IκBα Expression Research Articles

Flaxseed Effects on Inflammation Regulatory Gene Expressions in an Obese Animal Model.

Obesity is known to be partially influenced by low-grade inflammation caused by pro-inflammatory cytokines and excess adipose tissue. Flaxseed contains secoisolariciresinol diglucoside (SDG) and α-linolenic acid (ALA) that have been shown to prevent pro-inflammatory cytokine production and secretion. This study determined flaxseed effects on inflammation regulatory gene expressions and their relationships with weight gain in an obese animal model. C57BL/6J mice were fed with whole flaxseed, defatted flaxseed, or flaxseed oil supplemented high-fat diet for eight weeks. After eight weeks of dietary treatment, NF-κB, IκBα, IKKβ, IL-6, TNF-α, Akt2, and adiponectin gene expressions were measured. The result shows how health-promoting compounds in flaxseed assist in alleviating and preventing obesity-induced low-grade inflammation by actively working against IKKβ/NF-κB pathway.

Effects of Sodium Thiosulfate During Resuscitation From Trauma-and-Hemorrhage in Cystathionine Gamma Lyase (CSE) Knockout Mice.

Sodium thiosulfate (Na2S2O3) is a clinically established drug with antioxidant and sulphide-releasing properties. Na2S2O3 mediated neuro- and cardioprotective effects in ischemia/reperfusion models and anti-inflammatory effects in LPS-induced acute lung injury. Moreover, Na2S2O3 improved lung function during resuscitation from hemorrhagic shock in swine with pre-existing atherosclerosis, characterized by decreased expression of cystathionine γ-lyase (CSE), a major source of hydrogen sulfide (H2S) synthesis in the vasculature. Based on these findings, we investigated the effects of Na2S2O3 administration during resuscitation from trauma-and-hemorrhage in mice under conditions of whole body CSE deficit. After blast wave-induced blunt chest trauma and surgical instrumentation, CSE knockout (CSE-/-) mice underwent 1 h of hemorrhagic shock (MAP 35 ± 5 mm Hg). At the beginning of resuscitation comprising retransfusion, norepinephrine support and lung-protective mechanical ventilation, animals received either i.v. Na2S2O3 (0.45 mg g-1, n = 12) or vehicle (saline, n = 13). Hemodynamics, acid-base status, metabolism using stable isotopes, and visceral organ function were assessed. Blood and organs were collected for analysis of cytokines, mitochondrial respiratory capacity, and immunoblotting. Na2S2O3 treatment improved arterial paO2 (P = 0.03) coinciding with higher lung tissue glucocorticoid receptor expression. Norepinephrine requirements were lower in the Na2S2O3 group (P < 0.05), which was associated with lower endogenous glucose production and higher urine output. Na2S2O3 significantly increased renal tissue IκBα and heme oxygenase-1 expression, whereas it lowered kidney IL-6 and MCP-1 levels. Na2S2O3 exerted beneficial effects during resuscitation of murine trauma-and-hemorrhage in CSE-/- mice, confirming and extending the previously described organ-protective and anti-inflammatory properties of Na2S2O3. The findings make Na2S2O3 a potentially promising therapeutic option in the context of impaired CSE activity and/or reduced endogenous H2S availability.

Damage to intestinal barrier integrity in piglets caused by porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1β, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.

Protective Effects of Microbiome-Derived Inosine on Lipopolysaccharide-Induced Acute Liver Damage and Inflammation in Mice via Mediating the TLR4/NF-κB Pathway.

This research assessed the anti-inflammatory and hepatoprotective properties of inosine and the associated mechanism. Inosine pretreatment significantly reduced the secretion of several inflammatory factors and serum alanine transaminase (ALT) and aspartate amino transferase (AST) levels in a dose-dependent manner compared with the lipopolysaccharide (LPS) group. In LPS-treated mice, inosine pretreatment significantly reduced the ALT and malondialdehyde (MDA) concentration and significantly elevated the antioxidant enzyme activity. Furthermore, inosine pretreatment significantly altered the relative abundance of the genera, Bifidobacterium, Lachnospiraceae UCG-006, and Muribaculum. Correlation analysis showed that Bifidobacterium and Lachnospiraceae UCG-006 were positively related to the cecal short-chain fatty acids but negatively related to the serum IL-6 and hepatic AST and ALT levels. Notably, inosine pretreatment significantly modulated the hepatic TLR4, MYD88, NF-κB, iNOS, COX2, AMPK, Nfr2, and IκB-α expression. These results suggested that inosine pretreatment alters the intestinal microbiota structure and improves LPS-induced acute liver damage and inflammation through modulating the TLR4/NF-κB signaling pathway.

