Ginkgo biloba extract ameliorates hyperglycaemia-induced enteric glial cell injury via regulation of the TLR2-related pathway.

BACKGROUND: Diabetic gastrointestinal dysfunction (DGD) is a common complication in diabetic patients, and enteric glial cells (EGCs) found in the gastrointestinal tract have been shown to play an essential role in gastrointestinal dysfunction. Thus, targeting EGCs may be helpful for the control of DGD. This study aimed to evaluate the protective effect of Ginkgo biloba extract (GBE) from G. biloba dropping pills against hyperglycaemic stress-induced EGCs injury and its underlying mechanism. METHODS: In vitro, the protective effect of GBE on CRL-2690 cells was evaluated by MTT assay and TUNEL assay. The expression of related markers was evaluated by RNA sequencing and validated by using western blotting. In vivo, STZ-induced C57BL/6J WT mice were used as models to evaluate the effects of GBE on blood glucose, body weight, and EGCs' activity and relevant signalling pathways were validated by immunofluorescence. RESULTS: The results showed that GBE (25 µg/ml) treatment significantly attenuated hyperglycaemic stress-induced cytotoxicity and cell apoptosis in CRL-2690 cells, which was verified in an STZ-induced (100 mg/kg, 3 days) diabetic mouse model with continuous GBE administration (25/100 mg/kg/day, 6/12 weeks). Further mechanistic study based on transcriptomic data revealed that GBE exerted its beneficial effect by regulating immune-related pathways, and TLR2/BTK/NF-κB/IL-1α/IL-10 comprised the main targets of this drug. CONCLUSIONS: This study demonstrates the protective effect of GBE against hyperglycaemic stress-induced EGCs injury using both in vitro and in vivo models and further reveals that the effect was achieved by targeting TLR2 and its downstream molecules BTK/NF-κB/IL-1α/IL-10. This study may be helpful for expanding the clinical application of GBE in treating DGD.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses.

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

The modulatory effect of Artemisia annua L. on toll-like receptor expression in Acanthamoeba infected mouse lungs.

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4.

Screening of Cytotoxicity and Anti-Inflammatory Properties of Feijoa Extracts Using Genetically Modified Cell Models Targeting TLR2, TLR4 and NOD2 Pathways, and the Implication for Inflammatory Bowel Disease.

Feijoa has been increasingly studied in the recent decade, while investigations into its bioactivities including anti-inflammatory activity are lacking. In this article, the cytotoxicity and anti-inflammatory properties of feijoa extracts, from flesh, peel and whole fruit, from four cultivars namely APOLLO, UNIQUE, OPAL STAR and WIKI TU are presented. Three inflammatory pathways, Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerization domain-containing protein 2 (NOD2), were investigated using genetically modified cell models namely HEK-Blue™ hTLR2, HEK-Blue™ hTLR4, NOD2-WT and NOD2-G908R. Results show that feijoa peel extract induced higher cytotoxicity than flesh and whole fruit extracts, and the APOLLO cultivar was the most anti-inflammatory among the four tested cultivars. The anti-inflammatory activity of feijoa flesh was detected only through the TLR2 pathway, and the activity of feijoa peel and whole fruit was evident mainly through the TLR2 and NOD2 pathways. Most notably, feijoa anti-inflammatory activity was superior to ibuprofen particularly through the TLR2 pathway, with significantly lower secreted embryonic alkaline phosphatase IC50 concentrations (7.88, 12.81, 30.84 and 442.90 µg/mL for APOLLO flesh, peel, whole fruit extract and ibuprofen respectively). These findings indicate that feijoa has great potential to be used in the treatment and prevention of inflammation-related diseases including inflammatory bowel disease.

Influence of Artemisia annua L. on toll-like receptor expression in brain of mice infected with Acanthamoeba sp.

The treatment of acanthamoebiasis is a still a problem. Our previous studies showed that the application of extracts from Artemisia annua L. significantly prolonged the survival of mice infected by Acanthamoeba. This plant has medicinal properties in the treatment of human parasitic diseases. The aim of this study was to evaluate the effects of A. annua on expression of Toll-like receptors (TLRs) 2 and 4 in brain of mice with Acanthamoeba infection. Mice were infected with Acanthamoeba sp. strain Ac309 (KY203908) by intranasal inoculation without and after application of A. annua extract. The administration of extract from A. annua significantly reduced the level of expression of TLR2 and modified the level of expression of TLR4. A. annua extract is a natural substance that is well tolerated in animals and may be considered as a combination therapy in treatment of acanthamoebiasis. Our study suggested that A. annua extract may be used as an alternative therapeutic tool.

