A quantitative description of the compatibility of technetium-selective chromatographic technetium-99m separation with low specific activity molybdenum-99.

Technetium-99m generators employing a technetium-selective stationary phase are a chromatographic instrument developed for use with 99Mo having low specific activity (LSA); particularly, 99Mo produced by electron accelerators. This paper presents a mathematical description of technetium-selective chromatographic (TSC) 99mTc separation and analyzes its compatibility with LSA 99Mo. We developed a theoretical formula for TSC 99mTc separation by discretizing its pertechnetate selectivity, and validated it using an electron linear accelerator and activated carbon-based TSC (AC-TSC) 99mTc generators. We confirmed that the activity concentration of 99mTc obtained from a TSC 99mTc generator can be calculated directly from its input 99Mo activity regardless of the 99Mo specific activity. The formula corroborates that TSC 99mTc separation is compatible with LSA 99Mo, and has a practical application in estimating the number of TSC 99mTc generators required for 99mTc demand of interest.

A preliminary biodistribution study of [99mTc]sodium pertechnetate prepared from an electron linear accelerator and activated carbon-based 99mTc generator.

INTRODUCTION: Production of 99Mo/99mTc using an electron linear accelerator (linac) and activated carbon (AC)-based 99mTc generator (linac-AC) is an alternative approach to the conventional fission production of 99Mo/99mTc. As a preliminary investigation of the clinical applicability of a linac-AC-derived 99mTc radiopharmaceutical, the biodistribution of linac-AC-derived [99mTc]sodium pertechnetate ([99mTc]NaTcO4) was measured and compared against fission-derived [99mTc]NaTcO4 at one time point. METHODS: 99Mo was produced by irradiating nonenriched MoO3 targets with bremsstrahlung photons generated from 55.5-MeV linac electron beams. 99mTc was then separated and purified from the 99Mo using an AC-based 99mTc generator. Subsequently, biodistribution of the linac-AC-derived [99mTc]NaTcO4 in healthy female Slc:ICR mice (n = 6) was measured by dissection and compared with that of fission-derived [99mTc]NaTcO4 (n = 4) at 30 min after injection. RESULTS: The two types of [99mTc]NaTcO4 exhibited similar biodistribution in all the organs and tissues examined: the uptakes of [99mTc]NaTcO4 prepared from the linac-AC method and those prepared from the fission method were 138.9 ± 69.9%ID/g and 160.6 ± 49.2%ID/g in the thyroids, respectively, 33.4 ± 5.5%ID/g and 29.4 ± 9.1%ID/g in the salivary glands, respectively, and less than 10%ID/g in blood and all the other organs. No adverse effects were observed in the mice administered with either [99mTc]NaTcO4. CONCLUSION: The clinical applicability of linac-AC-derived [99mTc]NaTcO4 was suggested by its similar biodistribution with fission-derived [99mTc]NaTcO4 at one time point. Further biodistribution studies at multiple time points are encouraged to demonstrate the bioequivalence between linac-AC- and fission-derived [99mTc]NaTcO4.

