Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Integrative analysis of network pharmacology and proteomics reveal the protective effect of Xiaoqinglong Decotion on neutrophilic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoqinglong Decotion (XQLD) is a commonly used Chinese herbal formula in clinical practice, especially for allergic diseases such as asthma. However, its intrinsic mechanism for the treatment of neutrophilic asthma (NA) remains unclear. AIM OF THE STUDY: The aim of this study was to evaluate the efficacy and potential mechanisms of XQLD on NA using network pharmacology and in vivo experiments. MATERIALS AND METHODS: First, the active compounds, potential targets and mechanisms of XQLD against NA were initially elucidated by network pharmacology. Then, OVA/CFA-induced NA mice were treated with XQLD to assess its efficacy. Proteins were then analyzed and quantified using a Tandem Mass Tags approach for differentially expressed proteins (DEPs) to further reveal the mechanisms of NA treatment by XQLD. Finally, the hub genes, critical DEPs and potential pathways were validated. RESULTS: 176 active compounds and 180 targets against NA were identified in XQLD. Protein-protein interaction (PPI) network revealed CXCL10, CX3CR1, TLR7, NCF1 and FABP4 as hub genes. In vivo experiments showed that XQLD attenuated inflammatory infiltrates, airway mucus secretion and remodeling in the lungs of NA mice. Moreover, XQLD significantly alleviated airway neutrophil inflammation in NA mice by decreasing the expression of IL-8, MPO and NE. XQLD also reduced the levels of CXCL10, CX3CR1, TLR7, NCF1 and FABP4, which are closely associated with neutrophil inflammation. Proteomics analysis identified 28 overlapping DEPs in the control, NA and XQLD groups, and we found that XQLD inhibited ferroptosis signal pathway (elevated GPX4 and decreased ASCL3) as well as the expression of ARG1, MMP12 and SPP1, while activating the Rap1 signaling pathway. CONCLUSION: This study revealed that inhibition of ARG1, MMP12 and SPP1 expression as well as ferroptosis pathways, and activation of the Rap1 signaling pathway contribute to the therapeutic effect of XQLD on NA.

Sambucus nigra L. leaves inhibit TNF-α secretion by LPS-stimulated human neutrophils and strongly scavenge reactive oxygen species.

ETHNOPHARMACOLOGICAL RELEVANCE: Sambucus nigra (elderberry) leaves were used in folk medicine to treat skin inflammations, ulcers, burns or boils, as well as to treat wounds, including infected and chronic ones. For centuries, elderberry leaves have been used mainly in eastern and southern Europe, as well as in western Asia. AIM OF THE STUDY: The study aimed to investigate the anti-inflammatory and antioxidant activity of four different extracts, such as aqueous and ethanolic prepared at room temperature and the solvent's boiling point, from the leaves of elderberry. MATERIALS AND METHODS: The effect of extracts both on the secretion of cytokines (TNF-α, IL-1ß, and IL-8) and reactive oxygen species (ROS) by neutrophils stimulated with bacteria-derived products was investigated. The cytotoxicity of extracts was analyzed by staining with propidium iodide measured by flow cytometry. The anti-inflammatory activity of extracts was also investigated through their influence on lipoxygenase activity. The antioxidant properties, including scavenging superoxide anion, hydrogen peroxide, nitric oxide, and 2,2-diphenyl-1-picrylhydrazyl radical were investigated in cell-free systems. The total content of phenolic compounds was tested using the Folin-Ciocalteu reagent. The qualitative and quantitative determination of the content of individual phenolic acids and flavonoids was performed by HPLC-DAD-MSn and HPLC-DAD method, respectively. RESULTS: Elderberry leaves extracts turned out to affect the inflammatory response of neutrophils by inhibiting the secretion of TNF-α and ROS. The ethanolic and aqueous extracts at a concentration of 50 µg × mL-1 reduce the secretion of TNF-α by approximately 40% and 10%, respectively. ROS secretion was decreased by around 50% for all extracts at concentration of 5 µg × mL-1. All the extracts were able to inhibit the activity of lipoxygenase. The ethanolic extracts were characterized by a higher content of phenolic compounds and a higher antioxidant activity, especially against nitric oxide, compared to the aqueous extracts. CONCLUSIONS: Our research has confirmed that elderberry leaves are a plant material with anti-inflammatory activity, especially against reactive oxygen species, and a potentially rich source of antioxidants. Preliminary analyses performed in this study could be the first step in confirming the traditional use of elderberry leaves in relieving inflammation.