Paeoniflorin inhibits high glucose-induced macrophage activation through TLR2-dependent signal pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin(PF), extracted from the root peeled of Paeonia lactiflora Pall(Family: Ranunculaceae), has therapeutic potential in many animal models of inflammatory diseases. AIM OF THE STUDY: Although the anti-inflammatory efficacy of PF has been well illustrated in several animal models, whether it could attenuate diabetic nephropathy and detailed mechanisms are still obscure. Till now, accumulating evidence has proposed the pivotal role of toll-like receptors (TLRs) in renal inflammation in diabetic patients. In this setting, the current study aimed to investigate the effects and underlying mechanism of PF on high glucose-induced activation of toll like-receptor 2 (TLR2) signaling in macrophages. MATERIALS AND METHODS: Bone marrow-derived macrophages (BMDM) were isolated from male Tlr2tm1kir (TLR2-/-) mice and wild-type littermates (C57BL/6JWT). The level of TLR2 and activation of downstream signaling were evaluated in response to 30mmol/L high glucose (HG)-containing medium. Macrophages behaviors, which include cell viability, migration and inflammatory cytokines production, were also determined. RESULTS: PF suppressed HG-induced production of TLR2, activation of downstream signaling and synthesis of inducible nitric oxide synthase (iNOS). PF could further inhibit MyD88-dependent pathway in HG-induced models in which TLR2 was knocked out. Moreover, deletion of TLR2 inhibited the HG-induced activation of MyD88-dependent pathway, but not TIR domain containing adapter inducing interferon-ß (Trif) signal pathway in BMDMs. As HG stimulation polarizes macrophages into M1 phenotype, treatment of PF or knockout of TLR2 significantly reduces M1 markers on the membrane of macrophages. Additionally, levels of inflammatory cytokines and iNOS were remarkably reduced in response to PF or TLR2 deficiency. CONCLUSION: Collectively, these data demonstrated that HG activated macrophages primarily through TLR2-dependent mechanisms which aggravated the severity of renal inflammation and eventually contributed to DN. Additionally, PF might be applied as a potential therapeutic agent in the battle against progressive DN.

Protection of Gastrointestinal Mucosa from Acute Heavy Alcohol Consumption: The Effect of Berberine and Its Correlation with TLR2, 4/IL1ß-TNFα Signaling.

The purpose of the present study is to confirm the protective effect of berberine (BBR) on gastrointestinal injury caused by acute heavy alcohol exposure, an effect that has not been reported previously. Our research details how BBR protects against gastrointestinal injuries from acute alcohol exposure using both in vivo and in vitro experiments. Acute high alcohol concentrations lead to obvious damage to the gastrointestinal mucosa, resulting in necrosis of the intestinal mucosa. Oral administration of BBR was able to significantly reduce this alcohol-induced damage, inhibit increases of alcohol-induced TNFα and IL-1ß expression in gastrointestinal mucosa as well as their upstream signals TLR2 and TLR4, and regulate cytokines that modulate tight junctions. Alcohol consumption is a popular human social behavior worldwide, and the present study reports a comprehensive mechanism by which BBR protects against gastrointestinal injuries from alcohol stress, providing people with a novel application of BBR.

Effects of rhubarb on intestinal flora and toll-like receptors of intestinal mucosa in rats with severe acute pancreatitis.

