A new synthetic chalcone derivative, 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139), suppresses the Toll-like receptor 4-mediated inflammatory response through inhibition of the Akt/NF-κB pathway in BV2 microglial cells

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IκB and p65/RelA NF-κB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-κB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, and IL-1β, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant- active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IκB kinase (IKK)/NF-κB signaling pathway.     

Celastrol inhibits production of nitric oxide and proinflammatory cytokines through MAPK signal transduction and NF-kappaB in LPS-stimulated BV-2 microglial cells

Excessive production of nitric oxide (NO) and proinflammatory cytokines from activated microglia play an important role in human neurodegenerative disorders. Here, we investigated whether celastrol, which has been used as a potent anti-inflammatory and anti-oxidative agent in Chinese medicine, attenuates excessive production of NO and proinflammatory cytokines such as TNF-alpha and IL-1betal in LPS-stimulated BV-2 cells, a mouse microglial cell line. We report here that the LPS-elicited excessive production of NO, TNF-alpha, and IL-1beta in BV-2 cells was largely inhibited in the presence of celastrol, and the attenuation of inducible iNOS and these cytokines resulted from the reduced expression of mRNAs of iNOS and these cytokines, respectively. The molecular mechanisms that underlie celastrol-mediated attenuation were the inhibition of LPS-induced phosphorylation of MAPK/ERK1/2 and the DNA binding activity of NF-kappaB in BV-2 cells. The results indicate that celastrol effectively attenuated NO and proinflammatory cytokine production via the inhibition of ERK1/2 phosphorylation and NF-kappaB activation in LPS-activated microglia. Thus, celastrol may be an effective therapeutic candidate for use in the treatment of neurodegenerative human brain disorders.     

Antiviral furanosesquiterpenes from Commiphora erythraea

The crude methanolic extract obtained from C. erythraea resin was chromatographed on silica gel with solvent of increasing polarity. The extract and fractions were evaluated for cytotoxicity and antiviral activity [parainfluenza type 3 virus (PIV3)] by plaque forming units (PFU) reduction assay using HEp-2 cells (human larynx epidermoid carcinoma cell line). From the active fraction, five compounds were isolated and tested. Only two of these showed anti-PIV3 activity with a selectivity index (SI) of 66.6 and 17.5, respectively. Both the compounds are furanosesquiterpenoids.     

Genkwalathins A and B,new lathyrane-type diterpenes from Daphne genkwa

Screening for new natural anti-neuroinflammatory compounds was performed with the traditional folk medicine Genkwa Flos, which potently inhibited nitric oxide (NO) production by LPS-activated microglial BV-2 cells. Two new lathyrane-type diterpenes, genkwalathins A (1) and B (2), and 14 known daphnane-type diterpenes (3–16) were isolated. The lathyrane-type diterpenes were isolated for the first time from the Thymelaeaceae family in this study. Compounds 1 and 2 moderately inhibited LPS-induced NO production in BV-2 cells without affecting cell viability, while six daphnane-type diterpenes (3, 4, 6, 7, 9 and 10) potently reduced NO production with IC₅₀ values less than 1 μM, although they did display weak cytotoxicity. A structure–activity relationship study on the daphnane-type diterpenes indicated that the stereochemistry at C-19, the benzoate group at C-20, and the epoxide moiety could be important for their anti-neuroinflammatory effects.     

Central Activation of Alpha7 Nicotinic Signaling Attenuates LPS-Induced Neuroinflammation and Sickness Behavior in Adult but Not in Aged Animals

