Effects of insoluble dietary fibre differing in lignin on performance, gut microbiology, and digestibility in weanling piglets

The study aimed to investigate the effects of insoluble dietary fibre differing in lignin content on performance and parameters of gut microbiology in 48 weanling piglets (8.5 kg mean body weight) fed common diets ad libitum. Fibre sources tested were wheat bran (low lignin) and pollen from Chinese Masson pine (Pinus massoniana) as model of fibre rich in lignin, added to diets as follows: no addition (control), 3.0% wheat bran, 1.27% pine pollen and 2.55% pine pollen. In colonic chyme, bacterial colony counts remained unaffected by treatment, but ammonia contents were reduced by fibre additions (up to -38%). The effects on ammonia were best explained by added cellulose and lignin. Fibre additions reduced apparent (faecal) digestibility of dry matter (DM) and crude protein (CP) up to -3.5 and -4.3% units with cellulose and lignin being the major determinants for changes in CP digestibility.     

The TGFβ1 pathway is required for NFκB dependent gene expression in mouse keratinocytes

The transforming growth factor-beta 1 (TGFβ1) and NFκB pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGFβ1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFβ1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFβ1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFβ1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFβ1 induction of NFκB-luciferase. TGFβ1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFβ1 and this was blocked in NFκB+/− and −/− keratinocytes and by the IκB super-repressor. To test the effects of the TGFβ1 pathway on a biologically relevant activator of NFκB, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGFβ1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/− mice. These studies demonstrate that intact TGFβ1 signaling is required for NFκB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFβ1.     

NFkappaB activation is essential for miR-21 induction by TGFβ1 in high glucose conditions

Transforming growth factor beta1 (TGFβ1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFβ1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGFβ1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFβ1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGFβ1 in high glucose conditions. The induction by TGFβ1 was dependent on NFκB activation and subsequent ROS generation. TGFβ1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFβ1. ChIP assay revealed binding of p65 to miR-21 promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGFβ1–NFκB–miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.     

In vitro fermentability and physicochemical properties of fibre substrates and their effect on bacteriological and morphological characteristics of the gastrointestinal tract of newly weaned piglets

Abstract

Fermentability of fibre has a great impact on the bacterial flora along the gastrointestinal tract of newly weaned piglets. Therefore, this parameter was determined by incubating in vitro different fibre substrates (chicory roots, sugar beet pulp, wheat bran and corn cobs) with contents of jejunum or caecum sampled from slaughtered pigs. Incubating with small intestinal contents, lactic acid was the only fermentation product. Fermentability was highest for chicory roots, followed by wheat bran and sugar beet pulp, while corn cobs were not fermented. Based on SCFA formed in the incubations with caecal contents, ranking of the fermentability of the fibre substrates was in the same order. The effect of adding different fibre substrates to diets of newly weaned piglets on bacteriological and morphological aspects of the gastrointestinal tract was also investigated. In Experiment 1 three groups of five piglets, weaned at four weeks of age, received a control feed (C), C supplemented with corn cobs (50 g/kg) or with chicory roots (20 g/kg). In Experiment 2, diet C was supplemented with sugar beet pulp (120 g/kg) or with wheat bran (75 g/kg). After three weeks animals were euthanized and digesta were sampled from stomach, proximal and distal jejunum, caecum and colon. Furthermore, mucosal scrapings were prepared and tissue samples were taken from jejunum, caecum and colon. Viscosity was determined for jejunal, caecal and colon contents. Corn cobs in the feed increased the number of total bacteria, lactobacilli and bifidobacteria in the stomach and proximal duodenum, while a decreased count of streptococci in distal jejunum contents was noted. Chicory roots increased the counts of Escherichia coli in the distal jejunum and on the mucosa, while sugar beet pulp decreased the number of lactobacilli on the mucosa only. Wheat bran seemed to increase the count of E. coli in jejunal digesta and on the mucosa, and also the number of lactobacilli in the stomach and jejunum. Bifidobacterial numbers were increased but only in the proximal part of the jejunum. Fibre substrates affected the concentration of lactate and SCFA in different parts of the intestinal tract. Feeding corn cobs increased villus length in the proximal jejunum by 13%. The number of intra-epithelial lymphocytes in the villous epithelium of proximal and distal jejunum was decreased by corn cobs and chicory roots supplementation while beet pulp and wheat bran had the opposite effect. In Experiment 1, apoptotic index of the mucosa of the distal jejunum was very low and decreased when corn cobs were fed. Mitotic index in the crypts was only affected by the wheat bran diet and a small decrease was noted. It was concluded that the fermentability of fibre was not an ideal criterion for predicting its effects on the flora. The effect of fibres on viscosity of digesta was negligible probably explaining the lack of clear and consistent influences on the intestinal mucosa.     

