Chloroform Fraction of Prasiola japonica Ethanolic Extract Alleviates UPM 1648a-Induced Lung Injury by Suppressing NF-κB Signaling

Prasiola japonica is an edible alga, and the ethanol extract of P. japonica (Pj-EE) possesses various biological activities. Interestingly, in a recent study, we observed the potent anti-inflammatory activity of the chloroform fraction of Pj-EE (Pj-EE-CF). Thus, to extend the application of Pj-EE-CF, we further studied its effects on lung injury. To establish an experimental model of lung injury, we nasally administered urban particulate matter UPM 1648a (50 mg/kg) to mice. In addition, BEAS-2B cells were treated with 300 µg/mL of UPM 1648a for in vitro analysis. Intranasal administration of UPM 1648a increased lung injury score, macrophage infiltration, and upregulation of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in lung tissues. On the other hand, oral administration of Pj-EE-CF (25, 50, and 100 mg/kg) alleviated these pathological features as assessed by lung wet/dry ratio, lung injury score, bronchoalveolar lavage fluid (BALF) protein amount in the lung tissues up to 70%, 95%, and 99%, respectively. In addition, Pj-EE-CF down-regulated the release of inflammatory cytokines, interleukins (ILs), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ elevated by UPM 1648a in the lung tissues and lung BALF up to 95%. According to Western blot and luciferase assay, Pj-EE-CF (100 mg/kg in vivo or 50 and 100 µg/mL in vitro) significantly reduced the nuclear factor-κB (NF-κB) signal activated by UPM 1648a. Finally, UPM 1648a increased cellular reactive oxygen species (ROS) levels in BEAS-2B cells, while Pj-EE-CF reduced them. These results suggest that Pj-EE-CF alleviates UPM 1648a-induced lung damage via anti-inflammatory and antioxidant activities and by suppressing NF-κB signaling. In conclusion, these observations imply that Pj-EE-CF could be a practical component of food supplements to mitigate air pollution-derived lung damage.

Anti-Inflammatory Activities of the Ethanol Extract of Prasiola japonica, an Edible Freshwater Green Algae, and Its Various Solvent Fractions in LPS-Induced Macrophages and Carrageenan-Induced Paw Edema via the AP-1 Pathway.

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.

Anti-Inflammatory Functions of Alverine via Targeting Src in the NF-κB 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-ß (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.