Propolis Alleviates Acute Lung Injury Induced by Heat-Inactivated Methicillin-Resistant Staphylococcus aureus via Regulating Inflammatory Mediators, Gut Microbiota and Serum Metabolites.

Propolis has potential anti-inflammatory properties, but little is known about its efficacy against inflammatory reactions caused by drug-resistant bacteria, and the difference in efficacy between propolis and tree gum is also unclear. Here, an in vivo study was performed to study the effects of ethanol extract from poplar propolis (EEP) and poplar tree gum (EEG) against heat-inactivated methicillin-resistant Staphylococcus aureus (MRSA)-induced acute lung injury (ALI) in mice. Pre-treatment with EEP and EEG (100 mg/kg, p.o.) resulted in significant protective effects on ALI in mice, and EEP exerted stronger activity to alleviate lung tissue lesions and ALI scores compared with that of EEG. Furthermore, EEP significantly suppressed the levels of pro-inflammatory mediators in the lung, including TNF-α, IL-1ß, IL-6, and IFN-γ. Gut microbiota analysis revealed that both EEP and EEG could modulate the composition of the gut microbiota, enhance the abundance of beneficial microbiota and reduce the harmful ones, and partly restore the levels of short-chain fatty acids. EEP could modulate more serum metabolites and showed a more robust correlation between serum metabolites and gut microbiota. Overall, these results support the anti-inflammatory effects of propolis in the treatment of ALI, and the necessity of the quality control of propolis.

Caffeic acid phenethyl ester inhibits MDA-MB-231 cell proliferation in inflammatory microenvironment by suppressing glycolysis and lipid metabolism.

Caffeic acid phenethyl ester (CAPE) is one of the main active ingredients of propolis with good antitumor activities. However, the potential effects of CAPE on the glycolysis and lipid metabolism of tumor cells are unclear. Here, the anti-tumor effects of CAPE on MDA-MB-231 cells in an inflammatory microenvironment stimulated with lipopolysaccharide (LPS) were studied by estimating the inflammatory mediators and the key factors of glycolysis and lipid metabolism. The CAPE treatment obviously inhibited proliferation, migration, invasion, and angiogenesis, and the mitochondrial membrane potential was decreased in the LPS-stimulated MDA-MB-231 cells. Compared with the LPS group, pro-inflammatory mediators, including toll-like receptor 4 (TLR4), tumor necrosis factor alpha (TNF-α), NF-kappa-B inhibitor alpha (IκBα), interleukin (IL)-1ß, and IL-6, as well as interleukin-1 receptor-associated kinase 4 (IRAK4), declined after the CAPE treatment. Additionally, CAPE significantly down-regulated the levels of glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), and the key enzymes of glycolysis-hexokinase 2 (HK2), phosphofructokinase (PFK), pyruvate kinase muscle isozyme M2 (PKM2), and lactate dehydrogenase A (LDHA). Moreover, CAPE treatment decreased the levels of key lipid metabolism proteins, including acetyl coenzyme A carboxylase (ACC), fatty acid synthase (FASN), and free fatty acid (FFA)-transported-related protein CD36. After adding the glycolysis inhibitor 2-deoxy-D-glucose (2-DG), the inhibitory effects of CAPE on cell viability and migration were not significant when compared with the LPS group. In summary, the antitumor activity of CAPE in vitro was mainly via the modulation of the inflammatory mediators and the inhibition of key proteins and enzymes in glucose and lipid metabolism.

Integration with Transcriptomic and Metabolomic Analyses Reveals the In Vitro Cytotoxic Mechanisms of Chinese Poplar Propolis by Triggering the Glucose Metabolism in Human Hepatocellular Carcinoma Cells.

