Lipopolysaccharide-stimulated Macrophages Research Articles

Systems Biology and Multi‐Omics

Systems Biology and Multi‐Omics

A hybrid platform featuring nanomagnetic ligand fishing for discovering COX-2 selective inhibitors from aerial part of Saussurea laniceps Hand.-Mazz

Ethnopharmacological relevanceSaussurea laniceps Hand.-Mazz. (Compositae) is a representative “snow lotus” herb well known in Chinese folk medicine to treat inflammation-related diseases such as arthritis. S. laniceps (SL) shows anti-inflammatory and analgesic potencies and contains various constituents potentially with cyclooxygenase-2 (COX-2) selective inhibition. The herb is a valuable source of natural alternatives to synthetic COX-2 selective nonsteroidal anti-inflammatory drugs, a common medication for rheumatoid arthritis (RA) and osteoarthritis (OA) reported with serious cardiovascular side effects. Aim of the studyBased on an innovative drug screening platform, this study aimed to discover safe, effective COX-2 selective inhibitors from SL. Materials and methodsAn enzyme-anchored nanomagnetic fishing assay was developed to separate COX-2 ligands from SL. Cell and animal models of cardiomyocytes, lipopolysaccharide-stimulated macrophages, rat adjuvant-induced arthritis, and anterior cruciate ligament transection-induced OA rats, were adopted to screen the single/combined ligands regarding toxicity and bioactivity levels. Molecular docking was employed to unravel binding mechanisms of the ligands towards COX-1 and COX-2. ResultsFour COX-2 selective compounds were separated from SL using optimized COX-2-functionalized magnetic nanoparticles. All the four ligands were proved with evidently lower cardiotoxicity both in vitro and in vivo than celecoxib, a known COX-2 selective inhibitor. Two ligands, scopoletin and syringin, exhibited potent anti-arthritic activities in rat models of RA and OA by alleviating clinical statuses, immune responses, and joint pathological features; their optimum combination ratio was discovered with stronger remedial effects on rat OA than single administrations. The COX-1/2 binding modes of the two phytochemicals contributed to explain their cardiac safety and therapeutic performances. ConclusionsThe screened chemicals are promising to be developed as COX-2 selective inhibitors as part of treating RA and OA. The hybrid strategy for discovering therapeutic agents from SL is shown here to be efficient; it should be equally valuable for finding other active chemicals in other natural sources.

Polymerized Luteolin Nanoparticles: Synthesis, Structure Elucidation, and Anti-Inflammatory Activity.

Luteolin is an anti-inflammatory flavonoid commonly found in many edible plants. The compound is popularly consumed as a supplement regardless of its poor water solubility (27.8 μg/mL at 25 °C) and low bioavailability. Here, mild one-pot polymerization of luteolin into water-dispersible nanospheres, with an average dry size of 234.8 ± 101.6 nm, an aqueous size distribution of 379.1 ± 220.5 nm (PDI = 0.338), an average ζ-potential of −36.2 ± 0.2 mV, and an 89.3 ± 4.8% yield, is described. The nanospheres consist of polymerized luteolin (polyluteolin) with a weight-average molecular mass of around 410000 Da. The chemical structure of polyluteolin is identified through 1H–1H correlated spectroscopy (COSY), 1H–13C heteronuclear single-quantum coherence (HSQC), and 1H–13C heteronuclear multiple-bond correlation (HMBC) NMR spectroscopic analyses of the oligomers, and a polymerization mechanism is proposed. Unlike luteolin that showed both dose-dependent anti-inflammatory activity and cytotoxicity when tested in lipopolysaccharide-stimulated macrophages, the polyluteolin nanoparticles possess dose-dependent anti-inflammatory activity without causing cell death even at high concentrations.

In vitro and in vivo anti-inflammatory activity and chemical composition of Renealmia petasites Gagnep.