Hippo Pathway Counter-Regulates Innate Immunity in Hepatitis B Virus Infection.

Whether hepatitis B virus (HBV) activates or represses innate immunity continues to be debated. Toll-like receptor (TLR) 2 has been identified to recognize HBV particles in human hepatocytes. The Hippo pathway, known for growth control, is suggested to play a vital role in immune regulation. Here, molecular interactions between HBV-triggered TLR signaling and the Hippo pathway were comprehensively investigated. Reanalysis of GSE69590 data, in which human hepatocytes have been treated with cell culture-derived HBV particles, identified changes in Hippo and NF-κB signaling. Immunocytochemical staining and western blotting revealed time-dependent nuclear translocation of YAP and NF-κB in HBV-exposed primary human and murine hepatocytes (PMH). Analysis of PMH isolated from MyD88- or IRAK4-deficient mice and the inhibition of TLR2 and MST1/2 in vitro confirmed the relation between TLR2 and Hippo signaling in HBV-induced immunity. Loss and gain of function experiments implied that Hippo-downstream effector YAP directly regulated IκBα expression. Functional investigations confirmed the regulation of Nfkbia promoter activity by the YAP/TEAD4 transcription factor complex. Administration of TLR ligands to mice highlighted the relevance of the TLR2-MyD88-IRAK4-Hippo axis in hepatic immunity. Interestingly, reanalysis of gene expression pattern in liver biopsies of patients chronically infected with HBV (GSE83148, GSE65359) indicated an activation of TLR2 and however, an MST1-dominated Hippo control in the immune clearance phase of patients with chronic HBV infection. We demonstrated that MyD88-dependent TLR signaling activates NF-κB and Hippo signaling, with YAP prompting the IκBα-mediated negative feedback, alongside NF-κB. Imbalance between immune induction and Hippo activation may have implications for the safety of novel HBV cure strategies interfering with pathogen recognition receptors.

MicroRNA-146a-3p/HDAC1/KLF5/IKBα signal axis modulates plaque formation of atherosclerosis mice

BackgroundAtherosclerosis (AS) is a multifocal, smoldering immune inflammatory disease of medium and large arteries driven by lipids. The aim of this study is to discuss the mechanism of microRNA-146a-3p (miR-146a-3p)/histone deacetylase 1 (HDAC1)/Krüppel-like factor 5 (KLF5)/inhibitors of kappa B α (IKBα) signal axis in plaque formation of AS mice. MethodsApoE−/− mice were fed with high-fat feed for 12 weeks to establish AS mice model. The expression of miR-146a-3p, KLF5, HDAC1 and IKBα in aortic wall tissues of AS mice was tested. The targeting relationship between miR-146a-3p and HDAC1 was verified. AS mice were injected with miR-146a-3p antagomir or HDAC1 overexpression to verify the impacts of miR-146a-3p and HDAC1 on blood lipids and inflammatory factors in serum, aortic wall apoptotic cells, antioxidant stress capacity and the plaque area in AS mice. VECs proliferation and apoptosis were also measured in vitro. ResultsmiR-146a-3p and KLF5 were increased while HDAC1 and IKBα were reduced in aortic wall tissues of AS mice. miR-146a-3p directly targeted to HDAC1. Depletion of miR-146a-3p or restoration of HDAC1 was correlated to lower plasma lipid level, reduced inflammatory factors in serum, attenuated aortic wall apoptosis, increased antioxidant stress capacity and improved the stability of pathological plaque of AS mice. miR-146a-3p down-regulation or HDAC1 up-regulation promoted VECs proliferation and inhibited apoptosis. ConclusionFunctional studies show that depleted miR-146a-3p advances HDAC1 and IKBα expression as well as inhibits KLF5 expression to facilitate the stability of pathological plaques in AS mice.

