Magnolol and 5-fluorouracil synergy inhibition of metastasis of cervical cancer cells by targeting PI3K/AKT/mTOR and EMT pathways.

Objective: This study is designed to investigate the mode of action of the synergistic effect of 5-fluorouracil (5-FU) and magnolol against cervical cancer. Methods: Network pharmacological approach was applied to predict the molecular mechanism of 5-FU combined with magnolol against cervical cancer. CCK-8 assay, colony formation assay, immunofluorescence staining, adhesion assay, wound healing mobility assay, cell migration and invasion assay and Western blot analysis were conducted to validate the results of in silico study. Results: Phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was identified as the key pathway in silico study. The experimental results showed that 5-FU combined with magnolol strongly inhibited cervical cancer cell proliferation, induced the morphological change of HeLa cells by down-regulating the expression of α-actinin, tensin-2 and vinculin. Moreover, magnolol enhanced inhibitory effect of 5-FU on the cell adhesion, migration and invasion. The phosphorylation of AKT and PI3K and the expression of mTOR were strongly inhibited by the combination of 5-FU and magnolol. Moreover, the expression of E-cadherin and ß-catenin was upregulated and the expression of Snail, Slug and vimentin was down-regulated by the 5-FU together with magnolol. Conclusion: Taken together, this study suggests that 5-FU combined with magnolol exerts a synergistic anti-cervical cancer effect by regulating the PI3K/AKT/mTOR and epithelial-mesenchymal transition (EMT) signaling pathways.

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase.

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

Anti-inflammatory mechanism of the optimized active ingredients of Sargentodoxa cuneata and Patrinia villosa.

Pelvic inflammatory disease (PID) is a common gynecological infection. The combined use of Sargentodoxa cuneata (da xue teng) and Patrinia villosa (bai jiang cao) has been shown to inhibit PID progression. The active components of S. cuneata (emodin, Emo) and P. villosa (acacetin, Aca; oleanolic acid, OA; sinoacutine, Sin) have been identified but the mode of action of this combination of compounds against PID has not been clarified. Therefore, this study aims to investigate the mechanism of these active components against PID through network pharmacological, molecular docking and experimental validation. The results showed the optimal combination of components was 40 µM Emo + 40 µM OA, 40 µM Emo + 40 µM Aca, and 40 µM Emo + 150 µM Sin by cell proliferation and NO release. The potential key targets of this combination in the treatment of PID include SRC, GRB2, PIK3R1, PIK3CA, PTPN11, and SOS1, which act on signaling pathways such as EGFR, PI3K/Akt, TNF, and IL-17. Emo, Aca, OA, and their optimal combination inhibited the expression of IL-6, TNF-α, MCP-1, IL-12p70, IFN-γ, and the M1 phenotype markers CD11c and CD16/32, and promoted the expression of the M2 phenotype markers CD206 and arginase 1 (Arg1). Western blotting confirmed that Emo, Aca, OA, and their optimal combination significantly inhibited the expression of glucose metabolism-related proteins PKM2, PD, HK I, and HK II. This study proved the advantage of combination use of active components from S. cuneata and P. villosa, and clarified that they exert the anti-inflammatory effect by regulation of M1/M2 phenotype transition and regulation of glucose metabolism. The results provide a theoretical basis for the clinical treatment of PID.

Genistein Inhibits Proliferation and Metastasis in Human Cervical Cancer Cells through the Focal Adhesion Kinase Signaling Pathway: A Network Pharmacology-Based In Vitro Study in HeLa Cells.

