Design, synthesis and bioactivity evaluation of 4-hydroxycoumarin derivatives as potential anti-inflammatory agents against acute lung injury and colitis.

Acute lung injury (ALI) and inflammatory bowel disease (IBD) are common inflammatory illnesses that seriously affect people's health. Herein, a series of 4-hydroxylcoumarin (4-HC) derivatives were designed and synthesized. The inhibitory effects of these compounds on LPS-induced interleukin-6 (IL-6) release from J774A.1 cells were then screened via ELISA assay, compound B8 showed 3 times more active than the lead compound 4-HC. The most active compound B8 had the IC50 values of 4.57 µM and 6.51 µM for IL-6 release on mouse cells J774A.1 and human cells THP-1, respectively. Furthermore, we also found that B8 could act on the MAPK pathway. Based on the target prediction results of computer virtual docking, kinase inhibitory assay was carried out, and it revealed that targeting IRAK1 was a key mechanism for B8 to exert anti-inflammatory activity. Moreover, B8 exerted a good therapeutic effect on the dextran sulfate sodium (DSS)-induced colitis model and liposaccharide (LPS)-induced ALI mouse models. The acute toxicity experiments indicated that high-dose B8 caused no adverse reactions in mice, confirming its safety in vivo. Additionally, the preliminary pharmacokinetic (PK) parameters of B8 in SD rats were also examined, revealing a bioavailability (F) of 28.72 %. In conclusion, B8 is a potential candidate of drug for the treatment of ALI and colitis.

Anti-inflammatory discovery of sesquiterpenoids and a jasmonic acid derivative from Artemisia stolonifera.

Eleven previously undescribed sesquiterpenoids (8-18), one undescribed jasmonic acid derivative (35) and 28 known compounds were isolated from the leaves of Artemisia stolonifera. Undescribed compounds with their absolute configurations were determined by extensive spectroscopic analysis, single-crystal X-ray diffraction and ECD calculation. Compound 8 was identified as a rare sesquiterpenoid featuring a rearranged 5/8 bicyclic ring system, whereas compound 17 was found to be an unprecedented monocyclic sesquiterpenoid with methyl rearrangement. Evaluation of biological activity showed that compounds 1-5 and 7 displayed cytotoxicity against six tumor cells. In the meantime, compounds 11, 12, 18 and 35 exhibited inhibitory effects against LPS-stimulated NO production in RAW 264.7 macrophage cells and reduced the transcription of IL-6 and IL-1ß in a dose-dependent manner at 25, 50 and 100 µM. Moreover, the anti-inflammatory-based network pharmacology and molecular docking analyses revealed potential target proteins of 11, 12, 18 and 35.

Discovery of sesquiterpenoids from the roots of Chloranthus henryi Hemsl. var. hupehensis (Pamp.) K. F. Wu and their anti-inflammatory activity by IKBα/NF-κB p65 signaling pathway suppression.

Phytochemical analysis of Chloranthus henryi var. hupehensis roots led to the identification of a new eudesmane sesquiterpenoid dimer, 18 new sesquiterpenoids, and three known sesquiterpenoids. Among the isolates, 1 was a rare sesquiterpenoid dimer that is assembled by a unique oxygen bridge (C11-O-C8') of two highly rearranged eudesmane-type sesquiterpenes with the undescribed C16 carbon framework. (+)-2 and (-)-2 were a pair of new skeleton dinorsesquiterpenoids with a remarkable 6/6/5 tricyclic ring framework including one γ-lactone ring and the bicyclo[3.3.1]nonane core. Their structures were elucidated using spectroscopic data, single-crystal X-ray diffraction analysis, and quantum chemical computations. In the LPS-induced BV-2 microglial cell model, 17 suppressed IL-1ß and TNF-α expression with EC50 values of 6.81 and 2.76 µM, respectively, indicating its excellent efficacy in inhibiting inflammatory factors production in a dose dependent manner and without cytotoxicity. In subsequent mechanism studies, compounds 3, 16, and 17 could reduce IL-1ß and TNF-α production by inhibiting IKBα/p65 pathway activation.