Ubiquitin‐specific peptidase 53 inhibits the occurrence and development of clear cell renal cell carcinoma through NF‐κB pathway inactivation

BackgroundClear cell renal cell carcinoma (ccRCC) is one of the most prevalent malignant diseases in the urinary system with more than 140,000 related deaths annually. Ubiquitination–deubiquitination homeostasis is an important factor in ccRCC progression; ubiquitin‐specific peptidase 53 (USP53) belongs to the family of deubiquitinating enzymes, but its functions are rarely reported.MethodsDatabases obtained from GEO and TCGA were analyzed to reveal the role of USP53 in ccRCC. CCK‐8/BrdU and EDU assays were used to detect the proliferation of ccRCC after USP53 overexpression or knockdown. A tumor xenograft experiment was used to verify the effect of the proliferation of ccRCC after USP53 knockdown. Transwell assays were used to detect the metastasis of ccRCC after USP53 overexpression or knockdown. RNA sequencing and western blot analysis were employed to detect the change in genes after USP53 overexpression and knockdown. Then we tested the effect of USP53 on IκBα protein stability through western blot analysis. Detect the effect of USP53 on IκBα ubiquitination in vitro by immunoprecipitation method.ResultsUSP53 expression was downregulated in ccRCC tissues and USP53 expression was significantly negatively correlated with the tumor progression and clinical prognosis. The ability of growth and metastasis of ccRCC was inhibited after USP53 overexpression. In addition, USP53 knockdown promoted ccRCC growth and metastasis. Moreover, USP53 knockdown promoted the ability of clone formation of ccRCC in vivo. NF‐κB signaling pathway significantly enriched and downregulated in USP53 overexpressed cells, and genes in the NF‐κB pathway (such as IL1B, CXCL1‐3, RELA, RELB, etc.) were obviously downregulated in USP53 overexpressed cells. USP53 overexpression decreased the phosphorylation of IKKβ and P65 in both Caki‐1 and 786‐O cells, and the expression of IκBα was increased. Phosphorylation of IKKβ and P65 was increased in both Caki‐1 and 786‐O cells after USP53 knockdown. As the expression of USP53 increases, the protein expression of IκBα was also gradually increased and USP53 reduced the ubiquitination of IκBα.ConclusionIn summary, our data indicate that USP53 inhibits the inactivation of the NF‐κB pathway by reducing the ubiquitination of IκBα to further inhibit ccRCC proliferation and metastasis. These findings may help understand the pathogenesis of ccRCC and introduce new potential therapeutic targets for kidney cancer patients.

Rosiglitazone alleviates lipopolysaccharide-induced inflammation in RAW264.7 cells via inhibition of NF-κB and in a PPARγ-dependent manner.

Rosiglitazone is a synthetic peroxisome proliferator-activated receptor (PPAR)γ agonist widely used for the treatment of type 2 diabetes. Recent studies have demonstrated that rosiglitazone displays anti-inflammatory effects. The present study aimed to investigate whether rosiglitazone alleviates decreases in RAW264.7 cell viability resulting from lipopolysaccharide (LPS)-induced inflammation, as well as exploring the underlying mechanism. A macrophage inflammatory injury model was established by treating RAW264.7 cells with 100 ng/ml LPS. Cells were divided into LPS and rosiglitazone groups with different concentrations. Cell viability was assessed by performing an MTT assay. The expression of inflammatory cytokines was detected by conducting enzyme-linked immunosorbent assays and reverse transcription-quantitative PCR. Nitric oxidesecretion was assessed using the Griess reagent system. The expression levels of key nuclear factor-κB pathway-associated proteins were detected via western blotting. Rosiglitazone alleviated LPS-induced decrease in RAW264.7 cell viability and inhibited inflammatory cytokine expression in a concentration-dependent manner. Rosiglitazone significantly inhibited LPS-induced upregulation of p65 phosphorylation levels and downregulated IκBα expression levels. However, rosiglitazone-mediated inhibitory effects were reversed by PPARγ knockdown. The results of the present study demonstrated that rosiglitazone significantly inhibited LPS-induced inflammatory responses in RAW264.7 macrophage cells, which was dependent on PPARγ activation and NF-κB suppression.

