Hua-Shi-Bai-Du decoction inactivates NLRP3 inflammasome through inhibiting PDE4B in macrophages and ameliorates mouse acute lung injury.

BACKGROUND: Hua-Shi-Bai-Du decoction (HSBD) exerts significant effects on the prevention and treatment of COVID-19 in China. The activation of the NLRP3 inflammasome of macrophages plays a vital role in COVID-19 pathology. However, no previous studies have focused on this pathological process to explore the effect of HSBD. PURPOSE: Our aim is to uncover the effect of HSBD on NLRP3 inflammasome activation and the underlying mechanisms. METHODS: The NLRP3-activated J774A.1 cells primed by LPS and activated by nigericin/ATP/MSU were used to evaluate NLRP3 activation in vitro. ASC oligomerization and speck formation were assessed by western blot and immunofluorescence imaging. Intracellular K+ levels were determined by the colorimetric assay. Mitochondrial ROS (mtROS) level was detected by the flow cytometry and the fluorescence spectrophotometry. The intracellular cAMP level was determined by chemiluminescence method and ELISA, while phosphodiesterase (PDE) activity was measured using the fluorescent substrate MANT-cAMP. siRNA was applied to knockdown PDE4B. Two in vivo mouse models, MSU-induced peritonitis and LPS-induced acute lung injury (ALI), were used to evaluate the effects of HSBD on IL-1ß and other inflammatory cytokines. Pathological changes in lung tissue were observed by histopathological examination. RESULTS: HSBD not only decreased supernatant IL-1ß, caspase-1 p20, and cleaved gasdermin D (GSDMD) in NLRP3-activated J774A.1 cells, but also reduced IL-1ß in the peritoneal lavage fluid of mice with MSU-induced peritonitis, demonstrating the suppressive effect on NLRP3 inflammasome activation. The mechanism study showed that HSBD blocked ASC oligomerization and speck formation without affecting K+ efflux or mtROS production. Furthermore, it prevented the decrease of intracellular cAMP by inhibiting PDE4B activity. And in the PDE4B-deficient cells, its suppressive effect on IL-1ß release was abolished. In LPS-induced ALI mice, oral administration of HSBD decreased several proinflammatory cytokines (IL-1ß, IL-6, TNF-α, and CXCL-1) and attenuated the pathological damage to the lung. CONCLUSION: HSBD suppresses the activation of NLRP3 inflammasome by inhibiting PDE4B activity to counteract the decrease of intracellular cAMP, thereby blocking ASC oligomerization in macrophages. Our findings may provide new insight into the clinical effets of HSBD for the treatment of COVID-19.

Potentilla discolor ameliorates LPS-induced inflammatory responses through suppressing NF-κB and AP-1 pathways.

Potentilla discolor Bunge (PD) is a traditional Chinese medicine which has been widely used for the treatment of various inflammatory diseases (e.g., diarrhea, fever and furuncle). However, few studies focused on its effect on classical inflammation. This study aimed to investigate the anti-inflammatory effect and potential mechanism of the ethanol extract of the whole herbs of PD (EPD) in lipopolysaccharide (LPS)-induced inflammatory models. The obtained results showed that EPD decreased supernatant NO, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in LPS-activated RAW264.7 cells and mouse peritoneal macrophages. Moreover, its effect on NO was attributed to the suppression of iNOS expression rather than its activity. At the transcriptional level, EPD suppressed iNOS, TNF-α and MCP-1 mRNA expressions in LPS-stimulated RAW264.7 cells. Further study showed that EPD didn't affect the phosphorylation and degradation of IκBα, but yet impeded the nuclear translocation of p65 to inhibit NF-κB activation. Meanwhile, it also prevented JNK, ERK1/2 and p38 phosphorylation to dampen the activation of AP-1. In endotoxemia mouse model, EPD not only decreased interleukin-6, TNF-α and MCP-1 levels in serum, but also potently ameliorated diarrhea. These findings provide the theoretical basis for PD to treat inflammatory diseases, especially intestinal inflammation.

