IL-6 In RAW Research Articles

Integrating network pharmacology and transcriptomics to study the potential mechanism of Jingzhi Niuhuang Jiedu tablet in rats with accumulation of heat in the lungs and stomach

Ethnopharmacological relevanceAccumulation of heat in the lungs and stomach (AHLS) is an important syndrome within the realm of traditional Chinese medicine (TCM). It is the fundamental reason behind numerous illnesses, including mouth ulcers, dermatological conditions, acne, and pharyngitis. Jingzhi Niuhuang Jiedu tablet (JN) serves as the representative prescription for treatment of AHLS clinically. However, the effective components and mechanism of JN's impact on AHLS remain unexplored. Aim of the studyThe objective of this research was to analyze the effective components of JN and investigate the therapeutic effect and potential mechanism of JN on AHLS. Materials and methodsThe effective compounds of JN extract were analyzed and identified using UHPLC-Q-Exactive/HRMS. Utilizing network pharmacology to investigate JN's multi-target, multi-pathway process in treating AHLS. Subsequently, anti-inflammatory activities of JN extract were evaluated in the RAW264.7 cells stimulated by lipopolysaccharide (LPS). In addition, a rat AHLS model induced by LPS and dried ginger was established. Pathological changes in rat lung and stomach tissues observed by HE staining and Masson's trichrome staining. Additionally, the expression of TNF-α, IL-6, and IL-1β in bronchoalveolar lavage fluid (BALF) was identified through the ELISA assay. For a deeper understanding of how JN might affect AHLS, transcriptomics was utilized to examine differential genes and their underlying mechanisms. Concurrently, techniques like quantitative polymerase chain reaction (q-PCR), immunofluorescence, and western blotting (WB) were employed to confirm various mRNA and protein expression, including Il17ra, Il17re, IL-17A, IL-1β, IL-6, PPARγ, PGC1-α and UCP1. ResultsWe identified 178 potential effective components in the JN extract. Network pharmacology analysis showed that the 144 components in JN act on 200 key targets for the treatment of AHLS by suppressing inflammation, regulating energy metabolism, and gastric function. In addition, JN suppressed the LPS-stimulated generation of NO, TNF-α, IL-1β, and IL-6 in RAW264.7 cells. And JN treatment effectively alleviated lung and stomach injury and reduced inflammation in rats. Analysis of RNA-seq from lung tissues revealed JN's substantial control over crucial genes in the IL-17 signaling pathway, including Il1b and Il17ra. Likewise, RNA sequencing of stomach tissues revealed that JN markedly decreased crucial genes in the Thermogenesis pathway, including Ppargc1a and Ppara. Additional experimental findings confirmed that treatment with JN significantly reduced the expression levels of mRNA (Il17ra, Il17re, Il1b, Ppargc1a and Ucp1), and the expression levels of protein (IL-17A, IL-1β, IL-6, PPARγ, PGC1-α and UCP1). ConclusionThis study not only analyzes the effective components of JN but also reveals that JN could effectively ameliorate AHLS by inhibiting IL-17 signaling pathway and Thermogenesis pathway, which provides evidence for subsequent clinical studies and drug development.

Lipoteichoic Acid from Heyndrickxia coagulans HOM5301 Modulates the Immune Response of RAW 264.7 Macrophages.