Design, synthesis and molecular modeling of novel D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives as anti-inflammatory agents by inhibition of COX-2/iNOS production and down-regulation of NF-κB/MAPKs in LPS-induced RAW264.7 macrophage cells.

It has been reported that 4,5-dihydropyrazole and thiazole derivatives have many biological functions, especially in the aspect of anti-inflammation. According to the strategy of pharmacophore combination, we introduced thiazolinone and dihydropyrazole moiety into steroid skeleton to design and synthesize a novel series of D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives, and assessed their in vitro anti-inflammatory profiles against Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. The anti-inflammatory activities assay demonstrated that compound 12e was considered as the most effective anti-inflammatory drug, which suppressed the expression of pro-inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), it also dose-dependently inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 macrophage cells. Furthermore, the results of the Western blot analysis showed a correlation between the inhibition of the Nuclear factor-kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs) signaling pathways and the suppressive effects of compound 12e on pro-inflammatory cytokines. Molecular docking studies of compound 12e into the COX-2 protein receptor (PDB ID: 5IKQ) active site was performed to rationalize their COX-2 inhibitory potency. The results were found to be in line with the biological findings as they exerted more favorable interactions compared to that of dexamethasone (DXM), explaining their remarkable COX-2 inhibitory activity. The findings revealed that these candidates could be identified as potent anti-inflammatory agents, compound 12e could be a promising drug for the treatment of inflammatory diseases.

Synthesis of a new 2-prenylated quinoline as potential drug for metabolic syndrome with pan-PPAR activity and anti-inflammatory effects.

We have previously reported the total synthesis and structure-activity relationships (SAR) of 2-prenylated benzopyrans with PPAR agonist activity. Herein, we have described the synthesis and PPAR activity of 2-prenylated benzopyrans and 2-prenylated quinolines. The benzopyran nucleus was generated via enamine-catalyzed Kabbe condensation, and the quinoline nucleus via Friedländer condensation. Results demonstrated that both benzopyran (5a) and quinoline (4b) derivatives bearing a γ,δ-unsaturated ester displayed a pan-PPAR agonism. They were full PPARα agonists, but showed different preferences for PPARγ and PPARß/δ activation. It was noteworthy that quinoline 4b displayed full hPPARα activation (2-fold than WY-14,643), weak PPARß/δ and partial PPARγ activation. In addition, quinoline 4b showed anti-inflammatory effects on macrophages by reducing LPS-induced expression of both MCP-1 and IL-6. Therefore, 4b emerges as a first-in-class promising hit compound for the development of potential therapeutics aimed at treating metabolic syndrome, metabolic dysfunction-associated fatty liver disease (MAFLD), and its associated cardiovascular comorbidities.

1H NMR guided isolation of 3-arylisoquinoline alkaloids from Hypecoum erectum L. and their anti-inflammation activity.

Nine 3-arylisoquinoline alkaloids including five undescribed ones, hypectumines A-E (1-5), were isolated from the whole herb of Hypecoum erectum L. with the guidance of 1H-NMR. Their structures were established by a combination of 1D, 2D NMR, and HRESIMS spectrometry. Among them, hypectumines A and B possessed rare urea moieties while hypectumines C and D were characterized by 3-(methylamino)propanoic acid scaffolds. Biological assay demonstrated that alkaloids hypectumine B and 2,3-dimethoxy-N-formylcorydamine had anti-inflammatory effects by inhibiting NO production on LPS-induced RAW264.7 cells with IC50 values of 24.4 and 44.2 µM, respectively. Furthermore, hypectumine B could reduce the expression of pro-inflammatory cytokines TNF-α and IL-6, suggesting it might be a potential candidate for treating inflammatory disease.