Xuanfei Baidu Decoction reduces acute lung injury by regulating infiltration of neutrophils and macrophages via PD-1/IL17A pathway.

The pathogenic hyper-inflammatory response has been revealed as the major cause of the severity and death of the Corona Virus Disease 2019 (COVID-19). Xuanfei Baidu Decoction (XFBD) as one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, shows unique advantages in the control of symptomatic transition from moderate to severe disease states. However, the roles of XFBD to against hyper-inflammatory response and its mechanism remain unclear. Here, we established acute lung injury (ALI) model induced by lipopolysaccharide (LPS), presenting a hyperinflammatory process to explore the pharmacodynamic effect and molecular mechanism of XFBD on ALI. The in vitro experiments demonstrated that XFBD inhibited the secretion of IL-6 and TNF-α and iNOS activity in LPS-stimulated RAW264.7 macrophages. In vivo, we confirmed that XFBD improved pulmonary injury via down-regulating the expression of proinflammatory cytokines such as IL-6, TNF-α and IL1-ß as well as macrophages and neutrophils infiltration in LPS-induced ALI mice. Mechanically, we revealed that XFBD treated LPS-induced acute lung injury through PD-1/IL17A pathway which regulates the infiltration of neutrophils and macrophages. Additionally, one major compound from XFBD, i.e. glycyrrhizic acid, shows a high binding affinity with IL17A. In conclusion, we demonstrated the therapeutic effects of XFBD, which provides the immune foundations of XFBD and fatherly support its clinical applications.

Sambucus nigra: A traditional medicine effective in reducing inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sambucus nigra L. is a plant of European origin and popularly known as elder, elderberry, black elder, European elder, European elderberry, and European black elderberry, being described in pharmacopoeia of several countries. Its flowers and berries have been used in folk medicine to treat feverish conditions, coughing, nasal congestion, and influenza besides its popular use as anti-inflammatory, analgesic, and diuretic agent. AIM OF THE STUDY: The aim of this investigation was to elucidate the anti-inflammatory and the relaxant effect of the lyophilized aqueous extract obtained from S. nigra's flowers on in vivo and in vitro inflammation assays and on the isolated rat vascular and airway smooth muscle tissue. MATERIAL AND METHODS: The anti-inflammatory activity of the extract was investigated using carrageenan-induced inflammation model in the subcutaneous tissue of male Swiss mice orally treated with S. nigra extract (30, 100, 300 or 600 mg/kg). Leukocyte influx and the secretion of chemical mediators were quantified in the inflamed exudate. Additionally, histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1ß, IL-10 and NO production, and adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry) were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with LPS and treated with the extract (1, 10 or 100 µg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. Finally, the effect of the extract was evaluated in isolated rat aorta and trachea rings. RESULTS: The oral treatment with S. nigra promoted reduction in the neutrophil migration as well as the decrease of TNF, IL-1ß and IL-6 levels in the inflamed exudate. In vitro treatment with S. nigra decreased NO2-, TNF, IL-1ß and IL-6 and promoted increase of IL-10 in LPS-stimulated neutrophils. Similarly, the extract reduced the NO2-, TNF and IL-6 in LPS-stimulated macrophages. Rutin, the major constituent of S. nigra extract reduced NO2-, TNF, IL-1ß, and IL-6 and promoted the increase of IL-10 in LPS-stimulated neutrophils supernatant. The extract also shed CD62L and CD18 expressions. The extract was able to increase the efferocytosis of apoptotic neutrophils by increasing the IL-10 and decreasing the TNF levels. Additionally, the extract reduced the hypersensitivity induced by carrageenan and promoted a relaxant effect in isolated vascular and non-vascular rat tissue. CONCLUSIONS: S. nigra flowers extract presents anti-inflammatory effect by modulating macrophage and neutrophil functions including the production of inflammatory mediators and cell migration, by promoting efferocytosis and consequently the resolution of acute inflammation, besides exerting antinociceptive effects, scientifically proving its popular use as medicinal plant. Allied to the relaxant effect in both vascular and non-vascular smooth muscle tissue, S. nigra extract represents an important tool for the management of acute inflammation.