Previous studies have provided evidence that genistein exerts a therapeutic effect on different tumor cells. However, the mechanism of action of genistein against cervical cancer cells remains largely unknown. The aim of this study was to comprehensively decipher the anti-metastatic effect and molecular mechanism of genistein action on cervical cancer cells. We developed an integrated strategy from genotype to phenotype, combining network pharmacology and a transcriptome screening approach, to elucidate the underlying mechanism of action of genistein against human cervical cancer cells. In silico studies predicted that the focal adhesion pathway may be an important signaling cascade targeted by genistein treatment. Using RNA sequencing analysis, representative genes of the focal adhesion pathway were demonstrated to be significantly downregulated. Phenotypic studies revealed that genistein demonstrated strong anti-proliferative and anti-metastatic activity in HeLa cells. Moreover, genistein modulated this activity in a concentration-dependent manner. Genistein also inhibited both the activation and gene expression of FAK (Focal Adhesion Kinase) and paxillin. In addition, vimentin and ß-catenin protein expression, and Snail and Twist gene expression, were strongly inhibited by genistein. Our findings provide strong evidence for a pleiotropic effect of genistein on cervical cancer cells, mediated through the focal adhesion pathway.

Tanshinone I inhibits metastasis of cervical cancer cells by inducing BNIP3/NIX-mediated mitophagy and reprogramming mitochondrial metabolism.

BACKGROUND: Cervical cancer is the most common malignancy of the female lower genital tract. Tanshinone I (Tan I) is one of the crucial lipid-soluble components of red sage (Salvia miltiorrhiza). While its mode of action against cervical cancer is unclear. PURPOSE: Our study aimed to explore the role of Tan I on cervical cancer in vitro. STUDY DESIGN AND METHODS: Effects of Tan I on cervical cancer cells viability, migration and mitochondrial function were investigated by Cell Counting Kit-8, Transwell and Fluorescence laser confocal microscope assays respectively. The potential mechanism of Tan I was uncovered by an integrative approach combining RNA profiling and hydrogen nuclear magnetic resonance-based metabolic analysis, molecular docking and Western blot. RESULTS: Tan I significantly inhibited the growth and colony formation of HeLa and SiHa cells. It induced apoptosis and cell cycle S phase arrest at low (12.5-25 µM) but not high (50 µM) concentrations. It also altered the HeLa cell ultrastructure, decreased the membrane potential and increased the total mitochondrial content. Further, Tan I induced autophagic flux and the colocalization of mitochondria with lysosomes, led to decreased adhesion, invasion, and migration of cervical cancer cells. Transcriptomic analysis revealed that Tan I altered the RNA profile and signal processing in HeLa cells. Tan I significantly impacted "central carbon metabolism in cancer" and "mitophagy-animal" processes. A global metabolic analysis identified 25 metabolites affected by Tan I treatment in HeLa cells. Changes in the metabolic profile indicated that Tan I affected such processes as protein digestion and absorption, central carbon metabolism in cancer, and aminoacyl-tRNA biosynthesis in cervical cancer cells. Furthermore, Tan I significantly induced the expression of mitophagy-related proteins BNIP3, NIX and Optineurin and the conversion from LC3-I to LC3-II, inhibited the NDP52 and P62 level in a concentration-dependent manner. While CQ further increased the conversion of LC3-I to LC3-II and the expression of P62. Moreover, Tan I interacted with BNIP3 and NIX through hydrogen bond. Tan I induce mitophagy could be prevented by BNIP3 and NIX siRNA transfection. CONCLUSION: Tan I induced the BNIP3/NIX-mediated mitophagy, and reprogrammed the mitochondrial metabolism in cervical cancer cells, thus inhibiting metastasis.

Risk prediction of drug-drug interaction potential of phenytoin and miconazole topical formulations.

The drug-drug interaction (DDI) risk of phenytoin with several topical formulations of miconazole is still unclear. The present investigation conducted in vitro-in vivo extrapolation to predict the potential risks. Our data indicated that miconazole potently inhibited phenytoin hydroxylation in both pooled human liver microsomes (HLMs) and recombinant cytochrome P450 2C9 (CYP2C9) with the Ki values of 125 ± 7 nM and 30 ± 2 nM, respectively. Quantitative prediction of DDI risk suggests that, beside intravenous administration or swallowed tablet, combination of phenytoin and miconazole high dose oral gel or buccal tablet may also result in a clinically significant increase of phenytoin AUC (>53%) by the inhibition of miconazole against phenytoin hydroxylation, consequently a higher frequency of adverse events, while the coadministration of miconazole vaginal formulation and phenytoin will be safe.