Natural products serve as a valuable reservoir of anticancer agents. Chinese poplar propolis (CP) has exhibited remarkable antitumor activities, yet its precise mechanisms of action remain elusive. This study aims to elucidate the in vitro cytotoxic mechanisms of CP in human hepatocellular carcinoma cells (HepG2) through comprehensive transcriptomic and metabolomic analyses. Our evidence suggested that CP possesses a great potential to inhibit the proliferation of HepG2 cells by targeting the glucose metabolism. Notably, CP exhibited a dose- and time-dependent reduction in the viability of HepG2 cells. Transcriptome sequencing unveiled significant alterations in the cellular metabolism, particularly within glucose metabolism pathways. CP effectively restrained glucose consumption and lactic acid production. Moreover, the CP treatment led to a substantial decrease in the mRNA expression levels of key glucose transporters (GLUT1 and GLUT3) and glycolytic enzymes (LDHA, HK2, PKM2, and PFK). Correspondingly, CP suppressed some key protein levels. Cellular metabolomic analysis demonstrated a marked reduction in intermediary products of glucose metabolism, specifically fructose 1,6-bisphosphate and acetyl-CoA, following CP administration. Finally, key compounds in CP were screened, and apigenin, pinobanksin, pinocembrin, and galangin were identified as potential active agents against glycolysis. It indicates that the effectiveness of propolis in inhibiting liver cancer is the result of the combined action of several components. These findings underscore the potential therapeutic value of propolis in the treatment of liver cancer by targeting glycolytic pathways.

Conceptual functional-by-design optimisation of the antioxidant capacity of trans-resveratrol, quercetin, and chlorogenic acid: Application in a functional tea.

Designing functional foods as delivery systemsmay become a tailored strategy to decrease the risk of noncommunicable diseases. Therefore, this work aims to optimise a combination of t-resveratrol (RES), chlorogenic acid (CHA), and quercetin (QUE) based on antioxidant assays and develop a functional tea formulation enriched with the optimal polyphenol combination (OPM). Experimental results showed that the antioxidant capacity of these compounds is assay- and compound-dependent. A mixture containing 73% RES and 27% QUE maximised the hydroxyl radical scavenging activity and FRAP. OPM upregulated the gene expressions of heme oxygenase-1, superoxide dismutase, and catalase and decreased the reactive oxygen species generation in L929 fibroblasts. Adding OPM (100 mg/L)to a chamomile tea increased FRAP:39%, DPPH:59%; total phenolic content: 57%, iron reducing capacity: 41%, human plasma protection against oxidation: 67%. However, pasteurisation (63 °C/30 min) decreased onlythe DPPH. Combining technology, engineering, and cell biology was effective for functional tea design.

Rapid Identification of Corn Sugar Syrup Adulteration in Wolfberry Honey Based on Fluorescence Spectroscopy Coupled with Chemometrics.

Honey adulteration has become a prominent issue in the honey market. Herein, we used the fluorescence spectroscopy combined with chemometrics to explore a simple, fast, and non-destructive method to detect wolfberry honey adulteration. The main parameters such as the maximum fluorescence intensity, peak positions, and fluorescence lifetime were analyzed and depicted with a principal component analysis (PCA). We demonstrated that the peak position of the wolfberry honey was relatively fixed at 342 nm compared with those of the multifloral honey. The fluorescence intensity decreased and the peak position redshifted with an increase in the syrup concentration (10-100%). The three-dimensional (3D) spectra and fluorescence lifetime fitting plots could obviously distinguish the honey from syrups. It was difficult to distinguish the wolfberry honey from another monofloral honey, acacia honey, using fluorescence spectra, but it could easily be distinguished when the fluorescence data were combined with a PCA. In all, fluorescence spectroscopy coupled with a PCA could easily distinguish wolfberry honey adulteration with syrups or other monofloral honeys. The method was simple, fast, and non-destructive, with a significant potential for the detection of honey adulteration.

Antibacterial and antibiofilm activities of Chinese propolis essential oil microemulsion against Streptococcus mutans.

AIMS: To solve the shortcomings of poor solubility, easy volatilization, and decomposition, propolis essential oil microemulsion (PEOME) was prepared. The antibacterial, antibiofilm activities, and action mechanism of PEOME against Streptococcus mutans was analyzed. METHODS: PEOME was prepared using anhydrous ethanol and Tween-80 as the cosurfactant and surfactant, respectively. The antibacterial activity of PEOME against S. mutans was evaluated using the agar disk diffusion method and broth microdilution method. The effects of PEOME on S. mutans biofilm was detected through the assays of crystal violet (CV), XTT reduction, lactic dehydrogenase (LDH) and calcium ions leaking, live/dead staining and scanning electron microscopy (SEM). And the antibiofilm mechanism of PEOME was elaborated by the assays of extracellular polysaccharide (EPS) production and glucosyltransferase (GTF) activity. RESULTS: The inhibition zone diameter (DIZ) of PEOME against S. mutans was 31 mm, while the minimal inhibitory concentration (MIC) was 2.5 µL mL-1. CV and XTT assays showed that PEOME could prevent fresh biofilm formation and disrupt preformed biofilm through decreasing the activities and biomass of biofilm. The leaking assays for LDH and calcium ions, as well as the live/dead staining assay, indicated that PEOME was able to damage the integrity of bacterial cell membranes within the biofilm. SEM revealed that PEOME had a noticeable inhibitory effect on bacterial adhesion and aggregation through observing the overall structure of biofilm. The assays of EPS production and GTF activity suggested that PEOME could reduce EPS production by inhibiting the activity of GTFs, thus showing an antibiofilm effect. CONCLUSIONS: The significant antibacterial and antibiofilm activities against S. mutans of PEOME meant that PEOME has great potential to be developed as a drug to prevent and cure dental caries caused by S. mutans.