Bioflocs attenuates lipopolysaccharide-induced inflammation, immunosuppression and oxidative stress in Channa argus

The regulation of C/N in aquaculture water is an important means of environmental regulation, of which the most common is bioflocs technology (BFT). Here, an eight-week feeding experiment and a lipopolysaccharide (LPS) challenge test were proceed to investigate the growth, oxidative stress, immunosuppression and GR–NF–κB related genes expression of Channa argus rearing in the BFT. Four groups were set, the control group was a basic diet (C/N = 7.6:1), and the other three groups were adjusted by glucose, which was C/N 10: 1, C/N 15: 1 and C/N 20:1, respectively. And we detected the two-stage test indexes of C. argus before and after the LPS challenge. The results showed that the bioflocs of C/N 15:1 group significantly (P < 0.05) promoted the growth performance. Similarly, the trend of immune enzyme activity was the same before and after LPS challenge, but decreased after challenge (except AKP and IgM). The best group is still treatment C/N 15:1. Liver and intestine SOD, CAT, GPX, ASA and T-AOC levels of juveniles in treatment C/N 15:1 were markedly increased (P < 0.05) compared to control before and after the LPS challenge. Simultaneously, the content of MDA in control group was considerably higher than that in treatment C/N 15:1 (P < 0.05). Furthermore, C/N 15:1 group significantly down-regulated the expression level of pro-inflammatory factors (NF-κB, TNF-α, IL-1β and IL-8), and up-regulated IκBα, GR, HSP70 and HSP90 expression levels considerably (P < 0.05). Also, intestinal morphology appeared injury in control group, while intestinal integrity was better in treatment C/N 10:1 and C/N 15:1 after challenge. Taken together, these upshot indicated that bioflocs could enhance growth and alleviate C. argus from LPS-induced oxidative stress, immunosuppression and inflammation through restraining GR–NF–κB signaling pathway. The best C/N ratio for alleviating LPS is 15:1.

PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial ischemia/reperfusion injury.

Myocardial ischemia/reperfusion (I/R) injury induces cardiomyocyte apoptosis to deteriorate heart function. Thus, how to inhibit cardiomyocyte apoptosis is the focus of recent researches. Proteasome family member PSMB4 (proteasome subunit beta type-4) promotes cell survival. The relationship between PSMB4 and cardiomyocyte apoptosis during myocardial I/R is unknown. In this study, PSMB4 expression increased in rat myocardial I/R model, positively correlated with cleaved caspase-3 expression, negatively correlated with Bcl-2 expression. In vitro, neonatal ventricle cardiomyocyte hypoxia/reoxygenation (H/R) model was constructed to mimic myocardial I/R. PSMB4 silence promoted cardiomyocyte apoptosis and IκBα expression, inhibited the activation of NF-κB. On the contrary, PSMB4 overexpession inhibited cardiomyocyte apoptosis and IκBα expression, promoted the activation of NF-κB. Additionally, PSMB4-IκBα interaction was identified, suggesting that PSMB4 might participate in the proteasome dependent degradation of IκBα. The data indicates that PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial I/R, which can supply novel molecular target for the treatment of ischemic heart disease.

Fibrinogen activates IκBα signaling in neurons via binding to its receptors, ICAM‐1 and PrP C

Mild traumatic brain injury (TBI) is the most common type of brain injury that is a major health issue. The goal of the current study was to extend our previous observations that during TBI, extravasated fibrinogen (Fg) deposited in vasculo/astrocyte interfaces and was associated with neurodegeneration and short-term memory (STM) reduction. We have previously shown that an elevated level of Fg, called hyperfibrinogenemia, causes astrocyte activation via interactions with intercellular adhesion molecule-1 (ICAM-1) and cellular prion protein (PrPC). However, the direct effect of Fg on neurons is not known. Primary mouse brain cortical neurons from C57BL/6 mice (Lonza) were grown in Primary Neural Basal Medium and PNGMTM Single QuotsTM. Twenty-four well plates, with #1 glass-bottomed coverslips coated with Poly-D-Lysine (30 µg/ml) and laminin (200 µg/ml) were used for seeding neurons at the density of 200,000 cells/well. After the required 7-day growth in complete medium, neurons were treated with various doses of Fg for 24 hours unless otherwise indicated. Each experimental group contained hirudin (1 U/ml) to inhibit the conversion of Fg to fibrin via thrombin. In parallel, experiments to test neuronal viability using a live/dead assay were performed without the presence of hirudin. Interactions of Fg with neuronal ICAM-1 and PrPC were tested using a proximity ligation assay (PLA), which allows highly specific and sensitive immunofluorescent detection to be visualized and consequently protein-protein interactions in situ to be objectively quantified. Real time PCR was performed to investigate the expression of mRNAs for interleukin 6 (IL-6) and inhibitor of kappa Bα (IκBα). The levels of IκBα mRNA reflect stimulation of nuclear factor-kappa B (NF-κB) transcription. PLA confirmed Fg-ICAM-1 and Fg-PrPC interactions on the surface of neurons. An increased expression of neuronal IκBα and IL-6 was induced after treatment with Fg for 2hr or 24 hr, respectively. Data showed that Fg, in the presence or absence of hirudin, caused increased neuronal death. Thus, Fg binding to its neuronal receptors, ICAM-1 and PrPC may activate NF-κB, triggered by IκBα signaling, that leads to neuroinflammation and increased neuronal death. This study is a first attempt to define the molecular mechanisms by which Fg causes neuroinflammation and neurodegeneration that resulted in the cognitive decline previously seen during mild-to-moderate TBI in our studies.