Heyndrickxia coagulans (formerly Bacillus coagulans) has been increasingly utilized as an immunomodulatory probiotics. Oral administration of H. coagulans HOM5301 significantly boosted both innate and adaptive immunity in mice, particularly by increasing the phagocytic capacity of monocytes/macrophages. Lipoteichoic acid (LTA), a major microbe-associated molecular pattern (MAMP) in Gram-positive bacteria, exhibits differential immunomodulatory effects due to its structural heterogeneity. We extracted, purified, and characterized LTA from H. coagulans HOM5301. The results showed that HOM5301 LTA consists of a glycerophosphate backbone. Its molecular weight is in the range of 10-16 kDa. HOM5301 LTA induced greater productions of nitric oxide, TNFα, and IL-6 in RAW 264.7 macrophages compared to Staphylococcus aureus LTA. Comparative transcriptome and proteome analyses identified the differentially expressed genes and proteins triggered by HOM5301 LTA. KEGG analyses revealed that HOM5301 LTA transcriptionally and translationally activated macrophages through two immune-related pathways: cytokine-cytokine receptor interaction and phagosome formation. Protein-protein interaction network analysis indicated that the pro-inflammatory response elicited by HOM5301 LTA was TLR2-dependent, possibly requiring the coreceptor CD14, and is mediated via the MAPK and NF-kappaB pathways. Our results demonstrate that LTA is an important MAMP of H. coagulans HOM5301 that boosts immune responses, suggesting that HOM5301 LTA may be a promising immunoadjuvant.

Therapeutic effect of oxidized bletilla striata polysaccharide-natamycin eye drops on fungal keratitis.

Fungal keratitis (FK) usually develops to a poor clinical prognosis due to the fungal invasion and excessive inflammatory reaction. In order to enhance the therapeutic effect of natamycin (NAT), we used the anti-inflammatory biological polysaccharide bletilla striata polysaccharide (BSP) combined with NAT to prepare a new eye drop -- oxidized bletilla striata polysaccharide-natamycin (OBN). UV-vis, FT-IR, and fluorescence spectroscopy were used to identify the synthesis of OBN. Biocompatibility of OBN was determined by CCK-8, scratch assay, and corneal toxicity test. RAW264.7 cells and C57BL/6 mice were stimulated with A. fumigatus and treated with PBS, OBN, or NAT. The anti-inflammatory activity of OBN was detected by RT-PCR and ELISA. In mice with FK, the clinical scores were used to evaluate the effect of OBN; HE staining was performed to assess the corneal pathological changes; MPO assay and immunofluorescence staining were used to investigate neutrophil infiltration. OBN was synthesized by combining oxidized bletilla striata polysaccharide (OBSP) with NAT through Schiff base reaction. OBN did not affect cell viability at a concentration of 160μg/mL in HCECs, RAW264.7 cells, and mouse corneas. OBN versus NAT significantly improved the prognosis of A. fumigatus keratitis by reducing disease severity, neutrophil infiltration, and expression of inflammatory factors in vivo. Additionally, OBN treatment down-regulated the mRNA and protein expression levels of inflammatory factors IL-1β, TNF-α, and IL-6 in RAW264.7 and mouse models. OBN is a compound prepared by covalently linking OBSP to the imino group of NAT through Schiff base reaction. OBN treatment down-regulated inflammation and improved the prognosis of mice with A. fumigatus keratitis.

Megastigmane glycosides from the traditional Uighur medicine Cydonia oblonga Mill.

Phytochemical investigation on the fruits of Cydonia oblonga Mill., a traditional Uighur medicine, led to the isolation of seven undescribed and nine known megastigmane glycosides. Their structures including absolute configurations were characterized by an extensive analysis of spectroscopic data including HRESIMS and NMR, combined with ECD calculations. Additionally, compounds 1, 2, 4, and 6–16 exhibited anti-inflammatory activity by inhibiting the secretion of cytokines TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharides (LPS) with inhibitory rates of 10.79%–44.58% at 20 μM.

