Effects of polysaccharides from Fuzhuan brick tea on immune function and gut microbiota of cyclophosphamide-treated mice.

In this study, cyclophosphamide (Cy) was used to treat mice to establish an immunosuppressant model in mice, and the regulatory effects of polysaccharides from Fuzhuan brick tea (FBTPSs) including crude FBTPSs (CFBTPSs) and the purified fraction (FBTPSs-3) on the immune function and gut microbiota of mice were investigated. The results showed that CFBTPSs and FBTPSs-3 restored the levels of body weight, feed intake, immune organ index, cytokine and immunoglobulin A in mice. The Cy-induced injury of gut including intestinal morphology and expression of tight junction proteins were also restored. Furthermore, CFBTPSs and FBTPSs-3 could significantly modulate gut microbiota by increasing the relative abundance of Muribaculaceae and reduceing the relative abundances of Lachnospiraceae, Helicobacteraceae, Clostridaceae, Desulfovibrionaceae and Deferribacteraceae. Moreover, the gut microbiota derived short-chain fatty acids might play an important role in improvement of immune function by FBTPSs. Our results showed that FBTPSs could regulate the immune function of mice, which provided evidences for the development of FBTPSs as potentially functional foods to improve human health.

Effect of Garlic Organic Sulfides on Gene Expression Profiling in HepG2 Cells and Its Biological Function Analysis by Ingenuity Pathway Analysis System and Bio-Plex-Based Assays.

Garlic organic sulfides are dietary bioactive components with multiple biofunctions to prevent chronic diseases/inflammation and promote human health. DADS (diallyl disulfide), DATS (diallyl trisulfide), and DTS (diallyl tetrasulfide) are typical organic sulfides with similar structures from garlic. However, the structure-activity relationship of garlic organic sulfides remained unknown. The aim of the present study was to investigate the effect of DADS, DATS, and DTS on the gene expression profiling of human hepatocellular carcinoma cells (HepG2) by application of microarray and specialized analysis software, GO, Bio-Plex-based cytokines assay and IPA and analyze their structure-activity relationship according to antioxidant, anti-inflammatory, and metabolic-related properties. According to the microarray data, with the increase of S atom in garlic organic sulfides, its biological activity was gradually enhanced. In the general catalog of GO, garlic organic sulfides mainly affect biological process, molecular function, and cellular component. RT-qPCR results indicated that the microarray data is trustworthy, and the structure-activity analysis data found that more sulfur atoms have more powerful properties; thus, microarray data of DTS was preceded to the subsequent IPA analysis. The results of IPA analysis showed that the top 5 signaling pathways and molecular functions were disturbed by DTS; the molecular functions with the highest scores affected by DTS are cancer, cell apoptosis, and cell proliferation, which imply that the occurrence or metabolism of these diseases is related to the differential expression of the above-mentioned related genes and the activation of signaling channels, and the core of the most significant molecular network is inflammation. Finally, the results found that the secretions of 6 cytokines in macrophages were significantly inhibited by DTS treatment. This is the first study that analyzed the structure-activity relationship of garlic organic sulfides, which will provide useful genetic information for its multi-biofunction and promote their clinical application in the near future.

The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment.

Flavonols possess several beneficial bioactivities in vitro and in vivo. In this study, two flavonols galangin and quercetin with or without heat treatment (100 °C for 15-30 min) were assessed for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated rat intestinal epithelial (IEC-6) cells and whether the heat treatment caused activity changes. The flavonol dosages of 2.5-20 µmol/L had no cytotoxicity on the cells but could enhance cell viability (especially using 5 µmol/L flavonol dosage). The flavonols could decrease the production of prostaglandin E2 and three pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α, and simultaneously promote the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-ß. The Western-blot results verified that the flavonols could suppress the LPS-induced expression of TLR4 and phosphorylated IκBα and p65, while the molecular docking results also illustrated that the flavonols could bind with TLR4 and NF-κB to yield energy decreases of -(21.9-28.6) kJ/mol. Furthermore, an inhibitor BAY 11-7082 blocked the NF-κB signaling pathway by inhibiting the expression of phosphorylated IκBα/p65 and thus mediated the production of IL-6/IL-10 as the flavonols did, which confirmed the assessed anti-inflammatory effect of the flavonols. Consistently, galangin had higher anti-inflammatory activity than quercetin, while the heated flavonols (especially those with longer heat time) were less active than the unheated counterparts to exert these target anti-inflammatory effects. It is highlighted that the flavonols could antagonize the LPS-caused IEC-6 cells inflammation via suppressing TLR4/NF-κB activation, but heat treatment of the flavonols led to reduced anti-inflammatory efficacy.