Combination of aspirin with telmisartan suppresses the augmented TGFβ/smad signaling during the development of streptozotocin-induced type I diabetic nephropathy

Diabetic nephropathy (DN) is the most common indication for the development of end stage renal diseases. Inflammation is increasingly seen as the core process in the development of diabetes. Inflammatory markers e.g. NFκB (p65 levels), TNFα, COX-2 and TGFβ–smad signaling are the key elements in the development of DN. Renin–angiotensin system suppressors like telmisartan have been used to treat DN, but they are not able to prevent completely because of development of resistance against them. Anti-inflammatory agents like, aspirin acts through both COX dependent and COX independent pathways. Hence, we thought that combining aspirin with telmisartan will be better therapeutic option in preventing the progression of nephropathy in diabetes. In the present study we studied the effect of this combination on inflammatory markers [COX-2, NFκB (p65 levels), TNFα], TGFβ–smad expression in preventing the progression of streptozotocin-induced type I diabetic nephropathy. Treatment of aspirin significantly prevented the progression of nephropathy and inhibited the augmented COX-2, NFκB (p65 levels), TNFα, and TGFβ–smad expression. Combination of aspirin with telmisartan resulted in a further decrease in the development of nephropathy and inflammatory markers in comparison to aspirin alone treatment. This is the first report which shows that aspirin in combination with telmisartan is more proficient in the treatment of diabetic nephropathy than any single drug therapy and involves the change in expression of inflammatory markers and TGFβ–smad signaling.     

Activation of integrin β1-focal adhesion kinase-RasGTP pathway plays a critical role in TGF beta1-induced podocyte injury

The depletion of glomerular podocytes is the key mechanism of glomerulosclerosis and progressive renal failure. Transforming growth factor-β (TGFβ) is a central mediator of signaling networks that control a diverse set of cellular processes, such as cell proliferation, differentiation, and apoptosis. Though many key events in TGFβ1 signaling have been documented at cellular and molecular level in podocytes, the complete effects of TGFβ1 on podocyte integrity are still elusive. In this study, the function of adhesion protein integrin β1, focal adhesion kinase (FAK), and a small GTPase Ras was explored in TGFβ1-induced podocyte injury. In cultured mouse podocyte, caspase 3-positive cells were counted by flow cytometry to evaluate podocyte damage at different time points after TGFβ1 treatment. Immunoblotting assay showed that integrin β1, FAK, Src kinase, and an adaptor protein Grb2 were activated rapidly after TGFβ1 stimulation. Active Ras Pull-Down assay revealed that the active Ras (GTP-bound Ras) level was upregulated in TGFβ1-treated cell. Immunoprecipitation results displayed that TGFβ1 enhanced the complex formation of integrin β1, FAK and Src kinase, as well as FAK, Grb2 and Ras. The FAK inhibitor TAE226 and the specific knockdown of Grb2 remarkably alleviated TGFβ1-induced podocyte apoptosis. The activation of p38MAPK and Erk1/2, and the nuclear translocation of NFκB(p65) were increased evidently in TGFβ1-treated cell, which could be dramatically prohibited by the application of the p38MAPK inhibitor SB202190 and the Ras inhibitor FPT Inhibitor III. The Src kinase inhibitor PP2 obviously prevented the activation of FAK and Ras, as well as the translocation of NFκB(p65) from cytoplasm to nuclei. The PP2, FPT Inhibitor III, and SB202190 significantly decreased TGFβ1-induced podocyte apoptosis. Taken together, these data demonstrated that the activation of integrin β1/Src/FAK and Grb2/RasGTP should be responsible for TGFβ1-induced podocyte damage through the p38MAPK and Erk1/2-mediated nuclear translocation of NFκB(p65).     

USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation

Lys63-linked polyubiquitination of transforming growth factor-β-activated kinase 1 (TAK1) has an important role in tumor necrosis factor-α (TNFα)-induced NF-κB activation. Using a functional genomic approach, we have identified ubiquitin-specific peptidase 4 (USP4) as a deubiquitinase for TAK1. USP4 deubiquitinates TAK1 in vitro and in vivo. TNFα induces association of USP4 with TAK1 to deubiquitinate TAK1 and downregulate TAK1-mediated NF-κB activation. Overexpression of USP4 wild type, but not deuibiquitinase-deficient C311A mutant, inhibits both TNFα- and TAK1/TAB1 co-overexpression-induced TAK1 polyubiquitination and NF-κB activation. Notably, knockdown of USP4 in HeLa cells enhances TNFα-induced TAK1 polyubiquitination, IκB kinase phosphorylation, IκBα phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Moreover, USP4 negatively regulates IL-1β-, LPS- and TGFβ-induced NF-κB activation. Together, our results demonstrate that USP4 serves as a critical control to downregulate TNFα-induced NF-κB activation through deubiquitinating TAK1.     

Long‐term restricted feeding alters circadian expression and reduces the level of inflammatory and disease markers

The circadian clock in peripheral tissues can be entrained by restricted feeding (RF), a regimen that restricts the duration of food availability with no calorie restriction (CR). However, it is not known whether RF can delay the occurrence of age‐associated changes similar to CR. We measured circadian expression of clock genes, disease marker genes, metabolic factors and inflammatory and allergy markers in mouse serum, liver, jejunum and white adipose tissue (WAT) after long‐term RF of 4 months. We found that circadian rhythmicity is more robust and is phase advanced in most of the genes and proteins tested under RF. In addition, average daily levels of some disease and inflammatory markers were reduced under RF, including liver Il‐6 mRNA, tumour necrosis factor (TNF)‐α and nuclear factor κB (NF‐κB) protein; jejunum Arginase, Afp, Gadd45β, Il‐1α and Il‐1β mRNA, and interleukin (IL)‐6 and TNF‐α protein and WAT Il‐6, Il‐1β, Tnfα and Nfκb mRNA. In contrast, the anti‐inflammatory cytokine Il‐10 mRNA increased in the liver and jejunum. Our results suggest that RF may share some benefits with those of CR. As RF is a less harsh regimen to follow than CR, the data suggest it could be proposed for individuals seeking to improve their health.     

转化生长因子β激活性激酶在胶质细胞信号转导中的作用机制

丝裂原激活蛋白激酶（MAPK）和 NFκB介导了炎症细胞转录活性的信号转导过程.转化生长因子β激活性激酶（TGFβ-activated kinase 1,TAK1）是这些转导通路的上游激酶.通过在胶质细胞株中瞬时转染TAK1和它的结合蛋白因子（TAK1-binding protein1 TAB1）基因,或与iNOS(可诱导型氧化氮合酶基因)启动子报告基因（iNOS-Luc）质粒共转染,探讨中枢两类胶质细胞在炎症反应过程中TAK1诱导iNOS 和细胞因子表达的作用机制.结果显示,TAK1明显激活iNOS 和细胞因子（TNFα、IL-1、IL-6）的表达活性. 而且当使用它的下游激酶p38 MAPK、JNK和NFκB的抑制剂（SB203580、SP620125和CAPE）后,这些表达活性明显被抑制.用IκBα的磷酸化突变体质粒(IκBαM)共转染胶质细胞株,能完全抑制iNOS的表达活性.研究结果提示：在胶质细胞内的p38 MAPK、JNK和NFκB信号介导的iNOS和细胞因子的转录表达过程中,TAK1起着非常重要的调节作用.     