Effect of Vitamin D3 in combination with Omega-3 Polyunsaturated Fatty Acids on NETosis in Type 2 Diabetes Mellitus Patients.

An understanding of the consequences of oxidative/halogenative stress triggered by neutrophil activation is impossible without considering NETosis. NETosis, formation of neutrophil extracellular traps (NETs), is known to promote microthrombus formation and impair wound healing in type 2 diabetes mellitus (T2DM) patients. Therefore, there is a need to search for drugs and treatment approaches that could prevent excessive NET formation. We aimed to evaluate the effect of vitamin D3 in combination with omega-3 polyunsaturated fatty acids (vitamin D3/omega-3 PUFAs) on NETosis in T2DM patients with purulent necrotizing lesions of the lower extremities. Patients and healthy subjects had vitamin D3 deficiency. Patients received, beyond standard treatment, 6000 IU of vitamin D3 and 480 mg of omega-3 PUFAs, and healthy subjects 1000 IU of vitamin D3 and 240 mg of omega-3 PUFAs daily for seven days. Neutrophil activation in ex vivo blood by phorbol-12-myristate-13-acetate (PMA) was used as a NETosis model. The percentage of blood NETs relative to leukocytes (NETbackground) before vitamin D3/omega-3 PUFA supplementation was 3.2%-4.9% in healthy subjects and 1.7%-10.8% in patients. These values rose, respectively, to 7.7%-9.1% and 4.0%-17.9% upon PMA-induced NETosis. In addition, the leukocyte count decreased by 700-1300 per 1 µL in healthy subjects and 700-4000 per 1 µL in patients. For both patients and healthy subjects, taking vitamin D3/omega-3 PUFAs had no effect on NETbackground but completely inhibited PMA-induced NET formation, though neutrophils exhibited morphological features of activation. Also, leukocyte loss was reduced (to 500 per 1 µL). For patients on standard treatment alone, changes occurred neither in background NETs and leukocytes nor in their amount after PMA stimulation. The decreased ability of neutrophils to generate NETs, which can be achieved by vitamin D3/omega-3 PUFA supplementation, could have a positive effect on wound healing in T2DM patients and reduce the incidence and severity of complications.

Screening for the Active Anti-Inflammatory and Antioxidant Polyphenols of Gaultheria procumbens and Their Application for Standardisation: From Identification through Cellular Studies to Quantitative Determination.