Protective Effects of Glutamine and Leucine Supplementation on Sepsis-Induced Skeletal Muscle Injuries.

This study investigated the effects of l-glutamine (Gln) and/or l-leucine (Leu) administration on sepsis-induced skeletal muscle injuries. C57BL/6J mice were subjected to cecal ligation and puncture to induce polymicrobial sepsis and then given an intraperitoneal injection of Gln, Leu, or Gln plus Leu beginning at 1 h after the operation with re-injections every 24 h. All mice were sacrificed on either day 1 or day 4 after the operation. Blood and muscles were collected for analysis of inflammation and oxidative damage-related biomolecules. Results indicated that both Gln and Leu supplementation alleviated sepsis-induced skeletal muscle damage by reducing monocyte infiltration, calpain activity, and mRNA expression levels of inflammatory cytokines and hypoxia-inducible factor-1α. Furthermore, septic mice treated with Gln had higher percentages of blood anti-inflammatory monocytes and muscle M2 macrophages, whereas Leu treatment enhanced the muscle expressions of mitochondrion-related genes. However, there were no synergistic effects when Gln and Leu were simultaneously administered. These findings suggest that both Gln and Leu had prominent abilities to attenuate inflammation and degradation of skeletal muscles in the early and/or late phases of sepsis. Moreover, Gln promoted the switch of leukocytes toward an anti-inflammatory phenotype, while Leu treatment maintained muscle bioenergetic function.

AGSE: A Novel Grape Seed Extract Enriched for PP2A Activating Flavonoids That Combats Oxidative Stress and Promotes Skin Health.

Environmental stimuli attack the skin daily resulting in the generation of reactive oxygen species (ROS) and inflammation. One pathway that regulates oxidative stress in skin involves Protein Phosphatase 2A (PP2A), a phosphatase which has been previously linked to Alzheimer's Disease and aging. Oxidative stress decreases PP2A methylation in normal human dermal fibroblasts (NHDFs). Thus, we hypothesize agents that increase PP2A methylation and activity will promote skin health and combat aging. To discover novel inhibitors of PP2A demethylation activity, we screened a library of 32 natural botanical extracts. We discovered Grape Seed Extract (GSE), which has previously been reported to have several benefits for skin, to be the most potent PP2A demethylating extract. Via several fractionation and extraction steps we developed a novel grape seed extract called Activated Grape Seed Extract (AGSE), which is enriched for PP2A activating flavonoids that increase potency in preventing PP2A demethylation when compared to commercial GSE. We then determined that 1% AGSE and 1% commercial GSE exhibit distinct gene expression profiles when topically applied to a 3D human skin model. To begin to characterize AGSE's activity, we investigated its antioxidant potential and demonstrate it reduces ROS levels in NHDFs and cell-free assays equal to or better than Vitamin C and E. Moreover, AGSE shows anti-inflammatory properties, dose-dependently inhibiting UVA, UVB and chemical-induced inflammation. These results demonstrate AGSE is a novel, multi-functional extract that modulates methylation levels of PP2A and supports the hypothesis of PP2A as a master regulator for oxidative stress signaling and aging in skin.

Oxalate Alters Cellular Bioenergetics, Redox Homeostasis, Antibacterial Response, and Immune Response in Macrophages.