Paeonia lactiflora extract improves the muscle function of mdx mice, an animal model of Duchenne muscular dystrophy, via downregulating the high mobility group box 1 protein.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall. is an ethnopharmacological medicine with a long history of human use for treating various inflammatory diseases in many Asian countries. AIM OF THE STUDY: Duchenne muscular dystrophy (DMD) is an X-linked degenerative muscle disease affecting 1 in 3500 males and is characterized by severe muscle inflammation and a progressive decline in muscle function. This study aimed to elucidate the effects of an ethanol extract of the root of Paeonia lactiflora Pall. (PL) on the muscle function in the muscular dystrophy X-linked (mdx) mouse, the most commonly used animal model of DMD. MATERIALS AND METHODS: Male mdx mice and wild-type controls aged 5 weeks were orally treated with PL for 4 weeks. The corticosteroid prednisolone was used as a comparator drug. Muscle strength and motor coordination were assessed via the grip-strength and rotarod tests, respectively. Muscle damage was evaluated via histological examination and assessment of plasma creatine-kinase activity. Proteomic analyses were conducted to identify the muscle proteins whose levels were significantly affected by PL (ProteomeXchange identifier: PXD028886). Muscle and plasma levels of these proteins, and their corresponding mRNAs were measured using western blotting and ELISA, and quantitative reverse transcription-polymerase chain reaction, respectively. RESULTS: The muscle strength and motor coordination of mdx mice were significantly increased by the oral treatment of PL. PL significantly reduced the histological muscle damage and plasma creatine-kinase activity. Proteomic analyses of the muscle showed that PL significantly downregulated the high mobility group box 1 (HMGB1) protein and Toll-like receptor (TLR) 4, thus suppressing the HMGB1-TLR4-NF-κB signaling, in the muscle of mdx mice. Consequently, the muscle levels of proinflammatory cytokines/chemokines, which play crucial roles in inflammation, were downregulated. CONCLUSION: PL improves the muscle function and reduces the muscle damage in mdx mice via suppressing the HMGB1-TLR4-NF-κB signaling and downregulating proinflammatory cytokines/chemokines.

Wnt-Signaling Inhibitor Wnt-C59 Suppresses the Cytokine Upregulation in Multiple Organs of Lipopolysaccharide-Induced Endotoxemic Mice via Reducing the Interaction between ß-Catenin and NF-κB.

Sepsis is characterized by multiple-organ dysfunction caused by the dysregulated host response to infection. Until now, however, the role of the Wnt signaling has not been fully characterized in multiple organs during sepsis. This study assessed the suppressive effect of a Wnt signaling inhibitor, Wnt-C59, in the kidney, lung, and liver of lipopolysaccharide-induced endotoxemic mice, serving as an animal model of sepsis. We found that Wnt-C59 elevated the survival rate of these mice and decreased their plasma levels of proinflammatory cytokines and organ-damage biomarkers, such as BUN, ALT, and AST. The Wnt/ß-catenin and NF-κB pathways were stimulated and proinflammatory cytokines were upregulated in the kidney, lung, and liver of endotoxemic mice. Wnt-C59, as a Wnt signaling inhibitor, inhibited the Wnt/ß-catenin pathway, and its interaction with the NF-κB pathway, which resulted in the inhibition of NF-κB activity and proinflammatory cytokine expression. In multiple organs of endotoxemic mice, Wnt-C59 significantly reduced the ß-catenin level and interaction with NF-κB. Our findings suggest that the anti-endotoxemic effect of Wnt-C59 is mediated via reducing the interaction between ß-catenin and NF-κB, consequently suppressing the associated cytokine upregulation in multiple organs. Thus, Wnt-C59 may be useful for the suppression of the multiple-organ dysfunction during sepsis.

Wnt-C59 inhibits proinflammatory cytokine expression by reducing the interaction between ß-catenin and NF-κB in LPS-stimulated epithelial and macrophage cells.

