Anti-Inflammatory Effects of Huberia peruviana Cogn. Methanol Extract by Inhibiting Src Activity in the NF-κB Pathway.

There is a growing need to develop anti-inflammatory drugs to regulate inflammatory responses. An extract of Huberia peruviana Cogn. had the best inhibitory effect on nitric oxide (NO) production in screening process undertaken in our laboratory. However, the anti-inflammatory effect of Huberia peruviana Cogn. methanol extract (Hp-ME) has not been studied. In this study, the anti-inflammatory effect of Hp-ME was assessed by using an NO assay, RT-PCR, luciferase reporter gene activity assay, western blotting assay, HCl/EtOH-induced acute gastritis model, and LPS-induced acute lung injury model. The phytochemical components of Hp-ME were determined through LC-MS/MS analysis. When RAW264.7 and HEK293T cells were treated with Hp-ME, NO production was decreased dose-dependently without cytotoxicity and the mRNA levels of iNOS, COX-2, and TNF-α were decreased. In a luciferase assay, the activity of transcription factors, NF-κB in TRIF or MyD88-overexpressing HEK293T cells was extremely reduced by Hp-ME. The western blotting analysis indicated that Hp-ME has anti-inflammatory effects by inhibiting the phosphorylation of Src. Hp-ME showed anti-inflammatory effects on in vivo models of HCl/EtOH-induced gastritis and LPS-induced acute lung injury. LC-MS/MS revealed that Hp-ME contains several anti-inflammatory flavonoids. The final findings of this study imply that Hp-ME could be used as an anti-inflammatory drug in several inflammatory diseases.

Caragana rosea Turcz Methanol Extract Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Suppressing the TLR4/NF-κB/IRF3 Signaling Pathways.

Caragana rosea Turcz, which belongs to the Leguminosae family, is a small shrub found in Northern and Eastern China that is known to possess anti-inflammatory properties and is used to treat fever, asthma, and cough. However, the underlying molecular mechanisms of its anti-inflammatory effects are unknown. Therefore, we used lipopolysaccharide (LPS) in RAW264.7 macrophages to investigate the molecular mechanisms that underlie the anti-inflammatory activities of a methanol extract of Caragana rosea (Cr-ME). We showed that Cr-ME reduced the production of nitric oxide (NO) and mRNA levels of iNOS, TNF-α, and IL-6 in a concentration-dependent manner. We also found that Cr-ME blocked MyD88- and TBK1-induced NF-κB and IRF3 promoter activity, suggesting that it affects multiple targets. Moreover, Cr-ME reduced the phosphorylation levels of IκBα, IKKα/ß and IRF3 in a time-dependent manner and regulated the upstream NF-κB proteins Syk and Src, and the IRF3 protein TBK1. Upon overexpression of Src and TBK1, Cr-ME stimulation attenuated the phosphorylation of the NF-κB subunits p50 and p65 and IRF3 signaling. Together, our results suggest that the anti-inflammatory activity of Cr-ME occurs by inhibiting the NF-κB and IRF3 signaling pathways.

Cocculus hirsutus ameliorates gastric and lung injuries by suppressing Src/Syk.

BACKGROUND: Cocculus hirsutus (L.) W. Thedo., a traditionally well-known plant, has confirmed antitumor properties as well as acute and chronic diuretic effects. However, little is known about its inflammatory activities and the potential effect on inflammatory disease treatment. PURPOSE: Our aim in this study was to explore additional beneficial properties of C. hirsutus ethanol extract (Ch-EE) such as anti-inflammatory activity in vitro and in vivo as well as its underlying mechanisms and to provide a theoretical basis for its role as a candidate natural drug in clinical gastritis and lung disease therapy. STUDY DESIGN: RAW264.7 cells, HEK293T cells, peritoneal macrophages, and mouse models of acute gastritis and acute lung injury were used to assess the anti-inflammatory activity of Ch-EE. METHODS: Decreases in LPS-induced nitric oxide (NO) production and cytokine expression by RAW264.7 cells after Ch-EE treatment were evaluated by Griess assays and PCR, respectively. Transcription factor activity was assessed through luciferase reporter gene assay, and protein expression was determined by Western blotting analysis. Overexpression assays and cellular thermal shift assays were executed in HEK293T cells. Our two in vivo models were an HCl/EtOH-induced gastritis model and an LPS-induced lung injury model. Changes in stomach lesions, lung edema, and lung histology were examined upon treatment with Ch-EE. Components of Ch-EE were determined by liquid chromatography-mass spectrometry. RESULTS: LPS-induced nitric oxide production and Pam3CSK4- and L-NAME-induced NO production were inhibited by Ch-EE treatment of RAW264.7 cells. Furthermore, LPS-induced increases in transcript levels of iNOS, COX2, CCL12, and IL-1ß were reduced by Ch-EE treatment. Ch-EE decreased both MyD88- and TRIF-induced NF-κB promotor activity. Proteins upstream of NF-κB, namely p-p50, p-p65, p-IκBα, p-AKT1, p-Src, and p-Syk, were all downregulated by Ch-EE. Moreover, Src and Syk were targets of Ch-EE. Ch-EE treatment reduced the size of inflammatory stomach lesions induced by HCl/EtOH, lung edema, and accumulation of activated neutrophils caused by LPS. CONCLUSIONS: These results strongly suggest that Cocculus hirsutus can be developed as a promising anti-inflammatory remedy with Src- and Syk-inhibitory functions targeting diseases related to gastritis and lung injury.

Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages, HCl/EtOH-induced Gastritis, and LPS-induced Lung Injury via Attenuation of Src and TAK1.

Several Cissus species have been used and reported to possess medicinal benefits. However, the anti-inflammatory mechanisms of Cissus subtetragona have not been described. In this study, we examined the potential anti-inflammatory effects of C. subtetragona ethanol extract (Cs-EE) in vitro and in vivo, and investigated its molecular mechanism as well as its flavonoid content. Lipopolysaccharide (LPS)-induced macrophage-like RAW264.7 cells and primary macrophages as well as LPS-induced acute lung injury (ALI) and HCl/EtOH-induced acute gastritis mouse models were utilized. Luciferase assays, immunoblotting analyses, overexpression strategies, and cellular thermal shift assay (CETSA) were performed to identify the molecular mechanisms and targets of Cs-EE. Cs-EE concentration-dependently reduced the secretion of NO and PGE2, inhibited the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells, and decreased NF-κB- and AP-1-luciferase activity. Subsequently, we determined that Cs-EE decreased the phosphorylation events of NF-κB and AP-1 pathways. Cs-EE treatment also significantly ameliorated the inflammatory symptoms of HCl/EtOH-induced acute gastritis and LPS-induced ALI mouse models. Overexpression of HA-Src and HA-TAK1 along with CETSA experiments validated that inhibited inflammatory responses are the outcome of attenuation of Src and TAK1 activation. Taken together, these findings suggest that Cs-EE could be utilized as an anti-inflammatory remedy especially targeting against gastritis and acute lung injury by attenuating the activities of Src and TAK1.

Syk/NF-κB-targeted anti-inflammatory activity of Melicope accedens (Blume) T.G. Hartley methanol extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Melicope accedens (Blume) Thomas G. Hartley is a plant included in the family Rutaceae and genus Melicope. It is a native plant from Vietnam that has been used for ethnopharmacology. In Indonesia and Malaysia, the leaves of M. accedens are applied externally to decrease fever. AIM OF THE STUDY: The molecular mechanisms of the anti-inflammatory properties of M. accedens are not yet understood. Therefore, we examined those mechanisms using a methanol extract of M. accedens (Ma-ME) and determined the target molecule in macrophages. MATERIALS AND METHODS: We evaluated the anti-inflammatory effects of Ma-ME in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in an HCl/EtOH-triggered gastritis model in mice. To investigate the anti-inflammatory activity, we performed a nitric oxide (NO) production assay and ELISA assay for prostaglandin E2 (PGE2). RT-PCR, luciferase gene reporter assays, western blotting analyses, and a cellular thermal shift assay (CETSA) were conducted to identify the mechanism and target molecule of Ma-ME. The phytochemical composition of Ma-ME was analyzed by HPLC and LC-MS/MS. RESULTS: Ma-ME suppressed the production of NO and PGE2 and the mRNA expression of proinflammatory genes (iNOS, IL-1ß, and COX-2) in LPS-stimulated RAW264.7 cells without cytotoxicity. Ma-ME inhibited NF-κB activation by suppressing signaling molecules such as IκBα, Akt, Src, and Syk. Moreover, the CETSA assay revealed that Ma-ME binds to Syk, the most upstream molecule in the NF-κB signal pathway. Oral administration of Ma-ME not only alleviated inflammatory lesions, but also reduced the gene expression of IL-1ß and p-Syk in mice with HCl/EtOH-induced gastritis. HPLC and LC-MS/MS analyses confirmed that Ma-ME contains various anti-inflammatory flavonoids, including quercetin, daidzein, and nevadensin. CONCLUSIONS: Ma-ME exhibited anti-inflammatory activities in vitro and in vivo by targeting Syk in the NF-κB signaling pathway. Therefore, we propose that Ma-ME could be used to treat inflammatory diseases such as gastritis.

