Dietary high protein-induced diarrhea and intestinal inflammation by activation of NF-κB signaling in piglets

The present study aimed to investigate whether inflammation-associated responses in piglets are induced by high protein (HP) through activating nuclear factor kappa B (NF-κB) signaling. Sixteen piglets (35 d of age, Duroc × [Landrace × Yorkshire], weaned at d 21, initial BW = 9.70 ± 0.11 kg) were allocated to 18% and 26% CP (HP group) at random, comprising 8 replicate pens per treatment. The piglets were slaughtered to collect intestinal tissues when apparent, persistent, and stable diarrhea syndromes happened (on d 12). No significant differences were observed in their growth performance (P > 0.05), but reduction by 19.11%, 25.31%, 23.64% of ADFI, ADG, and G:F, respectively was detected in the HP group. The HP group had greater (P = 0.002) diarrhea rates. Furthermore, dietary HP had lower ileal villus height (VH; P = 0.048), ratio of villus height to crypt depth (VH/CD ratio; P = 0.016), and colonic CD (P = 0.034), as well as had the trend (P = 0.075) to reduce the ileal villus absorptive area. Moreover, HP diets significantly elevated the goblet cell numbers in the ileal villi (P = 0.016) and colonic crypts (P < 0.001) and up-regulated (P = 0.012) the mRNA expression of mucin2 (Muc2) in the ileum. In addition, HP diets increased the myeloperoxidase concentration in the ileum (P = 0.002) and colon (P = 0.007) of piglets. Dietary HP significantly down-regulated the mRNA expression of tumor necrosis factor-α (TNF-α; P < 0.001) in the ileum, induced nitric oxide synthase (iNOS; P = 0.040) and interleukin-22 (IL-22; P = 0.008) in the colon, and inclined to down-regulate interleukin-1β (IL-1β; P = 0.076) expression in the colon. The relative protein abundance of Galectin-3 (P = 0.046) in the colon and the ratio of phosphorylation NF-κB to NF-κB (p–NF–κB/NF-κB ratio) in the ileum of HP piglets were also greater (P = 0.038). These results suggest that dietary HP may cause diarrhea in piglets by activating NF-κB signaling induced intestinal inflammation.     

Inhibitory effects of recombinant porcine interferon-α on high- and low-virulence porcine reproductive and respiratory syndrome viruses

The inhibitory effects of recombinant porcine interferon alpha (rPoIFN-α) on the propagation of low-virulence PRRSV (lvPRRSV) in MARC-145 cells, and on the progress and severity of high virulence PRRSV (hvPRRSV)-induced infections in pigs, were determined. Pre-treatment of MARC-145 cells with increasing concentrations of rPoIFN-α prior to infection with lvPRRSV decreased the observed cytopathic effects (CPEs) in a concentration-dependent manner. Viral propagation and antibody response were temporarily delayed in swine treated with rPoIFN-α either at the same time as the hvPRRSV challenge was administered or post-challenge. Exposure of challenged animals to rPoIFN-α after the onset of disease symptoms alleviated associated hyperthermia. Variations in lymphocyte subsets indicated that rPoIFN-α treatment might alleviate damage to the immune system or enhance propagation of host cytotoxic T-lymphocytes when the treatment was applied simultaneously with the virus or 1dpc, respectively.     

Relationship between NF-κB expression and malignancy of canine mammary gland tumor tissues

In this study, the nuclear expression of nuclear factor kappa B (NF-κB) in 48 tissues specimens from 25 canine spontaneous mammary gland tumor (MGT) patients was assessed by immunohistochemistry to compare their levels with clinical features, histological types, prognostic outcomes and proliferative activities, including the mitotic index (MI) and cylcinD1 expression. Twelve of eighteen (66.7%) malignant tumor tissues showed greater than 10% nuclear staining, while benign tumor and hyperplastic tissues showed less than 10% nuclear staining. Higher nuclear expression of NF-κB was positively correlated with larger tumor size, lymph node metastasis and higher MI; however, no correlation was observed with distant metastasis and cyclin D1 expression. Higher NF-κB nuclear expression correlated with shorter patient survival. These findings suggest that NF-κB is a useful prognostic factor for canine MGT patients.     