M1 Macrophage-Targeted Curcumin Nanocrystals with l-Arginine-Modified for Acute Lung Injury by Inhalation

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) with clinical manifestations of respiratory distress and hypoxemia remains a significant cause of respiratory failure, boasting a persistently high incidence and mortality rate. Given the central role of M1 macrophages in the pathogenesis of acute lung injury (ALI), this study utilized the anti-inflammatory agent curcumin as a model drug. l-arginine (L-Arg) was employed as a targeting ligand, and chitosan was initially modified with l-arginine. Subsequently, it was utilized as a surface modifier to prepare inhalable nano-crystals loaded with curcumin (Arg-CS-Cur), aiming for specific targeting of pulmonary M1 macrophages. Compared with unmodified chitosan-curcumin nanocrystals (CS-Cur), Arg-CS-Cur exhibited higher uptake in vitro by M1 macrophages, as evidenced by flow cytometry showing the highest fluorescence intensity in the Arg-CS-Cur group (P < 0.01). In vivo accumulation was greater in inflamed lung tissues, as indicated by small animal imaging demonstrating higher lung fluorescence intensity in the DiR-Arg-CS-Cur group compared to the DiR-CS-Cur group in the rat ALI model (P < 0.05), peaking at 12 h. Moreover, Arg-CS-Cur demonstrated enhanced therapeutic effects in both LPS-induced RAW264.7 cells and ALI rat models. Specifically, treatment with Arg-CS-Cur significantly suppressed NO release and levels of TNF-α and IL-6 in RAW264.7 cells (p < 0.01), while in ALI rat models, expression levels of TNF-α and IL-6 in lung tissues were significantly lower than those in the model group (P < 0.01). Furthermore, lung tissue damage was significantly reduced, with histological scores significantly lower than those in the CS-Cur group (P < 0.01). In conclusion, these findings underscore the targeting potential of l-arginine-modified nanocrystals, which effectively enhance curcumin concentration in inflammatory environments by selectively targeting M1 macrophages. This study thus introduces novel perspectives and theoretical support for the development of targeted therapeutic interventions for acute inflammatory lung diseases, including ALI/ARDS.

An anti-inflammatory active ingredients in Poriae cutis: Screening based on network pharmacology.

Hyperuricemia (HUA) is a disorder of uric acid metabolism, which can lead to the formation of gouty arthritis, kidney inflammation and other damages. Previous studies have found that the alcohol extract of Poria cutis can reduce the level of uric acid and protect against kidney injury. Based on network pharmacology, the core targets and main active components of P. cutis intervention in HUA were determined. Most of the potential active ingredients are triterpenoid acids such as tumulosic acid (TA) and eburicoic acid (EA), and the potential targets are TNF and IL-6, which are associated with inflammation. In vitro experiments have shown that TA can significantly inhibit the release of NO, TNF-α and IL-6 in inflammatory RAW264.7 cell culture medium and the expression of TNF-α and IL-6 in RAW264.7 cells. This study suggests that TA based on network pharmacological screening has obvious anti-inflammatory effect on inflammatory RAW264.7 cells and is a promising anti-inflammatory compound.

Purification, structural characterization, and immunoregulatory activity of a polysaccharide from mulberry branch

In view of the fact that mulberry branch has not been effectively utilized and polysaccharide is one of the main active components in mulberry branch, this study aims to reveal the structure and immunomodulatory activity of its polysaccharide. A type of neutral polysaccharides, named mulberry branch polysaccharide-2 (MBP-2), was separated from mulberry branch using DEAE-52 and Sephadex G-100. As analyzed, they were mainly composed of glucose with a molecular weight of approximately 21.7 kDa. Methylation analysis demonstrated that MBP-2 primary contained a → 4)-α-d-Glcp-(1 → , α-d-Glcp-(1 → and → 4, 6)-α-d-Glcp-(1 → structure, which was validated by nuclear magnetic resonance (NMR). In addition, cellular experiments indicated that MBP-2 significantly enhanced the production of NO, TNF-α and IL-6 in RAW264.7 cells, unraveling the potential immunoregulatory activity of MBP-2. Further analysis showed that MBP-2 exerted their immunoregulatory activity mainly via binding with TLR4 to activate the downstream TRIF-dependent signaling pathways.Graphical

In vitro immune-enhancing effects of Platycodon grandiflorum combined with Salvia plebeian via MAPK and NF-κB signaling in RAW264.7 cells.