Aerial parts, leaves, and stems of Gaultheria procumbens are polyphenol-rich herbal medicines with anti-inflammatory and antioxidant effects. The present study focused on identifying active markers of the G. procumbens extracts in an integrated approach combining phytochemical and biological capacity tests. The target compounds, representing all classes of Gaultheria polyphenols, were pre-selected by LC-ESI-PDA-MS/MS. For unambiguous identification, the key analytes, including a rare procyanidin trimer (cinnamtannin B-1), miquelianin potassium salt, and two new natural products: quercetin and kaempferol 3-O-ß-d-xylopyranosyl-(1→2)-ß-d-glucuronopyranosides, were isolated by preparative HPLC and investigated by spectroscopy (HR-ESI-MS, UV-vis, CD, 1D- and 2D-NMR), thiolysis, flame photometry, optical rotation experiments, and absolute configuration studies. The significant contribution of the pre-selected compounds to the biological effects of the extracts was confirmed in vitro: the analytes significantly and in a dose-dependent manner down-regulated the pro-oxidant and pro-inflammatory functions of human neutrophils ex vivo (inhibited the release of reactive oxygen species, IL-1ß, TNF-α, and neutrophils elastase, ELA-2), inhibited two key pro-inflammatory enzymes (cyclooxygenase, COX-2, and hyaluronidase), and most of them, except gaultherin, exerted potent direct antioxidant activity (ferric reducing antioxidant power and superoxide anion scavenging capacity). Moreover, cellular safety was confirmed for all compounds by flow cytometry. Eventually, as these mechanisms have been connected to the health benefits of G. procumbens, 11 polyphenols were accepted as active markers, and a simple, accurate, reproducible, and fully validated RP-HPLC-PDA method for standardisation of the target extracts was proposed.

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

Targeting DCs for Tolerance Induction: Don't Lose Sight of the Neutrophils.

Chronic inflammatory disorders (CID), such as autoimmune diseases, are characterized by overactivation of the immune system and loss of immune tolerance. T helper 17 (Th17) cells are strongly associated with the pathogenesis of multiple CID, including psoriasis, rheumatoid arthritis, and inflammatory bowel disease. In line with the increasingly recognized contribution of innate immune cells to the modulation of dendritic cell (DC) function and DC-driven adaptive immune responses, we recently showed that neutrophils are required for DC-driven Th17 cell differentiation from human naive T cells. Consequently, recruitment of neutrophils to inflamed tissues and lymph nodes likely creates a highly inflammatory loop through the induction of Th17 cells that should be intercepted to attenuate disease progression. Tolerogenic therapy via DCs, the central orchestrators of the adaptive immune response, is a promising strategy for the treatment of CID. Tolerogenic DCs could restore immune tolerance by driving the development of regulatory T cells (Tregs) in the periphery. In this review, we discuss the effects of the tolerogenic adjuvants vitamin D3 (VD3), corticosteroids (CS), and retinoic acid (RA) on both DCs and neutrophils and their potential interplay. We briefly summarize how neutrophils shape DC-driven T-cell development in general. We propose that, for optimization of tolerogenic DC therapy for the treatment of CID, both DCs for tolerance induction and the neutrophil inflammatory loop should be targeted while preserving the potential Treg-enhancing effects of neutrophils.

Effect of the Fungal Immunomodulatory Protein FIP-fve in the Neutrophilic Asthma Animal Model.

BACKGROUND: Asthma animal models provide valuable information about the pathogenesis and the treatment of asthma. An ovalbumin (OVA)/complete Freund's adjuvant (CFA)-sensitized model was developed to induce neutrophil-dominant asthma and to investigate whether fungal immunomodulatory peptide-fve (FIP-fve) could improve asthma features in the OVA/CFA-sensitized model. METHODS: We used female BALB/c mice and sensitized them intraperitoneally with OVA/CFA on days 1, 2, and 3. On days 14, 17, 21, 24, and 27, they were challenged with intranasal OVA. The airway hyper-responsiveness (AHR) was detected by BUXCO, inflammatory cells were stained with Liu's stain, the cytokines were detected using ELISA, and the airway inflammation was analyzed with hematoxylin and eosin stain. RESULTS: According to the results, OVA/CFA sensitization could induce AHR, high levels of IgE, and inflammatory cells especially neutrophils infiltration in the lung and airway inflammation. IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-17, IL-25, IL-33, and transforming growth factor-ß (TGF-ß) increased in the OVA/CFA-sensitized mice. OVA/CFA-sensitized mice treated with FIP-fve not only increased IL-12 and IFN-γ but also decreased IL-4, IL-5, IL-6, IL-8, IL-13, IL-17, IL-25, IL-33, and TGF-ß in the bronchoalveolar lavage fluid. Moreover, FIP-fve significantly decreased neutrophil infiltration in the lung. CONCLUSION: The OVA/CFA model induced neutrophilic asthma successfully, and FIP-fve improved neutrophil-dominant asthma.