Individuals with calcium oxalate (CaOx) kidney stones can have secondarily infected calculi which may play a role in the development of recurrent urinary tract infection (UTI). Uropathogenic Escherichia coli (UPEC) is the most common causative pathogen of UTIs. Macrophages play a critical role in host immune defense against bacterial infections. Our previous study demonstrated that oxalate, an important component of the most common type of kidney stone, impairs monocyte cellular bioenergetics and redox homeostasis. The objective of this study was to investigate whether oxalate compromises macrophage metabolism, redox status, anti-bacterial response, and immune response. Monocytes (THP-1, a human monocytic cell line) were exposed to sodium oxalate (soluble oxalate; 50 µM) for 48 hours prior to being differentiated into macrophages. Macrophages were subsequently exposed to calcium oxalate crystals (50 µM) for 48 hours followed by UPEC (MOI 1:2 or 1:5) for 2 hours. Peritoneal macrophages and bone marrow-derived macrophages (BMDM) from C57BL/6 mice were also exposed to oxalate. THP-1 macrophages treated with oxalate had decreased cellular bioenergetics, mitochondrial complex I and IV activity, and ATP levels compared to control cells. In addition, these cells had a significant increase in mitochondrial and total reactive oxygen species levels, mitochondrial gene expression, and pro-inflammatory cytokine (i.e. Interleukin-1ß, IL-1ß and Interleukin-6, IL-6) mRNA levels and secretion. In contrast, oxalate significantly decreased the mRNA levels and secretion of the anti-inflammatory cytokine, Interleukin-10 (IL-10). Further, oxalate increased the bacterial burden of primary macrophages. Our findings demonstrate that oxalate compromises macrophage metabolism, redox homeostasis, and cytokine signaling leading to a reduction in anti-bacterial response and increased infection. These data highlight a novel role of oxalate on macrophage function.

Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.

Omega-3 polyunsaturated fatty acids ameliorated inflammatory response of mammary epithelial cells and mammary gland induced by lipopolysaccharide.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), essential fatty acids for humans and animals, have been reported to play a beneficial role in a variety of inflammatory diseases. In this study, we investigated the inhibitory effects and potential molecular mechanisms of n-3 PUFAs on the inflammatory response in lipopolysaccharide (LPS)-stimulated mammary alveolar cell line (MAC-T). Results showed that n-3 PUFAs could abate LPS-induced secretions of tumor necrosis factor-α, interleukin (IL)-6 and IL-1ß in MAC-T cells through the nuclear transcription factor kappa B (NF-κB) signal pathway. Meanwhile, n-3 PUFA intervention attenuated histopathologic changes of mammary glands, the white blood cell number decrease, and the alkaline phosphatase level decrease in the serum of mice challenged by LPS. Furthermore, n-3 PUFA intervention improved the ecological structure of the flora in terms of the structural disorder of the non-significant dominant flora induced by LPS in mice. Collectively, both in vitro and in vivo experiments revealed that n-3 PUFAs have a positive effect on LPS-induced inflammatory response, which was possibly mediated by the NF-κB signaling pathway and the intestinal microbiota.

Piperine Improves Experimental Autoimmune Encephalomyelitis (EAE) in Lewis Rats Through its Neuroprotective, Anti-inflammatory, and Antioxidant Effects.

Inflammation, demyelination, glial activation, and oxidative damage are the most pathological hallmarks of multiple sclerosis (MS). Piperine, a main bioactive alkaloid of black pepper, possesses antioxidant, anti-inflammatory, and neuroprotective properties whose therapeutic potential has been less studied in the experimental autoimmune encephalomyelitis (EAE) models. In this study, the efficiency of piperine on progression of EAE model and myelin repair mechanisms was investigated. EAE was induced in female Lewis rats and piperine and its vehicle were daily administrated intraperitoneally from day 8 to 29 post immunization. We found that piperine alleviated neurological deficits and EAE disease progression. Luxol fast blue and H&E staining and immunostaining of lumbar spinal cord cross sections confirmed that piperine significantly reduced the extent of demyelination, inflammation, immune cell infiltration, microglia, and astrocyte activation. Gene expression analysis in lumbar spinal cord showed that piperine treatment decreased the level of pro-inflammatory cytokines (TNF-α, IL-1ß) and iNOS and enhanced IL-10, Nrf2, HO-1, and MBP expressions. Piperine supplementation also enhanced the total antioxidant capacity (FRAP) and reduced the level of oxidative stress marker (MDA) in the CNS of EAE rats. Finally, we found that piperine has anti-apoptotic and neuroprotective effect in EAE through reducing caspase-3 (apoptosis marker) and enhancing BDNF and NeuN expressing cells. This study strongly indicates that piperine has a beneficial effect on the EAE progression and could be considered as a potential therapeutic target for MS treatment. Upcoming clinical trials will provide a deeper understanding of piperine's role for the treatment of the MS.

