Fuliang Hu

Miscellaneous standard methods for Apis mellifera research

Summary A variety of methods are used in honey bee research and differ depending on the level at which the research is conducted. On an individual level, the handling of individual honey bees, including the queen, larvae and pupae are required. There are different methods for the immobilising, killing and storing as well as determining individual weight of bees. The precise timing of developmental stages is also an important aspect of sampling individuals for experiments. In order to investigate and manipulate functional processes in honey bees, e.g. memory formation and retrieval and gene expression, microinjection is often used. A method that is used by both researchers and beekeepers is the marking of queens that serves not only to help to locate her during her life, but also enables the dating of queens. Creating multiple queen colonies allows the beekeeper to maintain spare queens, increase brood production or ask questions related to reproduction. On colony level, very useful techniques are the measurement of intra hive mortality using dead bee traps, weighing of full hives, collecting pollen and nectar, and digital monitoring of brood development via location recognition. At the population level, estimation of population density is essential to evaluate the health status and using beelines help to locate wild colonies. These methods, described in this paper, are especially valuable when investigating the effects of pesticide applications, environmental pollution and diseases on colony survival.

Recent advances in the chemical composition of propolis.

Propolis is a honeybee product with broad clinical applications. Current literature describes that propolis is collected from plant resins. From a systematic database search, 241 compounds were identified in propolis for the first time between 2000 and 2012; and they belong to such diverse chemical classes as flavonoids, phenylpropanoids, terpenenes, stilbenes, lignans, coumarins, and their prenylated derivatives, showing a pattern consistent with around 300 previously reported compounds. The chemical characteristics of propolis are linked to the diversity of geographical location, plant sources and bee species.

Anti-inflammatory effects of ethanol extracts of Chinese propolis and buds from poplar (Populus×canadensis).

ETHNOPHARMACOLOGICAL RELEVANCE Propolis is used widely in a number of cultures as a folk medicine and is gaining wider recognition for its potential therapeutic use, due to its wide range of biological properties and pharmacological activities, especially its anti-inflammatory effects. Despite an increasing number of studies focused on the biological activities of propolis together with its botanical sources, studies on Chinese propolis are insufficient. This study was designed to investigate the anti-inflammatory properties of ethanol extracts from Chinese propolis (EECP) and poplar buds (EEPB) from Populus×canadensis Moench (Salicaceae family). MATERIALS AND METHODS Phytochemical analysis of EECP and EEPB was performed via total phenolic and flavonoid content measurements followed by high-performance liquid chromatography (HPLC) analysis. DPPH and ABTS free-radical scavenging methods were used to evaluate their anti-oxidant properties. The anti-inflammatory effects of EECP and EEPB were investigated in vitro by evaluating their modulating effects on the key inflammatory cytokines and mediators in LPS/IFN-γ co-stimulated RAW 264.7 cells and by measuring nuclear factor (NF)-κB activation in TNF-α or IL-1β stimulation HEK 293 cells using reporter gene assays. Their effects on acute inflammatory symptoms (LPS-induced endotoxemia and acute pulmonary damage) were also examined in mice. RESULTS EECP and EEPB exhibited strong free-radical scavenging activity and significant in vitro anti-inflammatory effects by modulating key inflammatory mediators of mRNA transcription, inhibiting the production of specific inflammatory cytokines, and blocking the activation of nuclear factor (NF)-κB. The administration of EECP and EEPB (25 and 100 mg/kg) provided significant protective effects by attenuating lung histopathological changes and suppressing the secretion of LPS-stimulated inflammatory cytokines, such as interleukin-6 (IL-6), IL-10, MCP-1, TNF-α and IL-12p70 production in endotoxemic mice. CONCLUSIONS The results presented here reveal the potent anti-inflammatory properties of Chinese propolis and poplar buds, and provide biological information for developing suitable substitute(s) for propolis in the prevention and treatment of inflammatory diseases.

Next-generation small RNA sequencing for microRNAs profiling in the honey bee Apis mellifera.