Comparative interaction study of soy protein isolate and three flavonoids (Chrysin, Apigenin and Luteolin) and their potential as natural preservatives.

In this work, the potential of soy protein isolate (SPI)-luteolin (Lut)/apigenin (Ap)/chrysin (Chr) complexes as natural preservatives for food and cosmetics was evaluated by comparing their interactional and functional properties with structure-activity relationship. The results of spectrometry and molecular docking indicated that the B-ring hydroxylation of flavonoids affected their binding constants with SPI, which were determined as Lut (1.45 × 106 L/mol) > Ap (2.04 × 105 L/mol) > Chr (3.81 × 104 L/mol) at 298.15 K. It demonstrated that the hydrogen bonding force played an important role in binding flavonoids to SPI. Moreover, the anti-oxidation ability, antimicrobial effect, and foaming properties were positively correlated with increase in number of hydroxyl groups on the B-ring, but the amount and type of the preservative should be adjusted aimed at the nutrition components. This study provides a theoretical basis for the use of flavonoids and SPI-flavonoid complexes as natural preservatives for food and cosmetics.

Anti-Biofilm Activities of Chinese Poplar Propolis Essential Oil against Streptococcus mutans.

Streptococcus mutans (S. mutans) is a common cariogenic bacterium that secretes glucosyltransferases (GTFs) to synthesize extracellular polysaccharides (EPSs) and plays an important role in plaque formation. Propolis essential oil (PEO) is one of the main components of propolis, and its antibacterial activity has been proven. However, little is known about the potential effects of PEO against S. mutans. We found that PEO has antibacterial effects against S. mutans by decreasing bacterial viability within the biofilm, as demonstrated by the XTT assay, live/dead staining assay, LDH activity assay, and leakage of calcium ions. Furthermore, PEO also suppresses the total of biofilm biomasses and damages the biofilm structure. The underlying mechanisms involved may be related to inhibiting bacterial adhesion and GTFs activity, resulting in decreased production of EPSs. In addition, a CCK8 assay suggests that PEO has no cytotoxicity on normal oral epithelial cells. Overall, PEO has great potential for preventing and treating oral bacterial infections caused by S. mutans.

Antibacterial activity of Chinese propolis and its synergy with ß-lactams against methicillin-resistant Staphylococcus aureus.

Propolis has exhibited effective antibacterial activities in preventing the growth of multiple pathogenic bacteria. However, the antibacterial activity of Chinese propolis against methicillin-resistant Staphylococcus aureus (MRSA) is almost unknown. The present study aimed to explore the antibacterial activity and action mechanism of Chinese propolis ethanol extract (CPEE) against MRSA. Thirteen compounds of CPEE were identified using HPLC-DAD/Q-TOF-MS, and none of them showed better anti-MRSA activity than CPEE. The diameter of inhibition zone (DIZ) of CPEE was 20.1 mm. The minimal inhibitory concentration (MIC) of CPEE was 32 mg/L, while the minimal bactericidal concentration (MBC) against MRSA was 64 mg/L. Moreover, CPEE showed significant synergistic effects with ß-lactam antibiotics (ampicillin and oxacillin). Nucleic acid and protein leakage assays showed that CPEE can stimulate the release of intracellular macromolecules by damaging the cell membrane integrity of MRSA. Live/dead-staining and SDS-PAGE assays further confirmed that CPEE could inhibit bacterial activities by disrupting the membrane. The reduction in PBP2a expression and ß-lactamase activity, as shown by western blot and ß-lactamase detection assays, suggested that CPEE was able to reverse the drug resistance of MRSA. These results demonstrated the anti-MRSA activity of CPEE was mainly due to changing the cell membrane and reversing resistance, which indicates that CPEE could be an attractive candidate for use in future food and medical applications.