Antagonizing peroxisome proliferator‐activated receptor γ facilitates M1‐to‐M2 shift of microglia by enhancing autophagy via the LKB1–AMPK signaling pathway

Microglia‐mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator‐activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)‐induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS‐stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL‐4, IGF‐1, TGF‐β1, TGF‐β2, TGF‐β3, G‐CSF, and GM‐CSF, and reduced the expression of M1 markers, such as CD86, Cox‐2, iNOS, IL‐1β, IL‐6, TNF‐α, IFN‐γ, and CCL2, thereby inhibiting NFκB–IKKβ activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3‐II/LC3‐I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1–STRAD–MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1‐to‐M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1–AMPK signaling and inhibited NFκB–IKKβ activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1‐to‐M2 phenotypic shift in LPS‐induced microglia, which might be due to improved autophagy via the activation of the LKB1–AMPK signaling pathway.     

Green tea extract protects against hepatic NFκB activation along the gut-liver axis in diet-induced obese mice with nonalcoholic steatohepatitis by reducing endotoxin and TLR4/MyD88 signaling

Green tea extract (GTE) reduces NFκB-mediated inflammation during nonalcoholic steatohepatitis (NASH). We hypothesized that its anti-inflammatory activities would be mediated in a Toll-like receptor 4 (TLR4)-dependent manner. Wild-type (WT) and loss-of-function TLR4-mutant (TLR4m) mice were fed a high-fat diet containing GTE at 0 or 2% for 8 weeks before assessing NASH, NFκB-mediated inflammation, TLR4 and its adaptor proteins MyD88 and TRIF, circulating endotoxin, and intestinal tight junction protein mRNA expression. TLR4m mice had lower (P < .05) body mass compared with WT mice but similar adiposity, whereas body mass and adiposity were lowered by GTE regardless of genotype. Liver steatosis, serum alanine aminotransferase, and hepatic lipid peroxidation were also lowered by GTE in WT mice, and were similarly lowered in TLR4m mice regardless of GTE. Phosphorylation of the NFκB p65 subunit and pro-inflammatory genes (TNFα, iNOS, MCP-1, MPO) were lowered by GTE in WT mice, and did not differ from the lowered levels in TLR4m mice regardless of GTE. TLR4m mice had lower TLR4 mRNA, which was also lowered by GTE in both genotypes. TRIF expression was unaffected by genotype and GTE, whereas MyD88 was lower in mice fed GTE regardless of genotype. Serum endotoxin was similarly lowered by GTE regardless of genotype. Tight junction protein mRNA levels were unaffected by genotype. However, GTE similarly increased claudin-1 mRNA in the duodenum and jejunum and mRNA levels of occludin and zonula occluden-1 in the jejunum and ileum. Thus, GTE protects against inflammation during NASH, likely by limiting gut-derived endotoxin translocation and TLR4/MyD88/NFκB activation.     

Autophagy is required for the activation of NFκB

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.     

TNFα regulates the localization of CD40 in lipid rafts of glioma cells

Resistance of glioblastoma multiforme (GBM) to TNFα induced apoptosis is attributed to NFκB activation. As TNF-receptor family member CD40 regulates NFκB activation, we investigated the role of CD40 in NFκB activation in GBM. We observed elevated CD40 levels in human glioma samples as compared to the surrounding normal tissue. Treatment with TNFα elevated CD40 levels in glioma cells and inhibition of CD40 signaling failed to abrogate TNFα induced NFκΒ activity. While TNFα increased the interaction between TRAF2/6, IκBα, IKKα/β in the CD40 signalosome, the level of CD40 in the signalosome remained unaffected upon TNFα treatment. Interestingly, TNFα decreased the spatial localization of CD40 and increased TRAF2/6 co-localization with lipid raft marker Caveolin. As localization of CD40 signalosome in lipid raft is crucial for NFκB activation, TNFα mediated decreased clustering of CD40 in lipid rafts could have possibly contributed to its non-involvement in NFκB activation.     