Dysregulation of the Wnt pathway causes various diseases including cancer, Parkinson's disease, Alzheimer's disease, schizophrenia, osteoporosis, obesity and chronic kidney diseases. The modulation of dysregulated Wnt pathway is absolutely necessary. In the present study, we evaluated the anti-inflammatory effect and the mechanism of action of Wnt-C59, a Wnt signaling inhibitor, in lipopolysaccharide (LPS)-stimulated epithelial cells and macrophage cells. Wnt-C59 showed a dose-dependent anti-inflammatory effect by suppressing the expression of proinflammatory cytokines including IL6, CCL2, IL1A, IL1B, and TNF in LPS-stimulated cells. The dysregulation of the Wnt/ß-catenin pathway in LPS stimulated cells was suppressed by Wnt- C59 treatment. The level of ß-catenin, the executor protein of Wnt/ß-catenin pathway, was elevated by LPS and suppressed by Wnt-C59. Overexpression of ß-catenin rescued the suppressive effect of Wnt-C59 on proinflammatory cytokine expression and nuclear factor-kappa B (NF-κB) activity. We found that the interaction between ß-catenin and NF-κB, measured by co-immunoprecipitation assay, was elevated by LPS and suppressed by Wnt-C59 treatment. Both NF-κB activity for its target DNA binding and the reporter activity of NF-κB-responsive promoter showed identical patterns with the interaction between ß-catenin and NF-κB. Altogether, our findings suggest that the anti-inflammatory effect of Wnt-C59 is mediated by the reduction of the cellular level of ß-catenin and the interaction between ß-catenin and NF-κB, which results in the suppressions of the NF-κB activity and proinflammatory cytokine expression.

LGK974 suppresses lipopolysaccharide-induced endotoxemia in mice by modulating the crosstalk between the Wnt/ß-catenin and NF-κB pathways.

Endotoxemia, a type of sepsis caused by gram-negative bacterial endotoxin [i.e., lipopolysaccharide (LPS)], is associated with manifestations such as cytokine storm; failure of multiple organs, including the liver; and a high mortality rate. We investigated the effect and mechanism of action of LGK974, a Wnt signaling inhibitor, in mice with LPS-induced endotoxemia, an animal model of sepsis. LGK974 significantly and dose-dependently increased the survival rate and reduced plasma cytokine levels in mice with LPS-induced endotoxemia. Transcriptome analysis of liver tissues revealed significant changes in the expression of genes associated with the Wnt pathway as well as cytokine and NF-κB signaling during endotoxemia. LGK974 treatment suppressed the activation of NF-κB signaling and cytokine expression as well as the Wnt/ß-catenin pathway in the livers of endotoxemic mice. Coimmunoprecipitation of phospho-IκB and ß-transducin repeat-containing protein (ß-TrCP) was increased in the livers of endotoxemic mice but was reduced by LGK974 treatment. Moreover, LGK974 treatment decreased the coimmunoprecipitation and colocalization of ß-catenin and NF-κB, which were elevated in the livers of endotoxemic mice. Our results reveal crosstalk between the Wnt/ß-catenin and NF-κB pathways via interactions between ß-TrCP and phospho-IκB and between ß-catenin and NF-κB during endotoxemia. The results of this study strongly suggest that the crosstalk between the Wnt/ß-catenin and NF-κB pathways contributes to the mutual activation of these two pathways during endotoxemia, which results in amplified cytokine production, liver damage and death, and that LGK974 suppresses this vicious amplification cycle by reducing the crosstalk between these two pathways.

Paeonia lactiflora extract suppresses cisplatin-induced muscle wasting via downregulation of muscle-specific ubiquitin E3 ligases, NF-κB signaling, and cytokine levels.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Paeonia lactiflora Pall. (Radix Paeoniae) has been traditionally used to treat various inflammatory diseases in many Asian countries. AIM OF THE STUDY: Cisplatin is a broad-spectrum anticancer drug used in diverse types of cancer. However, muscle wasting is a common side effect of cisplatin chemotherapy. This study aimed to elucidate the effects of an ethanol extract of the root of Paeonia lactiflora Pall. (Radix Paeoniae, RP) on cisplatin-induced muscle wasting along with its molecular mechanism. MATERIAL AND METHODS: C57BL/6 mice were intraperitoneally injected with cisplatin and orally treated with RP. Megestrol acetate was used as a comparator drug. Skeletal muscle mass was measured as the weight of gastrocnemius and quadriceps muscles, and skeletal muscle function was measured by treadmill running time and grip strength. Skeletal muscle tissues were analyzed by RNAseq, western blotting, ELISA, and immunofluorescence microscopy. RESULTS: In mice treated with cisplatin, skeletal muscle mass and skeletal muscle function were significantly reduced. However, oral administration of RP significantly restored skeletal muscle mass and function in the cisplatin-treated mice. In the skeletal muscle tissues of the cisplatin-treated mice, RP downregulated NF-κB signaling and cytokine levels. RP also downregulated muscle-specific ubiquitin E3 ligases, resulting in the restoration of myosin heavy chain (MyHC) and myoblast determination protein (MyoD), which play crucial roles in muscle contraction and muscle differentiation, respectively. CONCLUSION: RP restored skeletal muscle function and mass in cisplatin-treated mice by restoring the muscle levels of MyHC and MyoD proteins via downregulation of muscle-specific ubiquitin E3 ligases as well as muscle NF-κB signaling and cytokine levels.