Exploring the chemical diversity of sesquiterpenes from the rarely studied south China sea soft coral Sinularia tumulosa assisted by molecular networking strategy.

Molecular networking strategy-based prioritization of the isolation of the rarely studied soft coral Sinularia tumulosa yielded 14 sesquiterpenes. These isolated constituents consisted of nine different types of carbon frameworks, namely asteriscane, humulane, capillosane, seco-asteriscane, guaiane, dumortane, cadinane, farnesane, and benzofarnesane. Among them, situmulosaols A-C (1, 3 and 4) were previously undescribed ones, whose structures with absolute configurations were established by the combination of extensive spectral data analyses, quantum mechanical-nuclear magnetic resonance and time-dependent density functional theory electronic circular dichroism calculations, the Snatzke's method, and the modified Mosher's method. Notably, situmulosaol C (4) was the second member of capillosane-type sesquiterpenes. The plausible biogenetic relationships of these skeletally different sesquiterpenes were proposed. All sesquiterpenoids were evaluated for their antibacterial, cytotoxic and anti-inflammatory effects. The bioassay results showed compound 14 exhibited significant antibacterial activities against a variety of fish and human pathogenic bacteria with MIC90 values ranging from 3.6 to 33.8 µg/mL. Moreover, moderate cytotoxic effects against HEL cells for components 13 and 14 and moderate inhibitory effect on lipopolysaccharide-induced inflammatory responses in RAW264.7 cells for substance 13 were also observed.

Discovery of 6-Acylamino/Sulfonamido Benzoxazolone with IL-6 Inhibitory Activity as Promising Therapeutic Agents for Ulcerative Colitis.

Ulcerative colitis has been widely concerned for its persistent upward trend, and the sustained overproduction of pro-inflammatory cytokines such as IL-6 remains a crucial factor in the development of UC. Therefore, the identification of new effective drugs to block inflammatory responses is an urgent and viable therapeutic strategy for UC. In our research, twenty-three 6-acylamino/sulfonamido benzoxazolone derivatives were synthesized, characterized, and evaluated for anti-inflammatory activity against NO and IL-6 production in LPS-induced RAW264.7 cells. The results demonstrated that most of the target compounds were capable of reducing the overexpression of NO and IL-6 to a certain degree. For the most active compounds 3i, 3j and 3 l, the inhibitory activities were superior or equivalent to those of the positive drug celecoxib with a dose-dependent relationship. Furthermore, animal experiments revealed that active derivatives 3i, 3j and 3 l exhibited definitive therapeutical effect on DSS induced ulcerative colitis in mice by mitigating weight loss and DAI score while decreasing levels of pro-inflammatory cytokines such as IL-6 and IFN-γ, simultaneously increasing production of anti-inflammatory cytokines IL-10. In addition, compounds 3i, 3j and 3 l could also inhibit the oxidative stress to alleviate ulcerative colitis by decreasing MDA and MPO levels. These finding demonstrated that compounds 3i, 3j and 3 l hold significant potential as novel therapeutic agents for ulcerative colitis.

Dual COX-2 and 15-LOX inhibition study of novel 4-arylidine-2-mercapto-1-phenyl-1H-imidazolidin-5(4H)-ones: Design, synthesis, docking, and anti-inflammatory activity.