In vitro fermentability and physicochemical properties of fibre substrates and their effect on bacteriological and morphological characteristics of the gastrointestinal tract of newly weaned piglets

Fermentability of fibre has a great impact on the bacterial flora along the gastrointestinal tract of newly weaned piglets. Therefore, this parameter was determined by incubating in vitro different fibre substrates (chicory roots, sugar beet pulp, wheat bran and corn cobs) with contents of jejunum or caecum sampled from slaughtered pigs. Incubating with small intestinal contents, lactic acid was the only fermentation product. Fermentability was highest for chicory roots, followed by wheat bran and sugar beet pulp, while corn cobs were not fermented. Based on SCFA formed in the incubations with caecal contents, ranking of the fermentability of the fibre substrates was in the same order. The effect of adding different fibre substrates to diets of newly weaned piglets on bacteriological and morphological aspects of the gastrointestinal tract was also investigated. In Experiment 1 three groups of five piglets, weaned at four weeks of age, received a control feed (C), C supplemented with corn cobs (50 g/kg) or with chicory roots (20 g/kg). In Experiment 2, diet C was supplemented with sugar beet pulp (120 g/kg) or with wheat bran (75 g/kg). After three weeks animals were euthanized and digesta were sampled from stomach, proximal and distal jejunum, caecum and colon. Furthermore, mucosal scrapings were prepared and tissue samples were taken from jejunum, caecum and colon. Viscosity was determined for jejunal, caecal and colon contents. Corn cobs in the feed increased the number of total bacteria, lactobacilli and bifidobacteria in the stomach and proximal duodenum, while a decreased count of streptococci in distal jejunum contents was noted. Chicory roots increased the counts of Escherichia coli in the distal jejunum and on the mucosa, while sugar beet pulp decreased the number of lactobacilli on the mucosa only. Wheat bran seemed to increase the count of E. coli in jejunal digesta and on the mucosa, and also the number of lactobacilli in the stomach and jejunum. Bifidobacterial numbers were increased but only in the proximal part of the jejunum. Fibre substrates affected the concentration of lactate and SCFA in different parts of the intestinal tract. Feeding corn cobs increased villus length in the proximal jejunum by 13%. The number of intra-epithelial lymphocytes in the villous epithelium of proximal and distal jejunum was decreased by corn cobs and chicory roots supplementation while beet pulp and wheat bran had the opposite effect. In Experiment 1, apoptotic index of the mucosa of the distal jejunum was very low and decreased when corn cobs were fed. Mitotic index in the crypts was only affected by the wheat bran diet and a small decrease was noted. It was concluded that the fermentability of fibre was not an ideal criterion for predicting its effects on the flora. The effect of fibres on viscosity of digesta was negligible probably explaining the lack of clear and consistent influences on the intestinal mucosa.     

In vitro binding of estrogens by dietary fiber and the in vivo apparent digestibility tested in pigs.

Within the framework of experiments related to the association between dietary fiber and breast cancer an in vitro test system was used to study the binding of estrogens to various fibers (e.g. cholestyramin, lignin and cellulose) and fiber sources (e.g. wheat bran, cereals, seeds and legumes). Furthermore, the in vivo apparent digestibility of the different fiber sources was tested using a mobile nylon bag technique in intestine-cannulated pigs. Estradiol-17 beta (E2) bound more strongly to the various fibers than did estrone (E1), estriol or estrone-3-glucuronide. At increasing pH (greater than 7) binding of both E1 and E2 to wheat bran decreased significantly. Cholestyramine and lignin bound almost all estrogens present in the medium. Linseed (91%), oats (83%), barley chaff (88%) and wheat bran (82%) are other excellent binders of E2. Corn, rye and white wheat flour showed lower binding capacity with a relatively low affinity. Cereals with the highest percentage of lignin in the fiber (greater than 3%) were also the fiber sources with the lowest apparent digestibility. Estrogens bound with the highest affinity (relative to bovine serum albumin) to these fiber sources. Together with wheat bran and lignin, oats, linseed and soybean seem to be products with good perspectives for in vivo evaluation of the lowering effect of dietary fiber on estrogen exposure of estrogen-sensitive tissues.     