Neuroprotective effect of combined use of nicotine and celecoxib by inhibiting neuroinflammation in ischemic rats.

INTRODUCTION: The contribution of neuroinflammation in cognitive impairment is increasingly recognized. Non-steroidal anti-inflammatory drugs had been proven that it could improve cognitive impairment in large dose but with more side effect, which limited the application. The main objective of this study was to investigate whether the combined use of nicotine and celecoxib could obtain synergistic neuroprotective effect in ischemic rats. METHODS: Twenty adult Sprague-Dawley (SD) rats underwent ischemic model surgery by injecting endothelin-1 into the left thalamus, which were classified into four groups with different interventions: nicotine (1.5 mg/kg/d), celecoxib (15 mg/kg/d), nicotine (1.5 mg/kg/d) +celecoxib (15 mg/kg/d), or saline after surgery. The other five SD rats also underwent same surgery by injecting saline instead of endothelin-1, as the control group. Morris water maze (MWM) test was adopted to assess the cognition. Micro PET/CT with 2-[18F]-A-85380 were performed for α4ß2-nAChRs detection in vivo. Western blot, real-time PCR and immunohistochemical staining were adopted to detect the expression of α4ß2-nAChRs and inflammatory factors which included TNF-α, IL-1ß, IL-6 in brain tissue. Microglial activation in the brain was monitored by immunofluorescence with IBA1 staining. RESULTS: The MWM test showed rats given with nicotine or celecoxib alone showed much better memory than rats with saline, no difference was observed between nicotine and celecoxib. The rat memory was recovered most significant when the nicotine and celecoxib were combined (p < 0.05). Micro-PET/CT showed much more tracer uptake in the left thalamus and whole brain in rats given with nicotine, or nicotine + celecoxib (nico + cele group) than saline treated rats, whereas the rats given celecoxib did not. Compared with saline treated rats, we found the proteins of α4nAChR and ß2nAChR in rats given nicotine or nico + cele increased significantly, and mRNA/proteins of TNF-α, IL-1ß and IL-6 decreased at the same time. The α 4nAChR and ß 2nAChR proteins in rats given celecoxib is the same as saline treated rats, whereas the inflammatory factors decreased obviously compared with saline treated rats. Microglial activation was confirmed in saline treated rats, which was inhibited in rats give nicotine, celecoxib or both. CONCLUSIONS: The study revealed the combined use of nicotine and celecoxib may improve the cognitive function in ischemic rats, with a better effect than either alone. Both nicotine and celecoxib can inhibit inflammation, but through different mechanisms: nicotine can activate α4ß2-nAChRs while celecoxib is cyclooxygenase-2 inhibitor. Our findings suggest the combined application of two drugs with different anti-inflammation mechanism could attenuate cognitive impairment more effectively in ischemic rats, which may hold therapeutic potential in the clinical practice.

Targeting ROS/NF-κB sigaling pathway by the seedless black Vitis vinifera polyphenols in CCl4-intoxicated kidney, lung, brain, and spleen in rats.

Carbon tetrachloride (CCl4) is an abundant environmental pollutant that can generate free radicals and induce oxidative stress in different human and animal organs like the kidney, lung, brain, and spleen, causing toxicity. The present study evaluated the alleviative mechanism of the isolated polyphenolic fraction from seedless (pulp and skin) black Vitis vinifera (VVPF) on systemic oxidative and necroinflammatory stress in CCl4-intoxicated rats. Here, we found that the administration of VVPF to CCl4-intoxicated rats for ten days was obviously ameliorated the CCl4-induced systemic elevation in ROS, NO and TBARS levels, as well as MPO activity. Also, it upregulated the cellular activities of the enzymatic (SOD, and GPx) and non-enzymatic (TAC and GSH) antioxidants. Furthermore, the gene expression of the ROS-related necroinflammatory mediators (NF-κB, iNOS, COX-2, and TNF-α) in the kidney, brain, and spleen, as well as IL-1ß, and IL-8 in the lung were greatly restored. The histopathological studies confirmed these biochemical results and showed a noticeable enhancing effect in the architecture of the studied organs after VVPF intake. Thus, this study indicated that VVPF had an alleviative effect on CCl4-induced necroinflammation and oxidative stress in rat kidney, lung, brain, and spleen via controlling the ROS/NF-κB pathway.