CD5 regulates the heterogenous depot of NFκB available for T cell activation

Abstract T cells bearing receptors to the same antigen often differ considerably in their ability to respond. We hypothesized that variations in intracellular signaling molecules in T cells may underlie these cell-to-cell heterogeneities. We found that the expression levels of PLCγ and IκBα, varied considerably between individual peripheral T cells. The ~1000-fold variation in IκBα levels, but that not of PLCγ, strictly correlated with surface levels of CD5. CD5 is typically used as a marker for T cell self-reactivity and differences in responses between CD5-high and -low cells is attributed to the self-awareness of the TCR. Our data suggested that CD5 directly regulates the NFκB pathway to modulate T cell responses. Accordingly, ablating CD5 using germline or conditional knock-out mice resulted in a significant reduction in IκBα. This regulation was independent of SHP-1 and casein kinase II and not evident in mRNA levels, suggesting a previously unknown post-translational mechanism. Overexpression of CD5 in TCR-deficient cells also increased basal IκBα levels indicating independence from TCR signaling. Intriguingly, cells with higher CD5 and IκBα expression maintained a proportionally greater amount of NFκBp65 in a cellular reservoir. This suggests that CD5-high T cells with a greater NFκB reservoir might allow for a more robust NFκB-dependent gene expression. Consistent with this postulate, we found that CD5-high thymocytes survived ex vivo better than CD5-low ones, but only when NFκB signaling was intact. These data suggest a key role for CD5 in the control of T cell heterogeneity, independent of the self-reactivity of a TCR itself.

Human Caspase 12 Enhances NF-κB Activity through Activation of IKK in Nasopharyngeal Carcinoma Cells.

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.

Bioflocs attenuate Mn-induced bioaccumulation, immunotoxic and oxidative stress via inhibiting GR-NF-κB signalling pathway in Channa asiatica

Manganese (Mn) is a relatively common element in aquatic ecosystems and can be bio-concentration, but the mechanism of manganese poisoning on fish health is unclear. Here, this study's objective was to evaluate the potential mechanisms of bioflocs in ameliorating Mn-induced toxicity in Channa asiatica. Three hundred sixty juveniles were randomly divided into 12 tanks. Four C:N ratios in triplicate tanks were tried: C/N = 7.6:1 with a commercial diet (control), C/N 10:1, C/N 15:1 and C/N 20:1, and the bio-accumulation, immunotoxic, oxidative stress, GR-NF-κB related genes expression and intestinal histomorphology were assessed in three different periods after Mn exposure (0 h, 48 h and 96 h). The results showed that bioflocs had a significant protective effect on Mn poisoning by preventing alterations in bio-accumulation levels, LSZ, AKP, C3, C4 and IgM, of which the C/N 15:1 group had the best relief effect. Furthermore, bioflocs also assisted in the recovery of liver T-SOD, CAT, GPX and T-AOC levels while decreasing the content of MDA. Moreover, C/N 15:1 group significantly down-regulated the expression level of NF-κB, TNF-α, IL-1β and IL-8 and up-regulated significantly IκBα, GR, HSP70 and HSP90 expression levels considerably (P < 0.05). From the intestinal section, the C/N 15:1 group resistance was the best one, and there was no difference between C/N 20:1 group and control group. These results revealed that administration of bioflocs (C/N 15:1) has the potential to combat Mn toxicity in C. asiatica, and the specific pathway may be GR-NF-κB.