Effects of propofol and its formulation components on macrophages and neutrophils in obese and lean animals.

We hypothesized whether propofol or active propofol component (2,6-diisopropylphenol [DIPPH] and lipid excipient [LIP-EXC]) separately may alter inflammatory mediators expressed by macrophages and neutrophils in lean and obese rats. Male Wistar rats (n = 10) were randomly assigned to receive a standard (lean) or obesity-inducing diet (obese) for 12 weeks. Animals were euthanized, and alveolar macrophages and neutrophils from lean and obese animals were exposed to propofol (50 µM), active propofol component (50 µM, 2,6-DIPPH), and lipid excipient (soybean oil, purified egg phospholipid, and glycerol) for 1 h. The primary outcome was IL-6 expression after propofol and its components exposure by alveolar macrophages extracted from bronchoalveolar lavage fluid. The secondary outcomes were the production of mediators released by macrophages from adipose tissue, and neutrophils from lung and adipose tissues, and neutrophil migration. IL-6 increased after the exposure to both propofol (median [interquartile range] 4.14[1.95-5.20]; p = .04) and its active component (2,6-DIPPH) (4.09[1.67-5.91]; p = .04) in alveolar macrophages from obese animals. However, only 2,6-DIPPH increased IL-10 expression (7.59[6.28-12.95]; p = .001) in adipose tissue-derived macrophages. Additionally, 2,6-DIPPH increased C-X-C chemokine receptor 2 and 4 (CXCR2 and CXCR4, respectively) in lung (10.08[8.23-29.01]; p = .02; 1.55[1.49-3.43]; p = .02) and adipose tissues (8.78[4.15-11.57]; p = .03; 2.86[2.17-3.71]; p = .01), as well as improved lung-derived neutrophil migration (28.00[-3.42 to 45.07]; p = .001). In obesity, the active component of propofol affected both the M1 and M2 markers as well as neutrophils in both alveolar and adipose tissue cells, suggesting that lipid excipient may hinder the effects of active propofol.

Anti-Inflammatory and Healing Activity of the Hydroalcoholic Fruit Extract of Solanum diploconos (Mart.) Bohs.

BACKGROUND: Solanum diploconos (Mart.) Bohs is a native Brazilian plant belonging to the Solanaceae family, popularly known as "tomatinho do mato" and poorly investigated. Herein, we presented for the first time evidence for the anti-inflammatory and wound healing activities of S. diploconos fruit hydroalcoholic extract. Material and Methods. In vitro fMLP-induced chemotaxis, LPS-induced inflammatory mediator levels (cytokines by ELISA and NO release by Griess reaction), and adhesion molecule expression (CD62L, CD49d, and CD18, by flow-cytometry) were assessed in neutrophils treated with different concentrations of the extract. Inflammation resolution was measured by the efferocytosis assay and the healing activity by in vivo and in vitro assays. The air pouch model of carrageenan-induced inflammation in Swiss mice was used to investigate the in vivo anti-inflammatory effects of the extract. Leukocyte influx (by optical microscopy) and cytokine release were quantified in the pouch exudates. Additionally, the acute and subacute toxic and genotoxic effects of the extract were evaluated. RESULTS: In vitro, the extract impaired neutrophil chemotaxis and its ability to produce and/or release cytokines (TNFα, IL-1ß, and IL-6) and NO upon LPS stimuli (p < 0.01). LPS-treated neutrophils incubated with the extract presented increased CD62L expression (p < 0.01), indicating a reduced activation. An enhanced efferocytosis of apoptotic neutrophils by macrophages was observed and accompanied by higher IL-10 and decreased TNFα secretion (p < 0.01). In vivo, similar results were noted, including reduction of neutrophil migration, protein exudation, and cytokine release (p < 0.01). Also, the extract increased fibroblast proliferation and promoted skin wound healing (p < 0.01). No signs of toxicity or genotoxicity were observed for the extract. CONCLUSION: S. diploconos fruit extract is anti-inflammatory by modulating neutrophil migration/activation as well macrophage-dependent efferocytosis and inflammatory mediator release. It also indicates its potential use as a healing agent. Finally, the absence of acute toxic and genotoxic effects reinforces its possible use as medicinal product.