The immune-enhancing activity of the combination of Platycodon grandiflorum and Salvia plebeian extracts (PGSP) was evaluated through macrophage activation using RAW264.7 cells. PGSP (250-1000 μg/mL) showed a higher release of NO in a dose-dependent manner. The results showed that PGSP could significantly stimulate the production of PGE2, COX-2, TNF-α, IL-1β, and IL-6 in RAW264.7 cells and promote iNOS, COX-2, TNF-α, IL-1β, IL-4, and IL-6 mRNA expression. Western blot analysis demonstrated that the protein expression of iNOS and COX-2 and the phosphorylation of ERK, JNK, p38, and NF-κB p65 were greatly increased in PGSP-treated cells. PGSP also promoted the phagocytic activity of RAW264.7 cells. All these results indicated that PGSP might activate macrophages through MAPK and NF-κB signaling pathways. Taken together, PGSP may be considered to have immune-enhancing activity and might be used as a potential immune-enhancing agent.

Antimicrobial and Anti-inflammatory Cyclic Tetrapeptides from the Co-cultures of Two Marine-Derived Fungi.

Violaceotides B-E (1-4), four new cyclic tetrapeptides, along with seven known compounds, were identified from the sponge-associated Aspergillus insulicola IMB18-072 co-cultivated with the marine-derived Alternaria angustiovoidea IMB20-805. Their structures were elucidated by extensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR, and MS/MS data. The absolute configurations were determined by the advanced Marfey's method. Compounds 2, 3, and violaceotide A (5) displayed selective antimicrobial activities against the aquatic pathogenic bacteria Edwardsiella tarda and E. ictaluri. In addition, compounds 1-5 showed inhibitory activities against the LPS-induced expression of the inflammatory mediator IL-6 in RAW264.7 cells at a concentration of 10 μM.

Echinatin attenuates acute lung injury and inflammatory responses via TAK1-MAPK/NF-κB and Keap1-Nrf2-HO-1 signaling pathways in macrophages.

Echinatin is an active ingredient in licorice, a traditional Chinese medicine used in the treatment of inflammatory disorders. However, the protective effect and underlying mechanism of echinatin against acute lung injury (ALI) is still unclear. Herein, we aimed to explore echinatin-mediated anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated ALI and its molecular mechanisms in macrophages. In vitro, echinatin markedly decreased the levels of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated murine MH-S alveolar macrophages and RAW264.7 macrophages by suppressing inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) expression. Furthermore, echinatin reduced LPS-induced mRNA expression and release of interleukin-1β (IL-1β) and IL-6 in RAW264.7 cells. Western blotting and CETSA showed that echinatin repressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways through targeting transforming growth factor-beta-activated kinase 1 (TAK1). Furthermore, echinatin directly interacted with Kelch-like ECH-associated protein 1 (Keap1) and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway to enhance heme oxygenase-1 (HO-1) expression. In vivo, echinatin ameliorated LPS-induced lung inflammatory injury, and reduced production of IL-1β and IL-6. These findings demonstrated that echinatin exerted anti-inflammatory effects in vitro and in vivo, via blocking the TAK1-MAPK/NF-κB pathway and activating the Keap1-Nrf2-HO-1 pathway.

Effects of Lactobacillus plantarum FM 17 fermentation on jackfruit polysaccharides: Physicochemical, structural, and bioactive properties