Simiao Wan attenuates monosodium urate crystal-induced arthritis in rats through contributing to macrophage M2 polarization

Ethnopharmacological relevanceSimiao Wan (SMW) is a classical traditional Chinese medicine (TCM) prescription to empirically treat gouty arthritis (GA) in TCM clinical practice. However, the potential mechanisms of SMW on GA are not fully evaluated. Aim of studyThe aim of this study is to investigate the role of macrophage polarization in the anti-GA activity of SMW. Materials and methodsRats were intragastricly treated with SMW for consecutive 7 days. On day 6, monosodium urate (MSU) crystal-induced arthritis (MIA) in the ankle joint was prepared. Paw volume, gait score and histological score were measured. Levels of interleukin (IL)-1β and IL-10 in serum were detected by enzyme-linked immunosorbent assay. Expressions of inducible nitric oxide synthase (iNOS), arginase (Arg)-1, phosphorylated (p)-p65, inhibitor of nuclear factor (NF)-κB (IκB)α, p-signal transducer and transcription activator (STAT)3 and p-Janus kinase (JAK)2 in synovial tissues were determined by Western blot. ResultsThe elevated paw volume, gait score and histological score in MIA rats were significantly decreased by SMW treatment. Meanwhile, SMW significantly decreased the IL-1β level and increased the IL-10 level in serum of MIA rats. Furthermore, SMW reduced the expressions of iNOS, p-p65 and enhanced the expressions of Arg-1, IκBα, p-STAT3 and p-JAK2 in synovial tissues of MIA rats. ConclusionsThe results suggest that SMW attenuates the inflammation in MIA rats through promoting macrophage M2 polarization.

Bioactive-guided isolation and identification of oligostilbenes as anti-rheumatoid arthritis constituents from the roots of Caragana stenophylla

Ethnopharmacological relevanceThe roots of Caragana stenophylla have been used as folk medicine due to the functions of activating blood, diuresis, analgesic and tonicity, especially in treating rheumatoid arthritis and hypertension. However, the anti-rheumatoid arthritis mechanisms and bioactive ingredients have not previously been fully investigated. Aim of the studyThe aim of this study was to assess the anti-rheumatoid arthritis effects of the roots of Caragana stenophylla ethanol extract (EC), elucidate its mechanism of action and identify its active substances. Materials and methodsAnti-rheumatoid arthritis activity of EC was assessed using type II-collagen induced arthritis in rats. Arthritis severity was evaluated by foot paw volume, arthritis index, joint swelling degree and histopathology. The serum inflammatory cytokines and matrix metalloproteinases (MMPs) were also detected by immunohistochemical analysis. In addition, the protein expression of IκB, p-IκB, iNOS and COX-2 was analyzed by western blot. RAW 264.7 macrophage cells were employed to assess the anti-inflammatory effects of fractions and compounds in vitro. UPLC-Q-TOF-MS was adopted to appraise the ingredients of the active fraction of the roots of C. stenophylla. Furthermore, various chromatographic techniques and spectroscopic methods were used for isolation and structure elucidation of compounds. ResultsThe results showed that EC could reduce type II collagen-induced rheumatoid arthritis model arthritic score and histopathology markedly at dose of 240 mg/kg. Besides, EC could suppress the levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17, and TNF-α) and matrix metalloproteinases (MMP-3, MMP-9), and the expression levels of COX-2, p-IκB and iNOS also were declined. While, the levels of IL-10 and IκB were increased. The ethyl acetate fraction exhibited potent inhibitory effects against nitric oxide production in RAW 264.7 macrophage cells. Eleven main components including 1 flavonoid and 10 oligostilbenes from active fraction were isolated by mass directed chromatographic techniques. Their structures were determined on the basis of various spectroscopic methods and by comparison with the published NMR data. ConclusionThe roots of C. stenophylla attenuated arthritis severity, restored serum cytokine imbalances by regulating NF-κB signaling pathway in type II collagen-induced rheumatoid arthritis model. Oligostilbenes were essential ingredients in ethyl acetate extract of C. stenophylla roots. Stilbenes and flavonoids should be responsible for its anti-rheumatoid arthritis activities.