Significance of PD1 Alternative Splicing in Celiac Disease as a Novel Source for Diagnostic and Therapeutic Target.

Background: We have focused on the alteration of the PD-1/PD-L1 pathway in celiac disease and discussed the roles of the PD1 pathway in regulating the immune response. We explored the idea that the altered mRNA splicing process in key regulatory proteins could represent a novel source to identify diagnostic, prognostic, and therapeutic targets in celiac disease. Methods: We characterized the PD1 mRNA variants' profile in CD patients and in response to gluten peptides' incubation after in vitro experiments. Total RNA from whole blood was isolated, and the coding region of the human PD-1 mRNA was amplified by cDNA PCR. Results: PCR amplification of the human PD-1 coding sequence revealed an association between the over-expression of the sPD-1 protein and the PD-1Δex3 transcript in celiac disease. Thus, we have found three novel alternative spliced isoforms, two of which result in a truncated protein and the other isoform with a loss of 14 aa of exon 2 and complete exon 3 (Δ3) which could encode a new soluble form of PD1 (sPD-1). Conclusions: Our study provides evidence that dietary gluten can modulate processes required for cell homeostasis through the splicing of pre-mRNAs encoding key regulatory proteins, which represents an adaptive mechanism in response to different nutritional conditions.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Xi Lei San Attenuates Dextran Sulfate Sodium-Induced Colitis in Rats and TNF-α-Stimulated Colitis in CACO2 Cells: Involvement of the NLRP3 Inflammasome and Autophagy.

OBJECTIVE: Inflammatory bowel disease (IBD) is a chronic nonspecific inflammatory bowel disease with an unclear etiology. The active ingredients of traditional Chinese medicines (TCMs) exert anti-inflammatory, antitumor, and immunomodulatory effects, and their multitarget characteristics provide them with a unique advantage for treating IBD. However, the therapeutic effects and underlying mechanisms of Xi Lei San in treatment of IBD remain unknown. This study was designed to investigate whether Xi Lei San exerted an anti-inflammatory effect in IBD via a mechanism involving NLRP3 inflammasomes and autophagy. METHODS: We successfully established a rat model of dextran sulfate sodium- (DSS-) induced colitis as well as a cellular model of TNF-α-induced colitis. Xi Lei San and indirubin were identified by HPLC analysis. Rats were treated with Xi Lei San or alum crystals, and their body weights and morphology of intestinal tissues were examined. A western blot analysis was performed to determine the expression levels of inflammasome-related proteins and autophagy-related proteins, ELISA was performed to analyze IL-1ß, IL-18, and IL-33 concentrations, and flow cytometry was used to monitor cell apoptosis and ROS levels. RESULTS: Xi Lei San and indirubin were identified by HPLC analysis. We found that Xi Lei San could significantly increase the weights of rats and improve the structure of the intestinal tissues in DSS-induced colitis model rats. We also found that Xi Lei San significantly inhibited NLRP3 inflammasome activity, reduced the levels of inflammatory cytokines, and suppressed autophagy in DSS-induced colitis model rats. In vitro experiments revealed that Xi Lei San could repress apoptosis as well as ROS and inflammatory cytokine production in TNF-α-induced CACO2 cells by reducing the activity of NLRP3 inflammasomes and autophagy. CONCLUSIONS: Our findings showed that Xi Lei San significantly ameliorated IBD by inhibiting NLRP3 inflammasome, autophagy, and oxidative stress.