LncRNA MIR22HG is downregulated in adenomyosis and upregulates miR-2861 through demethylation to inhibit endometrial cell proliferation.

AIM: Endometrial cell proliferation plays a critical role in adenomyosis. It has been reported that MIR22HG and miR-2861 play similar roles in regulating endometrial cell proliferation, indicating their involvement in adenomyosis. This study aimed to investigate the potential involvement of MIR22HG and miR-2861 in adenomyosis. METHODS: Endometrial biopsy was collected from both adenomyosis (n = 45) and the healthy controls (n = 45). The expression of MIR22HG and miR-2861 in biopsies were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The relationship between MIR22HG and miR-2861 in endometrial cells was analyzed by RT-qPCR. Methylation-specific PCR (MSP) was performed to analyze the effects of overexpression of MIR22HG on the expression of miR-2861. Western-blot assays were performed to illustrate the effect of MIR22HG, miR-2861, STAT3, and MMP2 on adenomyosis. Luciferase report assay was performed to analyze the interactions among miR-2861, STAT3, and MMP2. The role of MIR22HG and miR-2861 in regulating the proliferation of endometrial cells was analyzed by cell proliferation assay. RESULTS: The expression of MIR22HG and miR-2861 in adenomyosis did not change with menstrual cycle. MIR22HG and miR-2861 were significantly downregulated in adenomyosis and they were significantly and positively correlated with each other. In endometrial cells, overexpression of MIR22HG upregulated the expression of miR-2861 and decreased methylation of miR-2861 gene. MIR22HG and miR-2861 downregulated STAT3 and MMP2 to inhibit the proliferation of endometrial cells. In addition, overexpression of both MIR22HG and miR-2861 showed stronger effects. CONCLUSION: MIR22HG is downregulated in adenomyosis and upregulates miR-2861 through demethylation to inhibit endometrial cell proliferation.

Inhibition of Respiration of Candida albicans by Small Molecules Increases Phagocytosis Efficacy by Macrophages.

Candida albicans adapts to various conditions in different body niches by regulating gene expression, protein synthesis, and metabolic pathways. These adaptive reactions not only allow survival but also influence the interaction with host cells, which is governed by the composition and structure of the fungal cell wall. Numerous studies had shown linkages between mitochondrial functionality, cell wall integrity and structure, and pathogenicity. Thus, we decided to inhibit single complexes of the respiratory chain of C. albicans and to analyze the resultant interaction with macrophages via their phagocytic activity. Remarkably, inhibition of the fungal bc1 complex by antimycin A increased phagocytosis, which correlated with an increased accessibility of ß-glucans. To contribute to mechanistic insights, we performed metabolic studies, which highlighted significant changes in the abundance of constituents of the plasma membrane. Collectively, our results reinforce the strong linkage between fungal energy metabolism and other components of fungal physiology, which also determine the vulnerability to immune defense reactions.IMPORTANCE The yeast Candida albicans is one of the major fungal human pathogens, for which new therapeutic approaches are required. We aimed at enhancements of the phagocytosis efficacy of macrophages by targeting the cell wall structure of C. albicans, as the coverage of the ß-glucan layer by mannans is one of the immune escape mechanisms of the fungus. We unambiguously show that inhibition of the fungal bc1 complex correlates with increased accessibilities of ß-glucans and improved phagocytosis efficiency. Metabolic studies proved not only the known direct effects on reactive oxygen species (ROS) production and fermentative pathways but also the clear downregulation of the ergosterol pathway and upregulation of unsaturated fatty acids. The changed composition of the plasma membrane could also influence the interaction with the overlying cell wall. Thus, our work highlights the far-reaching relevance of energy metabolism, indirectly also for host-pathogen interactions, without affecting viability.