Downregulation of metallothionein-2 contributes to oxaliplatin-induced neuropathic pain

BackgroundWe previously reported a correlation between small doses of oxaliplatin penetrating onto the spinal cord and acute pain after chemotherapy. Here, we propose that MT2 within the spinal dorsal horns participates in the development of oxaliplatin-induced neuropathic pain and may be a pharmacological target for the prevention and treatment of chemotherapy-induced peripheral neuropathy (CIPN).MethodsThe rat model of CIPN was established by 5 consecutive injections of oxaliplatin (0.4 mg/100 g/day). Genetic restoration of neuron-specific metallothionein-2 was implemented 21 days before oxaliplatin treatment, and also, genetic inhibition by metallothionein-2 siRNA was performed. Mechanical allodynia and locomotor activity were assayed. Cell-specific expression of metallothionein-2, the mRNA levels of pro-inflammatory cytokines, nuclear translocation of NF-κB, the protein levels of expression of IκB-α, and interaction between IκB-α and P65 were evaluated in the spinal dorsal horns. Also, in vitro interaction of sequentially deleted IκB-α promoter with metallothionein-2 was used to assess the signal transduction mechanism.ResultsWe found that oxaliplatin induced downregulation of metallothionein-2 in rat spinal cord neurons. By contrast, genetic restoration of metallothionein-2 in the spinal dorsal horn neuron blocked and reversed neuropathic pain in oxaliplatin-treated rats of both sexes, whereas genetic inhibition of metallothionein-2 triggered neuropathic pain in normal rats. Overall locomotor activity was not impaired after the genetic alterations of metallothionein-2. At the molecular level, metallothionein-2 modulated oxaliplatin-induced neuroinflammation, activation of NF-κB, and inactive transcriptional expression of IκB-α promoter, and these processes could be blocked by genetic restoration of metallothionein-2 in the spinal dorsal horn neurons.ConclusionsMetallothionein-2 is a potential target for the prevention and treatment of CIPN. A reduction of NF-κB activation and inflammatory responses by enhancing the transcription of IκB-α promoter is proposed in the mechanism.

Astaxanthin inhibits inflammation of human periodontal ligament cells induced by lipopolysaccharide.

Human periodontal ligament cells (hPDLCs) are important source of periodontal tissue reconstruction. Under chronic inflammation, the multi-directional differentiation potential and chemotaxis in hPDLCs are decreased. Therefore, inhibiting inflammatory microenvironment and improving the functional characteristics of stem cells can better promote periodontal tissue reconstruction. This study was to investigate the effect of astaxanthin (AST) on lipopolysaccharide (LPS)-induced inflammation in hPDLCs and the underlying mechanisms. hPDLCs were isolated and cultured in vitro, and vimentin and keratin immunocytochemical staining were used to identify hPDLCs. CCK-8 assay was used to measure the effects of AST (1, 5, 10, 20, 50, 100, and 200 μmol/L) on proliferation of hPDLCs. Quantitative RT-PCR (RT-qPCR) and ELISA were used to measure the mRNA and protein expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in the control (Con) group, the LPS group, and the LPS+AST (5, 10, 20, and 50 μmol/L) group. Western blotting was used to detect the protein expression of IKBα, phosphorylated IKBα (p-IKBα), and p65 in the Con group, the LPS group, the AST (20 μmol/L) group, and the LPS+AST (20 μmol/L) group. After 10 μmol/L PDTC treatment, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α were detected by RT-qPCR and ELISA. Cell morphology and immunocytochemical staining showed that the cells were in line with the characteristics of hPDLCs. Treatment with AST could promote the proliferation of hPDLCs, which reached the peak at 20 μmol/L. The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS group were higher than those in the Con group (all P<0.05). Compared with the LPS group, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS+AST (5, 10, 20, and 50 μmol/L) group were down-regulated (all P<0.05). Compared with the Con group, the levels of IKBα and p65 in cytoplasm of the LPS group were significantly downregulated (both P<0.05), and the levels of p-IKBα in cytoplasm and p65 in nucleus of the LPS group were significantly up-regulated (both P<0.05). Compared with the LPS group, the levels of IKBα and p65 in cytoplasm of the LPS+AST (20 μmol/L) group were significantly upregulated (both P<0.05), and the levels of p-IKBα in cytoplasm and p65 in nucleus of the LPS+AST (20 μmol/L) group were significantly downregulated (both P<0.05). The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS+PDTC (10 μmol/L) group were lower than those in the LPS group (all P<0.05). AST promotes the proliferation of hPDLCs, which is related to suppression of LPS-induced the secretion of inflammatory factors via inhibiting the activation of NF-κB signaling pathway.

