nuclear expression after treatment with the ROS scavenger NAC. First, we observed that, as expected, ROS levels increased upon FHC silencing and return to basal levels upon NAC treatment. Interestingly, NAC was also able to decrease nuclear p65 amount in FHC-silenced K562 cells. Considering the effect of NF-kB activated pathway on cell survival, we analysed the effect of FHC silencing-mediated p65 increase in K562 cells upon treatment with increasing doses of Doxorubicin. Cell viability assay highlighted that FHC-silencing was accompanied by an increased resistance to the drug with an IC50 about doubled compared to that of the K562 control cells at each the time points. This resistance of FHC-silenced cells was reverted upon NF-kB inhibitor transfection.ConclusionFHC silencing induced NF-kB activation in K562 cells through the modulation of intracellular ROS content. This regulatory axis can be used to modulate K562 chemoresistance.

Expression changes of Notch and nuclear factor-κB signaling pathways in the rat heart with myocardial infarction

Objective: To observe the expression changes of Notch and nuclear factor-κB (NF-κB) signaling pathways in rat myocardium post myocardial infarction. Methods: Myocardial infarction was established by ligation of the left anterior descending coronary artery(MI group), sham rats (similar surgical procedure without coronary artery ligation) served as control, the rats were sacrificed at first week, 4th and 8th week after operation, the non-infarct myocardial tissue in both groups was obtained to detect the mRNA expression of Notch1, Dll4 and Hes1 by RT-PCR, the protein expression of NICD1 was detected by Western blot, the nuclear protein p65 content was detected to reflect the activation degree of NF-κB signaling in the cardiomyocytes. Results: The myocardial mRNA expression of Notch1 in MI group was significantly higher than in control group (1.68±0.35 vs. 0.47±0.12, P<0.05) at first week, and tended to be higher at the 4th week and 8th week (P>0.05). The mRNA expression of Dll4 and Hes1 was similar between the two groups at the three time points. NICD1 protein level was increased at the first week in MI group as compared with control group (1.31±0.33 vs.0.45±0.11, P<0.05), which tended also to be higher at the 4th week and 8th week post operation (P>0.05). For NF-κB activation study, the nuclear protein p65 content was higher at first week, 4th week and 8th week in MI group as compared with respective control groups (0.286±0.052 vs.0.049±0.016 (P<0.01), 0.247±0.056 vs. 0.043±0.018 (P<0.01), 0.120±0.033 vs. 0.044±0.009 (P<0.05)), the most significant increase was found in the first week. Conclusions: Notch and NF-κB signaling pathways are actively involved in the process of ventricular remodeling after myocardial infarction. Notch1 and NF-κB signaling pathways are both activated at the first week after myocardial infarction, NF-κB signaling pathway activation after myocardial infarction continues up to 8 weeks. These two signal transduction pathways may thus serve as new targets for future intervention studies to prevent heart failure.

LDOC1 inhibits proliferation and promotes apoptosis by repressing NF-κB activation in papillary thyroid carcinoma.

BackgroundThe incidence of thyroid cancer has progressively increased over the past few decades, and the most frequent types of this cancer are papillary thyroid carcinoma (PTC) and small primary tumors. In PTC, oncogene activation is known to occur at a high frequency. However, the potential roles of tumor suppressor genes in thyroid carcinogenesis remain unclear. LDOC1 was first identified as a gene encoding a leucine zipper protein whose expression was decreased in a series of pancreatic and gastric cancer cell lines. In this study, we aimed to determine the status of LDOC1 in PTC and identify its mechanistic role in PTC pathogenesis.MethodsLDOC1 expression was evaluated in fresh samples and stored specimens of human PTC and contralateral normal tissues by performing quantitative reverse transcription-PCR and immunohistochemical staining. The correlation to nuclear p65 content in the stored specimens was analyzed. Moreover, the basal level of LDOC1 in two human PTC-derived cell lines (BCPAP and TPC-1) compared with normal thyroid tissue was determined. Human LDOC1 cDNA was inserted into a lentiviral vector and transduced into TPC-1 cells. TPC-1 cells overexpressing LDOC1/GFP (Lv-LDOC1) or negative control GFP (Lv-NC) were stimulated with TNFα or recombinant TGF-β1, and then cell proliferation, cell cycle distribution, and apoptosis were assessed. Western blotting was used to examine the expression of p65, IκBα, c-Myc, Bax, and Bcl-xL, and a luciferase reporter assay was used to measure NF-κB activity stimulated by TNFα. Statistical significance was determined using Student’s t tests or ANOVA and Newman-Keuls multiple comparison tests. Pearson chi-square test was used to analyze possible associations.ResultsLDOC1 expression was significantly downregulated in PTC specimens as compared with the expression in normal thyroid tissues, and this downregulation was associated with an increase in tumor size (P < 0.05). There is a correlation between LDOC1 and nuclear P65 expression in human PTC tissues (P < 0.01). Lentivirus-mediated restoration of LDOC1 expression in TPC-1 cells characterized by low level of LDOC1 expression suppressed proliferation and induced apoptosis by inhibiting NF-κB activation, and LDOC1-overexpressing TPC-1 cells recovered responsiveness to TGF-β1 antiproliferative signaling.ConclusionsLDOC1 might function as a tumor suppressor gene in PTC by inhibiting NF-κΒ signaling, and thus might represent a promising therapeutic target in patients with PTC.