Network Pharmacology Analysis of ZiShenWan for Diabetic Nephropathy and Experimental Verification of Its Anti-Inflammatory Mechanism.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). The inflammatory response plays a critical role in DN. ZiShenWan (ZSW) is a classical Chinese medicinal formula with remarkable clinical therapeutic effects on DN, but its pharmacological action mechanisms remain unclear. AIM: In this study, a network pharmacology approach was applied to investigate the pharmacological mechanisms of ZSW in DN therapy. Based on the results of network analysis, the core targets and signaling pathways related to anti-inflammatory effect were verified via experiments in vivo. METHODS: The candidate chemical ingredients of ZSW as well as its putative targets and known therapeutic targets of DN were acquired from appropriate databases. The "herb-ingredient-target" network for ZSW in DN treatment was established. The protein-protein interaction (PPI) network of potential targets was constructed to screen the core targets. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. In addition to biochemical and pathological indicators, the core targets and signaling pathways associated with inflammation were partially validated in db/db mice at molecular level. RESULTS: A total of 56 active ingredients in ZSW and 166 DN-related targets were selected from databases. A high proportion of core targets and top signaling pathways participate in inflammation. ZSW markedly alleviated renal injuries pathologically and regulated related biomarkers. In particular, ZSW significantly inhibited the exaggerated release of inflammatory cytokines such as interleukin (IL)-1ß, IL-6, tumor necrosis factor receptor (TNF)-ɑ, and monocyte chemotactic protein (MCP)-1 as well as regulating p38 mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathways in db/db mice. CONCLUSION: This study first comprehensively investigated the active ingredients, potential targets, and molecular mechanism of ZSW as a therapy for DN. ZSW achieved renoprotective effects in DN via regulation of multiple targets and signaling pathways, especially by alleviating inflammation. Results indicate that ZSW is a promising multi-target therapeutic approach for DN treatment.

Colchicine acts selectively in the liver to induce hepatokines that inhibit myeloid cell activation.

Colchicine has served as a traditional medicine for millennia and remains widely used to treat inflammatory and other disorders. Colchicine binds tubulin and depolymerizes microtubules, but it remains unclear how this mechanism blocks myeloid cell recruitment to inflamed tissues. Here we show that colchicine inhibits myeloid cell activation via an indirect mechanism involving the release of hepatokines. We find that a safe dose of colchicine depolymerizes microtubules selectively in hepatocytes but not in circulating myeloid cells. Mechanistically, colchicine triggers Nrf2 activation in hepatocytes, leading to secretion of anti-inflammatory hepatokines, including growth differentiation factor 15 (GDF15). Nrf2 and GDF15 are required for the anti-inflammatory action of colchicine in vivo. Plasma from colchicine-treated mice inhibits inflammatory signalling in myeloid cells in a GDF15-dependent manner, by positive regulation of SHP-1 (PTPN6) phosphatase, although the precise molecular identities of colchicine-induced GDF15 and its receptor require further characterization. Our work shows that the efficacy and safety of colchicine depend on its selective action on hepatocytes, and reveals a new axis of liver-myeloid cell communication. Plasma GDF15 levels and myeloid cell SHP-1 activity may be useful pharmacodynamic biomarkers of colchicine action.

Discovery of novel reversible monoacylglycerol lipase inhibitors via docking-based virtual screening.

Monoacylglycerol lipase (MAGL) is the major enzyme that catalyzes the hydrolysis of monoacylglycerols (MAGs). MAGL is responsible for degrading 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain and specific tissues. The inhibition of MAGL could attenuate the inflammatory response. Here, we report a series of reversible non-covalent MAGL inhibitors via virtual screening combined with biochemical analysis. The hit, DC630-8 showed low-micromolar activity against MAGL in vitro, and exhibited significant anti-inflammatory effects.

The Effects of New Zealand Grown Ginseng Fractions on Cytokine Production from Human Monocytic THP-1 Cells.

Pro-inflammatory cytokines and anti-inflammatory cytokines are important mediators that regulate the inflammatory response in inflammation-related diseases. The aim of this study is to evaluate different New Zealand (NZ)-grown ginseng fractions on the productions of pro-inflammatory and anti-inflammatory cytokines in human monocytic THP-1 cells. Four NZ-grown ginseng fractions, including total ginseng extract (TGE), non-ginsenoside fraction extract (NGE), high-polar ginsenoside fraction extract (HPG), and less-polar ginsenoside fraction extract (LPG), were prepared and the ginsenoside compositions of extracts were analyzed by HPLC using 19 ginsenoside reference standards. The THP-1 cells were pre-treated with different concentrations of TGE, NGE, HPG, and LPG, and were then stimulated with lipopolysaccharide (LPS). The levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and anti-inflammatory cytokines, such as interleukin-10 (IL-10), and transforming growth factor beta-1 (TGF-ß1), were determined by enzyme-linked immunosorbent assay (ELISA). TGE at 400 µg/mL significantly inhibited LPS-induced TNF-α and IL-6 productions. NGE did not show any effects on inflammatory secretion except inhibited IL-6 production at a high dose. Furthermore, LPG displayed a stronger effect than HPG on inhibiting pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) productions. Particularly, 100 µg/mL LPG not only significantly inhibited the production of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6, but also remarkably enhanced the production of anti-inflammatory cytokine IL-10. NZ-grown ginseng exhibited anti-inflammatory effects in vitro, which is mainly attributed to ginsenoside fractions (particularly less-polar ginsenosides) rather than non-saponin fractions.

