Synthesis of Pyrazolo[1,5-a]pyridinyl, Pyrazolo[1,5-a]quinolinyl, and Pyrazolo[5,1-a]isoquinolinyl Sulfonyl Fluorides via a [3 + 2] Annulation.

A [3 + 2] cycloaddition reaction of N-aminopyridines, N-aminoquinolines, and N-aminoisoquinolines with 1-bromoethene-1-sulfonyl fluoride (BESF) was performed to obtain optimum yields of various useful pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]quinolinyl, and pyrazolo[5,1-a]isoquinolinyl sulfonyl fluorides (43-90% yield). The transformation process showed broad substrate specificity, mild reaction conditions, and operational simplicity. Therefore, the reaction has great applicable value in the field of medicinal chemistry and other disciplines.

Age-related differences in interferon regulatory factor-4 and -5 signaling in ischemic brains of mice.

Stroke is a disease that mainly affects the elderly. Since the age-related differences in stroke have not been well studied, modeling stroke in aged animals is clinically more relevant. The inflammatory responses to stroke are a fundamental pathological procedure, in which microglial activation plays an important role. Interferon regulatory factor-5 (IRF5) and IRF4 regulate M1 and M2 activation of macrophages, respectively, in peripheral inflammation; but it is unknown whether IRF5/IRF4 are also involved in cerebral inflammatory responses to stroke and whether age-related differences of the IRF5/IRF4 signaling exist in ischemic brain. Here, we investigated the influences of aging on IRF5/IRF4 signaling and post-stroke inflammation in mice. Both young (9-12 weeks) and aged (18 months) male mice were subjected to middle cerebral artery occlusion (MCAO). Morphological and biochemical changes in the ischemic brains and behavior deficits were assessed on 1, 3, and 7 d post-stroke. After MCAO, the aged mice showed smaller infarct sizes but higher neurological deficits and corner test scores than young mice. Young mice had higher levels of IRF4 and CD206 microglia in the ischemic brains, whereas the aged mice expressed more IRF5 and MHCII microglia. After MCAO, serum pro-inflammatory cytokines (TNF-α, iNOS, IL-6) were more prominently up-regulated in aged mice, whereas serum anti-inflammatory cytokines (TGF-ß, IL-4, IL-10) were more prominently up-regulated in young mice. Our results demonstrate that aging has a significant influence on stroke outcomes in mice, which is probably mediated by age-specific inflammatory responses.

Regulation of microglial activation in stroke.

When ischemic stroke occurs, oxygen and energy depletion triggers a cascade of events, including inflammatory responses, glutamate excitotoxicity, oxidative stress, and apoptosis that result in a profound brain injury. The inflammatory response contributes to secondary neuronal damage, which exerts a substantial impact on both acute ischemic injury and the chronic recovery of the brain function. Microglia are the resident immune cells in the brain that constantly monitor brain microenvironment under normal conditions. Once ischemia occurs, microglia are activated to produce both detrimental and neuroprotective mediators, and the balance of the two counteracting mediators determines the fate of injured neurons. The activation of microglia is defined as either classic (M1) or alternative (M2): M1 microglia secrete pro-inflammatory cytokines (TNFα, IL-23, IL-1ß, IL-12, etc) and exacerbate neuronal injury, whereas the M2 phenotype promotes anti-inflammatory responses that are reparative. It has important translational value to regulate M1/M2 microglial activation to minimize the detrimental effects and/or maximize the protective role. Here, we discuss various regulators of microglia/macrophage activation and the interaction between microglia and neurons in the context of ischemic stroke.