Fermentation is a novel technology for modifying polysaccharides in fruits and improving their bioactivities. In this work, we introduced Lactobacillus plantarum FM 17 to ferment jackfruit pulp and subsequently purified polysaccharides from unfermented (JP) and fermented jackfruit pulp (JP-F). Furthermore, the physicochemical, structural, and bioactive properties of JP and JP-F were investigated. Results showed fermentation dropped the glucuronic acid, molecular weight, and particle size of JP-F by 15.62 %, 23.92 %, and 39.43 %, respectively, compared with those of JP. JP-F showed higher solubility than JP but lower apparent viscosity and thermal stability. Additionally, FT-IR spectra and X-ray diffraction analysis showed that fermentation did not alter the different types of glycosidic bonds and the fundamental polysaccharide structure. Moreover, JP-F exhibited stronger DPPH and ABTS free radical scavenging properties than JP and stronger stimulation on macrophage secretion of NO and IL-6 in RAW 264.7 cells. Therefore, using L. plantarum FM 17 for fermentation can alter physical and chemical properties of jackfruit pulp polysaccharides, enhancing their bioactivities.

Lanostane triterpenoids from mycelia-associated Ganoderma sinense and their anti-inflammatory activity

Seven previously undescribed lanostane triterpenoids, ganoderic acid M1 (1), M2 (2), M3 (3), M4 (4), M5 (5), M6 (6), and M7 (7), together with eight known compounds, were isolated from mycelia of the basidiomycete Ganoderma sinense (Ganodermataceae). The structures of all compounds were elucidated by spectroscopic analysis. The possible biosynthetic pathway of these fifteen triterpenoids was proposed. Some of the compounds were evaluated for their anti-inflammatory activity by measuring the production of nitric oxide (NO), TNF-α, and IL-6 in RAW264.7 macrophage cells induced by lipopolysaccharide. Lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid (14) exhibited the strongest inhibition of NO production with an IC50 of 0.6 ± 0.1 μM and completely inhibited the secretion of TNF-α and IL-6 at 10 μM. The structure-activity relationship of the anti-inflammatory activity is discussed.

Simultaneous Presentation of Dexamethasone and Nerve Growth Factor via Layered Carbon Nanotubes and Polypyrrole to Interface Neural Cells.

The implantation of neural electrodes usually induces acute and chronic inflammation, which can result in the formation of glial scars encapsulating the implanted electrodes and the loss of neurons near the active electrode sites. Local presentation of anti-inflammatory drugs or neural protective factors has been evidenced as an effective strategy to modulate inflammatory responses and promote electrode-neuron integration. Here, a novel delivery system for the simultaneous presentation of both anti-inflammatory drugs (dexamethasone, Dex) and nerve-growth-promoting factors (nerve growth factor, NGF) from the electrode sites was developed via layer-structured carbon nanotubes and conductive polymers. The modified electrodes exhibited higher charge storage capacitance and lower electrochemical impedance compared to unmodified electrodes and electrodes coated with polypyrrole/Dex. Dex released from the functional coating under controlled electrochemical stimulation was able to inhibit the lipopolysaccharide-induced secretion or mRNA transcription of inflammatory cytokines, including nitric oxide, TNF-α, and IL-6 in RAW264.7 cells, and control the activation of cultured astrocytes. In addition, the functional coatings did not show a toxic effect on PC12 cells and primary neural cells but exhibited promoted activities on the adhesion, growth, and neurite extension of neural cells.

Therapeutic Role and Potential Mechanism of Resveratrol in Atherosclerosis: TLR4/NF-κB/HIF-1α.

Atherosclerosis, the main pathological basis of cardiovascular disease, is a chronic inflammatory disease that severely affects the quality of human life. Resveratrol (Res) is a natural polyphenol that is a major component of many herbs and foods. The present study analyzed resveratrol from the perspective of visualization and bibliometric analysis and found that resveratrol is closely related to the inflammatory response in cardiovascular diseases (associated with atherosclerosis). To explore the specific molecular mechanism of resveratrol, network pharmacology and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used, in which HIF-1α signaling may be a key pathway in the treatment of AS. Furthermore, we induced the polarization of macrophage RAW264.7 to M1 type to generate inflammatory response by the combination of lipopolysaccharide (LPS) (200 ng/mL) + interferon-γ (IFN-γ) (2.5 ng/mL). LPS and IFN-γ increased the inflammatory factor levels of IL-1β, TNF-α, and IL-6 in RAW264.7, and the proportion of M1-type macrophages also increased, but the expression of inflammatory factors decreased after resveratrol administration, which confirmed the anti-inflammatory effect of resveratrol in AS. In addition, we found that resveratrol downregulated the protein expression of toll-like receptor 4 (TLR4)/NF-κB/hypoxia inducible factor-1 alpha (HIF-1α). In conclusion, resveratrol has a significant anti-inflammatory effect, alleviates HIF-1α-mediated angiogenesis, and prevents the progression of AS through the TLR4/NF-κB signaling pathway.