Inhibitory effects of parthenolide on the activity of NF-κB in multiple myeloma via targeting TRAF6.

This study examined the mechanism of the inhibitory effect of parthenolide (PTL) on the activity of NF-κB in multiple myeloma (MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6 (TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.

Fuyuan Decoction inhibits nitric oxide production via inactivation of nuclear factor-κB in SW1353 chondrosarcoma cells

Ethnopharmacological relevanceFuyuan Decoction (FYD) is an empirical formula of treating Bi Zheng in traditional Chinese medicine (TCM). Despite the fact that the efficiency of FYD on treating osteoarthritis has been verified in clinic, the underlying mechanisms are not totally understood. This study was to investigate the effects and mechanisms of FYD on nitric oxide (NO) production and nuclear factor (NF)-κB activation in interleukin (IL)-1β-stimulated chondrocytes. Materials and methodsSW1353 human chondrosarcoma cells were pretreated with various concentrations of FYD-containing serum (FYD-CS), and then were stimulated by IL-1β. Amounts of NO were determined by Griess reaction assay. Inducible NO synthase (iNOS) expression, inhibitor-κBα (IκBα) degradation and nuclear translocation of p65 protein were determined by Western blot assay. DNA binding activity of NF-κB was determined by ELISA assay using Trans AM™ kit for p65. Results10% and 20% (v/v) FYD-CS significantly decreased NO production in a concentration-dependent manner (p<0.05 or p<0.01) as compared to control in IL-1β-induced SW1353 cells. Besides, 10% and 20% FYD-CS also significantly reduced iNOS protein expression by about 60% and 70% (both p<0.01), respectively. Furthermore, 10% and 20% FYD-CS markedly decreased IκBα degradation by about 45% and 26% (p<0.01 or p<0.05), lessened P65 content in the nucleus by about 28% and 60% (both p<0.01), and repressed DNA binding activity of P65 by about 30% and 45% (both p<0.01) in IL-1β-induced SW1353 cells. ConclusionsThese findings suggested that FYD could inhibit NO production and iNOS expression in IL-1β-induced chondrocytes through suppressing NF-κB activation.

Discovery of a Potential Anti-Inflammatory Agent: 3-Oxo-29-noroleana-1,9(11),12-trien-2,20-dicarbonitrile

Fifteen novel derivatives of glycyrrhetinic acid (GA) were synthesized and evaluated for anti-inflammatory activities. It was found that the introduction of 1-en-3-one and 9(11),12-diene and 2,20-dinitrile functionalities into the scaffold of GA led to the discovery of potent compound 19 for inhibition of LPS-induced NO production. Furthermore, 19 effectively inhibited the protein and mRNA expression of inducible NO synthase (iNOS) and the mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW 264.7 macrophages. Mechanistically, 19 exerted inhibitory effects on the activation of the three main MAPKs and phosphorylation and degradation of IκB-α, as well as the ratio of nuclear/cytosolic content of p65. Importantly, 19 significantly decreased the mortality rate in the mouse model of LPS-induced sepsis shock. It is noteworthy that inhibitory effect of 19 on NO production was not blocked by the glucocorticoid receptor antagonist mifepristone, indicating that it does not act through the glucocorticoid receptor.

Inflammation in Response to Glucose Ingestion Is Independent of Excess Abdominal Adiposity in Normal-Weight Women with Polycystic Ovary Syndrome