Effect of total flavonoids from the seeds of Astragali complanati on natural killer cell function.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Astragalus complanatus R.Br. (AC), a traditional Chinese medicine, have been extensively used for clinical treatment of liver and kidney complaints and tumors for more than a thousand years. It was believed that AC is warm and sweet in the most authoritative medical book of Ancient China "Compendium of Materia Medica". Our previous studies showed that the flavonoid component from the seeds of Astragalus complanatus (FAC) is mainly an active constituent and has the hepatoprotective effect, anti-liver fibrosis, and anti-tumor and immune enhancement. AIM: The aim of this study was to investigate in vitro the regulation effect of FAC on NK cells function and possible mechanism of action. METHODS: The effect of FAC on the proliferation ability of NK-92 cells and the cytolysis of NK-92 cells to K562 and SMMC-7721 were measured by MTT assay and lactase dehydrogenase (LDH)-releasing assay, respectively. The phenotypical characterization (CD3, CD16 and CD56) and activation markers (CD25, CD69 and CD95) of NK-92 cell were detected by flow cytometry analysis. IFN-γ production of NK-92 cells stimulating by K562 cells was quantitated using ELISA. To explore the mechanism of action, mRNA and protein expressions of activating receptors (NKp30, NKp44, NKp46 and NKG2D) in NK-92 cells were detected by quantitative real-time PCR and flow cytometry analysis. RESULTS: Our results demonstrated that FAC significantly promoted the proliferation and the cytotoxicity of NK-92 cells in a dose-dependent manner by enhancing IFN-γ and increasing the expression of the activation markers CD25 and CD69. In addition, FAC had not changed the NK-92 cells phenotypical characterization, but markedly enhanced the expression intensity of CD56. Furthermore, FAC significantly enhanced mRNA and protein expressions lever of NKp44 and NKG2D in NK-92 cells. CONCLUSION: These results suggest that FAC upregulate the expressions of NKG2D, NKp44, which in turn influence NK-92 cells activation. FAC may serve as an immunostimulatory of NK cells for tumor treatment.

[Assessment on dietary iodine intake in three areas of Zhejiang province, 2009].

OBJECTIVE: To assess the level of dietary iodine intake in three areas of Zhejiang and the related policy on universal salt iodization in the province. METHODS: The study involved 497 residents from 180 families living in Hangzhou, Taizhou, Zhoushan cities, representing coastal and inland areas in Zhejiang province in 2009. A total diet study was applied to obtain the typical diet samples at three study areas through food consumption, aggregation, sampling and preparation processes. The contents of iodine in diet samples were determined by tetramethylammonium hydroxide extraction-inductively coupled plasma-mass spectrometry. The amount of dietary iodine intake was calculated by timing the food consumption data and the iodine content in different dietary samples. The safety of dietary iodine intake was evaluated according to the recommended nutrient intake (RNI) and tolerable upper intake level (UL) published by the Chinese Nutrition Society in 2001. RESULTS: The dietary iodine intake of reference person in three areas of Zhejiang province was 421.0 µg/d. The levels of P5, P25, median, P75, P90, P95 dietary iodine intake were 145.7 µg/d, 267.6 µg/d, 358.5 µg/d, 495.6 µg/d, 774.1 µg/d and 1273.0 µg/d respectively. Daily dietary iodine intake at UL accounted for 5.2%, 87.5% and 7.2% of all the participants respectively. Without considering the loss through cooking, salt iodine provided 81.6% of the dietary iodine source. The resources of dietary iodine would include vegetables, cereals and marine food, proportionally, as 57.2%, 13.0% and 8.5%, respectively. Participants whose daily dietary iodine intake exceeded the UL level would consume more marine algae products than those whose dietary iodine intakes were lower than RNI or between RNI-UL. CONCLUSION: Dietary iodine intake among most residents and their average level were among reasonable ranges. Meanwhile, deficiency and excess of iodine intake coexisted. Salt iodine was the main resource of dietary iodine. Participants whose dietary iodine intakes exceeding the UL level, would prefer consume more marine algae products.

Protective effect of flavonoids from Astragalus complanatus on radiation induced damages in mice.

This study aimed to evaluate the radioprotective effect of flavonoids extracted from the seeds of Astragalus complanatus R.Br. (FAC) and their protective mechanism against radiation damage. FAC increased the survival rate of mice and made the damaged organ injured by (60)Co γ-irradiation recovered to normal appearance with the mechanism of enhancing immune function and blood-producing function in vivo. The molecule mechanism of FAC against radiation is involved in the reduction of DNA injury and mutation in vitro. Eleven monomers of the FAC were analyzed by HPLC. These results seem to support the use of FAC in relieving radiation damage.

Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus.

AIM OF THE STUDY: Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells. MATERIALS AND METHODS: Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis. RESULTS: The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased. CONCLUSIONS: These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.