Germacranolides from Carpesium divaricatum: Some New Data on Cytotoxic and Anti-Inflammatory Activity.

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.

Chemical Composition and Immunomodulatory Activity of Essential Oils from Rhododendron albiflorum.

Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REOFl) versus leaf (REOLv) essential oils revealed significant differences. REOFl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REOLv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REOLv and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N-formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca2+]i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.

Toxicological assessment of Opuntia dillenii (Ker Gawl.) Haw. cladode methanol extract, fractions and its alpha pyrones: Opuntiol and opuntioside.

ETHNOPHARMACOLOGICAL RELEVANCE: The edible plant Opuntia dillenii (Ker Gawl.) Haw. commonly known as Nagphana, belongs to the Cactaceae family. It is traditionally used to treat various ailments including inflammation, gastric ulcers, diabetes, hepatitis, asthma, whooping cough and intestinal spasm. AIM OF THE STUDY: Despite its traditional use in various countries, detailed toxicological studies of O. dillenii cladode are few. Thus in the current study, toxicity of O. dillenii cladode derived methanol extract, fractions and its α-pyrones: opuntiol and opuntioside have been addressed. METHODS: The test agents were assessed using both in vitro and in vivo toxicity assays. MTT on human embryonic kidney cell line (HEK-293), tryphan blue exclusion in rat neutrophils, Cytokinesis-B block micronucleus (CBMN) in human lymphocytes and genomic DNA fragmentation using agarose gel electrophoresis were performed. In acute toxicity test, mice orally received extract (5 g/kg) for 7 days followed by measurements of relative organ weight, biochemical (blood profile, liver and kidney function test) and histological studies (liver and kidney) were carried out. Rat bone marrow micronucleus genotoxicity assay was also conducted. RESULTS: O. dillenii derived test agents were non-cytotoxic and had no effect on the integrity of DNA. Methanol extract (5 g/kg) orally administered in mice did not cause any significant change in relative organ weights, biochemical parameters and liver and kidney histology as compared to vehicle control. In parallel, extract did not stimulate micronuclei formation in rat bone marrow polychromatic erythrocytes. CONCLUSION: These results led to conclude that edible O. dillenii extract is non-toxic via the oral route and appears to be non-cyto-, hepato-, nephro- or genotoxic, thereby supporting its safe traditional use against various ailments. Therefore, opuntiol and opuntioside may serve as lead compounds in designing new drug(s) derived from edible plants.

Berberine-Based Heterogeneous Linear Supramolecules Neutralized the Acute Nephrotoxicity of Aristolochic Acid by the Self-Assembly Strategy.

Aristolochic acid (AA) has been reported to cause a series of health problems, including aristolochic acid nephropathy and liver cancer. However, AA-containing herbs are highly safe in combination with berberine (Ber)-containing herbs in traditional medicine, suggesting the possible neutralizing effect of Ber on the toxicity of AA. In the present study, in vivo systematic toxicological experiments performed in zebrafish and mice showed that the supramolecule self-assembly formed by Ber and AA significantly reduced the toxicity of AA and attenuated AA-induced acute kidney injury. Ber and AA can self-assemble into linear heterogenous supramolecules (A-B) via electrostatic attraction and π-π stacking, with the hydrophobic groups outside and the hydrophilic groups inside during the drug combination practice. This self-assembly strategy may block the toxic site of AA and hinder its metabolism. Meanwhile, A-B linear supramolecules did not disrupt the homeostasis of gut microflora as AA did. RNA-sequence analysis, immunostaining, and western blot of the mice kidney also showed that A-B supramolecules almost abolished the acute nephrotoxicity of AA in the activation of the immune system and tumorigenesis-related pathways.