Inflammation and excess abdominal adiposity (AA) are often present in normal-weight women with polycystic ovary syndrome (PCOS). We determined the effects of hyperglycemia on nuclear factor-κB (NFκB) activation in mononuclear cells (MNC) of normal-weight women with PCOS with and without excess AA. This was a prospective controlled study. The study was conducted at an academic medical center. Fifteen normal-weight, reproductive-age women with PCOS (seven normal AA, eight excess AA) and 16 body composition-matched controls (eight normal AA, eight excess AA) participated in the study. Body composition was measured by dual-energy absorptiometry. Insulin sensitivity was derived from an oral glucose tolerance test (IS(OGTT)). Activated NFκB and the protein content of p65 and inhibitory-κB were quantified from MNC, and TNFα and C-reactive protein (CRP) were measured in plasma obtained from blood drawn while fasting and 2 h after glucose ingestion. Compared with controls, both PCOS groups exhibited lower IS(OGTT), increases in activated NFκB and p65 protein, and decreases in inhibitory-κB protein. Compared with women with PCOS with excess AA, those with normal AA exhibited higher testosterone levels and lower TNFα and CRP levels. For the combined groups, the percent change in NFκB activation was negatively correlated with IS(OGTT) and positively correlated with androgens. TNFα and CRP were positively correlated with abdominal fat. In normal-weight women with PCOS, the inflammatory response to glucose ingestion is independent of excess AA. Circulating MNC and excess AA are separate and unique sources of inflammation in this population.

Upregulation of Cystathionine Beta-Synthetase Expression by Nuclear Factor-Kappa B Activation Contributes to Visceral Hypersensitivity in Adult Rats with Neonatal Maternal Deprivation

BackgroundIrritable bowel syndrome (IBS) is characterized by chronic visceral hyperalgesia (CVH) that manifested with persistent or recurrent abdominal pain and altered bowel movement. However, the pathogenesis of the CVH remains unknown. The aim of this study was to investigate roles of endogenous hydrogen sulfide (H2S) producing enzyme cystathionine beta-synthetase (CBS) and p65 nuclear factor-kappa B subunits in CVH.ResultsCVH was induced by neonatal maternal deprivation (NMD) in male rats on postnatal days 2–15 and behavioral experiments were conducted at the age of 7–15 weeks. NMD significantly increased expression of CBS in colon-innervating DRGs from the 7th to 12th week. This change in CBS express is well correlated with the time course of enhanced visceromoter responses to colorectal distention (CRD), an indicator of visceral pain. Administration of AOAA, an inhibitor of CBS, produced a dose-dependent antinociceptive effect on NMD rats while it had no effect on age-matched healthy control rats. AOAA also reversed the enhanced neuronal excitability seen in colon-innervating DRGs. Application of NaHS, a donor of H2S, increased excitability of colon-innervating DRG neurons acutely dissociated from healthy control rats. Intrathecal injection of NaHS produced an acute visceral hyperalgesia. In addition, the content of p65 in nucleus was remarkably higher in NMD rats than that in age-matched controls. Intrathecal administration of PDTC, an inhibitor of p65, markedly reduced expression of CBS and attenuated nociceptive responses to CRD.ConclusionThe present results suggested that upregulation of CBS expression, which is mediated by activation of p65, contributes to NMD-induced CVH. This pathway might be a potential target for relieving CVH in patients with IBS.

Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice

AimsWe investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice. Main methodsThe effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages. Key findingsWTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver. SignificanceThese findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.

Glutamine induces nuclear degradation of the NF-κB p65 subunit in Caco-2/TC7 cells

In the intestine, NF-κB is the main transcription factor involved in the anti-inflammatory effect of glutamine and we previously demonstrated that glutamine via its conversion to glutamate diminished the p65 protein content in Caco-2/TC7 cell nuclei without affecting the stimulating effect of IL-1β on NF-κB [21]. However, the molecular mechanism by which glutamine acts is not established. We therefore tried to identify such a mechanism. Our results demonstrate that glutamine decreased the intracellular NF-κB through the nuclear ubiquitin-proteasome pathway requiring therefore the nuclear translocation of the factor. Indeed, time-course study revealed that glutamine induced an increase in the nuclear p65 content within the first 15 min of culture, the p65 nuclear and cytosolic content decreasing gradually thereafter to reach 50 % of the control value after 60 min. This translocation was initiated by the phosphorylation of IκBα by the IKKβ subunit inducing its degradation and the p65 translocation. In parallel, glutamine activated the IKKα subunit which in turn phosphorylates p65 at Ser 536 which was responsible for p65 degradation by the nuclear proteasome. We also demonstrate that p38 MAPK lies between glutamine and the NF-κB pathway. In conclusion, this study identified for the first time the signaling pathway by which glutamine may protect against inflammatory conditions.

Pre-Treatment of Recombinant Mouse MFG-E8 Downregulates LPS-Induced TNF-α Production in Macrophages via STAT3-Mediated SOCS3 Activation

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8−/− mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8−/− mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8−/− mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.