Inhibitory effects of pentosan polysulfate sodium on MAP-kinase pathway and NF-κB nuclear translocation in canine chondrocytes in vitro

Pentosan polysulfate sodium (PPS) has a heparin-like structure and is purificated from the plant of European beech wood. PPS has been used for the treatment of interstitial cystitis for human patients. Recent years, it was newly recognised that PPS reduce pain and inflammation of OA. The molecular biological mechanism of PPS to express its clinical effects is not fully understood. The purpose of the present study is to investigate a mechanism of action of PPS on inflammatory reaction of chondrocytes in vitro. It was evaluated that effects of PPS on interleukin (IL)-1β-induced phosphorylation of mitogen-actiated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), nuclear translocation of nuclear factor-kappa B (NF-κB), and matrix metalloproteinase (MMP)-3 production in cultured articular chondrocytes. As a result, in the presence of PPS existence, IL-1β-induced phosphorylation of p38 and ERK were certainly inhibited, while JNK phosphorylation was not affected. Nuclear translocation of NF-κB and MMP-3 production were suppressed by PPS pretreatment prior to IL-1β stimulation. In conclusion, it is strongly suggested that PPS treatment prevents inflammatory intracellular responses induced by IL-1 β through inhibition of phosphorylation of certain MAPKs, p38 and ERK and then nuclear translocation of NF-κB in cultured chondrocytes. These PPS properties may contribute to suppressive consequence of catabolic MMP-3 synthesis. These data might translate the clinical efficacy as PPS treatment could inhibit the cartilage catabolism and related clinical symptoms of OA in dogs.     

Preliminary Study on NF-κB Signaling Pathway Regulating Brucella Intracellular Survival Molecular Mechanisms

By the infection of Brucella virulent strain and attenuated strain in mice macrophage RAW264.7,the assay was aimed to explore the relationship between NF-κB signaling pathways and Brucella virulent strain and attenuated strain in intracellular survival.Use different MOI Brucella (2308,RB51,16M and M5) to infect mice macrophage RAW264.7,after 0,4,8 and 24 h infected,cracking cell and collecting supernatant,we detected the effect of Brucella on activation of NF-κB signaling pathway by Western blotting.Different concentrations of NF-κB signaling pathway inhibitor were incubated with mice macrophage RAW264.7,with different multiplicities of infection (MOI) of Brucella infecting cells,ELISA kits to detect the expressions of TNF-α,IL-1β and IL-6 cytokine;At the same time,count the number of intracellular bacteria of CFU.The results showed that rough cattle Brucella strains RB51 could strongly activate NF-κB signaling pathway,smooth cattle Brucella strains 2308 was weak in the activation;At the same time,the activation of NF-κB signaling pathway was concentration dependent.When the MOI was 80,infection time was 8 h,NF-κB activation degrees of rough cattle Brucella strains RB51 and smooth cattle Brucella strains 2308 were the strongest,and this pathway was involved in producing TNF-α and IL-6;NF-κB signaling pathway inhibitor BAY11-7082 affected Brucella intracellular survival.So rough cattle Brucella strains RB51 intracellular survival and NF-κB signaling pathway activity were closely related.The results laid the foundation for the further study of Brucella intracellular pathogenesis,also provided scientific basis for the research of new drugs to Brucella,and prevention and treatment of brucellosis.     

The Pseudomonas aeruginosa HSP70-like protein DnaK induces IL-1β expression via TLR4-dependent activation of the NF-κB and JNK signaling pathways

IL-1β expression is increased in response to P. aeruginosa infection, but the responsible proteins have not been clearly elucidated. Here, we demonstrate for the first time that IL-1β expression is induced in response to the heat shock protein 70-like protein DnaK. Treatment with recombinant DnaK (rDnaK) increased IL-1β expression in a dose- and time-dependent manner, and the release of mature IL-1β in response to rDnaK was detected to an extent similar to that stimulated by the well-known agonists, lipopolysaccharide and nigericin. rDnaK-mediated IL-1β expression was driven by the NF-κB signaling pathway. In addition, expression was controlled by the JNK signaling pathway, although these two signaling cascades act independently upon rDnaK stimulation. Finally, rDnaK-induced IL-1β expression was initiated via the action of TLR4. Taken together, the data reveal that P. aeruginosa-derived DnaK induces expression of IL-1β via TLR4-dependent activation of the NF-κB and JNK signaling pathways.     