Protective Effect of Anoectochilus formosanus Polysaccharide against Cyclophosphamide-Induced Immunosuppression in BALB/c Mice.

In this study, Anoectochilus formosanus polysaccharide (AFP) was acquired a via water extraction and alcohol precipitation method. The immunoregulatory activity of AFP was first evaluated on cyclophosphamide (Cy)-treated mice. Galacturonic acid, glucose and galactose were confirmed to be the main components of AFP. AFP demonstrated the ability to stimulate the production of TNF-α and IL-6 in RAW 264.7 macrophages. Not surprisingly, the activation of the NF-κB signaling pathway by AFP was validated via Western blot analysis. Furthermore, AFP could alleviate Cy-induced immunosuppression, and significantly enhance the immunity of mice via increasing the thymus index and body weight, stimulating the production of cytokines (IgA, IgG, SIgA, IL-2, IL-6 and IFN-γ). The improvement in the intestinal morphology of immunosuppressed mice showed that AFP could alleviate Cy-induced immune toxicity. These results have raised the possibility that AFP may act as a natural immunomodulator. Overall, the study of AFP was innovative and of great significance for AFP's further application and utilization.

New natural pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and iNOS inhibitors identified from Penicillium polonicum through in vitro and in vivo studies.

Overexpression of pro-inflammatory cytokines and iNOS have been found to be concomitant with several chronic inflammatory diseases and hence targeting their inhibition would be a useful therapy for inflammation. In view of this, study on discovery of natural pro-inflammatory cytokines inhibitory lead molecules from Penicillium polonicum, an endophytic fungus isolated from the fresh fruits of Piper nigrum was performed. When the culture broth extract of P. polonicum (EEPP) was subjected to LPS-induced cytokines expression (ELISA in RAW 264.7 cells), it exhibited inhibition of TNF-α, IL-6 and IL-1β and this encouraged us to do chemical investigation on EEPP to explore the bioactive components. Four compounds isolated and characterised as 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid (1), 2,4-di-tert-butyl phenol (2), indole 3-carboxylic acid (3) and tyrosol (4) were tested for their effect on the production of TNF-α, IL-1β and IL-6 in RAW 264.7 cells (ELISA). All the compounds exhibited a highly significant (P<0.0001) inhibition effect, particularly against IL-1β (IC50: 4-0.91μM, 1-2.81μM, 3-4.38μM, and 2-5.54μM). Tyrosol (4) was most active with IC50 values of 0.91, 2.67 and 4.60μM against IL-1β, IL-6 and TNF-α, respectively. On observing the potential activity of the compounds, two compositions C1 and C2 were prepared by mixing equimolar concentrations of compounds 1, 2, 3 & 4 (C1) and compounds 1, 2, 3, 4 & piperine (C2) in equal ratio. A synergistic effect was observed with C1 exhibiting potential suppression of IL-6 secretion (IC50 1.91μM) and C2 against IL-1β (IC50 5.98μM). Also, the individual compounds and C1 were effective in controlling iNOS expressions in RAW 264.7 cells (RTPCR). Further, the in vivo performance of the compounds and compositions were studied under two in vivo inflammatory models (LPS-induced endotoxaemia and carrageenan-induced paw oedema). Compounds 1, 2, 3, 4, C1 and C2 at 50mg/kg oral dose showed a significant control over the LPS-stimulated TNF-α, IL-1β and IL-6 levels in plasma. C1, C2 and 1 exhibited>50% pan-cytokine inhibition effect. Under the carrageenan-induced anti-inflammatory model, a significant reduction in the paw oedema measured in terms of the difference in the paw thickness was observed. Further, attenuation of pro-inflammatory cytokines levels following ELISA and RT-PCR experiments in the paw tissue homogenate was in agreement with paw thickness results. All compounds and C1 decreased the iNOS gene expression levels, and also the MPO activity and NO production in the paw tissue homogenate with tyrosol (4) as the most active molecule. Further, the mechanism of action was explored by testing the effect of the compounds on the expression of inflammatory markers using western blot analysis (in vitro). They were found to regulate the expression of pro-form and matured-form of IL-1β by inhibiting NFκB. Also, the compounds reduced the translocation of the NF-κB subunit p65 to the nucleus. Thus, compounds 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid (1), 2,4-di-tert-butyl phenol (2), indole 3-carboxylic acid (3) and tyrosol (4) are reported as new natural multiple pro-inflammatory cytokines inhibitory leads. The interesting results of C1 might lay a footing for the development of a new anti-inflammatory composition.