The effect of an isoquinoline alkaloid on treatment of periodontitis by regulating the neutrophils chemotaxis.

Neutrophil plays a critical role in the progression of periodontitis. In general, its chemotaxis and activation are benefit for the host defense of bacterial infection and inflammation. However, previous studies have reported that the hyperactive and reactive neutrophils appear to be one of the reasons for tissue destruction in periodontitis tissues. In this study, we investigated an isoquinoline alkaloid Litcubanine A (LA), which from the Traditional Chinese medicinal plant, Litsea cubeba. We found LA showed significant activity in inhibiting neutrophils chemotaxis in the zebrafish yolk sac microinjection model in vivo and in mouse neutrophils in vitro. Further investigation proved that LA could inhibit the expression levels of neutrophil respiratory burst-related and inflammation-related genes CYBB and NCF2, as well as inhibit the activation of MAPK signaling pathway. Moreover, using LA, we successfully achieved the effect of reducing periodontitis bone loss by regulating neutrophil chemotaxis and related functions in a mouse ligature-induced periodontitis model.

Tea tree oil extract causes mitochondrial superoxide production and apoptosis as an anticancer agent, promoting tumor infiltrating neutrophils cytotoxic for breast cancer to induce tumor regression.

The antitumor activity of the tea tree oil (TTO) derived product, Melaleuca Alternifolia Concentrate (MAC) was characterized mechanistically at the molecular and cellular level. MAC was analyzed for its anticancer activity against human prostate (LNCaP) and breast (MCF-7) cancer cell lines growing in vitro. MAC (0.02-0.06% v/v) dose-dependently induced the intrinsic (mitochondrial) apoptotic pathway in both the LNCaP and MCF-7 cell lines, involving increased mitochondrial superoxide production, loss of mitochondrial membrane potential (MMP), caspase 3/7 activation, as well as the presence of TUNEL+ and cleaved-PARP+ cell populations. At concentrations of 0.01-0.04% v/v, MAC caused cell cycle arrest in the G0/1-phase, as well as autophagy. The in vivo anticancer actions of MAC were examined as a treatment in the FVB/N c-Neu murine model for spontaneously arising breast cancers. Intratumoral MAC injections (1-4% v/v) significantly suppressed tumor progression in a dose-dependent manner and was associated with greater levels of tumor infiltrating neutrophils exhibiting anticancer cytotoxic activity. Induction of breast cancer cell death by MAC via the mitochondrial apoptotic pathway was also replicated occurring in tumors treated in vivo. In conclusion, our data highlights the potential for the Melaleuca-derived MAC product inducing anticancer neutrophil influx, supporting its application as a novel therapeutic agent.

Role of taraxerone isolated from Leucas lavandulifolia, as an immunomodulator.