The activation of the IFNβ induction/signaling pathway in porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus is variable

Background

It has been recognized that the expression of type I interferon (IFNα/β) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection.

Objective

Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFNβ) and downstream signaling events.

Methods

In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFNβ protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events.

Results

These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFNβ protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFNβ signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFNβ production.

Conclusion

The results of this study demonstrate that the induction of IFNβ and signaling in PAMs after PRRSV infection is variable.

     

The role of Bcl-xL and nuclear factor-��B in the effect of taxol on the viability of dendritic cells

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-κB (NF-κB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-_L-phenylalanine chloromethyl ketone confirmed that NF-κB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.     

猪繁殖与呼吸综合征病毒通过降解IκBα激活NF-κB信号通路

核转录因子κB(NF-κB)在调节细胞免疫功能及细胞增殖和存活方面均发挥着重要的作用。本研究采用高致病性猪繁殖与呼吸综合症病毒(HP-PRRSV)GD08-1株和经典株PRRSV GD-XH分别感染Marc-145细胞,于感染后不同时间提取胞浆蛋白和核蛋白,通过电泳迁移率(EMSA)检测NF-κB与DNA结合活性及western blot检测胞浆蛋白中NF-κB及其抑制因子(IκB)的表达情况。HP-PRRSV株和PRRSV经典株感染Marc-145核蛋白的EMSA结果中均出现NF-κB与DNA探针的结合条带;western blot结果表明,HP-PRRSV株和PRRSV经典株感染Marc-145细胞并未引起NF-κB表达量的变化,但两者均引起了IκB表达水平的下降,由此推测,HP-PRRSV株和经典PRRSV株感染均能够刺激Marc-145细胞NF-κB活化入核内,PRRSV感染Marc-145细胞引起的NF-κB的活化是通过IκB的降解来实现的。     

Seaweed polysaccharide mitigates intestinal barrier dysfunction induced by enterotoxigenic Escherichia coli through NF-κB pathway suppression in porcine intestinal epithelial cells

This study aimed to investigate the protective effects and underlying mechanism of seaweed polysaccharide (SWP) on intestinal epithelial barrier dysfunction induced by E. coli in an IPEC-J2 model. A preliminary study was done to screen optimum SWP concentrations by cell viability, cytotoxicity, apoptosis and proliferation evaluation. The regular study was conducted to evaluate the protective effects of SWP against E. coli challenge via the analysis of transepithelial electrical resistance (TEER), tight junction proteins, NF-κB signalling pathway, proinflammatory cytokines and the E. coli adhesion and invasion. Our results show that 4 h E. coli challenge down-regulated tight junction proteins expression, decreased TEER, activated NF-κB signalling pathway and increased proinflammatory response, which indicates that the E. coli infection model was well-established. Pre-treatment with 240 μg/ml SWP for 24 h alleviated the 4 h E. coli -induced intestinal epithelial barrier dysfunction, as evidenced by the up-regulated expression of Occludin, Claudin-1 and ZO-1 at both mRNA and protein level and the increased TEER of IPEC-J2 cells. Pre-incubation with 240 μg/ml SWP for 24 h inhibited the activation of the NF-κB signalling pathway by 4 h E. coli challenge, including the decreased mRNA expression of TLR-4, MyD88, IκBα, p-65, as well as the reduced ratio of protein expression of p-p65/p65. Also, pre-treatment with 240 μg/ml SWP for 24 h decreased proinflammatory response (IL-6 and TNF-α) induced by 4 h E. coli challenge and decreased the E. coli adhesion and invasion. In conclusion, SWP mitigated intestinal barrier dysfunction caused by E. coli through NF-κB pathway in IPEC-J2 cells and 240 μg/ml SWP exhibited better effect. Our results also provide a fundamental basis for SWP in reducing post-weaning diarrhoea of weaned piglets, especially under E. coli -infected or in-feed antibiotic-free conditions.     