The study aimed to investigate the chemical composition and the anti-inflammatory activity of the hydroethanolic rhizomes, stems, and leaf extracts of Renealmia petasites using in vitro and in vivo assays. The chemical composition of the extracts was characterized in a linear iron trap mass spectrometer. Total phenolic, flavonoid, and tannin content were determined by spectrophotometry analyses. In vitro anti-inflammatory activity was investigated in lipopolysaccharide-stimulated macrophages evaluating the influence on the production of superoxide anion (O2-), nitric oxide (NO), and the pro-inflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). In vivo effects were determined using the air pouch model in which were inoculated carrageenan and thereafter treated with 50mg/kg of the hydroethanolic extracts of R. petasites. After 4 and 24h, the cellular influx, protein exudation, cytokines, and nitric oxide were evaluated. Eight compounds were tentatively identified in the R. petasites extracts, suggesting five diarylheptanoids, one flavonoid, and two fatty alcohols. The in vitro results showed that the extracts were capable of blocking free radicals and/or inhibiting their intracellular actions by inhibiting the production of important mediators of the inflammatory process, such as NO, O2-, TNF-α, and IL-6. In vivo, R. petasites significantly decrease the influx of leukocytes, mainly neutrophils, protein exudation, NO, TNF-α, and IL-6 concentration in the air pouch model. The results evidenced that R. petasites can be considered a promising alternative therapy for the treatment and management of osteoarthritis and other inflammatory diseases.

Phytochemical Constituents of Citrus hystrix DC. Leaves Attenuate Inflammation via NF-κB Signaling and NLRP3 Inflammasome Activity in Macrophages.

Citrus hystrix DC. (CH) is found in many countries in Southeast Asia. This plant has been reported for anti-microbial, anti-cancer and anti-inflammatory bioactivities. However, the anti-inflammatory and anti-inflammasome properties of the leaves remain poorly understood. This study aimed to investigate the effect of CH leaves on NLRP3 and NF-κB signaling pathways. CH leaves were sequentially extracted using hexane, ethyl acetate and 95% ethanol to give three crude extracts. An active compound, lupeol was fractionated from the ethanolic extract using chromatographic techniques, and its structure was identified and confirmed by spectroscopic methods. Anti-inflammatory activities were observed on both lipopolysaccharide-stimulated and NLRP3 adenosine triphosphate-induced macrophages. The release of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) was analyzed by Enzyme-Linked Immunosorbent Assay (ELISA). Real-time qRT-polymerase chain reaction (PCR) was used to measure inflammatory-associated gene expression. NF-κB protein expressions were investigated using the immunoblotting technique. The active fraction of ethanolic CH leaves and lupeol significantly reduced the release of pro-inflammatory cytokines and suppressed the expression of both inflammasome genes and NF-κB proteins. The ethanolic extract of CH leaves and lupeol showed potent anti-inflammatory activities by targeting NF-κB and NLRP3 signaling pathways.

Chemical composition, antimycobacterial and anti-inflammatory activities of iridoids and triterpene from Psychotria suterella (Rubiaceae)

Background: Psychotria species are known for their medicinal properties and psychoactive activities. Extracts of Psychotria suterella showed anti-tuberculosis (TB) activity; however, the substances related to this activity are unknown. Objectives: The objective was to study on the chemical constituents of the leaves of plant and evaluate the anti-TB and anti-inflammatory activity. Materials and Methods: Solvent extraction, partition, and column chromatography were used to separate the compounds. The structures of these compounds were determined by extensive one dimensional-and two dimensional-Nuclear Magnetic Resonance, infrared and mass spectrometry spectroscopic analyses. Some compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis and their ability to inhibit nitric oxide (NO) production by lipopolysaccharide-stimulated macrophages. Results: This study led to the isolation and characterization of a new iridoid, named epi-geniposidic acid (1), together with nine known compounds: geniposidic acid (2), 3-O-acethyloleanolic acid (3), pomolic acid (4), spinolic acid (5), maslinic acid (6), tormentic acid (7), methyl oleanolate (8), lyalosidic acid (9), and strictosidinic acid (10). Triterpene 3-O-acethyloleanolic acid (3) was found to display antimycobacterial activity against M. tuberculosis H37Rv strain and hypervirulent strain (minimum inhibitory concentration 6.7 ± 0.1 and 89.1 ± 1.3 μg/mL, respectively). Epi-geniposidic acid (1), geniposidic acid (2), and 3-O-acethyloleanolic acid (3) showed promising inhibitory activities against NO production (IC50 range 4.12–5.12 μg/mL). The iridoid mixture showed no cytotoxicity against RAW 264.7 macrophages up to a concentration of 100 μg/mL. Conclusion: P. suterella presents relevant biological properties and should be considered for further in vivo studies using a pulmonary TB model.