Compound Kushen Injection Induces Immediate Hypersensitivity Reaction Through Promoting the Production of Platelet-Activating Factor via de Novo Pathway.

Compound Kushen Injection (CKI) is a bis-herbal formulation extracted from Kushen (Radix Sophorae Flavescentis) and Baituling (Rhizoma Heterosmilacis Yunnanensis). Clinically, it is used as the adjuvant treatment of cancer. However, with the increased application, the cases of immediate hypersensitivity reactions (IHRs) also gradually rise. In this study, we investigated the underlying mechanism(s) and active constituent(s) for CKI-induced IHRs in experimental models. The obtained results showed that CKI did not elevate serum total IgE (tIgE) and mouse mast cell protease 1 (MMCP1) after consecutive immunization for 5 weeks, but could induce Evans blue extravasation (local) and cause obvious hypothermia (systemic) after a single injection. Further study showed that alkaloids in Kushen, especially matrine, were responsible for CKI-induced IHRs. Mechanism study showed that various platelet-activating factor (PAF) receptor antagonists could significantly counter CKI-induced IHRs locally or systemically. In cell system, CKI was able to promote PAF production in a non-cell-selective manner. In cell lysate, the effect of CKI on PAF production became stronger and could be abolished by blocking de novo pathway. In conclusion, our study identifies, for the first time, that CKI is a PAF inducer. It causes non-immunologic IHRs, rather than IgE-dependent IHRs, by promoting PAF production through de novo pathway. Alkaloids in Kushen, especially matrine, are the prime culprits for IHRs. Our findings may provide a potential approach for preventing and treating CKI-induced IHRs.

Forsythiasides-Rich Extract From Forsythiae Fructus Inhibits Mast Cell Degranulation by Enhancing Mitochondrial Ca2+ Uptake.

Mast cells (MCs) activated via IgE/FcεRI or MAS-related G protein coupled receptor (Mrgpr)-mediated pathway can release granules that play prominent roles in hypersensitivity reactions. Forsythiae Fructus, a well-known traditional Chinese medicine, has been clinically used for allergic diseases. Although previous studies indicated that Forsythiae Fructus extract inhibited compound 48/80-induced histamine release from MCs, its effect on IgE-dependent MC degranulation and possible underlying mechanisms remain to be explored. Herein, we prepared the forsythiasides-rich extract (FRE) and investigated its action on MC degranulation and explored its underlying mechanism. Our data showed that FRE could dampen IgE/FcεRI- and Mrgpr-mediated MC degranulation in vitro and in vivo. Mechanism study indicated that FRE decreased cytosolic Ca2+ (Ca2+ [c]) level rapidly and reversibly. Moreover, FRE decreased Ca2+ [c] of MCs independent of plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). While, along with Ca2+ [c] decrease, the increase of mitochondrial Ca2+ (Ca2+ [m]) occurred simultaneously in FRE-treated RBL-2H3 cells. In the isolated mitochondria, FRE also promoted the subcellular organelle to uptake more extramitochondrial Ca2+. In conclusion, by increasing Ca2+ [m] uptake, FRE decreases Ca2+ [c] level to suppress MC degranulation. Our findings may provide theoretical support for the clinical application of Forsythiae Fructus on allergy and other MC-involved diseases.

Sophoraflavanone M, a prenylated flavonoid from Sophora flavescens Ait., suppresses pro-inflammatory mediators through both NF-κB and JNK/AP-1 signaling pathways in LPS-primed macrophages.