Paeonia lactiflora root extract suppresses cancer cachexia by down-regulating muscular NF-κB signalling and muscle-specific E3 ubiquitin ligases in cancer-bearing mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Paeonia lactiflora Pall. (Radix Paeoniae) has been traditionally used to treat various inflammatory diseases in many Asian countries. AIM OF THE STUDY: Cancer cachexia is a catabolic syndrome driven by inflammation and characterised by a loss of skeletal muscle. This study aimed to assess the effects of an ethanolic extract of Radix Paeoniae (RP) on cancer cachexia and elucidate its mechanism of action. MATERIAL AND METHODS: The anti-cachexic effect and mechanism of RP were examined in mouse models of cancer cachexia established in C57BL/6 mice by subcutaneously injecting Lewis lung carcinoma or MC38 colon carcinoma cells. Skeletal muscle tissues were analysed by RNAseq, real-time quantitative reverse transcription PCR, western blotting, and immunofluorescence microscopy. Megestrol acetate, which is recommended for the treatment of cachexia in cancer patients, was used as the comparator treatment in this study. RESULTS: In lung and colon cancer-bearing mice, RP significantly restored food intake and muscle mass, along with muscle function measured by grip strength and treadmill running time. In the skeletal muscle tissue of the cancer-bearing mice, RP suppressed NF-κB signalling and reduced inflammatory cytokines, including TNF-α, IL-6, and IL-1ß; it also down-regulated the muscle-specific E3 ubiquitin ligases MuRF1 and MAFbx. CONCLUSION: RP restored skeletal muscle function and mass in cancer-bearing mice by down-regulating the muscular NF-κB signalling pathway and muscle-specific E3 ubiquitin ligases. Our study indicates that RP is a potential candidate for development as a therapeutic agent against cancer cachexia.

XAV939, a Wnt/ß-catenin pathway modulator, has inhibitory effects on LPS-induced inflammatory response.

Aim: In this study, we report the anti-inflammatory activity of XAV939, a modulator of the Wnt/ß-catenin pathway. Methods: WNT/ß-catenin pathway and NF-κB signaling pathway were examined in LPS-stimulated human bronchial epithelial cells and effects of XAV939 on these pathways were analyzed. The effect of XAV939 was confirmed in human umbilical vein endothelial cells. Results: LPS-induced expressions of pro-inflammatory genes IL-6, IL-8, TNF-α, IL-1ß, MCP-1, MMP-9, iNOS and COX-2 were significantly and dose-dependently suppressed by XAV939. LPS-induced NF-κB signaling, such as IκB phosphorylation and degradation as well as nuclear translocation of NF-κB, was also suppressed by XAV939. Target DNA binding of NF-κB was significantly and dose-dependently suppressed by XAV939 during LPS-induced inflammatory response. The suppressive effects of XAV939 on NF-κB signaling, target DNA binding of NF-κB and pro-inflammatory gene expression were all rescued by over expression of ß-catenin, which shows that the anti-inflammatory effect of XAV939 is mediated by the modulation of ß-catenin, a central component of the WNT/ß-catenin pathway. Conclusion: The findings of this study showed that XAV939 exerts anti-inflammatory effects through the modulation of the Wnt/ß-catenin pathway.