Anti-inflammatory and analgesic potential of Amorphophallus commutatus var. wayanadensis and its inhibitory effect on inflammatory mediators in lipopolysaccharide-stimulated macrophages

Background: An edible tuber named Amorphophallus commutatus var. wayanadensis (ACW) is used by the local ethnic communities of Wayanad, India, for hemorrhoids while the health benefits remain unexplored to the scientific community. Objectives: Hence, our study was performed to screen the anti-inflammatory and analgesic activities of ACW. Materials and Methods: Screening for the in vitro anti-inflammatory potential on isolated peritoneal macrophages was performed using nitrite assay, nitroblue tetrazolium assay, tumor necrosis factor (TNF)-α production, and cyclooxygenase (COX) enzyme activity. Carrageenan- and formalin-induced paw edema assays were performed to determine anti-inflammatory potential, while tail immersion assays and acetic acid-induced writhing assays were carried out to explore antinociceptive activity in animal models. Results: Bio-molecular mechanistic investigation to evaluate the in-vitro anti-inflammatory activity confirmed the suppressive effect of MEAC on TNF-α, nitric oxide and COX-2 on lipopolysaccharide stimulated peritoneal macrophages, which benchmarked ACW as a potent medicinal plant against inflammation. Further, the efficacy of methanolic extract of A. commutatus var. wayanadensis (MEAC) as an anti-inflammatory agent in murine anti-inflammatory models was demonstrated by formalin- and carrageenan-induced paw edema assays. Administration of MEAC significantly increased the tail flicking latency in mice and also showed prominent reduction in the number of writhes induced by acetic acid, which establishes the real time application of ACW as an analgesic agent. Conclusion: The vital information regarding in vitro and in vivo action of MEAC provided scientific evidence for its traditional usage for hemorrhoids. Thus, this herbal medicine of ethnic tribes can be translated toward modern medicine for health maintenance and disease prevention.

Anti-inflammatory effect of IL-1ra-loaded dextran/PLGA microspheres on Porphyromonas gingivalis lipopolysaccharide-stimulated macrophages in vitro and in vivo in a rat model of periodontitis

Periodontitis is a multifactorial chronic infectious disease leading to a host immune response involving inflammatory cytokines, especially IL-1β, which is the main reason for further developing this disease. IL-1 receptor antagonist (IL-1ra) binds IL-1 receptor, inhibiting IL-1β signaling and reducing the levels of other cytokines closely related to periodontitis, such as IL-6 and TNF-α. Therefore, the use of IL-1ra to inhibit periodontitis development in a system, ensuring its sustained release, might be an effective way to combat this disease. Hence, in this study, a novel IL-1ra-loaded dextran/PLGA microsphere was developed to allow the sustained release of IL-1ra and enhance the anti-inflammatory properties. Therefore, this study’s purposes were to develop a novel periodontal treatment for inhibition and treatment of periodontitis and evaluate the sustained-release effect and anti-inflammatory properties of IL-1ra-loaded dextran/PLGA microspheres in vitro by cell experiments and in vivo by animal experiments. The results showed that IL-1ra-loaded dextran/PLGA microspheres were non-toxic both in vitro and in vivo and could be used as a safe and effective treatment. In addition, these microspheres could significantly prolong the half-life of IL-1ra drug, exerting a useful anti-inflammatory effect in macrophages stimulated with P. gingivalis lipopolysaccharide and in rats with periodontitis. In conclusion, IL-1ra-loaded dextran/PLGA microsphere might be a useful tool to combat periodontal disease.