Immunosuppressive diarylpropane dimer and spirocyclic-monomers from Horsfieldia kingii

Horsfiequinone G (1), a dimeric diarylpropane featuring an unprecedentedly oxo-6/7/6 fused ring system, a new flavane, horsfielenide F (2), three naturally occurring spirocyclic monomers containing all-carbon quaternary centers, horspirotone A (3), horspirotone B (4), and methyl spirobroussonin B (5), along with horsfiequinone A (6) were isolated from Horsfieldia kingii. Their structures and absolute configurations were determined by the inspection of extensive spectroscopic data and electronic circular dichroism (ECD) calculations. Biological evaluations of these isolates revealed that compounds 1 – 3 and 5 – 6 exhibited specifically immunosuppressive activities against Con A-induced T lymphocytes with IC50 values ranging from 2.07 to 12.34 μM (selectivity indices = 2.3–25.2). Compound 1 also suppressed the secretion of inflammatory factors like IL-1β and IL-6 in RAW264.7 cells which could present a new class of nonsteroidal anti-inflammatory agent. Finally, the primary structure–activity relationship (SAR) was also discussed.

Baicalin Relieves LPS-Induced Lung Inflammation via the NF-κB and MAPK Pathways

Baicalin is an active ingredient extracted from the Chinese medicine Scutellaria and has many beneficial effects. Pulmonary interstitial and alveolar edema are common symptoms of an acute lung injury (ALI). We investigated the effects of baicalin on LPS-induced inflammation and the underlying mechanisms in mice and cells. The protein contents and mRNA expression of TNF-α, IL-1β, and IL-6 in RAW264.7 cells and mice were detected using ELISA and qRT-PCR. Baicalin significantly suppressed TNF-α, IL-1β, and IL-6 levels and expression, both in vitro and in vivo, compared with the LPS group. Baicalin inhibits the expression of TLR4 and MyD88, resulting in significant decreases in p-p65, p-p38, p-ERK, and p-JNK, as measured by the Western blotting of RAW264.7 cells. A baicalin treatment for 12 h resulted in a rapid increasing of the white blood cell number and significantly improved the pathological changes in the lung. We also found that the baicalin pretreatment for 12 h could decrease the MPO content and wet/dry (W/D) weight ratio, which indicates that baicalin can significantly reduce pulmonary edema. Furthermore, the baicalin pretreatment also resulted in the recovery of TGF-β protein levels and decreased iNOS. Baicalin inhibits ALI inflammation in mice and cells and is a potential candidate for the treatment of ALI.

Attenuation of Lipopolysaccharide-Induced Inflammatory Responses through Inhibition of the NF-κB Pathway and the Increased NRF2 Level by a Flavonol-Enriched n-Butanol Fraction from Uvaria alba.

