Chemical composition and pharmacological mechanism of Qingfei Paidu Decoction and Ma Xing Shi Gan Decoction against Coronavirus Disease 2019 (COVID-19): In silico and experimental study.

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.

Structure-activity relationship study and biological evaluation of SAC-Garlic acid conjugates as novel anti-inflammatory agents.

A series of S-allyl-l-cysteine (SAC) with garlic acid conjugates as anti-inflammatory agents were designed and synthesized. Among the 40 tested compounds, SMU-8c exhibited the most potent inhibitory activity to Pam3CSK4-induced nitric oxide (NO) in RAW264.7 macrophages with IC50 of 22.54 ±â€¯2.60 µM. The structure-activity relationship (SAR) study suggested that the esterified carboxyl group, carbon chain extension and methoxylation phenol hydroxy could improve the anti-inflammatory efficacy. Preliminary anti-inflammatory mechanism studies showed that SMU-8c significantly down-regulated the levels of Pam3CSK4 triggered TNF-α cytokine in human THP-1 cells, mouse RAW 264.7 macrophages, as well as in ex-vivo human peripheral blood mononuclear cells (PBMC) with no influence on cell viability. SMU-8c specifically blocked the Pam3CSK4 ignited secreted embryonic alkaline phosphatase (SEAP) signaling with no influence to Poly I:C or LPS triggered TLR3 or TLR4 signaling. Moreover, SMU-8c suppressed TLR2 in HEK-Blue hTLR2 cells and inhibited the formation of TLR1-TLR2, and TLR2-TLR6 complex in human PBMC. In summary, SMU-8c inhibited the TLR2 signaling pathway to down-regulate the inflammation cytokines, such as NO, SEAP and TNF-α, to realize its anti-inflammatory activity.

Heterobasidion-growth inhibiting Bacillus subtilis A18 exhibits medium- and age-dependent production of lipopeptides.

Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps.

Targeting the deubiquitinase STAMBP inhibits NALP7 inflammasome activity.

Inflammasomes regulate innate immune responses by facilitating maturation of inflammatory cytokines, interleukin (IL)-1ß and IL-18. NACHT, LRR and PYD domains-containing protein 7 (NALP7) is one inflammasome constituent, but little is known about its cellular handling. Here we show a mechanism for NALP7 protein stabilization and activation of the inflammasome by Toll-like receptor (TLR) agonism with bacterial lipopolysaccharide (LPS) and the synthetic acylated lipopeptide Pam3CSK4. NALP7 is constitutively ubiquitinated and recruited to the endolysosome for degradation. With TLR ligation, the deubiquitinase enzyme, STAM-binding protein (STAMBP) impedes NALP7 trafficking to lysosomes to increase NALP7 abundance. STAMBP deubiquitinates NALP7 and STAMBP knockdown abrogates LPS or Pam3CSK4-induced increases in NALP7 protein. A small-molecule inhibitor of STAMBP deubiquitinase activity, BC-1471, decreases NALP7 protein levels and suppresses IL-1ß release after TLR agonism. These findings describe a unique pathway of inflammasome regulation with the identification of STAMBP as a potential therapeutic target to reduce pro-inflammatory stress.

Anti-inflammatory effects of the petasin phyto drug Ze339 are mediated by inhibition of the STAT pathway.

Ze339, an herbal extract from Petasites hybridus leaves is effective in treatment of allergic rhinitis by inhibition of a local production of IL-8 and eicosanoid LTB4 in allergen-challenged patients. However, the mechanism of action and anti-inflammatory potential in virally induced exacerbation of the upper airways is unknown. This study investigates the anti-inflammatory mechanisms of Ze339 on primary human nasal epithelial cells (HNECs) upon viral, bacterial and pro-inflammatory triggers. To investigate the influence of viral and bacterial infections on the airways, HNECs were stimulated with viral mimics, bacterial toll-like-receptor (TLR)-ligands or cytokines, in presence or absence of Ze339. The study uncovers Ze339 modulated changes in pro-inflammatory mediators and decreased neutrophil chemotaxis as well as a reduction of the nuclear translocation and phosphorylation of STAT molecules. Taken together, this study suggests that phyto drug Ze339 specifically targets STAT-signalling pathways in HNECs and has high potential as a broad anti-inflammatory drug that exceeds current indication. © 2016 BioFactors, 43(3):388-399, 2017.

Endoplasmic reticulum-derived reactive oxygen species (ROS) is involved in toxicity of cell wall stress to Candida albicans.

The cell wall is an important cell structure in both fungi and bacteria, and hence becomes a common antimicrobial target. The cell wall-perturbing agents disrupt synthesis and function of cell wall components, leading to cell wall stress and consequent cell death. However, little is known about the detailed mechanisms by which cell wall stress renders fungal cell death. In this study, we found that ROS scavengers drastically attenuated the antifungal effect of cell wall-perturbing agents to the model fungal pathogen Candida albicans, and these agents caused remarkable ROS accumulation and activation of oxidative stress response (OSR) in this fungus. Interestingly, cell wall stress did not cause mitochondrial dysfunction and elevation of mitochondrial superoxide levels. Furthermore, the iron chelator 2,2'-bipyridyl (BIP) and the hydroxyl radical scavengers could not attenuate cell wall stress-caused growth inhibition and ROS accumulation. However, cell wall stress up-regulated expression of unfold protein response (UPR) genes, enhanced protein secretion and promoted protein folding-related oxidation of Ero1, an important source of ROS production. These results indicated that oxidation of Ero1 in the endoplasmic reticulum (ER), rather than mitochondrial electron transport and Fenton reaction, contributed to cell wall stress-related ROS accumulation and consequent growth inhibition. Our findings uncover a novel link between cell wall integrity (CWI), ER function and ROS production in fungal cells, and shed novel light on development of strategies promoting the antifungal efficacy of cell wall-perturbing agents against fungal infections.