Interaction between caffeic acid phenethyl ester and protease: monitoring by spectroscopic and molecular docking approaches.

The interaction of one anticancer drug (caffeic acid phenethyl ester; CAPE) with three proteases (trypsin, pepsin and α-chymotrypsin) has been investigated with multispectral methods and molecular docking. As an active components in propolis, the findings are of great benefit to metabolism, design, and structural modification of drugs. The results show that CAPE has an obvious ability to quench the trypsin, pepsin, or α-chymotrypsin fluorescence mainly through a static quenching procedure. Trypsin has the largest binding affinity to CAPE, and α-chymotrypsin has the smallest binding affinity to CAPE. The data obtained from thermodynamic parameters and molecular docking prove that the spontaneously interaction between CAPE and each protease is mainly due to a combination of van der Waals (vdW) force and hydrogen bond (H-bond), controlled by an enthalpy-driven process. The binding force, strength, position, and the number of H-bond are further obtained from the results of molecular docking. Through ultraviolet spectroscopy, dynamic light scattering and circular dichroism experiments, the change in the protease secondary structure induced by CAPE was observed. Additionally, the addition of protease had a positive effect on the antioxidative activity of CAPE, and α-chymotrypsin has the greatest effect on the removal of 2,2-diphenyl-1-picrylhydrazyl free radicals by CAPE.

Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in Escherichia coli. In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using BsaI restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by one-step digestion-ligation reaction in a single tube, and even the FDI contains internal BsaI site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean Glyma.02g025400, Glyma.05g201700, Glyma.06g165700, and Glyma.17g095000 genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of AtMyb75/PAP1 in tomato and GUSPlus in soybean roots via Agrobacterium rhizogenes-mediated transformation, promoter activity analysis of AtGCSpro in Arabidopsis via A. tumefaciens-mediated transformation, and protein subcellular localization of the Vitis vinifera VvCEB1opt in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over)expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding.

Bioactive components and mechanisms of poplar propolis in inhibiting proliferation of human hepatocellular carcinoma HepG2 cells.

BACKGROUND: The aim of this study was to elucidate the bioactive components and anti-tumor mechanism of poplar propolis extract obtained from North China (CP) in human hepatocellular carcinoma HepG2 cells in vitro. METHODS: Cell viability and proliferation were measured by SRB assay and EdU proliferation test kit, respectively. Cell migration was evaluated by scratching test. Reactive oxygen species (ROS) production and mitochondrial membrane potential were investigated with the fluorescent probes, DCHF and JC-1, respectively. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were inspected by measurement kits. Apoptosis was assessed by acridine orange (AO) and Hoechst 33258 staining. Levels of Bax, Bcl-2, caspase 9, caspase 3, PARP, MMP-2, MMP-9, PI3K/p-PI3K, AKT/p-AKT, p38MAPK/p-p38 MAPK, ERK/p-ERK, LATS2, YAP, TAZ and TEAD1 were assessed by western blotting, respectively. RESULTS: The bioactive components of CP inhibiting HepG2 cells were mainly flavonoids, and esters. CP induced HepG2 apoptosis through a mitochondrial-dependent intrinsic pathway with elevated the levels of cleaved PARP, cleaved caspase 3, and Bax and decreased the expressions of Bcl-2 and procaspase 9. It seemed that CP triggered apoptosis by activation of the p38 MAPK and inactivation of p-ERK. More importantly, we found that CP suppressed the Hippo pathway, leading to inactivation of YAP/TAZ and TEAD1 and inhibition of PI3K/AKT signaling molecules. CONCLUSION: CP exerted excellent anti-proliferation and pro-apoptosis actions in HepG2 cells by inactivation of the loop between the Hippo/YAP and PI3K/AKT pathways, and may be a promising therapy for HCC.

Australian propolis ethanol extract exerts antibacterial activity against methicillin-resistant Staphylococcus aureus by mechanisms of disrupting cell structure, reversing resistance, and resisting biofilm.