Pathologic hyperreactive inflammatory responses occur when there is excessive activation of a proinflammatory NF-κB pathway and a reduced cytoprotective NRF2 cascade. The noncytotoxic, highly selective COX-2 inhibitory flavonol-enriched butanol fraction (UaB) from Uvaria alba (U. alba) was investigated for its inflammatory modulating potential by targeting NF-κB activation and NRF2 activity. Enzyme-linked immunosorbent assay was initially performed to measure levels of proinflammatory mediators [nitric oxide (NO), prostaglandin E2, and reactive oxygen species (ROS)] and cytokines [tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6], followed by reverse transcription-polymerase chain reaction and western blotting to determine mRNA and protein expression, respectively. Using immunofluorescence staining combined with western blot analysis, the activation of NF-κB was further investigated. NRF2 activity was also measured using a luciferase reporter assay. UaB abrogated protein and mRNA expressions of inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages, thereby suppressing the production of proinflammatory mediators and cytokines. This was further validated when a concentration-dependent decrease in NO and ROS production was observed in zebrafish (Danio rerio) larvae. UaB also increased NRF2 activity in HaCaT/ARE cell line and attenuated NF-κB activation by inhibiting the nuclear translocation of transcription factor p65 in RAW 264.7 macrophages. Nontargeted LC-MS analysis of UaB revealed the presence of the flavonols quercitrin (1), quercetin (2), rutin (3), kaempferol (4), and kaempferol 3-O-rutinoside (5). Molecular docking indicates that major flavonol aglycones have high affinity toward COX-2 NSAID-binding sites, TNF-α, and TNF-α converting enzyme, while the glycosylated flavonoids showed strong binding toward iNOS and IKK-all possessing dynamic stability when performing molecular dynamics simulations at 140 ns. This is the first report to have elucidated the mechanistic anti-inflammatory potential of the Philippine endemic plant U. alba.

Synergistic Anti-Inflammatory Effects of Ethanol Extracts from Chrysanthemum zawadskii Flower and Cudrania tricuspidata Fruit Occur via Inhibition of the NF-κB Signaling Pathway.

Chrysanthemum zawadskii (CZ) and Cudrania tricuspidata (CT) are both traditional Korea herbal medicines, which is widely used to treat fever, cough, gastritis, and women's diseases that may be linked to inflammatory response. Although it has been used to treat diseases related to inflammation, there has been no case of the synergistic anti-inflammatory properties of both extracts. Our data revealed that ethanol extracts of dried whole CZ exhibited free radical-scavenging capacity in vitro, reduced LPS-induced intracellular reactive oxygen species, and decreased the LPS-induced upregulations of the mRNAs encoding iNOS, COX-2, and IL-6 in RAW 264.7 cells, without significant cytotoxicity. This anti-inflammatory effect was most evident from flower extracts: ethanol extracts from flowers significantly reduced the LPS-induced upregulations of iNOS and COX-2 at a concentration of 100 μg/ml. An ethanol extract of the fruit from CT also exerted a radical scavenging capacity and suppressed LPS-induced proinflammatory gene expression: 5.5 μg/ml of the ethanol extract significantly reduced the ability of LPS to induce the mRNA expression levels of iNOS and IL-6 without apparent cytotoxicity. Furthermore, as little as 1.0 μg/ml of the combined ethanol extracts of CZ flower and CT fruit reduced the LPS-induced changes monitored herein, decreasing the upregulations of iNOS and IL-6, and decreasing the nuclear localization of NF-κB p65. These results suggest that the observed synergistic anti-inflammatory effects may be mediated via inhibition of NF-κB signaling. Taken together, these data suggest that ethanol extracts from CZ flowers and CT fruits have synergistic anti-inflammatory effects and that a combination of the two extracts could prove useful for the treatment of inflammation-related diseases.