ETHNOPHARMACOLOGICAL RELEVANCE: The Indian tradition system of medicine enlists a large number of plants for basic health care. Leucas lavandulifolia is mentioned in the ayurvedic medicinal system and also used among the folklores. The plant is used for the treatment of fever, asthma, psoriasis, dermatitis and healing snake bites. The scientific validation of the plant for their traditional use in different immune related disorders are yet to be explored. AIM OF THE STUDY: The study aims to isolate immunomodulatory active compound from Leucas lavandulifolia and evaluating its efficiency in immune related disorders. MATERIALS AND METHODS: The immunomodulatory activity of the phytocompound is evaluated through in vitro and in vivo studies. The compound purification and identification were done by chromatography and LC/Q-TOF respectively. Its immunomodulatory activity was evaluated in cells like PBMC, neutrophils and macrophages by MTT assay and cell cycle analysis. Animal studies were performed on Swiss albino mice. The levels of IL-4 and IL-6 cytokines were also evaluated in both in vitro and in vivo models. RESULTS: Leucas lavandulifolia stem portion was found to have good modulatory property. An active immunomodulator was isolated from the methanol extract of the plant. LC/Q-TOF data revealed the isolated compound to be taraxerone. In PBMC, the compound was capable of suppressing the proliferation rate of the compound indicated by a decrease in cell numbers. The activated IL-4 and IL-6 production was also suppressed actively at 25 µg/ml of taraxerone. Similar inhibitory effects were seen in RAW 264.7 and THP-1 macrophage cell lines. An IC50 value of 17.5 µg/ml was obtained for taraxerone in LPS stimulated RAW 264.7 macrophage cell lines. The NO level, IL-4, IL-6 and phagocytosis in the LPS stimulated macrophage was effectively lowered by 25 µg/ml of taraxerone. In PMA stimulated THP-1 Macrophage Cell Lines, taraxerone was capable of suppressing the cell number and IL-6. The compound didn't show any effect on IL-4 levels. The compound exhibited an immunosuppressive activity in PHA induced PMN cells by suppressing the respiratory burst and interleukins IL-4 and IL-6. TX could also suppress the proliferation of DNCB induced monocyte cells and IL-4. The haematological parameters exhibited a significant suppression for the high dose group of taraxerone. The antibody titre and phagocytic index was suppressed by the high dose group, whereas the low dose group did not have any effect. So taraxerone at 50 mg/kg body weight is capable of modulating the B-lymphocytes and macrophages. But the compound has exhibited insignificant effect on the DTH hypersensitivity response and organ index. CONCLUSION: Taraxerone at high concentration was capable of suppressing stimulated PBMC, macrophage and PMN. The activated nitric oxide, IL-4, IL-6 production and phagocytosis was also suppressed. The haematological parameters, antibody titre and phagocytic index was also lowered in antigenically challenged mice. The terpenoid taraxerone exhibits a good modulatory effect on the immune system and proves to be a potent drug for the treatment of many allergic disorders.

Randialic acid B and tomentosolic acid block formyl peptide receptor 1 in human neutrophils and attenuate psoriasis-like inflammation in vivo.

Psoriasis is a long-lasting inflammatory skin disease lacking proper cure. Dysregulated activation of neutrophils is a major pathogenic factor in psoriasis. Formyl peptide receptor 1 (FPR1) triggers neutrophil activation in response to bacteria- or mitochondria-derived N-formyl peptides, but its significance in neutrophilic psoriasis remains unknown. In this study, we discovered two derivatives of ursolic acid, 3ß-hydroxyurs-12,18-dien-28-oic acid (randialic acid B, RAB) and 3ß-hydroxyurs-12,19-dien-28-oic acid (tomentosolic acid, TA), as FPR1 inhibitors in human neutrophils with ability to suppress psoriatic symptoms in mice. Both RAB and TA, triterpenoids of traditional medicinal plant Ilex kaushue, selectively inhibited reactive oxygen species production, elastase release, and CD11b expression in human neutrophils activated by FPR1, but not non-FPR1 agonists. Importantly, RAB and TA inhibited the binding of N-formyl peptide to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells, indicating FPR1 antagonism. Moreover, in assays induced by various concentrations of FPR1 agonist, both RAB and TA acted competitively for its binding to the FPR1 receptor. The FPR1-downstream signaling such as Ca2+ mobilisation and activation of Akt and MAPKs was also competitively inhibited. In addition, imiquimod-induced psoriasis-like symptoms, including epidermal hyperplasia, desquamation with scaling, neutrophil skin infiltration, and transepidermal water loss were significantly reduced by both RAB and TA. The results illustrate a possible role of human neutrophils FPR1 receptor in psoriasis-like inflammation. Accordingly, triterpenoids RAB and TA represent novel FPR1 antagonists and exhibit therapeutic potential for treating neutrophilic inflammatory skin diseases.