(2R)-3α,7,4'-trihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone is a prenylated flavonoid isolated from the anti-inflammatory herb Sophora flavescens Ait. We firstly named it sophoraflavanone M (SFM) in accordance with trivial names of related constitutes from this plant. Although various studies investigated the anti-inflammatory properties of prenylated flavonoids from Sophora flavescens Ait., that of SFM remains unclear and is yet to be determined. In the current study, we assessed the anti-inflammatory effects of SFM in LPS-induced in vivo and in vitro models. In the serum of endotoxemia mice, SFM significantly suppressed LPS-elevated inflammatory cytokines. Furthermore, at nontoxic concentrations, SFM reduced LPS-induced production of inflammatory mediators NO, IL-6, TNF-α, and MCP-1 in mouse primary peritoneal macrophages. Accordingly, in LPS-primed RAW264.7 cell line, it also inhibited these mediators' expression at both transcriptional and translational levels without cytotoxicity. Mechanistically, SFM is found to concurrently inhibit two important inflammatory signaling pathways, NF-κB and JNK/AP-1. SFM restrained phosphorylation and degradation of IκBα as well as the subsequent p65 translocation to dampen NF-κB activity. Meanwhile, it also suppressed JNK phosphorylation to inhibit the transcriptional activity of AP-1. These results provide material basis for traditional application of the anti-inflammatory herb Sophora flavescens Ait. and suggest SFM is a promising natural candidate for alleviating inflammatory conditions.

Aloe-emodin, a naturally occurring anthraquinone, is a highly potent mast cell stabilizer through activating mitochondrial calcium uniporter.

Mast cells play a fundamental role in immune system. Upon stimulation, they are activated via IgE dependent or independent pathway and then release granules which contain plenty of preformed constituents. Mast cell stabilizers are commonly used clinically for inhibiting the degranulation of mast cells. In the current study, we firstly identified aloe-emodin, a naturally occurring anthraquinone, was a prominent mast cell stabilizer. It could strikingly dampen IgE/FcεRI- and MAS-related G protein coupled receptor (Mrgpr)-mediated mast cell degranulation in vitro and in vivo. Mechanism study indicated that aloe-emodin rapidly and reversibly decreased cytosolic Ca2+ (Ca2+[c]) concentration through enhancing the mitochondrial Ca2+ (Ca2+[m]) uptake. After genetically silencing or pharmacologic inhibiting mitochondrial calcium uniporter (MCU), the effects of aloe-emodin on the Ca2+[c] level and mast cell degranulation were significantly weakened. In contrast to six clinical drugs with mast cell stabilizing properties (amlexanox, tranilast, ketotifen, cromolyn disodium salt, dexamethasone and pimecrolimus), aloe-emodin showed an impressive and potent inhibitory action on the mast cell degranulation. Collectively, aloe-emodin is a highly potent mast cell stabilizer. By directly activating MCU, it decreases Ca2+[c] level to suppress mast cell degranulation. Our study may provide a promising candidate for the treatment of mast cell activation-related diseases.

Penicillin causes non-allergic anaphylaxis by activating the contact system.

Immediate hypersensitivity reaction (IHR) can be divided into allergic- and non-allergic-mediated, while "anaphylaxis" is reserved for severe IHR. Clinically, true penicillin allergy is rare and most reported penicillin allergy is "spurious". Penicillin-initiated anaphylaxis is possible to occur in skin test- and specific IgE-negative patients. The contact system is a plasma protease cascade initiated by activation of factor XII (FXII). Many agents with negative ion surface can activate FXII to drive contact system. Our data showed that penicillin significantly induced hypothermia in propranolol- or pertussis toxin-pretreated mice. It also caused a rapid and reversible drop in rat blood pressure, which did not overlap with IgE-mediated hypotension. These effects could be countered by a bradykinin-B2 receptor antagonist icatibant, and consistently, penicillin indeed increased rat plasma bradykinin. Moreover, penicillin not only directly activated contact system FXII-dependently, but also promoted bradykinin release in plasma incubated-human umbilical vein endothelial cells. In fact, besides penicillin, other beta-lactams also activated the contact system in vitro. Since the autoactivation of FXII can be affected by multiple-factors, plasma from different healthy individuals showed vastly different amidolytic activity in response to penicillin, suggesting the necessity of determining the potency of penicillin to induce individual plasma FXII activation. These results clarify that penicillin-initiated non-allergic anaphylaxis is attributed to contact system activation, which might bring more effective diagnosis options for predicting penicillin-induced fatal risk and avoiding costly and inappropriate treatment clinically.

Amlexanox exerts anti-inflammatory actions by targeting phosphodiesterase 4B in lipopolysaccharide-activated macrophages.