Heterocycle-linked phenylbenzyl amides as novel TRPV1 antagonists and their TRPV1 binding modes: constraint-induced enhancement of in vitro and in vivo activities.

A series of heterocycle-linked constrained phenylbenzyl amides were found to be TRPV1 antagonists with promising in vivo profiles. In particular, one of the analogues containing a furan linker exhibited excellent TRPV1 antagonistic activity and in vivo analgesic efficacy. In addition, the binding modes of dibenzyl thiourea, benzylphenethyl amide, and furan-linked phenylbenzyl amide were examined by using the flexible docking study within the rTRPV1 homology model.

Depigmenting activity of new kojic acid derivative obtained as a side product in the synthesis of cinnamate of kojic acid.

We synthesized cinnamate derivatives of kojic acid for use as depigmenting agents by various esterification methods. The cinnamate of 5-position of kojic acid (6) was obtained by EDC coupling, DCC coupling, acid chloride, and mixed anhydride methods. To obtain the cinnamate of the 2-position of kojic acid (7), we carried out the nucleophilic addition of the potassium salt of cinnamic acid to kojyl chloride. In this reaction, we discovered the occurrence of a side reaction and identified the structure of the side product thus formed. We evaluated the depigmenting activities of both the side product and the cinnamate derivatives of kojic acid. Interestingly, the side product (11) showed more potent depigmenting activity (IC(50)=23.51µM) than compound 7 (IC(50)>100µM) which is the mother compound of the side product. However, it has no tyrosinase inhibitory activity. Compound 6, the cinnamate of 5-position of kojic acid, also showed moderate depigmenting activity (IC(50)=46.64µM) without tyrosinase inhibitory activity. Production of this side product (11) may have originated from the proton exchange between the potassium salt of cinnamic acid and kojyl chloride. We then efficiently reduced the yield of the side product by controlling the equilibrium of the potassium salt of cinnamic acid. The addition of cinnamic acid greatly reduced the amount of the side product produced.

Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method.

PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C(18) column (4.6 mm × 75 mm, 3.5 µm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492→156; IS, 506→156, m/z). The calibration curve was linear over the range of 1.0-500 ng/ml (r(2)>0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ± 15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze-thaw cycles and 4-week storage in a freezer at -80°C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1h and 3.8h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application (F% 0.2-1.7%).

TRPV1 antagonist can suppress the atopic dermatitis-like symptoms by accelerating skin barrier recovery.

BACKGROUND: Transient receptor potential vanilloid type 1 (TRPV1) is a cation channel activated by diverse obnoxious stimuli like capsaicin, low pH or heat. Recently, it was revealed that TRPV1 might be deeply associated with skin permeability barrier function, suggesting that modulation of TRPV1 might be beneficial for the skin disorders with barrier damages. OBJECTIVE: We aimed to investigate whether the blockade of TRPV1 activation might accelerate skin barrier recovery and alleviate atopic dermatitis (AD)-like symptoms, employing a novel TRPV1 antagonist, PAC-14028. METHODS: TRPV1 antagonistic effects of PAC-14028 in human keratinocytes and skin were confirmed through capsaicin-evoked calcium influx assay and capsaicin-induced blood perfusion increase. Effects of PAC-14028 on skin barrier recovery were examined in vivo tape-stripping-induced barrier disruption in hairless mice. To determine the effects of PAC-14028 on AD, Dermatophagoides farina (Df)- and oxazolone (OXZ)-induced AD models were employed. RESULTS: PAC-14028 could inhibit capsaicin-evoked calcium influx in keratinocytes at sub-micromolar concentrations. This potent TRPV1 antagonistic activity in keratinocytes was manifested in vivo as the blockade of capsaicin-induced blood perfusion increase, and the accelerated barrier recovery from tape-stripping-induced barrier damages in hairless mice. PAC-14028 could also attenuate dermatitis-associated barrier damages in Df and OXZ models as determined by lower TEWL (trans-epidermal water loss), reformation of neutral lipid layer and reversion of changes in loricrin and filaggrin expression. Importantly, along with accelerated recovery of skin barrier function, PAC-14028 alleviated the general AD-like symptoms, including serum IgE increase, mast cell degranulation, scratching behavior and clinical severity of dermatitis. CONCLUSIONS: These results reflect that the blockade of TRPV1 activation can suppress the atopic dermatitis-like symptoms by accelerating skin barrier recovery.