The antibacterial activity and mechanisms of Australian propolis ethanol extract (APEE) against methicillin-resistant Staphylococcus aureus (MRSA) were investigated herein. The diameter of inhibition zones (DIZ) of APEE was 19.7 mm, while the minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC) of APEE were both 0.9 mg/mL against the tested strain of MRSA. Nucleic acid leakage and propidium iodide (PI) staining assays showed that APEE can stimulate the release of intracellular nucleic acids by disrupting the integrity of the cell wall and cytoplasmic membrane. Scanning electron microscopy (SEM) further confirmed that APEE could depress cellular activities via damaging the cell structure, including the cell wall and membrane. Western blot analysis and ß-lactamase activity assay showed that APEE could inhibit the expression of PBP2a and reduce the activity of ß-lactamase, suggesting that APEE is able to reverse the drug resistance of MRSA. XTT and crystal violet (CV) assays indicated that APEE had the capacity to prevent the formation of biofilms through decreasing cellular activities and biomass. Bacterial adhesion assay revealed that APEE could reduce the adhesive capacity of the strain, belonging to its antibiofilm mechanisms. Furthermore, nine main compounds of APEE were identified and quantified by HPLC-DAD/Q-TOF-MS. The results above all verified that the antibacterial activity of APEE against MRSA was mainly due to disrupting cell structure, reversing resistance, and resisting biofilm formation, which indicates that APEE is expected to be an efficient functional ingredient with great potential application in the field of medicine and food.

Chinese Poplar Propolis Inhibits MDA-MB-231 Cell Proliferation in an Inflammatory Microenvironment by Targeting Enzymes of the Glycolytic Pathway.

Propolis is rich in flavonoids and has excellent antitumor activity. However, little is known about the potential effects of propolis on glycolysis in tumor cells. Here, the antitumor effects of propolis against human breast cancer MDA-MB-231 cells in an inflammatory microenvironment stimulated with lipopolysaccharide (LPS) were investigated by assessing the key enzymes of glycolysis. Propolis treatment obviously inhibited MDA-MB-231 cell proliferation, migration and invasion, clone forming, and angiogenesis. Proinflammatory mediators, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and IL-6, as well as NLRP3 inflammasomes, were decreased following propolis treatment when compared with the LPS group. Moreover, propolis treatment significantly downregulated the levels of key enzymes of glycolysis-hexokinase 2 (HK2), phosphofructokinase (PFK), pyruvate kinase muscle isozyme M2 (PKM2), and lactate dehydrogenase A (LDHA) in MDA-MB-231 cells stimulated with LPS. After treatment with 2-deoxy-D-glucose (2-DG), an inhibitor of glycolysis, the inhibitory effect of propolis on migration was not significant when compared with the LPS group. In addition, propolis increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential. Taken together, these results indicated that propolis targeted key enzymes of glycolysis to suppress the proliferation of MDA-MB-231 cells in an inflammatory microenvironment. These studies provide a molecular basis for propolis as a natural anticancer agent against breast cancer.

Galangin Protects against Symptoms of Dextran Sodium Sulfate-induced Acute Colitis by Activating Autophagy and Modulating the Gut Microbiota.

Galangin is a natural flavonoid that has been reported to provide substantial health benefits. Nevertheless, little is known about the potential effects of galangin against inflammatory bowel diseases. Here, an in vivo study was performed to investigate the preventive effects of galangin against dextran sulphate sodium (DSS)-induced acute murine colitis, which mimics the symptoms of human ulcerative colitis (UC). Pre-treatment with galangin (15 mg/kg, p.o.) resulted in a significant decreased in the macroscopic signs of DSS-induced colitic symptoms, including a decreased disease activity index, prevention of the colon length shortening, and alleviation of the pathological changes occurring in the colon. Colonic pro-inflammatory mediators, including tumor necrosis factor-alpha, interleukin (IL)-1 beta, and IL-6, as well as myeloperoxidase activities were decreased following galangin pre-treatment when compared with the DSS control group. Moreover, galangin pre-treatment significantly increased the expressions of autophagy-related proteins and promoted the formation of autophagosome in the colon. Galangin pre-treatment increased the diversity of the gut microbiota, and this was accompanied by increased levels of short-chain fatty acids. These observed changes could involve the modulating effects conferred by galangin in relation to some specific bacteria populations, including the recovery of Lactobacillus spp., and increased Butyricimonas spp. Overall, these results support the use of galangin in the prevention of UC.