Amlexanox, an anti-inflammatory agent, is widely used for treating aphthous ulcers. Recently, amlexanox has received considerable attention because of its efficacy in mitigating metabolic inflammation via directly suppressing IKKε/TBK1. However, because the knockdown of IKKε/TBK1 has no anti-inflammatory effect on lipopolysaccharide (LPS)-primed RAW264.7 cells, the mechanism of amlexanox against classical inflammation is independent of IKKε/TBK1. In this study, we aim to examine the effects of amlexanox on LPS-treated macrophages and in a mouse model of endotoxemia. We found that amlexanox significantly inhibited the production of pro-inflammatory mediators, both in vitro and in vivo, while increased interleukin-10 level in LPS-activated macrophages. Mechanistically, amlexanox down-regulated nuclear factor κB and extracellular signal-regulated kinase/activator protein-1 signaling by elevating intracellular 3',5'-cyclic adenosine monophosphate (cAMP) level and subsequently activating protein kinase A. Molecular docking along with fluorescence polarization and enzyme inhibition assays revealed that amlexanox bound directly to phosphodiesterase (PDE) 4B to inhibit its activity. The anti-inflammatory effects of amlexanox could be abolished by the application of cAMP antagonist or PDE4B siRNA. In addition to PDE4B, the activities of PDE1C, 3A, and 3B were directly inhibited by amlexanox. Our results provide mechanistic insight into the clinical utility of amlexanox for the treatment of inflammatory disorders and might contribute to extending the clinical indications of amlexanox.

Isodeoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber Linn., inhibits pro-inflammatory mediators' production through both NF-κB and AP-1 pathways in LPS-activated macrophages.

Isodeoxyelephantopin (IDET) has been identified as an anti-tumor natural constituent whose anti-tumor activity and mechanism have been widely investigated. Since the occurrence and development of cancer usually accompany with inflammation, and tumor signaling shares many components with inflammation signaling, the agents with anti-tumor activity are likely to possess anti-inflammation potential. Thus, the current study aims to demonstrate the anti-inflammatory activity along with the underlying mechanism of IDET in lipopolysaccharide (LPS)-primed macrophages. By using Griess method and ELISA, we found that in both bone marrow derived macrophages and alveolar macrophage cell line, IDET, at relatively low concentrations (0.75, 1.5 and 3 µM), could inhibit LPS-induced expression of various pro-inflammatory mediators including nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS), interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) and IL-1ß. Meanwhile, in activated MH-S cells, the inhibitory action of IDET on mRNA expression levels of these cytokines was also detected using qPCR. Mechanistically, the effects of IDET on two key inflammatory signalings, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways, were determined in LPS-activated MH-S cells by reporter gene along with western blot assays. On the one hand, IDET suppressed NF-κB signaling via down-regulating phosphorylation and degradation of inhibitor of NF-κB (IκB)-α and the subsequent p65 translocation. On the other hand, IDET dampened AP-1 signaling through attenuating phosphorylation of both c-jun N-terminal kinase 1/2 (JNK1/2) and extracellular signal regulated kinase 1/2 (ERK1/2). Our study indicates that IDET might be a promising constituent from the anti-inflammatory herb Elephantopus scaber Linn. in mitigating inflammatory conditions, especially respiratory inflammation.

Ethanol extract of Elephantopus scaber Linn. Attenuates inflammatory response via the inhibition of NF-κB signaling by dampening p65-DNA binding activity in lipopolysaccharide-activated macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Elephantopus scaber Linn. (E.scaber) is a widely-used traditional herb whose use has been documented for various inflammatory diseases such as fever, sore throat, dysentery, carbuncle and so on. However, the effect and mechanism of E.scaber in LPS-activated macrophages remain unclear. AIM: This study aims to investigate the anti-inflammatory mechanism of the ethanol extract of E.scaber (ESE) in lipopolysaccharide (LPS)-induced inflammatory models. MATERIALS AND METHODS: Griess reagent was used to determine NO production, and the levels of TNF-α, IL-6, MCP-1 and IL-1ß were determined by ELISA kits. The molecular mechanism research was performed by RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA). LPS-induced endotoxemia mouse model was used for evaluating the in vivo anti-inflammatory action of ESE. RESULTS: ESE suppressed LPS-induced iNOS, TNF-α, IL-6, MCP-1 and IL-1ß transcription as well as supernatant NO, TNF-α, IL-6, MCP-1 and IL-1ß production in macrophages. Although ESE inhibited NF-κB activation, it did not affect the IκBα phosphorylation and degradation and the NF-κB p65 nuclear translocation. The result of EMSA revealed that ESE inhibited the NF-κB p65-DNA binding activity. Additionally, ESE also decreased the proinflammatory cytokines in serum and peritoneal lavage fluid of LPS-induced endotoxemic mice. CONCLUSION: ESE has a potently anti-inflammatory effect through inhibiting the NF-κB p65-DNA binding activity in LPS-activated macrophages.

