Aqueous extract of Chrysanthemum morifolium Ramat. inhibits RANKL-induced osteoclast differentiation by suppressing the c-fos/NFATc1 pathway.

OBJECTIVE: The flower of chrysanthemum, used worldwide as a medicinal and edible product, has shown various bioactivities, such as anti-inflammatory, antioxidant, anti-tumorigenic, and hepatoprotective activities, as well as cardiovascular protection. However, the effect of Chrysanthemum morifolium Ramat. on the regulation of osteoclast differentiation has not yet been reported. In this study, we aimed to investigate the inhibitory effect of Chrysanthemum morifolium Ramat. water extract (CME) on RANKL-induced osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN: Bone marrow-derived macrophages (BMMs) isolated from the C57BL/6 J mice. The viability of BMMs was detected with MTT assays. Inhibitory effects of CME on osteoclast differentiation and bone resorption was measured by TRAP staining and Pit assay. Osteoclast differentiation-associated gene expression were assessed by Real-time quantitative polymerase chain reaction. Intracellular signaling molecules was assessed by western blot. RESULTS: CME significantly inhibited osteoclast differentiation in BMMs without cytotoxicity, besides inhibiting MAPK/c-fos and PLCγ2/CREB activation. The inhibitory effects of CME on differentiation-related signaling molecules resulted in significant repression of NFATc1 expression, which is a key transcription factor in osteoclast differentiation, fusion, and activation. CONCLUSION: Our results confirmed the inhibition of RANKL-induced PLCγ2/CREB/c-fos/NFATc1 activation by CME during osteoclast differentiation. The findings collectively suggested CME as a traditional therapeutic agent for osteoporosis, RA, and periodontitis.

Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1.

Sirtuin 2 (Sirt2) is known to negatively regulate anoxia-reoxygenation injury in myoblasts. Because protein levels of Sirt2 are increased in ischemia-reperfusion (I/R)-injured liver tissues, we examined whether Sirt2 is protective or detrimental against hepatic I/R injury. We overexpressed Sirt2 in the liver of C57BL/6 mice using a Sirt2 adenovirus. Wild-type and Sirt2 knockout mice were subjected to a partial (70%) hepatic ischemia for 45 minutes, followed by various periods of reperfusion. In another set of experiments, wild-type mice were pretreated intraperitoneally with AGK2, a Sirt2 inhibitor. Isolated hepatocytes and Kupffer cells from wild-type and Sirt2 knockout mice were subjected to hypoxia-reoxygenation injury to determine the in vitro effects of Sirt2. Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Prior injection with Sirt2 adenovirus aggravated liver injury, as demonstrated by increases in serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration relative to control virus-injected mice. Pretreatment with AGK2 resulted in significant improvements in serum aminotransferase levels and histopathologic findings. Similarly, experiments with Sirt2 knockout mice also revealed reduced hepatocellular injury. The molecular mechanism of Sirt2's involvement in this aggravation of hepatic I/R injury includes the deacetylation and inhibition of mitogen-activated protein kinase phosphatase-1 and consequent activation of mitogen-activated protein kinases. CONCLUSION: Sirt2 is an aggravating factor during hepatic I/R injury. (Hepatology 2017;65:225-236).

Regulation of apoptosis and inflammatory responses by insulin-like growth factor binding protein 3 in fibroblast-like synoviocytes and experimental animal models of rheumatoid arthritis.

OBJECTIVE: Insulin-like growth factor binding protein 3 (IGFBP-3) is known to interfere with the NF-κB signaling pathway, and it effectively promotes apoptosis in tumor cells by a variety of mechanisms. NF-κB activation and apoptosis resistance of fibroblast-like synoviocytes (FLS) play pivotal roles in rheumatoid arthritis (RA). This study was undertaken to evaluate whether IGFBP-3 has antiarthritic effects. METHODS: To deliver IGFBP-3, we used an adenovirus containing IGFBP-3 complementary DNA (AdIGFBP-3) or IGFBP-3 mutant that is devoid of IGF binding affinity but retains IGFBP-3 receptor binding ability (AdmtIGFBP-3). The regulatory roles of IGFBP-3 in inflammation and bone destruction were investigated in mice with collagen-induced arthritis (CIA). RESULTS: IGFBP-3 levels were significantly higher in patients with RA than in those with osteoarthritis (OA) and were notably higher in patients with active RA. AdIGFBP-3 suppressed NF-κB activation, chemokine production, and matrix metalloproteinase secretion induced by tumor necrosis factor α (TNFα) in RA FLS. AdIGFBP-3 sensitized RA FLS to TNFα-induced apoptosis in vitro and also significantly increased apoptosis in an in vivo model of Matrigel implants engrafted into immunodeficient mice. AdIGFBP-3-injected mice with CIA had attenuated arthritis severity and reduced radiologic and pathologic abnormalities. Moreover, AdIGFBP-3 down-regulated local and systemic levels of NF-κB-targeted proinflammatory cytokines. Of note, RA FLS and mice with CIA treated with AdmtIGFBP-3 exhibited similar effects as those treated with AdIGFBP-3. CONCLUSION: Our results suggest that both the inflammatory response and bone destruction are reduced with blockage of NF-κB activation and induction of apoptosis in RA FLS by IGFBP-3. Therefore, IGFBP-3 may have therapeutic potential in RA.