The Extraction Solvent Influences the Anti-Inflammatory Effects of Jakyakgamcho-Tang in Lipopolysaccharide-Stimulated Macrophages and Mice with Gouty Arthritis.

Jakyakgamcho-Tang (JGT) is a traditional medicine used to treat muscular tension, spasm, and pain. Several studies have reported its clinical use as an anti-inflammatory and in gynaecological treatment. This study aimed to compare the anti-inflammatory effects of JGT according to extraction solvent, water (JGTW) and 30% EtOH (JGTE) on lipopolysaccharide (LPS)—stimulated macrophages and in mice with monosodium urate (MSU)—induced gouty arthritis. We evaluated the production of inflammatory mediators and cytokines and the expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. We also examined oedema, pain, and inflammation in MSU-induced mice by measuring affected hind paw swelling, weight-bearing, pro-inflammatory cytokines levels, and myeloperoxidase (MPO) activity. In LPS-stimulated RAW264.7 cells, JGTW and JGTE significantly decreased prostaglandin (PG) E2(PGE2) production via suppressing COX-2 expression and cytokines interleukin-1β and interleukin-6. Only JGTE reduced the production of NO and cytokines and the mRNA levels of iNOS and cytokines. In MSU-induced mice, JGTE and JGTW efficiently decreased paw swelling and attenuated joint pain. JGTE (200 and 300 mg/kg) effectively suppressed inflammation by downregulating pro-inflammatory cytokines (tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) and MPO activity, which were only slightly reduced by JGTW. Our data demonstrate the anti-inflammatory activity of JGT in macrophage and gouty arthritis animal models and show that JGTE is more effective than JGTW at lower concentrations.

Identification of Paracentrone in Fucoxanthin-Fed Mice and Anti-Inflammatory Effect against Lipopolysaccharide-Stimulated Macrophages and Adipocytes.

Fucoxanthin is converted to fucoxanthinol and amarouciaxanthin A in the mouse body. However, further metabolism such as cleavage products (i.e., apocarotenoids) remains unclear. The fucoxanthin-derived apocarotenoid in vivo is investigated and the anti-inflammatory effect of apocarotenoids with fucoxanthin partial structure such as allenic bond and epoxide residue against activated macrophages and adipocytes in vitro is evaluated. LC-MS analysis indicates the presence of paracentrone, a C31 -allenic-apocarotenoid, in white adipose tissue of diabetic/obese KK-Ay and normal C57BL/6J mice fed 0.2% fucoxanthin diet for 1 week. In lipopolysaccharide-activated RAW264.7 macrophages, paracentrone as well as C26 - and C28 -allenic-apocarotenoids suppresses the overexpression of inflammatory factors. Further, apo-10'-fucoxanthinal, a fucoxanthin-derived apocarotenoid which retained epoxide residue, exhibits a most potent anti-inflammatory activity through regulating mitogen-activated protein kinases and nuclear factor-κB inflammatory signal pathways. In contrast, β-apo-8'-carotenal without allenic bond and epoxide residue lacks suppressed inflammation. In 3T3-L1 adipocytes, paracentrone, and apo-10'-fucoxanthinal downregulate the mRNA expression of proinflammatory mediators and chemokines induced by co-culture with RAW264.7 cells. Dietary fucoxanthin accumulates as paracentrone as well as fucoxanthinol and amarouciaxanthin A in the mouse body. Allenic bond and epoxide residue of fucoxanthin-derived apocarotenoids have pivotal roles for anti-inflammatory action against activated macrophages and adipocytes.