OBJECTIVE: The aim of this study was to examine the effects of rhubarb on intestinal flora and toll-like receptors (TLRs) of intestinal mucosa in rats with severe acute pancreatitis (SAP). METHODS: Healthy male Sprague-Dawley rats were randomly allocated into sham-operated surgical model of SAP without or with postoperative rhubarb treatment groups (7 in each group). Rats in with rhubarb group received 10% rhubarb decoction (1 mL/200 g) through tube feeding at every 8 hours during postoperative 24 hours. Serum amylase, amount of intestinal flora, and TLR2/TLR4 messenger RNA expression in intestinal mucosa were tested among 3 groups at postoperative 24 hours. RESULTS: TLR2 and TLR4 messenger RNA expression levels in intestinal mucosa in SAP without rhubarb group were significantly higher than those in sham-operated or SAP with rhubarb groups (P < 0.05). The amount of intestinal lactobacilli and bifidobacteria in SAP without rhubarb group were significantly fewer than in those sham-operated group (P < 0.05) but not significantly different from those in SAP with rhubarb group (P > 0.05). The amount of intestinal Escherichia coli was relatively higher in SAP group than in sham-operated group (P > 0.05) but lesser in rhubarb treatment group (P > 0.05). CONCLUSIONS: Rhubarb might maintain the intestinal mucosal barrier through regulating intestinal flora and inhibiting intestinal inflammatory response in rats with SAP.

Antitumor activity of mushroom polysaccharides: a review.

Mushrooms were considered as a special delicacy by early civilizations and valued as a credible source of nutrients including considerable amounts of dietary fiber, minerals, and vitamins (in particularly, vitamin D). Mushrooms are also recognized as functional foods for their bioactive compounds offer huge beneficial impacts on human health. One of those potent bioactives is ß-glucan, comprising a backbone of glucose residues linked by ß-(1→3)-glycosidic bonds with attached ß-(1→6) branch points, which exhibits antitumor and immunostimulating properties. The commercial pharmaceutical products from this polysaccharide source, such as schizophyllan, lentinan, grifolan, PSP (polysaccharide-peptide complex) and PSK (polysaccharide-protein complex), have shown evident clinical results. The immunomodulating action of mushroom polysaccharides is to stimulate natural killer cells, T-cells, B-cells, neutrophils, and macrophage dependent immune system responses via differing receptors involving dectin-1, the toll-like receptor-2 (a class of proteins that play a role in the immune system), scavengers and lactosylceramides. ß-Glucans with various structures present distinct affinities toward these receptors to trigger different host responses. Basically, their antitumor abilities are influenced by the molecular mass, branching configuration, conformation, and chemical modification of the polysaccharides. This review aims to integrate the information regarding nutritional, chemical and biological aspects of polysaccharides in mushrooms, which will possibly be employed to elucidate the correlation between their structural features and biological functions.

Cyptoporus polysaccharide prevents lipopolysaccharide-induced acute lung injury associated with down-regulating Toll-like receptor 2 expression.

AIM OF THE STUDY: To evaluate the effects and the possible mechanism of Cryptoporus polysaccharides (CP) extracted from fruiting body of Cryptoporus volvatus in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and mice. MATERIALS AND METHODS: Acute lung injury was induced by intratracheally instillation of LPS into lung in either rats or mice, assessing leukocyte numbers and myeloperoxidase activity in bronchoalveolar lavage fluid, as well as evaluating cytokines mRNA and protein expressions, and Toll-like receptor 2 (TLR(2)) and nuclear factor (NF)-κB mRNA levels in the lung tissues of mice. Vascular permeability and edema of lung in mice, and arterial blood gas in rats were also performed. RESULTS: In ALI, CP-treated mice and rats exhibited significantly reduced leukocyte invasion, myeloperoxidase activity, vascular permeability, edema of lung, as well as tumor necrosis factor-α and Interleukin-1ß mRNA and protein expressions in the lung tissues compared with vehicle-treated mice. TLR(2) and NF-κB mRNA levels of the lung tissues were decreased in CP-treated mice in response to LPS. And decline in arterial blood gas was recovered in CP-treated rats. CONCLUSIONS: Our results supported a protective role of CP in ALI and suggested that the reduction of the activation of TLR(2) and NF-κB signal pathway in lung injury may be relavant to the pretreatment of CP.

Double blind clinical trial in a series of 115 patients with seborrheic dermatitis: prevention of relapses using a topical modulator of Toll like receptor 2.