Canscora lucidissima, a Chinese folk medicine, exerts anti-inflammatory activities by inhibiting the phosphorylation of ERK1/2 in LPS-activated macrophages.

BACKGROUND: Canscora lucidissima (Levl. & Vaniot) Hand.-Mazz. (C. lucidissima), mainly distributed in southern China, has been shown to be effective in the treatment of inflammatory diseases. However, the underlying mechanism of its anti-inflammatory effect is not fully understood. METHODS: In this study, we investigated the anti-inflammatory mechanism of ethanol extract of C. lucidissima (Cl-EE) in lipopolysaccharide (LPS)-induced inflammatory models. ELISA, real-time PCR, Western blot and luciferase reporter assay were used for the experiments in vitro, and ICR mouse endotoxemia model was used for in vivo test. RESULTS: Our data showed that Cl-EE reduced the production of NO by down-regulating the mRNA and protein expression of inducible nitric oxide synthase (iNOS) in LPS-activated RAW 264.7 cells. Meanwhile, it potently decreased other proinflammatory mediators, such as TNF-α, IL-6, MCP-1 and IL-1ß at the transcriptional and translational levels. Further study indicated that Cl-EE did not affect NF-κB signaling pathway but significantly suppressed the phosphorylation of ERK1/2, rather than JNK or p38. In a LPS-induced endotoxemia mouse model, a single intraperitoneal injection of Cl-EE (75-300 mg/kg) could lower circulatory TNF-α, IL-6 and MCP-1 levels. CONCLUSIONS: Collectively, our results indicated that Cl-EE suppressed the phosphorylation level of ERK1/2 thus reducing the transcription and translation of inflammatory genes, thereby exerted anti-inflammatory activity. This study reveals the anti-inflammatory mechanism of C. lucidissima and may provide an effective treatment option for a variety of inflammatory diseases.

Shuang-Huang-Lian Attenuates Airway Hyperresponsiveness and Inflammation in a Shrimp Protein-Induced Murine Asthma Model.

Shuang-Huang-Lian (SHL), an herbal formula of traditional Chinese medicine, is clinically used for bronchial asthma treatment. Our previous study found that SHL prevented basophil activation to suppress Th2 immunity and stabilized mast cells through activating its mitochondrial calcium uniporter. Sporadic clinical reports that SHL was used for the treatment of bronchial asthma can be found. Thus, in this study, we systematically investigated the effects of SHL on asthmatic responses using a shrimp protein (SP)- induced mouse model. SHL significantly inhibited airway inspiratory and expiratory resistance, and histological studies suggested it reduced thickness of airway smooth muscle and infiltration of inflammation cells. It also could alleviate eosinophilic airway inflammation (EAI), including reducing the number of eosinophils and decreasing eotaxin and eosinophil peroxidase levels in the bronchoalveolar lavage fluid (BALF). Further studies indicated that SHL suppressed SP-elevated mouse mast cell protease-1 and IgE levels, prevented Th2 differentiation in mediastinal lymph nodes, and lowered Th2 cytokine (e.g., IL-4, IL-5, and IL-13) production in BALF. In conclusion, SHL attenuates airway hyperresponsiveness and EAI mainly via the inhibition of mast cell activation and Th2 immunity, which may help to elucidate the underlying mechanism of SHL on asthma treatment and support its clinical use.