Novel arylidene-5(4H)-imidazolone derivatives 4a-r were designed and evaluated as multidrug-directed ligands, that is, inflammatory, proinflammatory mediators, and reactive oxygen species (ROS) inhibitors. All of the tested compounds showed cyclooxygenase (COX)-1 inhibitory effect more than celecoxib and less than indomethacin and also demonstrated an improved inhibitory activity against 15-lipoxygenase (15-LOX). Compounds 4f, 4l, and 4p exhibited COX-2 selectivity comparable to that of celecoxib, while 4k was the most selective COX-2 inhibitor. Interestingly, the screened results showed that compound 4k exhibited a superior inhibition effect against 15-LOX and was found to be the most selective COX-2 inhibitor over celecoxib, whereas compound 4f showed promising COX-2 and 15-LOX inhibitory activities besides its inhibitory effect against ROS production and its lowering effect of both tumor necrosis factor-α and interleukin-6 levels by ∼80%. Moreover, compound 4f attenuated the lipopolysaccharide-mediated increase in NF-κB activation in RAW 264.7 macrophages. The preferred binding affinity of these molecules was confirmed by docking studies. We conclude that arylidene-5(4H)-imidazolone scaffolds provide promising hits for developing new synthons with anti-inflammatory and antioxidant activities.

Prunetinoside Inhibits Lipopolysaccharide-Provoked Inflammatory Response via Suppressing NF-κB and Activating the JNK-Mediated Signaling Pathway in RAW264.7 Macrophage Cells.

Inflammation is a multifaceted response of the immune system at the site of injury or infection caused by pathogens or stress via immune cells. Due to the adverse effects of chemical drugs, plant-based compounds are gaining interest in current research. Prunetinoside or prunetin-5-O-glucoside (PUG) is a plant-based active compound, which possesses anti-inflammatory effects on immune cells. In this study, we investigate the effect of PUG on mouse macrophage RAW264.7 cells with or without stimulation of lipopolysaccharide (LPS). Cytotoxicity results showed that PUG is non-cytotoxic to the cells and it reversed the cytotoxicity in LPS-stimulated cells. The levels of nitric oxide (NO) and interleukin-6 (IL-6) were determined using a NO detection kit and IL-6 ELISA kit, respectively, and showed a significant decrease in NO and IL-6 in PUG-treated cells. Western blot and qRT-PCR were performed for the expression of two important pro-inflammatory cytokines, COX2 and iNOS, and found that their expression was downregulated in a dose-dependent manner. Other pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNFα, had reduced mRNA expression after PUG treatment. Furthermore, a Western blot was performed to calculate the expression of NF-κB and MAPK pathway proteins. The results show that PUG administration dramatically reduced the phosphorylation of p-Iκbα, p-NF-κB 65, and p-JNK. Remarkably, after PUG treatment, p-P38 and p-ERK remain unchanged. Furthermore, docking studies revealed that PUG is covalently linked to NF-κB and suppresses inflammation. In conclusion, PUG exerted the anti-inflammatory mechanism by barring the NF-κB pathway and activating JNK. Thus, prunetinoside could be adopted as a therapeutic compound for inflammatory-related conditions.

Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22 Exhibit Anti-Inflammatory Effect by Attenuation of NF-κB and MAPK Signaling in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-κB (NF-κB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.

Microbial transformation and inhibitory effect assessment of uvaol derivates against LPS and HMGB1 induced NO production in RAW264.7 macrophages.

For the discovery of new pentacyclic triterpenes as a potential anti-inflammatory agent, microbial transformation of uvaol by Penicilium griseofulvum CICC 40293 and Streptomyces griseus ATCC 13273 was investigated. Stereoselective hydroxylation and epoxidation reactions were observed in the biotransformation. Moreover, six new metabolites were isolated and structurally elucidated by HR-ESI-MS and NMR spectrum. All the compounds were evaluated upon the inhibitory effects of nitric oxide (NO) release in RAW 264.7 cells induced by lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1). Among them, compound 3 (13, 28-epoxy-3ß, 7ß, 21ß-trihydroxy-urs-11-ene) with the unique epoxy structure and compound 5 (3ß, 21ß, 24, 28-tetrahydroxy-urs-12-en-30-oic acid), exhibited a considerable inhibitory effect on both models while compound 2 (urs-12-ene-3ß, 7ß, 21ß, 28-tetraol) showed a significant bias in the LPS-induced inflammatory response with IC50 value of 2.22 µM. Therefore, this study could provide some insights on the discovery of the pentacyclic triterpene leads for the treatment of either DAMPs or PAMPs triggered inflammation.