2型糖尿病食蟹猴白细胞NF-κB和IκBα表达相关性分析

比较了食蟹猴2型糖尿病(T2DM)不同发病阶段与正常对照组的空腹血糖值(FPG)、甘油三酯(TG)、胆固醇(CHOL),低密度脂蛋白胆固醇(LDL-C),高密度脂蛋白胆固醇(HDL-C)在血液中含量,及荧光定量PCR方法测定的NF-κB和IκBα在外周血白细胞中表达量.结果显示,FPG、CHOL和LDL-C随疾病进行呈...     

Impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing “professional IFN-α-producing cells”. Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS.     

Inhibition of NF-κB signaling pathway by astaxanthin supplementation for prevention of heat stress–induced inflammatory changes and apoptosis in Karan Fries heifers

Present study was conducted on 12 Karan Fries (Holstein Friesian X Tharparkar) heifers (10–12 months) to assess the effect of astaxanthin supplementation on heat stress amelioration and inhibition of NF-κB signaling pathway for prevention of heat stress–induced inflammatory changes and apoptosis in the cell during the summer season. The heifers were randomly and equally divided into two groups, i.e., control (fed as per ICAR 2013) and treatment groups (additionally supplemented astaxanthin at a dose rate of 0.25 mg/kg BW/day/animal). Temperature humidity index used to assess the levels of summer stress during the experimental period. Blood samples were collected at the fortnightly interval for quantification of plasma cortisol and IL-12 from both the groups of the heifers and from collected blood samples, RNA was isolated and transcribed into cDNA for real time PCR, for genes expression of NF-κB, IL-2, caspase-3, and Bcl-2. Plasma cortisol, IL-12 levels, and expression pattern of NF-κB, IL-2, and caspase-3 were significantly (P ≤ 0.05) lower in treatment group of Karan Fries heifers than control group, whereas, Bcl-2 was higher (P ≤ 0.05) in astaxanthin supplemented group. The temperature humidity index had a positive correlation (P ≤ 0.05) with plasma cortisol and IL-12 and expression pattern of NF-κB, IL-2, and caspase-3. However, it was negatively correlated with Bcl-2. The supplementation of astaxanthin can ameliorate the impact of summer stress through NF-κB downregulation, might be due to the quenching of free radicals, which regulates the expression of pro-inflammatory mediators and apoptotic genes.

     

PHA复方制剂对鸡体内NF-κB的调节作用研究

为了探讨植物血凝素(PHA)复方制剂对鸡免疫调节的作用与核因子-κB(NF-κB)的关系,试验联合应用La Sota疫苗,在不同免疫时期,采用RT-PCR法对胸腺、脾脏、法氏囊中NF-κB mR-NA的表达进行测定,采用ELISA法测定血清中NF-κB的浓度变化。结果表明:在不同免疫时期,适当剂量的PHA复方制剂有上调胸腺中NF-κB mRNA表达、降低脾脏和法氏囊中NF-κB mRNA表达的作用;在外周血中,PHA复方制剂有降低NF-κB浓度的作用。说明PHA复方制剂的作用机理与调节NF-κB有关,在不同免疫阶段及免疫脏器中作用趋势不同。     

不同物种NF-κB1基因编码区生物信息分析

为了解不同物种NF-κB1基因编码区（CDS）的遗传变异,本试验使用生物信息学的方法比较分析了小家鼠、褐家鼠、人、黑猩猩、狨、毛猩猩、猕猴、家马、大熊猫、野猪、家短尾负鼠、牛、狼、非洲象和白颊长臂猿NF-κB1基因编码区的遗传多样性,并对该基因的氨基酸序列、跨膜结构域、导肽、信号肽和结构域进行了预测和分析,对有关NF-κB1基因功能的一些研究热点进行了回顾。结果表明：在15个物种36条基因序列中共检测到968个多态位点,生成24种单倍型,NF-κB1基因序列编码区在物种间存在丰富的遗传多样性;理论等电点均低于5.5,NF-κB1编码的蛋白呈酸性,N端无信号肽、导肽,无跨膜结构域,肽链表现为亲水性;这些蛋白均含有1个Rel同源域,6～7个Ankyrin重复,在结构上保守。