Qing-Kai-Ling Injection Induces Immediate Hypersensitivity Reaction via the Activation of Anaphylatoxin C3.

Background and Objective: Qing-Kai-Ling (QKL) is derived from a famous ancient Chinese patent medicine Angong Niuhuang pills (ANP) which has been used across Asia, especially in China, for the treatment of "febrile disease," such as stroke, encephalitis and meningitis for hundreds of years. As an extract of ANP without heavy metal, the clinical applicability of QKL is more intensive, of which its injection is commonly used in acute and serious diseases. This study aims to clarify the potential mechanisms of immediate hypersensitivity reaction (IHR) induced by QKL injection (QKLI). Methods: ß-hexosaminidase release assay was performed on the human mast cell line LAD2 and mouse peritoneal mast cells. T helper 2 (Th2) immunity-amplified mice were prepared by aluminum adjuvant. Anaphylactic shock was detected by measuring rectal thermometry in propranolol-pretreated mice. For evaluating microvascular permeability, Evans Blue extravasation assay was used. Serum total IgE (tIgE) and the activated complement-derived anaphylatoxin C3 (C3a) levels were measured by ELISA. Results: QKLI was unable to elevate serum tIgE level in the Th2 immunity-amplified mice, but can increase vasopermeability and trigger anaphylaxis after the first injection. By screening seven fractions of QKLI, only the extract of Isatidis Radix (Isatis tinctoria L.) induced hindpaw Evans Blue extravasation, which was disappeared in Isatidis Radix-free QKLI. Mechanism study indicated that QKLI or Isatidis Radix-caused IHR could be blocked by the antagonists for histamine or C3a, rather than PAF or C5a. Consistently, QKLI and Isatidis Radix could also directly activate human serum complement-derived anaphylatoxin 3 (C3) in vitro with the half effective concentration values of 0.69% and 218.6 µg/ml, respectively. Conclusion: QKLI-IHR is complement activation-related pseudoallergy, rather than an IgE-mediated allergy. QKLI activates C3 and might consequently provoke mast cells to release histamine, which is a principal effector of its IHR. The pseudoallergic reaction induced by QKLI was attributed to the extract of Isatidis Radix. This study suggests a potential therapeutic strategy for the prophylaxis and treatment of QKLI-IHR.

Shuang-Huang-Lian injection induces an immediate hypersensitivity reaction via C5a but not IgE.

Among traditional Chinese medicine injections, intravenous Shuang-Huang-Lian (IV-SHL) has the highest incidence of injection-induced immediate hypersensitivity reactions (IHRs). The precise mechanisms of IV-SHL-induced IHRs remain ambiguous. In this study, we investigated the mechanisms of SHL injection (SHLI)-induced IHRs. Our data showed that serum total IgE and mouse mast cell protease 1 (MMCP1) levels were higher in the SHLI antiserum; however, these effects of SHLI disappeared in the antibiotic-treated mice. SHLI caused intraplantar vasopermeability and shock during the first local or systemic injection. SHLI-induced nonallergic IHRs were attributed to its intermediate fraction F2 (the extract of Lonicerae Japonicae Flos and Fructus forsythiae), and could be blocked by antagonists for histamine or C5a, rather than PAF or C3a. Eight constituents of F2 were able to directly activate C5 to promote local vasopermeability at the mg/mL level. In conclusion, SHLI-induced IHRs are not mediated by IgE. SHLI or its F2 can directly activate blood C5. Subsequently, C5a is likely to provoke histamine release from its effector cells (e.g., mast cells and basophils), indicating that histamine is a principal effector of IHRs induced by SHLI.

Shuang-Huang-Lian prevents basophilic granulocyte activation to suppress Th2 immunity.