Gangjihwan, a polyherbal composition, inhibits fat accumulation through the modulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα.

ETHNOPHARMACOLOGICAL RELEVANCE: Gangjihwan (DF) which is composed of Ephedra intermedia, Lithospermum erythrorhizon, and Rheum palmatum has been used for the treatment of obesity in traditional medical clinics in Korea. AIM OF THE STUDY: This study was conducted to standardize DF and elucidate its mechanism of action for inhibiting fat accumulation in adipocytes and adipose tissues. MATERIALS AND METHODS: The herbal ingredients of DF were extracted in water, 30% ethanol or 70% ethanol and freeze-dried followed by HPLC analyses. 3T3-L1 adipocytes and high-fat diet-induced obese mice were treated with each of the three DF preparations. Messenger RNA and protein expression levels were measured by real-time qPCR and Western blotting. RNA-Seq analyses were conducted to examine the effects of DF treatment on whole transcriptome of adipocyte. RESULTS: (-)-Ephedrine and (+)-pseudoephedrine from E. intermedia, aloe-emodin and chrysophanol from R. palmatum and shikonin from L. erythrorhizon were identified as phytochemical components of DF. DF caused dose-dependent inhibition of fat accumulation in 3T3-L1 adipocytes. It also significantly reduced adipose tissue mass and adipocyte size in high-fat diet-induced obese mice. DF was found to down-regulate the expressions of the lipogenic transcription factors such as sterol regulatory element binding protein 1C (SREBP1C), peroxisome proliferator activated receptor gamma (PPARγ), and CCAAT/enhancer binding protein alpha (C/EBPα). Among the three preparations of DF, the 70% ethanol extract was the most effective. RNA-Seq analyses showed that DF treatment decreased the expression levels of up-regulators and increased those of down-regulators of lipogenic transcription factors. CONCLUSIONS: DF preparations, among which 70% ethanol extract was the most effective, reduced fat accumulation in 3T3-L1 adipocytes and high-fat diet-induced obese mice through the down-regulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα.

A calpain inhibitor protects against fractalkine production in lipopolysaccharide-treated endothelial cells.

BACKGROUND: Fractalkine (CX3CL1) is a chemokine with a unique CX3C motif and is produced by endothelial cells stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interferon-γ. There have been several reports that the caspase/calpain system is activated in endotoxemia, which leads to cellular apoptosis and acute inflammatory processes. We aimed to determine the role of the caspase/calpain system in cell viability and regulation of fractalkine production in LPS-treated endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with 0.01-100 µg/mL of LPS to determine cell viability. The changes of CX3CL1 expression were compared in control, LPS (1 µg/mL)-, IL-1α (1 µg/mL)-, and IL-1ß (1 µg/mL)-treated HUVECs. Cell viability and CX3CL1 production were compared with 50 µM of inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. RESULTS: Cell viability was significantly decreased from 1 to 100 µg/mL of LPS. Cell viability was significantly restored with inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. The expression of CX3CL1 was highest in IL-1ß-treated HUVECs. CX3CL1 production was highly inhibited with a calpain inhibitor and significantly decreased with the individual inhibitors of caspase-1, caspase-3, and caspase-9. CONCLUSION: The caspase/calpain system is an important modulator of cell viability and CX3CL1 production in LPS-treated endothelial cells.

Berberine activates AMPK to suppress proteolytic processing, nuclear translocation and target DNA binding of SREBP-1c in 3T3-L1 adipocytes.