A Systematic Study of Mechanism of Sargentodoxa cuneata and Patrinia scabiosifolia Against Pelvic Inflammatory Disease With Dampness-Heat Stasis Syndrome via Network Pharmacology Approach.

Objective: To investigate the mechanism of Sargentodoxa cuneata (Oliv.) Rehder & E.H.Wilson (SC) and Patrinia scabiosifolia (PS) against Pelvic Inflammatory Disease with Dampness-Heat Stasis Syndrome via network pharmacological approach and experimental validation. Methods: The active compounds with OB ≥ 30% and DL ≥ 0.18 were obtained from TCMSP database and further confirmed by literature research. The targets of the compounds and disease were acquired from multiple databases, such as GeneCards, CTD and TCMSP database. The intersection targets were identified by Venny software. Cytoscape 3.7.0 was employed to construct the protein-protein interaction (PPI) network and compound-target network. Moreover, GO enrichment and KEGG pathway analysis were analyzed by DAVID database. Finally, CCK-8, Griess assay and a cytometric bead array (CBA) immunoassay were used for experimental validation by detecting the influence of the active compounds on proliferation of macrophage, release of NO and TNF-α after LPS treatment. Results: 9 bioactive compounds were identified from SC and PS. Those compounds corresponded to 134 targets of pelvic inflammatory disease with dampness-heat stasis syndrome. The targets include vascular endothelial growth factor A (VEGFA), von willebrand factor (VWF), interleukin 6 (IL6), tumor necrosis factor (TNF) and nuclear transcription factor 1 (NFκB1). They act on the signaling pathways like advanced glycation end products-receptor of advanced glycation end products (AGE-RAGE), focal adhesion (FA), Toll-like receptor (TLR) and nuclear transcription factor κB (NF-κB). In addition, by in vitro validation, the selected active components of SC and PS such as acacetin, kaempferol, linarin, isovitexin, sinoacutine could significantly inhibit the release of NO induced by LPS, respectively. Moreover, different dose of acacetin, kaempferol, isovitexin and sinoacutine significantly inhibits the TNF-α production. Conclusion: This study provides solid evidence for the anti-inflammatory mechanism of SC and PS against pelvic inflammatory disease with dampness-heat stasis syndrome, which will provide a preliminary evidence and novelty ideas for future research on the two herbs.

A network pharmacology approach to investigate the anti-inflammatory mechanism of effective ingredients from Salvia miltiorrhiza.

Salvia miltiorrhiza, known as Danshen in Chinese, has been widely used to treat cardiovascular diseases in China. Tanshinone I (Tan I) and cryptotanshinone (CST) are the lipid-soluble and effective components from Salvia miltiorrhiza. However, the molecular mechanism of Tan I and CST for treating inflammation is still not known. Therefore, this study was designed to use network pharmacology-based strategy to predict therapeutic targets of Tan I and CST against inflammation, and further to investigate the pharmacological molecular mechanism in vitro. Inflammation targets were identified and followed by acquisition of verified targets of Tan I and CST. After constructing target-functional protein interaction network of Tan I and CST against inflammation, the core therapeutic targets of Tan I and CST against inflammation were obtained. Further, pathway enrichment analyses were performed on core therapeutic targets to evaluate key signaling pathways of Tan I and CST against inflammation. As revealed in network pharmacology analysis, 8 key hub targets for Tan I and CST against inflammation were identified, respectively: JUN, VEGFA, IL-6, TNF, MAPK8, CXCL8, and PTGS2 for Tan I, while STAT3, AKT1, CCND1, MAPK14, VEGFA, ESR1, MAPK8 and AR for CST. Pathway enrichment analysis by DAVID database indicated that Tan I and CST principally regulated the inflammation-associated pathway, such as TLR, JAK-STAT signaling pathway, focal adhesion, apoptosis, mTOR signaling pathway. In vitro, we found that both Tan I and CST exerts significantly effect on LPS stimulated NO secretion and iNOS expression in macrophages. Taken together, our data elucidate that anti-inflammatory pharmacological activities of Tan I and CST may be predominantly related to inhibition of TLR signaling pathway and regulating iNOS synthesis. These findings highlight the predicted therapeutic targets may be potential targets of Tan I and CST for anti-inflammation treatment.

