Inhibitory effects of indole α‐lipoic acid derivatives on nitric oxide production in LPS/IFNγ activated RAW 264.7 macrophages

Alpha‐lipoic acid (α‐lipoic acid) is a potent antioxidant compound that has been shown to possess anti‐inflammatory effects. RAW 264.7 macrophages produce various inflammatory mediators such as nitric oxide, IL‐1β, IL‐6 and TNF‐alpha upon activation with LPS ( Lipopolysaccharide) and IFNγ (interferon gamma). In this study, the effect of 12 synthetic indole α‐lipoic acid derivatives on nitric oxide production and iNOS (inducible nitric oxide synthase) protein expression in LPS/IFNγ activated RAW 264.7 macrophages was determined. Cell proliferation, nitric oxide levels and iNOS protein expression were examined with thiazolyl blue tetrazolium blue test, griess assay and western blot, respectively. Our results showed that all of the indole α‐lipoic acid derivatives showed significant inhibitory effects on nitric oxide production and iNOS protein levels (p < 0.05). The most active compounds were identified as compound I‐4b, I‐4e and II‐3b. In conclusion, these indole α‐lipoic acid derivatives may have the potential for treatment of inflammatory conditions related with high nitric oxide production. Copyright © 2015 John Wiley & Sons, Ltd.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Synthesis of new heterocyclic lupeol derivatives as nitric oxide and pro-inflammatory cytokine inhibitors

A series of heterocyclic derivatives including indoles, pyrazines along with oximes and esters were synthesized from lupeol and evaluated for anti-inflammatory activity through inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 and J774A.1 cells. All the synthesized molecules of lupeol were found to be more active in inhibiting NO production with an IC₅₀ of 18.4–48.7 μM in both the cell lines when compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC₅₀ = 69.21 and 73.18 μM on RAW 264.7 and J774A.1 cells, respectively). The halogen substitution at phenyl ring of indole moiety leads to potent inhibition of NO production with half maximal concentration ranging from 18.4 to 41.7 μM. Furthermore, alkyl (11, 12) and p-bromo/iodo (15, 16) substituted compounds at a concentration of 20 μg/mL exhibited mild inhibition (29–42%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (10–22%) towards interleukin 1-beta (IL-1β) production in both the cell lines. All the derivatives were found to be non-cytotoxic when tested at their IC₅₀ (μM). These findings suggest that the derivatives of lupeol could be a lead to potent inhibitors of NO.     

Anti-tubercular activities of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine analogues endowed with high activity toward non-replicative Mycobacterium tuberculosis

Thirty three derivatives of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine analogues were synthesized by molecular modification of a reported antimycobacterial molecule (GSK163574A). Compounds were evaluated in vitro against actively replicative and nutrient starved non-replicative Mycobacterium tuberculosis (MTB), enzymatic screening and cytotoxicity against RAW 264.7 cell line. Among the compounds, 2-ethyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (5c) was found to be the most active compound against non-replicative MTB with 2.7 log reduction of bacteria at 10 μg/mL and was more potent than isoniazid (1.2 log reduction) and rifampicin (2.0 log reduction) at same dose level. Compound 5c also showed activity against MTB alanine dehydrogenase enzyme with IC₅₀ of 1.82 ± 0.42 μM and showed 25% cytotoxicity against RAW 264.7 cell line at 50 μg/mL.     

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.     