Characterization and immunomodulatory effect of an alkali-extracted galactomannan from Morchella esculenta.

In our continuous exploration for bioactive polysaccharides, a novel polysaccharide FMP-2 was isolated and purified from the fruiting bodies of Morchella esculenta by alkali-assisted extraction. FMP-2 had an average molecular weight of 1.09 × 106 Da and contained mannose, glucuronic acid, glucose, galactose, and arabinose in a molar ratio of 4.10:0.22:1.00:5.75:0.44. The backbone of FMP-2 mainly consisted of 1,2-α-D-Galp, 1,6-α-D-Galp, and 1,4-α-D-Manp, with branches of 1,4,6-α-D-Manp and 1,2,6-α-D-Galp. FMP-2 can stimulate phagocytosis and promote the secretion of NO, ROS, and cytokines like IL-6, IL-1ß, and TNF-α in RAW264.7 cells ranging from 25 to 400 µg/mL. FMP-2 had great repairing effect on the immune injury of zebrafish induced by chloramphenicol. The phagocytosis ability of zebrafish macrophages and the proliferation of neutrophils can be greatly enhanced by polysaccharide FMP-2 with concentrations from 50 to 200 µg/mL. These findings suggest that FMP-2 might be used as a potential immunomodulator in the food and pharmaceutical industries.

Synthesis of sorbicillinoid analogues with anti-inflammation activities.

Recently, we demonstrated potential anti-inflammatory effects of sorbicillinoids isolated from marine fungi. Here, we report the synthesis of a series of new sorbicillinoid analogues and assessed their anti-inflammatory activities. Our results reveal that side chain substitution with (E)-2-butenoyl, (E)-3-(4-fluorophenyl)-2-propenoyl, and (E)-3-(3,4,5-trimethoxyphenyl)-2-propenoyl significantly enhanced the inhibitory effects of the derivatives on nitric oxide (NO) production and inducible NO synthesis (iNOS) expression stimulated by lipopolysaccharides (LPS) in mouse macrophage. Further chemical derivatization shows that the monomethylresorcinol skeleton worked better than the dimethylresorcinol skeleton in inhibiting LPS-induced inflammatory response in cultured cells. Among the 29 synthesized sorbicillinoid analogues, compounds 4b and 12b exhibited the strongest anti-inflammatory activities, holding the promise of being developed into lead compounds that can be explored as potent anti-inflammation agents.

Structure elucidation, biogenesis, and bioactivities of acylphloroglucinol-derived meroterpenoid enantiomers from Dryopteris crassirhizoma.

Twenty-four racemic acylphloroglucinol meroterpenoids including eighteen unusual stuctures (3 âˆ¼ 10, 13, 14, and 17 âˆ¼ 24), and a major component filixic acid ABA (25), were isolated from Dryopteris crassirhizoma. Structurally, the dimeric acylphloroglucinol derivatives possess unprecedented skeletons of mixed acylphloroglucinol and sesquiterpene biosynthetic origin. The stereochemistries of six reported meroterpenoids with undefined chiral centers were reassigned. Two intriguing methods by analyzing a) the regularity of chemical shift variation of protons and carbons around the stereogenic centers, and b) pyridine-induced deshielding effect of hydroxy groups, to discriminate relative configurations of flexible long-chain alcohol with chiral centers separated by three or seven covalent bonds, were successfully applied. A non-enzymatic biosynthesis of 1 âˆ¼ 24 was assumed based on a rare single-crystal cluster formed with two diastereomeric enantiomer pairs (±1/±2) and chiral HPLC analyses. Meroterpenoids 13 and 14 showed obvious inhibitory effects on NO production in LPS-induced RAW264.7, and suppressed the expression of iNOS, COX-2, IL-1ß, and IL-18. Their anti-inflammatory activity was closely related to the inhibition of the formation and function of inflammasomes. Additionally, the known 25 showed antiviral efficacy against the influenza viruse A/Puerto Rico/8/1934 (H1N1).