Quercetin inhibits LPS-induced macrophage migration by suppressing the iNOS/FAK/paxillin pathway and modulating the cytoskeleton.

The natural flavonoid quercetin has antioxidant, anti-inflammatory, and anticancer effects. We investigated the effect of quercetin on lipopolysaccharide (LPS)-induced macrophage migration. Quercetin significantly attenuated LPS-induced inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) production in RAW264.7 cells without affecting their viability. Additionally, quercetin altered the cell size and induced an elongated morphology and enlarged the vacuoles and concentrated nuclei. Quercetin significantly disrupted the F-actin cytoskeleton structure. Furthermore, quercetin strongly inhibited LPS-induced macrophage adhesion and migration in a dose-dependent manner. Moreover, quercetin inhibited the LPS-induced expression of p-FAK, p-paxillin, FAK, and paxillin as well as the cytoskeletal adapter proteins vinculin and Tensin-2. Therefore, quercetin suppresses LPS-induced migration by inhibiting NO production, disrupting the F-actin cytoskeleton, and suppressing the FAK-paxillin pathway. Quercetin may thus have potential as a therapeutic agent for chronic inflammatory diseases.

Genistein inhibits the growth and regulates the migration and invasion abilities of melanoma cells via the FAK/paxillin and MAPK pathways.

Genistein is one of the main components of soy-based foods, which are widely known for their many benefits, including anti-cancer, anti-inflammatory, and antioxidant effects. In this study, we investigated the anti-metastasis effects of genistein on B16F10 melanoma cells. Our results showed that genistein strongly inhibited B16F10 cell proliferation and induced apoptosis in time- and concentration-dependent manners. Genistein altered the morphology of B16F10 cells to an elongated shape with slim pseudopodia-like protrusions. Moreover, genistein inhibited the invasion and migration abilities of B16F10 cells in a dose-dependent manner. On one hand, a high concentration of genistein (100 µM) significantly inhibited cell adhesion and migration, as shown by wound healing assays and transwell-migration and invasion assays. Furthermore, the expression levels of p-FAK, p-paxillin, tensin-2, vinculin, and α-actinin were decreased by genistein. As a result, genistein is believed to strongly downregulate the migration and invasion abilities of B16F10 cells via the FAK/paxillin pathway. Moreover, p-p38, p-ERK, and p-JNK levels were also dramatically decreased by treatment with genistein. Finally, genistein significantly decreased the gene expression of FAK, paxillin, vimentin, and epithelial-to-mesenchymal transition-related transcription factor Snail, as shown by real-time PCR (qPCR) analysis. On the other hand, a lower concentration of genistein (12.5 µM) significantly promoted both invasion and migration by activating the FAK/paxillin and MAPK signaling cascades. Taken together, this study showed for the first time that genistein exerts dual functional effects on melanoma cells. Our findings suggest that genistein regulates the FAK/paxillin and MAPK signaling pathways in a highly concentration-dependent manner. Patients with melanoma should therefore be cautious of consuming soy-based foods in their diets.

Regulation of Candida albicans Interaction with Macrophages through the Activation of HOG Pathway by Genistein.

The severity of infections caused by Candida albicans, the most common opportunistic human fungal pathogen, needs rapid and effective antifungal treatments. One of the effective ways is to control the virulence factors of the pathogen. Therefore, the current study examined the effects of genistein, a natural isoflavone present in soybeans, on C. albicans. The genistein-treated C. albicans cells were then exposed to macrophages. Although no inhibition effect on the growth rates of C. albicans was noted an enhancement of the immune response to macrophages has been observed, indicated by phagocytosis and release of cytokines TNF-α and IL-10. The effect of genistein on the enhanced phagocytosis can be mimicked by the fungicides fludioxonil or iprodione, which inhibit the histidine kinase Cos1p and lead to activation of HOG pathway. The western blot results showed a clear phosphorylation of Hog1p in the wild type strain of C. albicans after incubation with genistein. In addition, effects of genistein on the phosphorylation of Hog1p in the histidine kinase mutants Δcos1 and Δsln1 were also observed. Our results thus indicate a new bio-activity of genistein on C. albicans by activation of the HOG pathway of the human pathogen C. albicans.