AIM: Seborrheic dermatitis is a chronic inflammatory disease aggravated by Malassezia species. Toll-like receptors (TLR) are part of innate immune system that can be activated by yeasts. Previous studies showed that an association of Umbelliferae extract with a lipid (TLR2-Regul™) decreases the IL-8 expression in human skin in contact with M. furfur. The aim of this study was to assess the activity of a topical formulated with TLR2-Regul™ in the prevention of seborrheic dermatitis (SD) relapses. METHODS: Immune-competent SD adult patients were treated for SD (topical imidazoles or steroids). Cleared patients were randomized and received a topical containing TLR2-Regul™ (A) or its vehicle (B). Erythema, scales and pruritus were assessed during two months. RESULTS: The study included 115 patients, mean age 43.4, sex ratio m/f 1.5. At week 4 the relapse rate was 26% (N.=15) in group A and 43% (N.=25) in group B. At W8 the relapse rate was 21% (N.=12) in group A and 40% (N.=23) (P=0.0309). CONCLUSION: In this series of 115 adults with seborrheic dermatitis, patients treated with a topical containing TLR-Regul™ showed a significantly less relapse rate compared with the excipient group (P<0.05). TLR modulation could represent a new therapeutic approach in the prevention of seborrheic dermatitis relapses.

Stimulants of Toll-like receptor (TLR)-2 and TLR-4 are abundant in certain minimally-processed vegetables.

Stimulants of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 have been shown to promote insulin resistance and atherosclerosis in animal models of these diseases. As minimally processed vegetables (MPV) can contain a relatively large bacterial load compared to other foodstuffs, we aimed to quantify the abundance of stimulants of TLR2 and TLR4 in MPV using a transfection-based bioassay calibrated with Escherichia coli LPS and the synthetic lipopeptide Pam(3)CSK(4). Of 5 classes of MPV and 3 classes of related vegetable products considered to be likely to contain a high microbial load, diced onion and bean sprouts contained the highest levels of stimulants of TLR2 (up to 18.5 µg Pam(3)CSK(4)-equivalents per g) and TLR4 (up to 11.4 µg LPS-equivalents per g). By contrast, the majority of fresh whole vegetables examined reproducibly contained minimal or undetectable levels of TLR2- or TLR4-stimulants. The accumulation of TLR-stimulants in MPVs correlated well with growth of enterobacterial spoilage organisms. In conclusion, the modern trend towards eating minimally processed vegetables rather than whole foods is likely to be associated with increased oral exposure to stimulants of TLR2 and TLR4.

Glutamine as a modulator of the immune system of critical care patients: effect on Toll-like receptor expression. A preliminary study.

OBJECTIVE: We evaluated the expression of Toll-like receptors 2 and 4 (TLR-2 and TLR-4) in circulating monocytes from peripheral blood of critical care patients treated with and without glutamine. Because no research has been published to date on the effect of glutamine on TLR receptors in critical patients, it was determined in an initial sample of 30 patients. METHODS: This was a prospective, randomized, single-blind study with 15 patients assigned to receive parenteral nutrition with a daily glutamine supplement of 0.35 g/kg. The control group received isocaloric-isonitrogenous parenteral nutrition. Blood samples were extracted before beginning the treatment and at 5 and 14 d. Expressions of CD14, TLR-2, and TLR-4 were determined by flow cytometry. Levels of TLRs were expressed as mean fluorescence intensity (mfi). RESULTS: Basal characteristics were similar in both groups. The expressions of TLR-2 in the treatment group with glutamine were 4.67 +/- 3.82 mfi before treatment, 3.91 +/- 2.04 mfi at 5 d, and 4.28 +/- 2.47 mfi at 14 d. The expressions of TLR-2 in the control group were 5.49 +/- 3.20 mfi before treatment, 4.48 +/- 2.15 mfi at 5 d, and 4.36 +/- 2.36 mfi at 14 d. The expressions of TLR-4 in the treatment group were 1.65 +/- 1.89 mfi before treatment, 1.23 +/- 1.10 mfi at 5 d, and 1.77 +/- 1.97 at 14 d. The expressions of TLR-4 in the control group were 1.51 +/- 1.76 mfi before treatment, 1.36 +/- 0.99 mfi at 5 d, and 1.26 +/- 0.59 mfi at 14 d. Infections were detected in 11 patients who received glutamine and 13 control patients (P = 0.51). CONCLUSION: In critical care patients, parenteral nutrition supplemented with glutamine does not increase the expression of TLR-2 or TLR-4 in peripheral blood monocytes.