AMP-activated protein kinase (AMPK) and sterol regulatory element binding protein (SREBP)­1c are major therapeutic targets in the treatment of metabolic diseases. In the present study, the fat­reducing mechanisms of berberine (BBR), a natural isoquinoline, was investigated by examining the AMPK­mediated modulation of SREBP­1c in 3T3­L1 adipocytes. BBR activated AMPK in a dose­ and time­dependent manner, and increased the phosphorylation of the 125­kDa precursor form of SREBP­1c, which suppressed its proteolytic processing into the mature 68­kDa form and its subsequent nuclear translocation. The binding of nuclear SREBP­1c to its E­box motif­containing target DNA sequence was decreased following treatment with BBR, which led to a decrease in the expression of lipogenic genes and subsequently reduced intracellular fat accumulation. Transfection with AMPKα1 siRNA, and not control siRNA, inhibited BBR­induced phosphorylation of the 125­kDa SREBP­1c, which confirmed that AMPK was responsible for phosphorylating SREBP­1c. AMPKα1 siRNA transfection rescued the proteolytic processing, nuclear translocation and target DNA binding of SREBP­1c that had been suppressed by BBR. In addition, BBR­induced suppression of lipogenic gene expression and intracellular fat accumulation were rescued by AMPKα1 siRNA transfection. In conclusion, the results of the present study demonstrate that BBR activates AMPK to induce phosphorylation of SREBP­1c, thereby suppressing proteolytic processing, nuclear translocation and target DNA binding of SREBP­1c, which leads to a reduction in lipogenic gene expression and intracellular fat accumulation. The results of the present study indicate that BBR may be a potential candidate for the development of drugs to treat obesity.

WNT/ß-catenin pathway modulates the TNF-α-induced inflammatory response in bronchial epithelial cells.

In this study, TNF-α was found to activate the WNT/ß-catenin pathway in BEAS-2B human bronchial epithelial cells. Levels of phospho-LRP6, Dvl-2, and phospho-GSK-3ß were elevated, while that of Axin was reduced by TNF-α treatment. Nuclear translocation of ß-catenin and the reporter activity of a ß-catenin-responsive promoter were increased by TNF-α treatment. Under the same experimental conditions, TNF-α activated the NF-κB signaling, which includes the phosphorylation and degradation of IκB and nuclear translocation and target DNA binding of NF-κB, and it was found that an inhibitor of NF-κB activation, JSH-23, inhibited TNF-α-induced Wnt signaling as well as NF-κB signaling. It was also found that recombinant Wnt proteins induced NF-κB nuclear translocations and its target DNA binding, suggesting that Wnt signaling and NF-κB signaling were inter-connected. TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression were significantly suppressed by the transfection of ß-catenin siRNA compared to that of control siRNA. Transfection of a ß-catenin expression plasmid augmented the TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression. These results clearly demonstrated that the WNT/ß-catenin pathway modulates the inflammatory response induced by TNF-α, suggesting that this pathway may be a useful target for the effective treatment of bronchial inflammation.

LPS-induced inflammatory response is suppressed by Wnt inhibitors, Dickkopf-1 and LGK974.

In this study, LPS-induced inflammatory responses in BEAS-2B human bronchial epithelial cells and human umbilical vein endothelial cell (HUVEC)s were found to be prevented by Dickkopf-1 (DKK-1), a secreted Wnt antagonist, and LGK974, a small molecular inhibitor of the Wnt secretion. LPS-induced IκB degradation and NF-κB nuclear translocation as well as the expressions of pro-inflammatory genes including IL-6, IL-8, TNF- α, IL-1ß, MCP-1, MMP-9, COX-2 and iNOS, were all suppressed by DKK-1 and LGK974 in a dose-dependent manner. The suppressive effects of LGK974 on NF-κB, IκB, and pro-inflammatory gene expression were rescued by ectopic expression of ß-catenin, suggesting that the anti-inflammatory activity of LGK974 is mediated by modulation of the Wnt/ß-catenin pathway and not by unrelated side effects. When Wnt recombinant proteins were treated to cells, Wnt3a and Wnt5a significantly induced pro-inflammatory gene expressions, while Wnt7a and Wnt10b showed little effects. It was also found that Wnt3a and Wnt5a expressions were significantly induced by LPS treatment. Consistently, knockdown of Wnt3a and Wnt5a blocked LPS-induced inflammatory responses, while treatment of recombinant Wnt3a and Wnt5a proteins rescued the inhibition of inflammatory responses by LGK974. Findings of this study showed that DKK-1 and LGK974 suppress LPS-induced inflammatory response by modulating Wnt/ß-catenin pathway.