Design,synthesis and anti-inflammatory evaluation of novel pyrrolo[2,3-d]pyrimidin derivatives as potent JAK inhibitors

Aiming to develop potent JAK inhibitors, two series of 4-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives (8a–8p and 11a–11i) were designed and synthesized by coalescing various N-acylpiperidine motifs with baricitinib. The pharmacological results based on enzymatic and cellular assays identified the optimized compound 11e, which exerted over 90% inhibition rates against JAK1 and JAK2, and displayed the most compelling anti-inflammatory efficacy superior to baricitinib by inhibiting NO generation from LPS-induced RAW264.7 macrophages. Importantly, low cytotoxity of 11e was revealed by the IC₅₀ value of 88.2 μM against normal RAW264.7 cells. The binding mode of 11e with JAK1 and JAK2 identified the essential structural bases in accord with SARs analysis. Furthermore, cellular morphology observation and western blot analysis disclosed the ability of 11e to relieve cells inflammatory damage by significantly down-regulating LPS-induced high expression of JAK1, JAK2, as well as pro cytokine IL-1β. Together, 11e was verified as a promising lead for JAK inhibitors for the treatment of inflammatory diseases.     

Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages

Aims

Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages.

Main methods

RAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine (pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viability/proliferation and various inflammatory parameters were measured by the established methods including ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also measured fluorescently.

Key findings

All the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited the secretion of all these inflammatory mediators even at the lowest dose (200 μg/ml). PyroGlu-Leu dose-dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface.

Significance

Our results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking of NF-κB and MAPK pathways in RAW 264.7 macrophages.     

Inhibitory effects of triarylpyrazole derivatives on LPS-induced nitric oxide and PGE2 productions in murine RAW 264.7 macrophages

In this article, a series of 22 triarylpyrazole derivatives were evaluated for in vitro antiinflammatory activity as inhibitors of nitric oxide (NO) and prostaglandin E₂ (PGE₂) release induced by lipopolysaccharide (LPS) in murine RAW 264.7 macrophages. The synthesized compounds 1a-h, 2a-f and 3a-h were first examined for their cytotoxicity for determination of the non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE₂ production were not caused by non-specific cytotoxicity. Compounds 1h and 2f were the most active PGE₂ inhibitors with IC₅₀ values of 2.94 μM and 4.21 μM, respectively. Western blotting and cell-free COX-2 screening revealed that their effects were due to inhibition of COX-2 protein expression. Moreover, compound 1h exerted strong inhibitory effect on the expression of COX-2 mRNA in LPS-induced murine RAW 264.7 macrophages.     

(S)-tetrahydroisoquinoline alkaloid inhibits LPS-induced arachidonic acid release through downregulation of cPLA2 expression

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A₂ (cPLA₂) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA₂ and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA₂ expression in RAW 264.7 cells.     

Synthesis of indolyl-3-acetonitrile derivatives and their inhibitory effects on nitric oxide and PGE2 productions in LPS-induced RAW 264.7 cells

Arvelexin is one of major constituents of Brassica rapa that exerts anti-inflammatory activities. Several indolyl-3-acetonitrile derivatives were synthesized as arvelexin analogs and evaluated for their abilities to inhibit NO and PGE₂ productions in LPS-induced RAW 264.7 cells. Of the indolyl-3-acetonitriles synthesized, compound 2k, which possesses a hydroxyl group at C-7 position of the indole ring and an N-methyl substituent, more potently inhibited NO and PGE₂ productions and was less cytotoxic than arvelexin on macrophage cells.     

A role of mitogen and stress-activated protein kinase 1/2 in survival of lipopolysaccharide-stimulated RAW 264.7 macrophages

The effect of inhibition of mitogen and stress-activated protein kinases 1/2 (MSK1/2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was investigated. Pretreatment with Ro 31-8220, an inhibitor of MSK1/2, induced cell death in LPS-stimulated RAW 264.7 cells. In contrast, calphostin C, another inhibitor of protein kinase C, did not cause cell death. Cell death was not mediated by the release of pro-inflammatory mediators from LPS-stimulated RAW 264.7 cells. Cell death was accompanied by DNA fragmentation and annexin V binding, suggesting apoptotic cell death. Further, several caspase inhibitors did not prevent LPS-induced cell death of Ro 31-8220-pretreated RAW 264.7 cells. Nuclear translocation of apoptosis-inducing factor (AIF) was detected in Ro 31-8220-pretreated cells after LPS stimulation. Cell death was due to mitochondrial damage. Ro 31-8220 exclusively inhibited the phosphorylation of cAMP-responsive element binding protein (CREB), a substrate of MSK1/2. RAW 264.7 cells transfected with the dominant-negative MSK1 clones underwent cell death in response to LPS. Hence, it was suggested that MSK1/2 might play a critical role in the survival of LPS-stimulated RAW 264.7 cells.     