N-Ethyl-2-Pyrrolidinone-Substituted Flavan-3-Ols with Anti-inflammatory Activity in Lipopolysaccharide-Stimulated Macrophages Are Storage-Related Marker Compounds for Green Tea.

Fresh green tea (GT) is commonly considered to have better sensory flavor and higher commercial value than long-term-stored GT; however, the chemical variations during storage are unclear. In this study, the chemical profiles of stored GT were surveyed among time-series samples from 0 to 19 months using a nontargeted metabolomics method. Seven N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) increased from 0.022 ± 0.019 to 3.212 ± 0.057 mg/g within 19 months (correlation coefficients with storage duration ranging from 0.936 to 0.965), and they were the most significantly increased compounds among the 127 identified compounds. Two representative EPSFs (R-EGCG-cThea and S-EGCG-cThea) possess potential anti-inflammatory properties by suppressing the expression, phosphorylation, and nuclear translocation of nuclear factor kappa-B (NF-κB) p65 in lipopolysaccharide-stimulated macrophages based on western blotting and immunofluorescence results. In conclusion, EPSFs were found to be marker compounds for stored GT and showed potential anti-inflammatory activity by regulating the NF-κB signaling pathway.

ZCPG, a cysteine protease from Zingiber montanum rhizome exhibits enhanced anti-inflammatory and acetylcholinesterase inhibition potential

A 48 kDa Zingiber montanum cysteine protease glycoprotein (ZCPG) purified previously was studied for anti-inflammatory and acetylcholinesterase inhibitory activity. The lipoxygenase inhibition by ZCPG was linear, with an IC50 value of 2.25 μM. MTT, LDH, and cell cycle analysis in THP-1 derived macrophages corroborate no significant cytotoxicity at a lower concentration. ZCPG inhibited the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in lipopolysaccharide-stimulated THP-1 macrophages. In contrast, an increase in the production of interleukin-10, an anti-inflammatory cytokine, was observed. A reverse-transcription polymerase chain reaction study further confirmed that ZCPG inhibited the expression of IL-1β, inducible nitric oxide synthase, and TNF-α by suppressing their mRNA transcription and expression in LPS stimulated THP-1 macrophages. Furthermore, the nature of acetylcholinesterase (AChE) inhibition by ZCPG is dose-dependent, competitive, and reversible. The AChE inhibitory activity was stable in a broad range of temperatures and pH. In vitro data were further validated by molecular interaction studies with a detailed inspection of the ZCPG probable binding modes in the active sites of AChE that provides the lead to deliver the structural determinants necessary for the activity towards AChE.

Combined Targeted Proteomics and Oxylipin Metabolomics for Monitoring of the COX-2 Pathway.

The important role of inducible cyclooxygenase-2 (COX-2) in several diseases necessitates analytical tools enabling thorough understanding of its modulation. Analysis of a comprehensive oxylipin pattern provides detailed information about changes in enzyme activities. In order to simultaneously monitor gene expression levels, a targeted proteomics method for human COX-2 is developed. With limits of detection and quantification down to 0.25 and 0.5 fmol (on column) the method enables sensitive quantitative analysis via LC-MS/MS within a linear range up to 2.5pmol. Three housekeeping proteins are included in the method for data normalization. A tiered approach for method development comprised of in silico and experimental steps is described for choosing unique peptides and selective and sensitive SRM transitions while avoiding isobaric interferences. This method combined with a well-established targeted oxylipin metabolomics method allows to investigate the role of COX-2 in the human colon carcinoma cell lines HCT-116, HT-29, and HCA-7. Moreover, the developed methodology is used to demonstrate the time-dependent prostanoid formation and COX-2 enzyme synthesis in lipopolysaccharide-stimulated human primary macrophages. The described approach is a helpful tool which will be further used as standard operation procedure, ultimately aiming at comprehensive targeted proteomics/oxylipin metabolomics strategies to examine the entire arachidonic acid cascade.