Overexpression of sirtuin 6 suppresses inflammatory responses and bone destruction in mice with collagen-induced arthritis.

OBJECTIVE: Sirtuin 6 (SIRT-6) is an NAD(+) -dependent deacetylase and mono-ADP-ribosyltransferase. It is known to interfere with the NF-κB signaling pathway and thereby has an antiinflammatory function. Due to the central role of NF-κB in rheumatoid arthritis (RA) development, we undertook this study to test our hypothesis that SIRT-6 could have antiarthritic effects. METHODS: An adenovirus containing SIRT-6 complementary DNA (Ad-SIRT6) was used to deliver SIRT-6 to human RA fibroblast-like synoviocytes in vitro as well as to mice with collagen-induced arthritis (CIA) in vivo via bilateral intraarticular injections into the ankle joints. RESULTS: In vitro experiments demonstrated that SIRT-6 overexpression suppressed NF-κB target gene expression induced by tumor necrosis factor α. SIRT-6 overexpression inhibited osteoclast differentiation induced by macrophage colony-stimulating factor and RANKL in bone marrow-derived macrophages. Mice with CIA had an increased incidence of disease and developed arthritis in the hind paws. In contrast, mice injected with Ad-SIRT6 showed attenuated severity of arthritis based on clinical scores, hind paw thickness, and radiographic and pathologic findings. Moreover, the injection of Ad-SIRT6 down-regulated local and systemic levels of proinflammatory cytokines. After induction of CIA, mice injected with Ad-SIRT6 showed significantly decreased arthritis severity, from the onset of clinical signs to the end of the study. CONCLUSION: These results suggest that blocking the NF-κB pathway by SIRT-6 in rheumatoid joints reduces both the inflammatory response and tissue destruction. Therefore, the development of an immunoregulatory strategy based on SIRT-6 may have therapeutic potential for the treatment of RA.

Aggravation of post-ischemic liver injury by overexpression of A20, an NF-κB suppressor.

BACKGROUND & AIMS: A20 is an intracellular ubiquitin-editing enzyme that plays an important role in the negative feedback regulation of NF-κB activation in response to a diverse range of stimuli. Liver ischemia/reperfusion injury is associated with rapid activation of NF-κB signaling, but the role of NF-κB in hepatic ischemia/reperfusion injury remains controversial. The NF-κB signaling pathway mediates both protective and deleterious effects in the liver. Here, we examined whether A20 inhibited or aggravated hepatic ischemia/reperfusion injury. METHODS: We used IκBα super-repressor as a positive control and overexpressed A20 and IκBα super-repressor in the liver of C57BL/6 mice. Mice underwent 45min of partial hepatic ischemia and were then reperfused. RESULTS: Protein level of A20 was increased after reperfusion. Mice subjected to ischemia/reperfusion injury showed increased NF-κB activation, as evidenced by phosphorylation of IκBα and nuclear translocation of NF-κB. Prior transfection with Ad-A20 or Ad-IκBα super-repressor attenuated NF-κB activation and aggravated liver injury. Serum aminotransferases and proinflammatory cytokines, hepatocellular necrosis, and hepatic neutrophil infiltration were markedly increased compared to those of uninfected or control virus infected mice. In addition, A20 abolished the beneficial effect of ischemic preconditioning. CONCLUSIONS: Our results suggest that inhibition of NF-κB activation by A20 aggravated partial hepatic ischemia/reperfusion injury. Understanding how the NF-κB pathway plays a role in directing a clinical outcome may lead to better prospects of more rational approaches to reduce post-ischemic liver injury.

Effect of university physical education courses on intention for physical activity adherence in Korea.

Individuals' intentions of adopting physical activity as part of their lifestyle changed after university physical education courses in Korea. Male students (N = 264) taking physical education courses at a university in Korea were tested on the first and last day of a semester using a physical activity adherence questionnaire. The results showed that the intention to continue physical activity increased after taking the courses.

Analysis of salicylic acid based on the fluorescence enhancement of the As(III)-salicylic acid system.

A new, simple and sensitive spectrofluorimetric method for the determination of salicylic acid (lambda(ex)=315 nm, lambda(em) = 408 nm) using As(III) as a sensitizing reagent has been investigated by measuring the increase of fluorescence intensity of salicylic acid due to the complexation of As(III)-salicylic acid in presence of sodium dodecyl sulfate (SDS) 10(-3) M. Under optimum conditions, a significant relationship was obtained between the fluorescence intensity and salicylic acid concentration. A linear calibration curve was obtained in the range 13.8-13812 microg l(-1) with product-moment correlation coefficient (R) 0.99985 and detection limit 4.2 microg l(-1). The R.S.D. is 2.35% (n=5). The method was applied successfully to the determination of salicylic acid in human serum.