Prostaglandin E2 production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line,RAW 264.7, by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E₂ production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE₂ production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC₅₀ of 1.0 μM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE₂ production by activated macrophages.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Cytotoxic and Anti-Inflammatory Activity of 3,19-Isopropylidene-/Arylidene-Andrographolide Analogs

A series of 3,19-isopropylidene-/or arylidene-andrographolide analogs were synthesized and their structures were confirmed by NMR spectroscopic methodology. Twenty-five analogs were evaluated for their in vitro cytotoxic activity against HT-29, HepG2 and LNCaP cancer cell lines based on the sulforhodamine B (SRB) assay. Analog 2 f exhibited the most potent cytotoxic activity, with IC₅₀ values of 11.14 and 9.25 μM on HepG2 and LNCaP cancer cell lines, respectively. Esterification of hydroxy functional group at position C-14 in andrographolide analogs, 2 a and 2 b _, showed somewhat higher cytotoxicity than the precursor. In addition, andrographolide analogs ( 2 a – 2 d , 2 f , 3 a , 4 a and 4 h ) were evaluated for the NO inhibitory activity in the LPS stimulated RAW264.7 macrophages. The most active analog 2 a significantly reduced nitric oxide (NO) production from LPS stimulated RAW264.7 cells, with IC₅₀ values of 0.34±0.02 μM providing encouraging results for anti-inflammatory compound development.     

Role of p38 mitogen-activated protein kinase (MAPK) for vacuole formation in lipopolysaccharide (LPS)-stimulated macrophages

The role of p38 mitogen-activated protein kinase (MAPK) on vacuole formation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was examined. LPS definitely induced the formation of vacuoles in RAW 264.7 cells and SB202190 as a p38 specific inhibitor also induced slight vacuole formation. The simultaneous treatment with LPS and SB202190 induced many more vacuoles in RAW 264.7 cells than the treatment with LPS or SB202190 alone, and the vacuoles were extraordinarily large in size. On the other hand, an inactive inhibitor of p38 MAPK did not augment LPS-induced vacuole formation. Further, the inhibitors of other MAPKs and nuclear factor (NF)-kappaB pathways did not affect it. The extraordinarily large vacuoles in RAW 264.7 cells treated with LPS and SB202190 were possibly formed via fusion of small vacuoles. However, SB202190 did not augment vacuole formation in CpG DNA or interferon (IFN)-gamma-stimulated RAW 264.7 cells. The role of p38 MAPK in the vacuole formation in LPS-stimulated macrophages is discussed.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line,RAW 264.7, activated by lipopolysaccharide and interferon-γ

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC₅₀ of 10.7 μM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-γ for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Synthesis of New Lathyrane Diterpenoid Derivatives from Euphorbia lathyris and Evaluation of Their Anti‐Inflammatory Activities

Euphorbia factor L₃, a lathyrane diterpenoid extracted from Euphorbia lathyris, was found to display good anti‐inflammatory activity with very low cytotoxicity. To find more potent anti‐inflammatory drugs, two series of Euphorbia factor L₃ derivatives with fatty and aromatic acids were designed and synthesized. Among them, lathyrane derivative 5n exhibited most potent inhibition on LPS‐induced NO production in RAW264.7 cells with no obvious cytotoxicity. To determine the key characteristics of Euphorbia factor L₃ derivatives that contribute to anti‐inflammatory activity, we conducted a structure‐activity relationship study of these compounds.