We previously reported that lipopolysaccharide (LPS) challenge caused microglial-mediated neuroinflammation and sickness behavior that was amplified in aged mice. As α7 nAChRs are implicated in the “Cholinergic anti-inflammatory pathway”, we aimed to determine how α7 nAChR stimulation modulates microglial phenotype in an LPS-induced neuroinflammation model in adult and aged mice. For this, BALB/c mice were injected intraperitoneally with LPS (0.33 mg/kg) and treated with the α7 nAChR agonist PNU282987, using different administration protocols. LPS challenge reduced body weight and induced lethargy and social withdrawal in adult mice. Peripheral (intraperitoneal) co-administration of the α7 nAChR agonist PNU282987 with LPS, attenuated body weight loss and sickness behavior associated with LPS challenge in adult mice, and reduced microglial activation with suppression of IL-1β and TNFα mRNA levels. Furthermore, central (intracerebroventricular) administration of the α7 nAChR agonist, even 2 h after LPS injection, attenuated the decrease in social exploratory behavior and microglial activation induced by peripheral administration of LPS, although this recovery was not achieved if activation of α7 nAChRs was performed peripherally. Finally, we observed that the positive results of central activation of α7 nAChRs were lost in aged mice. In conclusion, we provide evidence that stimulation of α7 nAChR signaling reduces microglial activation in an in vivo LPS-based model, but this cholinergic-dependent regulation seems to be dysfunctional in microglia of aged mice.     

Isolation and structural elucidation of 5-formyl-2,3-dihydroisocoumarin from Centaurium erythraea aerial parts

An isocoumarin was isolated from the aerial parts of Centauriwn erythraea Rafn. (Gentianaceae). Its structure was established by spectroscopic means as 5-formyl-2,3-dihydroisocoumarin.     

Protein kinase A mediates microglial activation induced by plasminogen and gangliosides

In the injured brain, microglia is known to be activated and produce proinflammatory mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). We investigated the role of protein kinase A (PKA) in microglial activation by both plasminogen and gangliosides in rat primary microglia and in the BV2 immortalized murine microglial cell line. Both plasminogen and gangliosides induced IL-1beta, TNF-alpha and iNOS mRNA expression, and that this expression was inhibited by the addition of the PKA inhibitors, KT5720 and H89. Both plasminogen and gangliosides activated PKA and increased the DNA binding activity of the cAMP response element- binding protein (CREB). Furthermore, KT5720 and H89 reduced the DNA binding activities of CREB and NF-kappaB in plasminogen-treated cells. These results suggest that PKA plays an important role in plasminogen and gangliosides- induced microglial activation.     

Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE₂ production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-κB DNA binding activity, IκBα degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.     

A novel vanadium complex VO(p-dmada) inhibits neuroinflammation induced by lipopolysaccharide

Uncontrolled microglial activation is decisively involved in the neuroinflammatory pathogenesis of brain diseases. Consequently, suppression of microglial overactivation appears to be a strategy for the prevention of nerve injury. In this paper, a novel vanadium complex, vanadyl N-(p-N,N-dimethylaminophenylcarbamoylmethyl)iminodiacetate (VO(p-dmada)), was synthesized from vanadyl sulfate and N,N-dimethyl-p-phenylenediamine, which was structurally characterized by Fourier transform infrared spectrum and ESI-MS analysis. The effect of VO(p-dmada) on neuroinflammation was investigated by using the models of lipopolysaccharide (LPS)-induced BV2 microglial cells and BALB/c mice. Our data demonstrated that VO(p-dmada) significantly suppressed microglial activation by downregulating inflammatory mediators and associated proteins, and inactivating nuclear factor-κB (NF-κB) signaling pathway. VO(p-dmada) also upregulated peroxisome proliferator activated receptor gamma (PPARγ) by reducing transglutaminase 2 and heat shock protein 60 expression. Co-treatment with PPARγ antagonist GW9662 significantly impeded the inhibitory effect of VO(p-dmada) on LPS-induced neuroinflammation. These cumulative findings demonstrated that VO(p-dmada) is a potential new drug for the treatment of neuroinflammation-related neurodegenerative diseases.     