[Effect of genistein on the TLR and MAPK transduction cascades in lipopolysaccharide -stimulated macrophages].

OBJECTIVE: To delineate and confirm the signaling processes involving mitogen-activated protein kinase (MAPK) and Toll-like receptor (TLR) in the regulation of lipopolysaccharide (LPS)-induced activity of macrophages by genistein at the protein and transcriptional levels. METHODS: RAW264.7 macrophages were treated with genistein and then stimulated with LPS (100 ng/mL) for different time duration. We evaluated the induction of MAPK phosphorylation by Western blot analysis; RT2 Profiler(TM); PCR array was used to investigate the expressions of TLR pathway-related genes after different treatments. RESULTS: LPS led to the phosphorylation of Raf, MEK1/2 and ERK1/2 at 15 minutes post-stimulation and it lasted till 30 minutes. Phosphorylation of p38 was also observed, but it was not as obvious as p-ERK1/2. Addition of genistein further increased the phosphorylation of ERK1/2, its downstream protein p90rsk and p38. Cells treated with LPS and LPS together with genistein demonstrated significant number of genes to be differentially regulated as compared with control cells. Genistein alone could up-regulate the gene CD80, MEKK1, c-fos, Rela, and Ticam2. LPS could up-regulate 20 genes including cytokines IFN-ß, IL-10, IL-1α, IL-1ß, IL-6, TNF-α, colony stimulating factor 2 (CSF-2) and CSF-3, chemokines CCL2 and CXCL10, transcription factor NF-κB1, IκB-α and cyclooxygenase 2 (COX-2). The presence of genistein led to a strong inhibition of the expressions of these genes and up-regulated the transcription factors IκB-ß and c-Rel, a subunit of NF-κB. CONCLUSION: Genistein strongly enhances the LPS-induced activities of MAPK transduction cascades and inhibits TLR pathway.

Genistein induces morphology change and G2/M cell cycle arrest by inducing p38 MAPK activation in macrophages.

Genistein is a well known natural compound which is present in soy foods and exerts many beneficial functions such as anticancer, anti-inflammatory and antioxidant. However, until now little is known about the effects of genistein on the function of macrophages. The murine macrophage cell line RAW264.7 was used as target cell line. The results show that at concentrations of 50-100µM, genistein reduced cell viability to 70%-80% (after 24h) and 50%-60% (after 48h), which was due to G2/M phase cell cycle arrest. Treatment of the macrophages with genistein for 24 or 48h also led to significant morphological changes, such as elongation of the cells and development of long pseudopodia-like protrusions. By staining the F-actin cytoskeleton, we observed accumulation of actin-filaments at the edges of the cells. The morphology change and G2/M phase arrest after genistein treatment is due to the activation of the phosphorylation of MAP kinase p38. The morphology change and cell cycle arrest can be significantly reverted when treatment is combined with p38 inhibitor SB203580. Moreover, after treatment of the macrophages with genistein for 24 and 48h, the phagocytotic efficiency for Candida albicans was decreased in a time- and dose-dependent manner which correlates to the morphology change. The production of cytokines (TNF-α) stimulated by C. albicans was strongly inhibited by genistein. In conclusion, genistein showed a strong immune modulatory effect on the macrophages.

[Anti-inflammatory effect of Syk inhibitor in LPS stimulated macrophages].