Hostaflavone A from Hosta plantaginea (Lam.) Asch. blocked NF-κB/iNOS/COX-2/MAPKs/Akt signaling pathways in LPS-induced RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Hostaflavone A (HA) is a new flavonoid component isolated from the flower of Hosta plantaginea (Lam.) Asch., which is commonly used as a folk herbal to treat inflammatory diseases in China. Nevertheless, the anti-inflammatory effect of HA remains unknown. AIM OF THE STUDY: This work aimed to evaluate the HA with anti-inflammatory activity and mechanism in RAW 264.7 macrophages activated by lipopolysaccharide (LPS). MATERIALS AND METHODS: Anti-inflammatory effect of HA was evaluated by measuring of cell viability, nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and IL-6 levels in RAW 264.7 cells. In parallel, the HA action mechanism of nuclear factor kappa B (NF-κB) p65, inhibitor of NF-κB (IκB), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (Erk), p38, and protein kinase B (Akt) were detected by Western blot analysis. RESULTS: HA has no cytotoxicity at concentrations as high as 40 µM. Besides, HA concentration-dependently clearly suppressed the overproduction of NO, PGE2, TNF-α, IL-1ß and IL-6 in RAW 264.7 cells induced by LPS. In addition, HA remarkably reduced the upregulation of phosphorylated NF-κB p65, phosphorylated IκB, phosphorylated JNK, phosphorylated Erk and phosphorylated p38, together with iNOS and COX-2 protein expressions in a concentration-dependent manner. CONCLUSION: HA blocked the LPS activated inflammation via suppressing NF-κB, iNOS, COX-2, mitogen-activated protein kinases (MAPKs) and Akt pathways in RAW 264.7 cells, and might be a new anti-inflammatory agent.

A novel modified chitosan/collagen coated-gold nanoparticles for 5-fluorouracil delivery: Synthesis, characterization, in vitro drug release studies, anti-inflammatory activity and in vitro cytotoxicity assay.

We report herein the development of the novel nanohybrids of gold nanoparticles reduced/stabilized/coated with collagen (AuNPs@collagen) in the first layer and subsequently modified with biotin-quat188-chitosan (Bi-QCS) in the outer layer for 5-fluorouracil (5-FU) delivery to improve cellular uptake and promote specific cell targeting of the nanocarrier. The fabrication of the layer-by-layer technique on the surface of gold nanoparticles (AuNPs) can overcome the limitation of poor drug loading capacity of the classic AuNPs from 64.67% to 87.46%. The AuNPs@collagen coated by the Bi-QCS exhibits strong electrostatic interactions between drug anion (5-FU) and amine groups of the modified chitosan as well as hydrogen bonding. Furthermore, the Bi-QCS-AuNPs@collagen demonstrated a significantly higher anti-inflammatory activity in RAW264.7 macrophage cell line. The Bi-QCS-AuNPs@collagen enhanced the activity of 5-FU approximately 3.3-fold (HeLa) and 6.2-fold (A549), compared to the free 5-Fluorouracil. According to these results, it is very promising that Bi-QCS-AuNPs@collagen can be used as an effective drug delivery carrier in the future.

Inhibition of Lipopolysaccharide-Induced Inflammatory Bone Loss by Saikosaponin D is Associated with Regulation of the RANKL/RANK Pathway.