Brazilian Green Propolis Inhibits Ox-LDL-Stimulated Oxidative Stress in Human Umbilical Vein Endothelial Cells Partly through PI3K/Akt/mTOR-Mediated Nrf2/HO-1 Pathway.

Propolis has been widely used as a dietary supplement for its health benefits, including cardiovascular protective effects. The aim of this study was to investigate the cytoprotective effects of Brazilian green propolis (BP) against oxidized low-density lipoprotein (Ox-LDL) induced human umbilical vein endothelial cells (HUVECs) damage. Our results suggested that treatment with BP rescued Ox-LDL-stimulated HUVECs cell viability losses, which might be associated with its inhibitive effects on the cell apoptosis and autophagy. We also noticed that BP restored Ox-LDL-stimulated HUVECs oxidative stress, by induced antioxidant gene expressions, including Heme oxygenase-1 and its upstream mediator, Nrf2, which were mediated by the activation of the phosphorylation of PI3K/Akt/mTOR. Pretreatment with wortmannin, PI3K/AKT inhibitor, abolished BP induced Nrf2 nuclear translocation and HO-1 level. Our results demonstrated that BP protected HUVECs against oxidative damage partly via PI3K/Akt/mTOR-mediated Nrf/HO-1 pathway, which might be applied into preventing Ox-LDL mediated cardiovascular diseases.

Anti-inflammatory effects of Chinese propolis in lipopolysaccharide-stimulated human umbilical vein endothelial cells by suppressing autophagy and MAPK/NF-κB signaling pathway.

This study aimed to investigate the possible benefits of Chinese poplar propolis (CP) in inhibiting inflammation using vascular endothelial cells (VECs) cultured in a nutrient-rich condition exposed to lipopolysaccharide (LPS). Cell proliferation was detected by sulforhodamine B assay and EdU kit. The production of reactive oxygen species (ROS) and level of mitochondrial membrane potential were determined with fluorescent probe DCHF and JC-1, respectively. Protein expression was examined by immunofluorescence staining and western blotting. The results showed that CP (6.25, 12.5, and 25 µg/mL) significantly reduced LPS-induced cytotoxicity, and when challenged with CP substantially suppressed ROS overproduction and protected mitochondrial membrane potential. CP treatment significantly inhibited autophagy by inhibiting LC3B distribution and accumulation, and elevating the p62 level in an mTOR-independent manner but mainly by suppressing the translocation of p53 from the cytoplasm to the nucleus. Furthermore, CP treatment markedly reduced protein levels of TLR4 at 12 and 24 h and significantly suppressed nuclear translocation of NF-κB p65 from cytoplasm to nucleus. In addition, CP treatment significantly reduced the phosphorylation of JNK, ERK1/2, and p38 MAPK. Our findings demonstrated that CP protects VECs from LPS-induced oxidative stress and inflammation, which might be associated with depressing autophagy and MAPK/NF-κB signaling pathway. The results provided novel insights for the potential use of nutrient-rich propolis against inflammation.

Novel metabolic disorders in skeletal muscle of Lipodystrophic Bscl2/Seipin deficient mice.

Bscl2-/- mice recapitulate many of the major metabolic manifestations in Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) individuals, including lipodystrophy, hepatosteatosis, muscular hypertrophy, and insulin resistance. Metabolic defects in Bscl2-/- mice with regard to glucose and lipid metabolism in skeletal muscle have never been investigated. Here, we identified Bscl2-/- mice displayed reduced intramyocellular triglyceride (IMTG) content but increased glycogen storage predominantly in oxidative type I soleus muscle (SM). These changes were associated with increased incomplete fatty acid oxidation and glycogen synthesis. Interestingly, SM in Bscl2-/- mice demonstrated a fasting duration induced insulin sensitivity which was further confirmed by hyperinsulinemic-euglycemic clamp in SM of overnight fasted Bscl2-/- mice but reversed by raising circulating NEFA levels through intralipid infusion. Furthermore, mice with skeletal muscle-specific inactivation of BSCL2 manifested no changes in muscle deposition of lipids and glycogen, suggesting BSCL2 does not play a cell-autonomous role in muscle lipid and glucose homeostasis. Our study uncovers a novel link between muscle metabolic defects and insulin resistance, and underscores an important role of circulating NEFA in regulating oxidative muscle insulin signaling in BSCL2 lipodystrophy.