BACKGROUND: Basophilic granulocytes (BGs) not only initiate the induction of Th2 cell differentiation, but also amplify the ongoing Th2 response. Shuang-Huang-Lian (SHL) is clinically used for relieving type I hypersensitivity by continuous treatment for several weeks. METHODS: ELISA, flow cytometry, magnetic activated cell sorting, isoelectric precipitation, hybridoma technique, transfection and luciferase reporter assay were used in this study. The statistical analysis was performed using a one-way ANOVA. RESULTS: Our recently published study demonstrated that SHL exerted a remarkable effect on mast cell stabilization. Herein, we sought to elucidate the effect of SHL on shrimp tropomyosin (ST)-induced Th2 immunity and its underlying mechanisms. The obtained data showed that continuous treatment with SHL significantly suppressed ST-stimulated Th2-cytokines release and IgE synthesis. A mechanistic study indicated that SHL not only reduced BG early IL-4 release before ST-specific IgE (sIgE) production, but also inhibited BG activation in the presence of sIgE, including suppressing CD200R surface expression and decreasing IL-4 production. Moreover, SHL markedly decreased the cytosolic Ca2+ (Ca2+[c]) level and inhibited the nuclear factor of activated T cells (NFAT) activation in RBL-2H3 cells. CONCLUSIONS: Collectively, SHL potently reduces ST-induced Th2 immunity by inhibiting the BG Ca2+-NFAT pathway and, thus, suppressing the early IL-4 release before sIgE synthesis and inhibiting BG activation in the presence of sIgE. This study provides the pharmacological basis for the clinical use of SHL to relieve type I hypersensitivity by a successive dose regimen.

Obacunone causes sustained expression of MKP-1 thus inactivating p38 MAPK to suppress pro-inflammatory mediators through intracellular MIF.

Obacunone (OBA) is a highly oxygenated triterpenoid with various pharmacological activities. In this study, we explored its anti-inflammatory effect and underlying mechanisms in LPS-activated macrophages. Our data showed that OBA potently decreased pro-inflammatory mediators (eg, NO, IL-6, IL-1ß, and MCP-1) at the transcriptional and translational levels without cytotoxicity. A mechanism study showed that OBA significantly suppressed p38-mediated AP-1 signaling by stabilizing the mRNA of mitogen-activated protein kinase phosphatase 1 (MKP-1), thus prolonging the expression time of the MKP-1 protein. Next, we used computational target-fishing technology to predict the possible target of OBA. Only one potential target, macrophage migration inhibitory factor (MIF), was presented. Experimentally, the interaction between OBA and MIF was also confirmed. By using an anti-mouse MIF antibody, extracellular MIF (exMIF) was neutralized. Our results showed that autocrine MIF had slight influence on the pro-inflammatory mediator production. Correspondingly, the anti-inflammatory activity of OBA was also not affected. Accordingly, we knocked down the MIF gene in RAW 264.7 cells and obtained stable MIF deficient cells MIF(-), in which the effects of OBA on p38 phosphorylation, AP-1 activation, and pro-inflammatory mediator production in response to LPS nearly disappeared. In contrast to MIF(+) cells, the MKP-1 protein expression time of the MIF(-) cells was markedly prolonged. We conclude that OBA exerts its anti-inflammatory effect by targeting intracellular MIF (inMIF) inhibition to regulate the MKP-1/p38/AP-1 pathway. Our findings also provide a chain of evidence that the inhibition of inMIF, rather than exMIF, may become a novel target for inflammation.

Dietary Flavone Tectochrysin Exerts Anti-Inflammatory Action by Directly Inhibiting MEK1/2 in LPS-Primed Macrophages.

SCOPE: In this study, we investigate the underlying molecular mechanism of the effect of tectochrysin on LPS-primed macrophages. METHODS AND RESULTS: As measured by western blot, RT-PCR, and ELISA, tectochrysin inhibits extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation and sequentially suppressed downstream inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and IL-6 transcription as well as the NO, TNF-α, and IL-6 in supernatant, but it does not affect extracellular signal-regulated kinase (MEK) phosphorylation levels. An enzyme reaction study shows that tectochrysin exerted an inhibitory effect on ERK1/2 phosphorylation by inactivating phosphorylated MEK1/2. Moreover, tectochrysin decreases arginase II expression in LPS-primed RAW264.7 macrophages via reduction of NO production. Tectochrysin also suppresses inflammatory mediator release in peritoneal lavage fluid and in the serum of LPS-induced endotoxemia mice. CONCLUSION: Our data indicate that by directly inactivating p-MEK1/2, tectochrysin decreases the phosphorylation level of ERK and subsequently suppresses activator protein-1 (AP-1) activation to reduce pro-inflammatory mediator production, suggesting that tectochrysin has great potential for use in a nutritional preventive strategy against inflammation-related diseases.