BACKGROUND: Osteolytic diseases such as osteoporosis are featured with accelerated osteoclast differentiation and strong bone resorption. Considering the complications and other limitations of current drug treatments, it is necessary to develop a safer and more reliable drug to deal with osteoclast-related diseases. Saikosaponin D (SSD) is the active extract of Bupleurum, which has anti-inflammation, anti-tumor and liver protection functions. However, the role of SSD in regulating the differentiation and function of osteoclasts is not clear. PURPOSE: To explore whether SSD could prevent osteoclast differentiation and bone resorption induced by M-CSF and RANKL, and further evaluate the potential therapeutic properties of SSD in LPS-induced inflammatory bone loss mouse models. METHODS: BMMs were cultured in complete medium stimulated by RANKL with different concentrations of SSD. TRAP staining, bone resorption determination, qRT-PCR, immunofluorescence and Western blotting were performed. A mouse model of LPS-induced calvarial bone loss was established and treated with different doses of SSD. The excised calvaria bones were used for TRAP staining, micro-CT scan and histological analysis. RESULTS: SSD inhibited the formation and bone resorption of osteoclasts induced by RANKL in vitro. SSD suppressed LPS-induced inflammatory bone loss in vivo. CONCLUSION: SSD inhibited osteoclastogenesis and LPS-induced osteolysis in mice both which served as a new potential agent for the treatment of osteoclast-related conditions.

The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment.

Flavonols possess several beneficial bioactivities in vitro and in vivo. In this study, two flavonols galangin and quercetin with or without heat treatment (100 °C for 15-30 min) were assessed for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated rat intestinal epithelial (IEC-6) cells and whether the heat treatment caused activity changes. The flavonol dosages of 2.5-20 µmol/L had no cytotoxicity on the cells but could enhance cell viability (especially using 5 µmol/L flavonol dosage). The flavonols could decrease the production of prostaglandin E2 and three pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α, and simultaneously promote the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-ß. The Western-blot results verified that the flavonols could suppress the LPS-induced expression of TLR4 and phosphorylated IκBα and p65, while the molecular docking results also illustrated that the flavonols could bind with TLR4 and NF-κB to yield energy decreases of -(21.9-28.6) kJ/mol. Furthermore, an inhibitor BAY 11-7082 blocked the NF-κB signaling pathway by inhibiting the expression of phosphorylated IκBα/p65 and thus mediated the production of IL-6/IL-10 as the flavonols did, which confirmed the assessed anti-inflammatory effect of the flavonols. Consistently, galangin had higher anti-inflammatory activity than quercetin, while the heated flavonols (especially those with longer heat time) were less active than the unheated counterparts to exert these target anti-inflammatory effects. It is highlighted that the flavonols could antagonize the LPS-caused IEC-6 cells inflammation via suppressing TLR4/NF-κB activation, but heat treatment of the flavonols led to reduced anti-inflammatory efficacy.

Meta-substituted piperlongumine derivatives attenuate inflammation in both RAW264.7 macrophages and a mouse model of colitis.

Piperlongumine (PL) has been showed to have multiple pharmacological activities. In this study, we reported the synthesis of three series of PL derivatives, and evaluation of their anti-inflammatory effects in both lipopolysaccharide (LPS)-induced Raw264.7 macrophages and a dextran sulfate sodium (DSS)-induced mouse model of colitis. Our results presented that two meta-substituent containing derivatives 1-3 and 1-6, in which γ-butyrolactam replaced α,ß-unsaturated δ-valerolactam ring of PL, displayed low cytotoxicity and effective anti-inflammatory activity. Molecular docking also showed that the meta-substituted derivative, compared with the corresponding ortho- or para-substituted derivative, had significant interactions with the amino acid residues of CD14, which was the core receptors recognizing LPS. In vitro and in vivo studies, 1-3 and 1-6 could inhibit the expression of pro-inflammatory cytokines, and the excessive production of reactive nitrogen species and reactive oxygen species. Oral administration of 100 mg/kg/day of 1-3 or 1-6 alleviated the severity of clinical symptoms of colitis in mice, and significantly reduced the colonic tissue damage to protect the colonic tissue from the DSS-induced colitis. These results suggested that meta-substituted derivatives 1-3 and 1-6 were potential anti-inflammatory agents, which may lead to future pharmaceutical development.