AMPK and SREBP-1c mediate the anti-adipogenic effect of ß-hydroxyisovalerylshikonin.

ß-hydroxyisovalerylshikonin (ß-HIVS), which is a natural naphthoquinone compound, is one of the main chemicals isolated from a therapeutic plant, Lithospermum erythrorhizon. In the present study, we demonstrated that ß-HIVS inhibited the adipogenesis of 3T3-L1 cells through AMP-activated protein kinase (AMPK)-mediated modulation of sterol regulatory element binding protein (SREBP)­1c. The anti-adipogenic effect of ß-HIVS was accompanied by the increased phosphorylation of AMPK and precursor SREBP­1c. In ß-HIVS-treated 3T3-L1 cells, AMPK was activated and phosphorylated precursor SREBP­1c, preventing the cleavage of precursor SREBP­1c to mature SREBP­1c. Expression of the fat-forming enzymes, acetyl-CoA carboxylase (ACC)1, fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD)1, which are transcribed by mature SREBP­1c, were downregulated, resulting in reduced intracellular fat accumulation. The anti-adipogenic effect of ß-HIVS was significantly attenuated by AMPK knockdown. Knockdown of AMPK using siRNA decreased the phosphorylation of precursor SREBP­1c and increased the levels of mature SREBP. The levels of the fat-forming enzymes, ACC1, FAS and SCD1, as well as intracellular fat accumulation were also significantly increased by AMPK knockdown. These results suggest that ß-HIVS activated AMPK, which was followed by the downregulation of mature SREBP­1c and fat-forming enzymes, leading to the inhibition of adipogenesis.

Lipoteichoic acid upregulates NF-κB and proinflammatory cytokines by modulating ß-catenin in bronchial epithelial cells.

Lipoteichoic acid (LTA) is a major cell wall component and virulence factor of gram-positive bacteria. The present study investigated the LTA­induced inflammatory response of BEAS­2B human bronchial epithelial cells, and detected the expression levels of proinflammatory cytokines interleukin (IL)­6, IL­8, IL­1ß, tumour necrosis factor­α and monocyte chemotactic protein­1, the upregulation of NF­κB, and the phosphorylation and degradation of I­κB. During the LTA­induced inflammatory response of the BEAS­2B human bronchial epithelial cells, the activity levels of the ß­catenin­dependent promoter, and the protein expression levels of ß­catenin were significantly upregulated, whereas ß­catenin phosphorylation and the expression levels of AXIN were significantly downregulated. Following knockdown of ß­catenin by small interfering (si)RNA transfection, both the LTA-induced protein expression levels of NF­κB and the LTA-induced activity levels of the NF­κB­dependent promoter were significantly reduced. Similarly, a marked reduction in I­κB phosphorylation and degradation was observed following ß­catenin knockdown. The expression levels of the LTA­induced proinflammatory cytokines were also significantly reduced following ß­catenin siRNA. These results suggest that ß­catenin has a significant role in the regulation of NF­κB activity and proinflammatory cytokine expression during the LTA-induced inflammatory response of bronchial epithelial cells.

Β-catenin regulates NF-κB activity and inflammatory cytokine expression in bronchial epithelial cells treated with lipopolysaccharide.