Biscaesalmins A and B from Caesalpinia minax, highly oxidized dimeric cassane diterpenoids as interleukin-1β inhibitors

To search naturally occurring interleukin-1β (IL-1β) inhibitors, biscaesalmins A (1) and B (2), two highly oxidized dimeric cassane diterpenoids with a newly formed alicyclic skeleton, have been isolated from the traditional Chinese medicine Kushilian (Caesalpinia minax). Their full structures were determined by comprehensive spectroscopic analysis and quantum chemical TD-DFT (time-dependent density functional theory) calculation. Biosynthetically, 1 and 2 were formed via an intermolecular [4 + 2] Diels-Alder cycloaddition of two monomers, affording an additional six-membered carbon ring linkage. Compounds 1 and 2 inhibited nitric oxide production on lipopolysaccharide-stimulated THP-1 macrophages, with IC50 values being at 1.20 ± 0.23 and 2.30 ± 0.15 μmol/L, respectively. Furthermore, compound 1 inhibited NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome-mediated IL-1β production and blocked the migration of macrophages towards adipocyte conditioned medium. Biscaesalmins A and B might be candidates for treating inflammation-related metabolic diseases.

Protective effects of HY1702 on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice

Acute respiratory distress syndrome is an inflammatory disease with no effective pharmacological treatment. We investigated the therapeutic effect of HY1702, a new small molecule diterpene obtained from the processing and modification of Glaucocalyxin A and may exhibit anti-inflammatory activity. Specifically, we studied the anti-inflammatory effects of HY1702 on lipopolysaccharide-induced inflammatory responses in RAW264.7 and THP-1 cells in vitro and its protective efficacy on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice.Our results showed that HY1702 significantly decreased lipopolysaccharide-induced inflammatory cytokine expression in RAW264.7 and THP-1 cells and attenuated the secretion of nitric oxide and prostaglandin E2 by down-regulating the expression of inducible nitric oxide synthase and cyclooxygenase 2 in RAW264.7 cells. In mice with lipopolysaccharide-induced mild acute respiratory distress syndrome, HY1702 alleviated histological alterations in the lungs and reduced the alveolar cavity protein leakage and inflammatory cytokine expression in murine bronchial alveolar lavage fluid. HY1702 decreased the myeloperoxidase activity and lung wet to dry weight ratio. In our mechanism studies in lipopolysaccharide-exposed RAW264.7 cells, HY1702 suppressed the inflammation stimulated by lipopolysaccharide through inhibiting phosphorylation of inhibitor of nuclear factor κB kinase subunit α/β (IKKα/β) and inhibitor of nuclear factor κB subunit α (IκBα), further affecting the nuclear transfer of phosphorylated p65. Meanwhile, phosphorylation of p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) was inhibited. These data suggest that HY1702 can reduce inflammation on lipopolysaccharide-stimulated macrophages and attenuate the symptoms of mild acute respiratory distress syndrome in a murine model by regulating the nuclear factor κB and MAP kinase signalling pathways.

Luteolin attenuates imiquimod–induced psoriasis-like skin lesions in BALB/c mice via suppression of inflammation response

Psoriasis is considered as a common chronic immune-mediated skin disorder characterized by abnormal keratinocyte proliferation. Luteolin, an anti-inflammatory natural flavonoid with well-accepted inhibition effect against keratinocyte proliferation, was hypothesized to have a potential therapeutic effect for psoriasis. In this paper, we investigated the relieving effect of luteolin against imiquimod-induced psoriasis-like lesions on BALB/c mice and its possible anti-inflammatory mechanisms in lipopolysaccharide-stimulated macrophages (RAW264.7 cells). We found that luteolin ameliorated psoriasis-like skin lesions, suppressed the cutaneous infiltration of macrophages, T cells and neutrophils, and downregulated the expression of cytokines like IL-6, IL-1β, TNF-α, IL-17A and IL-23 in both skin lesions and eyeball blood of model mice. In vitro, we observed luteolin significantly suppressed the levels of psoriasis-related pro-inflammatory cytokines, such as IL-17A, IL-6, TNF-α and IL-23, and inflammatory mediators like nitric oxide NO, inducible NOS, COX-2 in RAW264.7 cells. The anti-inflammatory activity was accomplished by inhibiting NF-κB expression and activation. This study demonstrates luteolin is effective in alleviating psoriasis-like skin lesions and downregulating inflammatory response via NF-κB pathway, suggesting luteolin as a potential molecule for further therapeutic research of inflammation-related skin diseases like psoriasis.