Isobavachalcone attenuates lipopolysaccharide-induced ICAM-1 expression in brain endothelial cells through blockade of toll-like receptor 4 signaling pathways.

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte-endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-ß, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.

Anti-Candida-biofilm activity of micafungin is attenuated by voriconazole but restored by pharmacological inhibition of Hsp90-related stress responses.

We have conducted an in vitro evaluation of the efficacy of a voriconazole-micafungin combination against Candida albicans. When used alone, both micafungin and voriconazole decreased the metabolic activity of planktonic cells, but only micafungin displayed potent anti-biofilm activity. Their combination appeared to have an additive effect against planktonic cells. However, voriconazole significantly antagonized the fungicidal effect of micafungin against Candida biofilms. Time-lag experiments showed that pre-treatment with voriconazole induced resistance to micafungin in Candida biofilms. The micafungin-antagonizing effect of voriconazole persisted even when the biofilm was no longer exposed to voriconazole. In contrast, voriconazole addition after 24 h of micafungin treatment did not alter micafungin sensitivity. To investigate the mechanism of antagonism, we used inhibitors of Hsp90 and its effectors because Hsp90 seems to be implicated in the resistance to micafungin. These molecules reversed the voriconazole-induced resistance to micafungin which suggests that Hsp90-related stress responses are involved in the antagonism. Our results may provide clues as to the mechanism of increased drug resistance in Candida biofilms and raises concerns about the use of the voriconazole-micafungin combination in clinical settings.

TLR triggering on tolerogenic dendritic cells results in TLR2 up-regulation and a reduced proinflammatory immune program.

Tolerogenic dendritic cells (TDC) offer a promising therapeutic potential to ameliorate autoimmune diseases. Reported to inhibit adaptive immune responses, little is known about their innate immunity receptor repertoire. In this study, we compared three types of human TDC (IL-10-DC, dexamethasone (DX)-DC, and 1,25(OH)(2)D(3)-DC) by their TLR expression and response to a set of TLR ligands. TDC are endowed with the same TLR set as standard monocyte-derived dendritic cells but respond differentially to the TLR stimuli Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and flagellin. TDC expressed low or no IL-12-related cytokines and remarkably elevated IL-10 levels. Interestingly, only TDC up-regulated the expression of TLR2 upon stimulation. This boosted the tolerogenic potential of these cells, because IL-10 production was up-regulated in TLR2-stimulated, LPS-primed DX-DC, whereas IL-12 and TNF-alpha secretion remained low. When comparing the TDC subsets, DX-DC and 1,25(OH)(2)D(3)-DC up-regulated TLR2 irrespective of the TLR triggered, whereas in IL-10-DC this effect was only mediated by LPS. Likewise, DX-DC and 1,25(OH)(2)D(3)-DC exhibited impaired ability to mature, reduced allostimulatory properties, and hampered capacity to induce Th1 differentiation. Therefore, both DX-DC and 1,25(OH)(2)D(3)-DC display the strongest tolerogenic and anti-inflammatory features and might be most suitable tools for the treatment of autoimmune diseases.

Pseudomonas aeruginosa Inhibition of Flagellin-activated NF-kappaB and interleukin-8 by human airway epithelial cells.

Pseudomonas aeruginosa-induced activation of NF-kappaB and secretion of proinflammatory cytokines by airway epithelial cells require that the bacteria express flagellin. We tested whether P. aeruginosa and human airway epithelial cells secrete factors that modulated this response. Experiments were performed with both the Calu-3 cell line and primary cultures of tracheal epithelial cells. P. aeruginosa strain PAK DeltafliC (flagellin knockout) did not activate NF-kappaB or interleukin-8 (IL-8) but inhibited flagellin-activated NF-kappaB by 40 to 50% and IL-8 secretion by 20 to 25%. PAK DeltafliC also inhibited NF-kappaB induced by IL-1beta and Toll-like receptor 2 agonist Pam3CSK4. Similar inhibitions were observed with strains PAK, PAO1, and PA14. The inhibitory factor was present in conditioned medium isolated from PAK DeltafliC or Calu-3 plus PAK DeltafliC, but it was not present in conditioned medium isolated from Calu-3 cells alone or from PAK DeltafliC that had been heat treated. Inhibition by PAK DeltafliC-conditioned medium was exerted from either the apical or the basolateral side of the epithelium, was enhanced in simple Ringer's solution over that in tissue culture medium, and did not result from altered pH or depletion of glucose. The inhibitory effect of conditioned medium was abolished by boiling and appeared from filtration studies to result from effects of a factor with a molecular mass of <3 kDa. These and further studies with isogenic mutants led to the conclusion that the NF-kappaB and IL-8 response of airway epithelial cells to P. aeruginosa results from a balance of proinflammatory effects of flagellin and antiinflammatory effects of a small (<3-kDa), heat-sensitive factor(s) that is not lipopolysaccharide, C12 homoserine lactone, alginate, CIF, or exotoxin A, S, T, U, or Y.