Bioactive components and mechanisms of Chinese poplar propolis alleviates oxidized low-density lipoprotein-induced endothelial cells injury.

BACKGROUND: Propolis, a polyphenol-rich natural product, has been used as a functional food in anti-inflammation. However, its bioactive components and mechanisms have not been fully elucidated. To discover the bioactive components and anti-inflammatory mechanism, we prepared and separated 8 subfractions from ethyl acetate extract of Chinese propolis (EACP) and investigated the mechanism in oxidized low density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) damage. METHODS: Eight subfractions were prepared and separated from ethyl acetate extract of Chinese propolis (EACP) with different concentrations of methanol-water solution, and analysed its chemical constituents by HPLC-DAD/Q-TOF-MS. Then 80% confluent HUVECs were stimulated with 40 µg/mL ox-LDL. Cell viability and apoptosis were evaluated by Sulforhodamine B (SRB) assay and Hoechst 33,258 staining, respectively. Levels of caspase 3, PARP, LC3B, p62, p-mTOR, p-p70S6K, p-PI3K, p-Akt, LOX-1 and p-p38 MAPK were assessed by western blotting and immunofluorescence assay, respectively. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with fluorescent probes. RESULTS: Each subfraction exhibited similar protective effect although the contents of chemical constituents were different. EACP attenuated ox-LDL induced HUVECs apoptosis, depressed the ratio of LC3-II/LC3-I and enhanced the p62 level. In addition, treatment with EACP also activated the phosphorylation of PI3K/Akt/mTOR, and deactivated the level of LOX-1 and phosphorylation of p38 MAPK. The overproduction of ROS and the damage of MMP were also ameliorated after ECAP treatment. CONCLUSIONS: These findings indicated that the bioactive component of propolis on anti-inflammatory activity was not determined by a single constituent, but a complex interaction including flavonoids, esters and phenolic acids. EACP attenuated ox-LDL induced HUVECs injury by inhibiting LOX-1 level and depressed ROS production against oxidative stress in ox-LDL induced HUVECs, further to activate PI3K/Akt/mTOR pathway and deactivate p38 MAPK to inhibit apoptosis and autophagy, which provide novel insights into the potential application of propolis on modulating chronic inflammation.

Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis and autophagy.

BACKGROUND: Propolis and its major constituent - caffeic acid phenethyl ester (CAPE) have good abilities on antitumor and anti-inflammation. However, little is known about the actions of propolis and CAPE on tumor in inflammatory microenvironment, and inflammatory responses play decisive roles at different stages of tumor development. To understand the effects and mechanisms of ethanol-extracted Chinese propolis (EECP) and its major constituent - CAPE in inflammation-stimulated tumor, we investigated their effects on Toll-like receptor 4 (TLR4) signaling pathway which plays a crucial role in breast cancer MDA-MB-231 cell line. METHODS: 80% confluent breast cancer MDA-MB-231 cells were stimulated with 1 µg/mL lipopolysaccaride (LPS). Then the cells were divided for treatment by CAPE (25 µg/mL) and EECP (25, 50 and 100 µg/mL), respectively. Cell viability, nitric oxide (NO) production and cell migration were measured by sulforhodamine B assay, chemical method and scratch assay. The levels of TLR4, MyD88, IRAK4, TRIF, caspase 3, PARP, LC3B and p62 were investigated through western blotting. The expression of TLR4, LC3B and nuclear factor-κB p65 (NF-κB p65) were tested by immunofluorescence microscopy assay. RESULTS: Treatment of different concentrations of EECP (25, 50 and 100 µg/mL) and CAPE (25 µg/mL) significantly inhibited LPS-stimulated MDA-MB-231 cell line proliferation, migration and NO production. Furthermore, EECP and CAPE activated caspase3 and PARP to induce cell apoptosis, and also upregulated LC3-II and decreased p62 level to induce autophagy during the process. TLR4 signaling pathway molecules such as TLR4, MyD88, IRAK4, TRIF and NF-κB p65 were all down-regulated after EECP and CAPE treatment in LPS-stimulated MDA-MB-231 cells. CONCLUSIONS: These findings indicated that EECP and its major constituent - CAPE inhibited breast cancer MDA-MB-231 cells proliferation in inflammatory microenvironment through activating apoptosis, autophagy and inhibiting TLR4 signaling pathway. EECP and CAPE may hold promising prospects in treating inflammation-induced tumor.