The Three-Herb Formula Shuang-Huang-Lian stabilizes mast cells through activation of mitochondrial calcium uniporter.

Mast cells (MCs) are key effector cells of IgE-FcεRI- or MrgprX2-mediated signaling event. Shuang-Huang-Lian (SHL), a herbal formula from Chinese Pharmacopoeia, has been clinically used in type I hypersensitivity. Our previous study demonstrated that SHL exerted a non-negligible effect on MC stabilization. Herein, we sought to elucidate the molecular mechanisms of the prominent anti-allergic ability of SHL. MrgprX2- and IgE-FcεRI-mediated MC activation in vitro and in vivo models were developed by using compound 48/80 (C48/80) and shrimp tropomyosin (ST), respectively. Our data showed that SHL markedly dampened C48/80- or ST-induced MC degranulation in vitro and in vivo. Mechanistic study indicated that cytosolic Ca2+ (Ca2+[c]) level decreased rapidly and sustainably after SHL treatment, and then returned to homeostasis when SHL was withdrawn. Moreover, SHL decreases Ca2+[c] levels mainly through enhancing the mitochondrial Ca2+ (Ca2+[m]) uptake. After genetically silencing or pharmacologic inhibiting mitochondrial calcium uniporter (MCU), the effect of SHL on the Ca2+[c] level and MC degranulation was significantly weakened. Simultaneously, the activation of SHL on Ca2+[m] uptake was completely lost. Collectively, by activating MCU, SHL decreases Ca2+[c] level to stabilize MCs, thus exerting a remarkable anti-allergic activity, which could have considerable influences on clinical practice and research.

Highly specific and sensitive immunoassay for the measurement of prostaglandin E2 in biological fluids.

BACKGROUND: Lack of specificity of anti-PGE2 antibodies is a long-standing problem. Given quite a few analogs and low PGE2 content in biological fluids, it is quite important to simultaneously meet the demands of high specificity and sensitivity. RESULTS: Highly specific anti-PGE2 antibodies were obtained by combined use of cationic carrier protein and Mannich reaction. The cross-reactivity values of the resultant polyclonal and monoclonal antibodies against eight analogs were <14 and <5%, respectively. Furthermore, we established a highly sensitive ELISA, which could be applied to direct analysis of PGE2 at the pg/ml level (LOQ = 15.6 pg/ml). CONCLUSION: We provide an appropriate strategy to develop a highly-specific and sensitive immunoassay for measuring low PGE2 content in biological samples.

Shuang-huang-lian attenuates lipopolysaccharide-induced acute lung injury in mice involving anti-inflammatory and antioxidative activities.

Shuang-Huang-Lian (SHL) is a common traditional Chinese preparation extracted from Lonicerae Japonicae Flos, Scutellariae Radix, and Fructus Forsythiae. In this study, we demonstrate the anti-inflammatory and antioxidative effects of SHL on lipopolysaccharide- (LPS-) induced acute lung injury (ALI) in mice. SHL reduced the lung wet/dry weight ratio, lowered the number of total cells in the bronchoalveolar lavage fluid, and decreased the myeloperoxidase activity in lung tissues 6 h after LPS treatment. It also inhibited the overproduction of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) in the bronchoalveolar lavage fluid. Histological studies demonstrated that SHL attenuated LPS-induced interstitial edema, hemorrhage, and the infiltration of neutrophils into the lung tissue. Moreover, SHL could also enhance the superoxide dismutase and catalase activities, increase the reduced glutathione content, and decrease the malondialdehyde content. The present results suggest that SHL possesses anti-inflammatory and antioxidative properties that may protect mice against LPS-induced ALI.