A novel peptide identified from skin secretions of Bombina maxima possesses LPS-neutralizing activity.

Lipopolysaccharide (LPS) is a major pathogenic factor in endotoxin shock or sepsis. Most antibiotics have little clinical anti-endotoxin activity, but some antimicrobial peptides (AMPs) have been shown to be effective in blocking LPS. We identified a novel peptide from the skin secretions of Bombina maxima (B. _maxima) by challenging the skin of frogs with an LPS solution. Peptide 2 has an amino acid sequence of LVGKLLKGAVGDVCGLLPIC. Peptide 2 possesses low hemolytic activity, low cytotoxicity against RAW 264.7 cells, and strong anti-inflammatory activity. Moreover, peptide 2 plays an anti-inflammatory role by inhibiting inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). A biolayer interferometry (BLI) assay indicated that peptide 2 binds to LPS with strong affinity and that this interaction has an affinity constant (KD) value of 1.05 × 10-9 M. A survival study showed that peptide 2 possesses potent LPS-neutralizing activity to protect LPS-treated mice from death. In conclusion, we have identified a potent peptide with LPS neutralizing activity, which lays a foundation for future research and development.

Tetrahydroxystilbene glycoside improves endothelial dysfunction and hypertension in obese rats: The role of omentin-1.

RATIONALE: Hypertension in obesity has become a major threat for public health. Omentin-1, a novel adipokine, is down-regulated in obesity. Tetrahydroxystilbene glycoside (TSG) is the main ingredient extracted from Polygonum multiflorum Thunb (PMT), a traditional Chinese medicinal herb safely used for protecting cardiovascular systems over bimillennium. This study aims to examine (i) the impact of omentin-1 downregulation on obesity-related hypertension in murine models and the underlying mechanisms; (ii) whether tetrahydroxystilbene glycoside (TSG) improved endothelial dysfunction and obesity-associated hypertension via the increase of omentin-1. METHODS: (TSG-treated) male Zucker diabetic fatty (ZDF) rats and omentin-1 knockout (OMT-/-) mice were used. In vitro, human umbilical vein endothelial cells (HUVECs) and mature adipocytes differentiated from human visceral preadipocyte (HPA-v) were maintained in a co-culture system. RESULTS: TSG was the main active component of PMT reducing systolic blood pressure and improving endothelial vasodilation. Fortnight-TSG treatment (100 mg/kg/day) increased serum omentin-1 level, also activated Akt/eNOS signaling and enhanced NO bioactivity; decreased expression of NOX2 and p22phox, suppressed production of superoxide and peroxynitrite anion. OMT-/- mice showed elevated blood pressure and impaired endothelial vasorelaxation, whereas hypotensive effect of TSG was blunted. In co-culture system, TSG incubation promoted binding of peroxisome proliferator-activated receptor-γ (PPAR-γ) and Itln-1 promoter in adipocytes, activated Akt/eNOS/NO signaling and attenuated oxidative/nitrative stress in HUVECs. Suppression of Itln-1 with siRNA significantly blocked the protective effect of TSG in vitro. CONCLUSIONS: Down-regulation of omentin-1 induces endothelial dysfunction and hypertension in obesity. TSG treatment (at least partially) increases omentin-1 via promoting binding of PPAR-γ and Itln-1 promoter in adipose tissues, subsequently exerts protective effects on endothelial function via activating Akt/eNOS/NO signaling and attenuating oxidative/nitrative stress. These results suggest that TSG could be developed as a promising anti-hypertension agent that protects against endothelial dysfunction and obesity-associated cardiovascular diseases.