NF-κB activation in organs from STZ-treated rats

Nuclear factor kappa B (NF-κB) is a ubiquitously expressed transcription factor comprised of various subunits (p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), RelB, and c-Rel). Activation of certain NF-κB subunits appears to foster an inflammatory state that may promote the development of disease. Thus characterizing the specific NF-κB subunits may provide insight into the pathogenesis of certain diseases. The purpose of this study was to determine if 1 month of a diabetic state, induced by streptozotocin (STZ) treatment, alters the constitutive level of NF-κB activation, its subunit composition, or the content of NF-κB-related proteins in rodent liver, kidney, spleen, and heart. Diabetes was induced in male Sprague-Dawley rats by a single tail vein injection of STZ (55mg·kg-1 body weight). After 30days, the heart, liver, spleen, and kidney were assessed for NF-κB activation and subunit composition with electrophoretic mobility shift assay (EMSA), and p50 and p65 subunit content was assessed with Western blotting. In diabetic animals, the constitutive level of NF-κB activation was reduced in liver, but was unchanged in kidney, spleen, and heart. EMSA supershifts showed the predominant subunit in the activated NF-κB complexes from both diabetic and control animals to be p50, although the p65 subunit was detected in NF-κB complexes from diabetic hearts. The content of p50 was unaltered in all diabetic tissues examined, whereas the content of p65 was increased only in hearts from diabetic animals. These findings support the idea that a diabetic state may induce specific changes in NF-κB subunit composition in certain tissues.

Protocatechuic Acid, a Metabolite of Anthocyanins, Inhibits Monocyte Adhesion and Reduces Atherosclerosis in Apolipoprotein E-Deficient Mice

Polyphenols, including anthocyanins, from various plant foods are effective in the prevention of atherosclerosis in animal and human studies. Protocatechuic acid (PCA), a major metabolite of anthocyanins, has been found to possess the anti-carcinogenic effect, whereas the in vivo effect of PCA as an anti-atherosclerotic agent remains unknown. We demonstrated herein that PCA inhibited monocyte adhesion to tumor necrosis factor-α (TNF-α)-activated mouse aortic endothelial cells, associated with the inhibition of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression. Furthermore, PCA inhibited the nuclear content of p65, a subunit of nuclear factor-κB (NF-κB), along with reduced NF-κB binding activity. Finally, PCA administration in the apolipoprotein E (ApoE)-deficient mouse model reduced aortic VCAM-1 and ICAM-1 expression, NF-κB activity, and plasma-soluble VCAM-1 and ICAM-1 levels, with inhibiting atherosclerosis development. We suggest that PCA possesses the anti-atherogenic effect at least partially via its anti-inflammatory activity.

MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-κB–dependent lymphoma

AbstractMLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell–like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIκBα, decrease in nuclear p65 content, reduction of nuclear factor-κB (NF-κB) transcriptional activity, and G1 arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-κB pathway inhibition. Treatment of germinal-center B cell–like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-κB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-κB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-κB–dependent lymphomas.

STZ-induced skeletal muscle atrophy is associated with increased p65 content and downregulation of insulin pathway without NF-κB canonical cascade activation

Type 1 diabetes mellitus (DM)-induced skeletal muscle atrophy is associated with an increased incidence in morbidity and mortality. Although the precise mechanism of diabetes-induced skeletal muscle atrophy remains to be established, several NF-κB-dependent pro-inflammatory genes have been identified as potential therapeutic targets. Moreover, activation of NF-κB has previously been shown to be required for cytokine-induced loss of skeletal muscle proteins. Therefore, we investigated activation of the NF-κB canonical pathway, concomitant to insulin signaling activation in skeletal muscle from diabetes-induced rats. Ten rats injected with streptozotocin (STZ) 4 weeks prior to tissue extraction were compared to 10 control rats. Using total, cytosolic and nuclear protein extracts from hindlimb muscles: soleus (SOL), extensor digitorum longus (EDL), gastrocnemius (GM) and liver tissue, we assessed key proteins important for the activation of both NF-κB and insulin pathways. Insulin blood concentration decreased to 3.9 ± 1.2 mU/ml following STZ-injection resulting in hyperglycemia (17.9 ± 0.7 mmol/l). SOL, EDL and GM mass decreased, and liver mass increased following STZ injection. NF-κB/p65 content in SOL, GM and liver increased in STZ-injected rats, without any change in IκB degradation or IKK phosphorylation. Muscle NF-κB/p65 remained bound to IκB and did not translocate or bind to DNA. Although the canonical NF-κB cascade was not activated, STZ induced a decrease in insulin pathway proteins including insulin receptor (IR) and substrate (IRS-1) content and phosphorylation compared to control animals. A downregulation of insulin pathway proteins and muscle atrophy occurred in response to STZ administration, and despite increased p65 content, STZ treatment did not activate the canonical NF-κB cascade. Therefore, it is unlikely that hyperglycemia initiates skeletal muscle atrophy via activation of the NF-κB canonical pathway.