Prasiola japonica possesses several biological activities. However, reports on the anti-inflammatory activities and molecular mechanisms of its different solvent fractions remain limited. In this study, we investigated the potential anti-inflammatory activities of P. japonica ethanol extract (Pj-EE) and four solvent fractions of Pj-EE made with hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), or water (Pj-EE-WF) in both in vitro (LPS-induced macrophage-like RAW264.7 cells) and in vivo (carrageenan-induced acute paw edema mouse models) experiments. The most active solvent fraction was selected for further analysis. Various in vitro and in vivo assessments, including nitric oxide (NO), cytokines, luciferase assays, real-time polymerase chain reactions, and immunoblotting analyses were performed to evaluate the underlying mechanisms. In addition, the phytochemical constituents were characterized by Liquid chromatography-tandem mass spectrometry. In in vitro studies, the highest inhibition of NO production was observed in Pj-EE-CF. Further examination revealed that Pj-EE-CF decreased the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells and suppressed subsequent AP-1-luciferase activity by inhibition of phosphorylation events in the AP-1 signaling pathway. Pj-EE-CF treatment also demonstrated the strongest reduction in thickness and volume of carrageenan-induced paw edema, while Pj-EE-BF showed the lowest activity. Furthermore, Pj-EE-CF also reduced gene expression and cytokines production in tissue lysates of carrageenan-induced paw edema. These findings support and validate the evidence that Pj-EE, and especially Pj-EE-CF, could be a good natural source for an anti-inflammatory agent that targets the AP1 pathway.

Alverine, a smooth muscle relaxant, is used to relieve cramps or spasms of the stomach and intestine. Although the effects of alverine on spontaneous and induced contractile activity are well known, its anti-inflammatory activity has not been fully evaluated. In this study, we investigated the anti-inflammatory effects of alverine in vitro and in vivo. The production of nitric oxide (NO) in RAW264.7 cells activated by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly (I:C)) was reduced by alverine. The mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) was also dose-dependently inhibited by treatment with alverine. In reporter gene assays, alverine clearly decreased luciferase activity, mediated by the transcription factor nuclear factor κB (NF-κB) in TIR-domain-containing adapter-inducing interferon-β (TRIF)- or MyD88-overexpressing HEK293 cells. Additionally, phosphorylation of NF-κB subunits and upstream signaling molecules, including p65, p50, AKT, IκBα, and Src was downregulated by 200 μM of alverine in LPS-treated RAW264.7 cells. Using immunoblotting and cellular thermal shift assays (CETSAs), Src was identified as the target of alverine in its anti-inflammatory response. In addition, HCl/EtOH-stimulated gastric ulcers in mice were ameliorated by alverine at doses of 100 and 200 mg/kg. In conclusion, alverine reduced inflammatory responses by targeting Src in the NF-κB pathway, and these findings provide new insights into the development of anti-inflammatory drugs.

Ultraviolet (UV) irradiation induces ROS production, which activates activator protein (AP)-1 and nuclear factor (NF)-κB signaling and downstream molecules, ultimately triggering the generation of matrix metalloproteinases (MMPs) and degradation of collagen. The aim of this study was to investigate the protective effect of methanol extract from Malus baccata (L.) Borkh (Mb-ME) against aging. DPPH and ABTS assays showed that Mb-ME had a significant antioxidant capacity. Flow cytometry results indicated that Mb-ME attenuated UVB and H₂O₂-stimulated apoptosis and reactive oxygen species (ROS) generation. RT-PCR analysis in HaCaT and HDF cells suggested that Mb-ME treatment blocked the expression of MMPs, COX-2, IL-1β, IL-6, HYALs, and p53 while promoting the levels of TGM1, FLG, HASs, Sirt1, and Col1A1. Mechanically, Mb-ME inhibited the phosphorylation of MAP kinases and NF-κB signaling. Overall, these results strongly suggest that Mb-ME can be developed as an antiaging therapy.

Many species in the genus Guettarda are known to exert anti-inflammatory effects and are used as traditional medicinal plants to treat various inflammatory symptoms. However, no studies on the inflammatory activities of Guettarda crispiflora Vahl have been reported. The aim of the study was to investigate in vitro and in vivo the anti-inflammatory effects of a methanol extract of Guettarda crispiflora Vahl (Gc-ME). To determine the anti-inflammatory activity of Gc-ME, lipopolysaccharide (LPS)-, poly(I:C)-, or Pam3CSK4-treated RAW264.7 cells, HCl/EtOH- and LPS-treated mice were employed for in vitro and in vivo tests. LPS-induced nitric oxide production in RAW264.7 cells was determined by Griess assays and cytokine gene expression in LPS-activated RAW264.7 cells, confirmed by RT- and real-time PCR. Transcriptional activation was evaluated by luciferase reporter gene assay. Target protein validation was assessed by Western blot analysis and cellular thermal shift assays (CETSA) with LPS-treated RAW264.7 and gene-transfected HEK293 cells. Using both a HCl/EtOH-induced gastritis model and an LPS-induced lung injury model, inflammatory states were checked by scoring or evaluating gastric lesions, lung edema, and lung histology. Phytochemical fingerprinting of Gc-ME was observed by using liquid chromatography–mass spectrometry. Nitric oxide production induced by LPS and Pam3CSK4 in RAW264.7 cells was revealed to be reduced by Gc-ME. The LPS-induced upregulation of iNOS, COX-2, IL-6, and IL-1β was also suppressed by Gc-ME treatment. Gc-ME downregulated the promotor activities of AP-1 and NF-κB triggered by MyD88- and TRIF induction. Upstream signaling proteins for NF-κB activation, namely, p-p50, p-p65, p-IκBα, and p-Src were all downregulated by Ch-EE. Moreover, Src was revealed to be directly targeted by Gc-ME. This extract, orally treated strongly, attenuated the inflammatory symptoms in HCl/EtOH-treated stomachs and LPS-treated lungs. Therefore, these results strongly imply that Guettarda crispiflora can be developed as a promising anti-inflammatory remedy with Src-suppressive properties.

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.