RvE1 Attenuates Polymicrobial Sepsis-Induced Cardiac Dysfunction and Enhances Bacterial Clearance.

The development of cardiac dysfunction caused by microbial infection predicts high mortality in sepsis patients. Specialized pro-resolving mediators (SPMs) mediate resolution of inflammation in many inflammatory diseases, and are differentially expressed in plasma of sepsis patients. Here, we investigated whether the levels of SPMs are altered in the murine septic heart following polymicrobial sepsis-induced cardiac dysfunction. Ten weeks-old male C57BL/6 mice were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP), which is a clinically relevant sepsis model receiving analgesics, antibiotics, and fluid resuscitation. CLP caused a significant systolic dysfunction assessed by echocardiography. The hearts were subjected to LC-MS/MS based lipid mediator profiling. Many SPMs were significantly reduced in septic hearts, among which RvE1 had a ~93-fold reduction. Treatment of CLP mice with synthetic RvE1 (1 μg/mouse i.v.) at 1 h after CLP increased peritoneal macrophages number, particularly MHC II− macrophages. RvE1 reduced pro-inflammatory gene expression (interleukin-1β, interleukin-6, and CCL2) in lipopolysaccharide-stimulated bone marrow-derived macrophages (BMDMs) in vitro. RvE1 attenuated cardiac dysfunction in septic mice and increased cardiac phosphorylated Akt; decreased cardiac phosphorylated IκB kinase α/β, nuclear translocation of the NF-κB subunit p65, extracellular signal–regulated kinase 1/2, and c-Jun amino-terminal kinases 1/2. Most notably, RvE1 treatment reduced peritoneal bacterial load and promoted phagocytosis activity of BMDMs. In conclusion, cardiac SPMs, particularly RvE1, are substantially reduced in mice with polymicrobial sepsis. Delayed therapeutic administration of RvE1 to mice with polymicrobial sepsis attenuates the cardiac dysfunction through modulating immuno-inflammatory responses. In addition to the above effects, the ability to enhance bacterial clearance makes RvE1 an ideal therapeutic to reduce the sequalae of polymicrobial sepsis.

The Development of Hyaluronan/Fucoidan-Based Nanoparticles as Macrophages Targeting an Epigallocatechin-3-Gallate Delivery System.

The aim of this study was to develop a macrophage-targeted nanoparticle composed of hyaluronan/fucoidan complexes with polyethylene glycol-gelatin to encapsulate and deliver epigallocatechin-3-gallate (EGCG), a compound that can regulate macrophage activation and pro-inflammatory mediator production. We show that our nanoparticles can successfully bond to macrophages and deliver more EGCG than an EGCG solution treatment, confirming the anti-inflammatory effects of these nanoparticles in lipopolysaccharide-stimulated macrophages. The prepared nanoparticles were established with a small mean particle size (217.00 ± 14.00 nm), an acceptable polydispersity index (0.28 ± 0.07), an acceptable zeta potential value (−33.60 ± 1.30 mV), and a high EGCG loading efficiency (52.08% ± 5.37%). The targeting abilities of CD44 binding were increased as the hyaluronan concentration increased and decreased by adding a competitor CD44 antibody. Moreover, we found that fucoidan treatment significantly reduced macrophage migration after lipopolysaccharide treatment in a dose-responsive manner. In summary, we successfully created macrophage-targeted nanoparticles for effective targeted delivery of EGCG, which should aid in the development of future anti-inflammatory drugs against macrophage-related diseases.