Exogenous regucalcin stimulates osteoclastogenesis and suppresses osteoblastogenesis through NF-κB activation

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss, and bone marrow from these animals exhibits significantly elevated osteoclast formation. Consistent with these effects exogenous regucalcin promotes osteoclastogenesis in mouse bone marrow cultures, but interestingly regucalcin suppresses the differentiation and mineralization of MC3T3 osteoblast precursors. However, the molecular mechanisms involved are presently unclear. As the nuclear factor-kappa B (NF-κB) signal transduction pathway is critical to osteoclastogenesis but inhibitory of osteoblastogenesis, we hypothesized that regucalcin may promote osteoclastogenesis and suppress osteoblastogenesis upregulating NF-κB signal transduction. In this study, we examined the effect of regucalcin on receptor activator of NF-κB (RANK) ligand (RANKL) -induced osteoclast formation using the RAW264.7 monocytic cell line and osteoblast formation using the pre-osteoblastic cell line MC3T3. As expected, culture with exogenous regucalcin was found to enhance RANKL-induced osteoclastogenesis. Consistent with this effect regucalcin increased basal and RANKL-induced NF-κB activation as assessed by NF-κB luciferase assay. The capacity of regucalcin to augment RANKL-induced NF-κB activity was inhibited by menaquinone-7, a potent NF-κB antagonist, while the Erk inhibitor PD98059 and staurosporine had no effect, demonstrating a specific effect on NF-κB signaling. By contrast, regucalcin inhibited mineralization of MC3T3 cells and enhanced tumor necrosis factor-α (TNFα)-induced NF-κB activation. As with NF-κB induction in osteoclasts, NF-κB activation was abolished by addition of the NF-κB antagonist menaquinone-7, but not by PD98059 and staurosporine. Transforming growth factor-β (TGFβ) and bone morphogenic protein-2 (BMP2) are potent early commitment and late osteoblast differentiation factors, respectively, and both mediate their actions through the Smad-signal transduction pathway, a system that is extremely sensitive to and inhibited by TNFα-induced NF-κB. We consequently examined the effect of regucalcin on TGFβ and BMP2-induced Smad activation in the presence and absence of TNFα. While regucalcin had no effect on basal Smad activation by TGFβ and BMP2, it enhanced the suppressive effect of TNFα on both TGFβ- and BMP2-induced Smad activations. Taken together, present data suggest that regucalcin may induce bone loss in vivo by promoting osteoclasts and simultaneously suppressing osteoblasts through amplification of basal and/or cytokine-induced NF-κB activation. Regucalcin may have a role as a modulator in NF-κB activation.     

Effect of high fibre diets formulated with different fibrous ingredients on performance,nutrient digestibility and faecal microbiota of weaned piglets

The aim of the experiment on 180 weaned piglets (8.9 kg body weight) was to investigate the influence of high fibre diets formulated with different fibrous ingredients on performance, nutrient digestibility, diarrhoea incidence and numbers of faecal microbiota. The dietary treatments included a Control diet and five high fibre diets formulated with different fibre sources including wheat bran, soybean hulls, naked oat hulls, palm kernel expeller and bamboo fibre. The high fibre diets averaged 14.6% neutral detergent fibre with different non-starch polysaccharides (NSP) components and were fed ad libitum for 28 d. Faecal samples were collected during the last 3 d of the experiment and the apparent total tract digestibility of nutrients and fibre components were determined. Pigs fed the Control and wheat bran diets had a higher (p ≤ 0.05) average daily gain (ADG) than pigs fed the palm kernel expeller and bamboo meal diets. The reduced ADG for pigs appeared to be related to reductions in the digestibility of gross energy and dry matter, respectively. The feed-to-gain ratio was significantly higher (p ≤ 0.05) for pigs fed the fibre diets. The digestibility of NSP components was different among the treatments. The diarrhoea incidence was not affected by treatments. The abundance of faecal bifidobacteria was significantly higher (p ≤ 0.05) for pigs fed the wheat bran diet than for pigs fed the bamboo meal diet. It was concluded that the diets formulated with different fibre sources when fed to weaned piglets have different effects on pig performance, nutrient digestibility and numbers of faecal microbiota. The wheat bran diet rich in arabinoxylans enabled a better performance than the other tested diets with fibre addition.