Anti-Neuroinflammatory Components from Clausena lenis Drake

Clausena lenis Drake (C. lenis) is a folk medicinal herb to treat influenza, colds, bronchitis, and malaria. The 95% and 50% ethanol extract of C. lenis showed significant nitric oxide (NO) inhibition activity in BV-2 microglial cells stimulated by lipopolysaccharide (LPS). Bio-guided isolation of the active extract afforded five new compounds, including a chlorine-containing furoquinoline racemate, (±)-claulenine A (1), an amide alkaloid, claulenine B (2), a prenylated coumarin, claulenin A (3), a furocoumarin glucoside, clauleside A (4), and a multi-prenylated p-hydroxybenzaldehyde, claulenin B (5), along with 33 known ones. Their structures were determined via spectroscopic methods, and the absolute configurations of new compounds were assigned via the electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction analysis. Compounds 2, 23, 27, 28, 33, and 34 showed potent anti-neuroinflammatory effects on LPS-induced NO production in BV-2 microglial cells, with IC₅₀ values in the range of 17.6–40.9 μM. The possible mechanism was deduced to interact with iNOS through molecular docking.     

Anti-Inflammatory Effects of Mitrephora sirikitiae Leaf Extract and Isolated Lignans in RAW 264.7 Cells

Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders has been reported as a rich source of lignans that contribute to biological activities and health benefits. However, cellular anti-inflammatory effects of M. sirikitiae leaves and their lignan compounds have not been fully elucidated. Therefore, this study aimed to investigate the anti-inflammatory activities of methanol extract of M. sirikitiae leaves and their lignan constituents on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 mouse macrophage cells. Treatment of RAW 264.7 cells with the methanol extract of M. sirikitiae leaves and its isolated lignans, including (−)-phylligenin (2) and 3′,4-O-dimethylcedrusin (6) significantly decreased LPS-induced prostaglandin E₂ (PGE₂) and nitric oxide (NO) productions. These inhibitory effects of the extract and isolated lignans on LPS-induced upregulation of PGE₂ and NO productions were derived from the suppression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) production, respectively. In addition, treatment with 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3) and mitrephoran (5) was able to suppress LPS-induced tumor necrosis factor alpha (TNF-α) secretion and synthesis in RAW 264.7 cells. These results demonstrated that M. sirikitiae leaves and some isolated lignans exhibited potent anti-inflammatory activity through the inhibition of secretion and synthesis of PGE₂, NO, and TNF-α.     

Lignans from Arctium lappa and their inhibition of LPS-induced nitric oxide production

A new butyrolactone sesquilignan, isolappaol C (1), together with four known lignans, lappaol C (2), lappaol D (3), lappaol F (4), and diarctigenin (5), were isolated from the methanolic extract of the seeds from the Arctium lappa plant. The structure of isolappaol C (1) was determined by spectral analysis including 1D- and 2D-NMR. All the isolates were evaluated for their inhibitory effects on the LPS-induced nitric oxide production using murine macrophage RAW264.7 cells. Lappaol F (4) and diarctigenin (5) strongly inhibited NO production in the LPS-stimulated RAW264.7 cells with IC(50) values of 9.5 and 9.6 microM, respectively.     

Cell type-specific upregulation of myristoylated alanine-rich C kinase substrate and protein kinase C-alpha, -beta I, -beta II, and -delta in microglia following kainic acid-induced seizures

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed protein kinase C (PKC) substrate and has been implicated in actin cytoskeletal rearrangement in response to extracellular stimuli. Although MARCKS was extensively examined in various cell culture systems, the physiological function of MARCKS in the central nervous system has not been clearly understood. We investigated alterations of cellular distribution and phosphorylation of MARCKS in the hippocampus following kainic acid (KA)-induced seizures. KA (25 mg/kg, i.p.) was administered to eight to nine week-old C57BL/6 mice. Behavioral seizure activity was observed for 2 h after the onset of seizures and was terminated with diazepam (8 mg/kg, i.p.). The animals were sacrificed and analyzed at various points in time after the initiation of seizure activity. Using double-labeling immunofluorescence analysis, we demonstrated that the expression and phosphorylation of MARCKS was dramatically upregulated specifically in microglial cells after KA-induced seizures, but not in other types of glial cells. PKC alpha, beta I, beta II and delta, from various PKC isoforms examined, also were markedly upregulated, specifically in microglial cells. Moreover, immunoreactivities of phosphorylated MARCKS were co-localized in the activated microglia with those of the above isoforms of PKC. Taken together, our in vivo data suggest that MARCKS is closely linked to microglial activation processes, which are important in pathological conditions, such as neuroinflammation and neurodegeneration.     