Stomopneulactone D from long-spined sea urchin Stomopneustes variolaris: Anti-inflammatory macrocylic lactone attenuates cyclooxygenase-2 expression in lipopolysaccharide-activated macrophages

Cyclooxygenase-2 is one of the prominent enzymes to cause an increased production of prostaglandins during inflammation and immune responses. Cyclooxygenase-2 expression is up-regulated in inflammatory conditions owing to the induction by different inflammatory stimuli including cytokines, and therefore, the expression studies of cyclooxygenase-2 in lipopolysaccharide-induced macrophage cells (RAW 264.7 cell line) could be used for screening of the compounds with anti-inflammatory potential. The present study evaluated the anti-inflammatory properties of four homologous stomopneulactones A-D, classified under the class of macrocyclic lactones isolated from the solvent extract of the long-spined sea urchin Stomopneustes variolaris (familyStomopneustidae) in the lipopolysaccharide-induced macrophages. The structures of these isolated compounds were assigned using detailed spectroscopic techniques. Stomopneulactone D bearing 5-butyl-4-hydroxy-12-oxo-1-oxa-5,9-cyclododecadienyl moiety exhibited relatively greater anti-inflammatory potentials against cyclooxygenase-2 (IC50 ~ 2 mM) and 5-lipoxygenase (IC50 2.6 mM) than those displayed by other macrocyclic lactones. The studied compounds displayed higher selectivity index values (anti-cyclooxygenase-1IC50/anti-cyclooxygenase-2IC50 > 1), which designated the selective anti-inflammatory potentials of the macrocyclic lactones against inducible inflammatory mediators than those exhibited by the anti-inflammatory agent ibuprofen (0.43). The in silico molecular modelling analyses of the stomopneulactones with cyclooxygenase-2/5-lipoxygenase enzymes recorded lowest binding energy (−7.71 and −9.60 kcal mol−1, respectively) and docking score (−8.82 and −11.12 kcal mol−1, respectively) for stomopneulactone D along with its higher electronic parameter (topological polar surface area of 72.83), which further confirmed its greater anti-inflammatory potential than other compounds in the series. Stomopneulactone D also inhibited the generation of inducible nitric oxide synthase, intracellular reactive oxygen species, along with 5-lipoxygenase and cyclooxygenase-2 in the lipopolysaccharide-stimulated macrophage cells. Additionally, the studied macrocyclic lactone decreased the mRNA expression of cyclooxygenase-2 in the inflammatory cells in dose-dependent manner, which demonstrated the therapeutic potential of stomopneulactone D in down-regulating the inflammatory pathogenesis.

Antibacterial and Anti-Inflammatory Effects of Novel Peptide Toxin from the Spider Pardosa astrigera.

The prevalence of antibiotic-resistant bacteria has become an immediate threat to public health. Antimicrobial peptides are attracting attention as a new source of antibiotics due to their ability to prevent drug-resistances with fewer side effects. Spider venom is composed of various bioactive substances with multiple functionalities such as antimicrobial and anti-inflammatory effects. Here, RNA sequencing was conducted on the venom gland of the spider Pardosa astrigera, and a potential toxin peptide with antibacterial properties was selected via homology and in silico analysis. A novel toxin, Lycotoxin-Pa4a, inhibited both gram-negative and gram-positive bacteria by disrupting the outer and bacterial cytoplasmic membrane. Moreover, the peptide downregulated the expression of pro-inflammatory mediators while upregulating the level of anti-inflammatory cytokine by inactivating mitogen-activated protein kinase signaling in a lipopolysaccharide-stimulated murine macrophage cell line. In this research, we identified a novel peptide toxin, Lycotoxin-pa4a, with antibacterial and anti-inflammatory properties, suggesting its potential for the development of a new antibiotics, as well as offering insights into the utilization of biological resources.