MicroRNAs (miRNAs) are key regulators in various physiological and pathological processes via post‐transcriptional regulation of gene expression. The honey bee (Apis mellifera) is a key model for highly social species, and its complex social behaviour can be interpreted theoretically as changes in gene regulation, in which miRNAs are thought to be involved. We used the SOLiD sequencing system to identify the repertoire of miRNAs in the honey bee by sequencing a mixed small RNA library from different developmental stages. We obtained a total of 36 796 459 raw sequences; of which 5 491 100 short sequences were fragments of mRNA and other noncoding RNAs (ncRNA), and 1 759 346 reads mapped to the known miRNAs. We predicted 267 novel honey bee miRNAs representing 380 182 short reads, including eight miRNAs of other insects in 14 107 583 genome‐mapped sequences. We verified 50 of them using stem‐loop reverse‐transcription PCR (RT‐PCR), in which 35 yielded PCR products. Cross‐species analyses showed 81 novel miRNAs with homologues in other insects, suggesting that they were authentic miRNAs and have similar functions. The results of this study provide a basis for studies of the miRNA‐modulating networks in development and some intriguing phenomena such as caste differentiation in A. mellifera.

Molecular Mechanisms Underlying the In Vitro Anti-Inflammatory Effects of a Flavonoid-Rich Ethanol Extract from Chinese Propolis (Poplar Type)

China produces the greatest amount of propolis but there is still lack of basic studies on its pharmacological mechanisms. Our previous study found that ethanol extract from Chinese propolis (EECP) exerted excellent anti-inflammatory effects in vivo but mechanisms of action were elusive. To further clarify the possible mechanisms underlying the anti-inflammatory effects of Chinese propolis (poplar type), we utilized EECP to analyze its chemical composition and evaluated its potential anti-inflammatory effects in vitro. High-performance liquid chromatography (HPLC) profile indicated that EECP contained abundant flavonoids, including rutin, myricetin, quercetin, kaempferol, apigenin, pinocembrin, chrysin, and galangin. Next we found that EECP could significantly inhibit the production of NO, IL-1β, and IL-6 in lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells and suppress mRNA expression of iNOS, IL-1β, and IL-6 in a time- and dose-dependent manner. Furthermore, we found that EECP could suppress the phosphorylation of IκBα and AP-1 but did not affect IκBαs degradation. In addition, using a reporter assay, we found that EECP could block the activation of NF-κB in TNF-α-stimulated HEK 293T cells. Our findings give new insights for understanding the mechanisms involved in the anti-inflammatory effects by Chinese propolis and provide additional references for using propolis in alternative and complementary therapies.

Effects of Encapsulated Propolis on Blood Glycemic Control, Lipid Metabolism, and Insulin Resistance in Type 2 Diabetes Mellitus Rats

The present study investigates the encapsulated propolis on blood glycemic control, lipid metabolism, and insulin resistance in type 2 diabetes mellitus (T2DM) rats. The animal characteristics and biological assays of body weight, fasting blood glucose (FBG), fasting serum insulin (FINS), insulin act index (IAI), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured and euglycemic hyperinsulinemic glucose clamp technique were used to determine these effects. Our findings show that oral administration of encapsulated propolis can significantly inhibit the increasing of FBG and TG in T2DM rats and can improve IAI and M value in euglycemic hyperinsulinemic clamp experiment. There was no significant effects on body weight, TC, HDL-C, and LDL-C in T2DM rats treated with encapsulated propolis. In conclusion, the results indicate that encapsulated propolis can control blood glucose, modulate lipid metabolism, and improve the insulin sensitivity in T2DM rats.

Changes in composition of royal jelly harvested at different times: consequences for quality standards

Most of the studies on royal jelly (RJ) composition or properties as well as quality standards of commercially available royal jelly are based on RJ harvested three days (72 h) after grafting. In China, some beekeepers produce RJ harvested one (24 h) or two (48 h) days after grafting. There is a lack of knowledge about the quality of the royal jelly harvested earlier than 72 h. This study compared 32 colonies for their chemical compositions of RJ harvested at 24, 48 and 72 h after grafting, according to the proportion of moisture, protein, 10-HDA, total sugar and the value of acidity and superoxide dismutase activity. The analysis of RJ samples revealed that the composition varied significantly (for both fresh and dehydrated samples) and on some occasions above and below the range of present Chinese and Swiss standards. The results suggest that harvesting time should be considered when defining new quality standards of RJ.