Involvement of Rho-kinase/IκB-α/NF-κB activation in IL-1β-induced inflammatory response and oxidative stress in human chondrocytes.

It has been clearly indicated that osteoarthritis (OA) is an inflammatory and degenerative disease that could be promoted by Rho-kinase (ROCK); however, little is known about the role of ROCK/inhibitor κBalpha (IκB-α)/nuclear factor-κB (NF-κB) p65 pathway activation in interleukin-1β (IL-1β) induced inflammatory response and oxidative stress in primary human chondrocytes. To test this hypothesis, we focused on determining ROCK-II, IκB-α, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), p22phox, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subtype 4 (NOX4) protein expression, ROCK-II activity, NADPH oxidase levels, and total antioxidant capacity (TAC) in the presence and absence of ROCK-inhibitor fasudil. IL-1β (2ng·mL-1, 24h) increased the expression of ROCK-II, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, TNF-α, COX-2, and p22phox proteins, and decreased the expression of IκB-α, and the NOX4 protein level did not alter. ROCK activity and NADPH oxidase levels were increased, whereas the TAC was decreased by IL-1β. Fasudil (10-5-10-7M) reversed all these changes induced by IL-1β. These results demonstrate that ROCK/IκB-α/NF-κB p65 pathway activation contributes to the IL-1β-induced inflammatory response and oxidative stress, and thus, ROCK inhibition might be a beneficial treatment option for OA patients mainly based on its anti-inflammatory and antioxidant effects.

Jian-Pi-Yi-Shen Formula Ameliorates Oxidative Stress, Inflammation, and Apoptosis by Activating the Nrf2 Signaling in 5/6 Nephrectomized Rats

Chronic kidney disease (CKD) is an increasing global public health problem, with high morbidity and mortality. Jian-Pi-Yi-Shen (JPYS) formula is a representative traditional Chinese medicine formula in the treatment of CKD, which is widely used in clinical practice in China. However, the underlying mechanism has not been well elucidated. In the present study, we measured the markers of apoptosis, inflammation, oxidative stress, and nuclear factor erythroid 2–related factor 2 (Nrf2) signaling to investigate the effects of JPYS formula on renal function and fibrosis and its molecular mechanism in an established animal model of 5/6 nephrectomized (5/6Nx) rats. The results demonstrated that the JPYS formula exerted a significant preventive effect on renal dysfunction and fibrosis, based on analysis of correlative parameters such as urinary protein, SCr, BUN, glomerular sclerosis index, and tubulointerstitial fibrosis score and renal histopathology and ultrastructural pathology of CKD rats. JPYS formula also induced downregulation of gene expression associated with fibrosis, such as TGF-β and type I, III, and IV collagen. Moreover, the JPYS formula showed a significant protective effect in suppressing cell apoptosis according to the results of apoptotic indexes, including increased gene expression of Bcl-2, decreased gene expression of Bax, caspase 3, caspase 9, and the number of TUNEL-positive cells. JPYS formula also ameliorated the activation of the NF-κB-mediated inflammatory pathway, as manifested by the downregulation of gene expression of TNF-α, IL-1β, IκBα, NF-κB p65, MCP-1, CXCL1, COX-2, and iNOS in the kidney. Our evidence also suggested that the JPYS formula ameliorates oxidative stress by promoting antioxidant function according to antioxidant index indicators as an indicator of GSH, SOD, CAT, and GPx and abating excessive accumulation of the reactive oxygen species biomarkers, including ROS, TBARS, 8-oxo-dG, and MDA. The data also suggested that the JPYS formula reversed the downregulation of HO-1 and Nrf2 level and upregulation of Keap1 expression. Together, our data highlighted that the JPYS formula relieved renal oxidative injury mediated by activation of Nrf2 signaling by inhibiting inflammation and apoptosis in CKD rats.