In the present study, we demonstrate that lipopolysaccharide (LPS) induces the expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, IL-1ß, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in BEAS-2B human bronchial epithelial cells in a dose- and time-dependent manner. This increase was accompanied by an increased activity of nuclear factor (NF)­κB. When the expression of ß-catenin was analyzed following treatment with LPS, the mRNA level was unaltered; however, the ß-catenin protein levels increased with a decrease in phosphorylation at the serine 33/37 residues. Nuclear ß-catenin protein levels also increased along with the reporter activity of a ß-catenin-responsive TOPFlash vector. To elucidate the regulatory role of ß-catenin in the LPS-induced inflammatory response of bronchial epithelial cells, ß-catenin production was knocked down using siRNA. Our results revealed that ß-catenin protein levels and TOPFlash vector reporter activity were reduced to basal levels by siRNA transfection. In this experimental condition, NF-κB activity, measured by enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay (EMSA) and an NF-κB responsive reporter assay, was reduced to basal levels. Similarly, LPS-induced inflammatory cytokine expression was reduced almost to basal levels following transfection with ß-catenin siRNA. These results demonstrate that ß-catenin positively regulates NF-κB activity, as well as the expression of inflammatory cytokines in the inflammatory response of LPS-treated bronchial epithelial cells.

ß-catenin mediates the inflammatory cytokine expression induced by the Der p 1 house dust mite allergen.

The modulations of ß-catenin were analyzed during the inflammatory response induced by the Der p 1 house dust mite allergen. Der p 1 induced the dose-dependent expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in THP-1 human monocytic cells. The mRNA expression levels of ß-catenin were not altered, however protein levels increased following Der p 1 treatment, demonstrating that ß-catenin was modulated by post-transcriptional processes. It was also revealed that nuclear ß-catenin levels were significantly increased while cytoplasmic ß-catenin levels were reduced, which demonstrated the nuclear translocation of ß-catenin by the Der p 1 allergen. Glycogen synthase kinase 3ß (GSK3ß), a regulator of ß-catenin stability, was demonstrated to be phosphorylated following Der p 1 treatment. When ß-catenin was knocked down by the transfection of its small interfering RNA (siRNA), inflammatory cytokine expression as well as nuclear factor-κB (NF-κB) activity, which were induced by Der p 1 treatment, were all significantly reduced. The results demonstrated that Der p 1-induced inflammatory responses were mediated by ß-catenin.

CD53, a suppressor of inflammatory cytokine production, is associated with population asthma risk via the functional promoter polymorphism -1560 C>T.

BACKGROUND: In this study, the association of asthma with CD53, a member of the tetraspanin family, was assessed for the first time in a mechanism-based study. METHODS: Genetic polymorphisms of CD53 were analyzed in 591 subjects and confirmed in a replication study of 1001 subjects. CD53 mRNA and protein levels were measured in peripheral blood leukocytes, and the effects of the promoter polymorphisms on nuclear factor binding were examined by electrophoretic mobility shift assay. Cellular functional studies were conducted by siRNA transfections. RESULTS: Among tagging SNPs of CD53, the -1560 C>T in the promoter region was significantly associated with asthma risk. Compared with the CC genotype, the CT and TT genotypes were associated with a higher asthma risk, with odd ratios of 1.74 (P=0.009) and 2.03 (P=0.004), respectively. These findings were confirmed in the replication study with odd ratios of 1.355 (P=0.047) and 1.495 (P=0.039), respectively. The -1560 C>T promoter SNP had functional effects on nuclear protein binding as well as mRNA and protein expression levels in peripheral blood leukocytes. When CD53 was knocked down by siRNA in THP-1 human monocytic cells stimulated with house dust mite, the production of inflammatory cytokines as well as NFκB activity was significantly over-activated, suggesting that CD53 suppresses over-activation of inflammatory responses. CONCLUSIONS: The -1560 C>T SNP is a functional promoter polymorphism that is significantly associated with population asthma risk, and is thought to act by directly modulating nuclear protein binding, thereby altering the expression of CD53, a suppressor of inflammatory cytokine production.