Antitumor Activity of Chinese Propolis in Human Breast Cancer MCF-7 and MDA-MB-231 Cells.

Chinese propolis has been reported to possess various biological activities such as antitumor. In present study, anticancer activity of ethanol extract of Chinese propolis (EECP) at 25, 50, 100, and 200 μg/mL was explored by testing the cytotoxicity in MCF-7 (human breast cancer ER(+)) and MDA-MB-231 (human breast cancer ER(−)) cells. EECP revealed a dose- and time-dependent cytotoxic effect. Furthermore, annexin A7 (ANXA7), p53, nuclear factor-κB p65 (NF-κB p65), reactive oxygen species (ROS) levels, and mitochondrial membrane potential were investigated. Our data indicated that treatment of EECP for 24 and 48 h induced both cells apoptosis obviously. Exposure to EECP significantly increased ANXA7 expression and ROS level, and NF-κB p65 level and mitochondrial membrane potential were depressed by EECP dramatically. The effects of EECP on p53 level were different in MCF-7 and MDA-MB-231 cells, which indicated that EECP exerted its antitumor effects in MCF-7 and MDA-MB-231 cells by inducing apoptosis, regulating the levels of ANXA7, p53, and NF-κB p65, upregulating intracellular ROS, and decreasing mitochondrial membrane potential. Interestingly, EECP had little or small cytotoxicity on normal human umbilical vein endothelial cells (HUVECs). These results suggest that EECP is a potential alternative agent on breast cancer treatment.

Effect of Brazilian propolis on human umbilical vein endothelial cell apoptosis

Brazilian propolis has been widely studied in recent years. Considering the lack of data concerning the effects of Brazilian propolis on human umbilical vein endothelial cells (HUVECs), we examined the effects of ethanol-extracted Brazilian propolis (EEBP) at 12.5, 25 and 50 μg/ml on apoptosis of HUVECs deprived of basic fibroblast growth factor (FGF-2) and serum. A high concentration of the extract induced HUVEC apoptosis at 24h. Furthermore, we investigated the molecular mechanisms of HUVEC apoptosis induced by EEBP by testing the levels of integrin β4, p53, reactive oxygen species (ROS) and mitochondrial membrane potential. A low concentration of EEBP (12.5 μg/ml) could decrease the expression of integrin β4, p53 and ROS levels, whereas high concentrations (25 and 50 μg/ml) could increase the levels of integrin β4, p53 and ROS at 24h and depress mitochondrial membrane potential level at all times. Considering the doses and the results obtained in this study, Brazilian propolis at high concentrations may be an apoptosis-inducing agent associated with the signal pathway mediated by integrin β4, p53, ROS and mitochondrial membrane potential, thus, propolis should be used in safer levels for human health.

Transcriptome comparison between inactivated and activated ovaries of the honey bee Apis mellifera L.

Ovarian activity not only influences fertility, but is also involved with the regulation of division of labour between reproductive and behavioural castes of female honey bees. In order to identify candidate genes associated with ovarian activity, we compared the gene expression patterns between inactivated and activated ovaries of queens and workers by means of high‐throughput RNA‐sequencing technology. A total of 1615 differentially expressed genes (DEGs) was detected between ovaries of virgin and mated queens, and more than 5300 DEGs were detected between inactivated and activated worker ovaries. Intersection analysis of DEGs amongst five libraries revealed that a similar set of genes (824) participated in the ovary activation of both queens and workers. A large number of these DEGs were predominantly related to cellular, cell and cell part, binding, biological regulation and metabolic processes. In addition, over 1000 DEGs were linked to more than 230 components of Kyoto Encyclopedia of Genes and Genomes pathways, including 25 signalling pathways. The reliability of the RNA‐sequencing results was confirmed by means of quantitative real‐time PCR. Our results provide new insights into the molecular mechanisms involved in ovary activation and reproductive division of labour.