OBJECTIVE: To investigate the role of spleen tyrosine kinase (Syk)on the Toll-like receptor 4 (TLR4) signal transduction cascade. METHODS: RAW264.7 macrophages were treated with different concentrations of Syk inhibitor and then stimulated with 100 ng/mL lipopolysaccharide (LPS). The cell viability was tested by the Alarmarblue assay. The NO production was detected by Griess reagents and cytokines in culture supernatants were quantified by ELISA. The expression of iNOS was detected by Western blotting. Moreover, the activation of the transcription factors NF-κB and activator protein 1 (AP-1) was investigated using the reporter cell line RAW-Blue(TM);. RESULTS: The Syk inhibitor had no effect on the cell proliferation even at concentrations of 14 µmol/L. However, the NO production (P<0.01) and iNOS expression resulting from LPS stimulation were inhibited, when the inhibitor concentration exceeded 1.5 µmol/L, and the TNF-α and IL-6 production were inhibited even at lower concentrations (P<0.01). Moreover, inhibition of the activation of both the transcription factors NF-κB and AP-1 was observed. CONCLUSION: The Syk inhibitor shows a significant anti-inflammatory property due to its inhibitory effect on NO and cytokine production by LPS stimulated macrophages. This effect correlated with the reduced activation of both transcription factors NF-κB and AP-1, which places Syk downstream of the TLR4 signal pathway but upstream of the point of divergence of both transcription factors.

Inhibitive effect on phagocytosis of Candida albicans induced by pretreatment with quercetin via actin cytoskeleton interference.

OBJECTIVE: To investigate the anti-inflammatory effect of pretreatment with quercetin on macrophages after Candida albicans infection. METHODS: RAW 264.7 macrophages were used as a target cell line. Cell viability after treatment with quercetin at different time points was detected by Carboxyfluorescein diacetate, succinimidyl ester. Phagocytic function of macrophages was determined by a fluorometric assay. Cytokine tumor necrosis factor alpha (TNF-alpha) production was measured by enzyme-linked immunosorbent assay. F-actin cytoskeleton of L929 cells was stained by Alexa Fluor 488-phalloidin. RESULTS: Pretreatment with quercetin decreased cell viability only at the highest concentration of 37 microg/mL 2, 24, and 48 h after the treatment. The phagocytic efficiency of macrophages pretreated with quercetin was significantly decreased in a time- and dose-dependent manner. F-actin labeling showed that the actin cytoskeleton of the cells started to break down 2 h after treatment. Moreover, it notably inhibited cytokine TNF-alpha production after Candida albicans infection. CONCLUSION: Pretreatment with quercetin induced an anti-inflammatory effect against Candida albicans infection in macrophages.

Deletion of the Candida albicans histidine kinase gene CHK1 improves recognition by phagocytes through an increased exposure of cell wall beta-1,3-glucans.

The pathogenic fungus Candida albicans is able to cover its most potent proinflammatory cell wall molecules, the ß-glucans, underneath a dense mannan layer, so that the pathogen becomes partly invisible for immune cells such as phagocytes. As the C. albicans histidine kinases Chk1p, Cos1p and CaSln1p had been reported to be involved in virulence and cell wall biosynthesis, we investigated whether deletion of the respective genes influences the activity of phagocytes against C. albicans. We found that among all histidine kinase genes, CHK1 plays a prominent role in phagocyte activation. Uptake of the deletion mutant Δchk1 as well as the acidification of Δchk1-carrying phagosomes was significantly increased compared with the parental strain. These improved activities could be correlated with an enhanced accessibility of the mutant ß-1,3-glucans for immunolabelling. In addition, any inhibition of ß-1,3-glucan-mediated phagocytosis resulted in a reduced uptake of Δchk1, while ingestion of the parental strain was hardly affected. Moreover, deletion of CHK1 caused an enhanced release of interleukins 6 and 10, indicating a stronger activation of the ß-1,3-glucan receptor dectin-1. In conclusion, the Chk1p protein is likely to be involved in masking ß-1,3-glucans from immune recognition. As there are no homologues of fungal histidine kinases in mammals, Chk1p has to be considered as a promising target for new antifungal agents.