Sesquiterpene esters from the fruits of Celastrus orbiculatus

Three new beta-dihydroagarofuran sesquiterpene esters, 6 alpha-acetoxy-9 beta-benzoyloxy-1 beta-cinnamoyloxy-8 beta-butanoyloxy-beta-dihydroagarofuran (1), 6 alpha-acetoxy-9 beta-benzoyloxy-1 beta-cinnamoyloxy-8 beta-(2-methylbutanoyloxy)-beta-dihydroagarofuran (2), and 6 alpha-acetoxy-1 beta,8 beta-dibenzoyloxy-9 beta-hydroxy-beta-dihydroagarofuran (6), together with three known compounds (3-5) were isolated from the fruits of Celastrus orbiculatus THUNB. Their structures were elucidated on the basis of spectroscopic methods. Compound 4 showed moderate activity in inhibiting LPS-induced nitric oxide production in murine macrophage RAW264.7 cells with an IC(50) value of 43.7 microM.     

In vivo trapping of polyketide intermediates from an assembly line synthase using malonyl carba(dethia)-N-acetyl cysteamines

Early-stage intermediates in the biosynthesis of erythromycin A by Saccharopolyspora erythraea were intercepted by malonyl carba(dethia)-N-acetyl cysteamines, generated in vivo from the hydrolysis of the corresponding methyl esters.     

Proteomic analysis of macrophages stimulated by lipopolysaccharide: Lipopolysaccharide inhibits the cleavage of nucleophosmin

Lipopolysaccharide (LPS) is a complex glycolipid composed of a hydrophilic polysaccharide and a hydrophobic domain that is responsible for the biological activity of LPS. There are many reports about LPS stimulation, and many activated proteins have been detected after LPS stimulation in various cell types. Furthermore, most of the LPS signaling pathways are clear. However, we were interested in examining the changes of LPS-induced total cytosolic proteins expression and the LPS signaling pathway by the proteomics technique during LPS-induced macrophage activation. Our study employed two-dimensional gel electrophoresis and mass spectrometry to analyze the proteins involved in LPS-induced activation in RAW 264.7 cells. We found 11 protein spots whose expression was different between untreated cells and LPS-treated cells. Ten protein spots were identified, seven of which, tubulin beta-4 chain (49.6 kDa, pI 4.78), nucleophosmin (32.6 kDa, pI 4.62, two spots), 40S ribosomal protein SA (P40) (32.7 kDa, pI 4.74), transforming protein RhoA (21.8 kDa, pI 5.83), nucleolin (76.6 kDa, pI 4.69), and T-complex protein 1 zeta subunit (58 kDa, pI 6.63) were down-regulated, and three of which, nucleophosmin (32.6 kDa, pI 4.62, two spots) and proteosome subunit alpha type-1 (29.5 kDa, pI 6.00), were up-regulated. The suppression of the proteolytic degradation of nucleophosmin was associated with LPS-induced RAW 264.7 cell activation. Cleaved caspase-3 decreased, thus it might be involved in proteolysis of nucleophosmin in LPS-induced macrophage activation. Our study also demonstrated that there was no change of the expression of nucleophosmin at the mRNA level.     

Three new eudesmane sesquiterpenes from Artemisia vestita

Three eudesmane sesquiterpene lactones,namely artemivestinolides A–C(1–3),along with three known 11-epimeric lactones(4–6),were isolated from the aerial parts of Artemisia vestita.Their structures were elucidated on the basis of extensive spectroscopic analyses(IR,HR-ESIMS,1D NMR and 2D NMR),and the absolute confgurations were determined by single-crystal X-ray diffraction(with copper radiation).Furthermore,in an in vitro assay,the three new compounds exhibited a moderate inhibition of the lipopolysaccharide(LPS)-induced nitric oxide(NO)production in BV-2 microglial cells.