fraxin and Inflammation

Inflammation and oxidative stress are associated with the pathogenesis of cerebral ischemia-reperfusion (I/R) injury. Fraxin, one of the primary active ingredients of Cortex Fraxini, may have potent anti-inflammatory activity. This study intended to investigate the function and mechanism of fraxin in a middle cerebral artery occlusion (MCAO) model.. A middle cerebral artery occlusion (MCAO) rat model was engineered. Both in-vivo and in-vitro models were dealt with Fraxin. The profiles of inflammation-concerned cytokines, proteins and oxidative stress factors were determined by RT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA), and neuronal apoptosis and reactive oxygen species (ROS) levels were measured. The neurological functions of rats were evaluated by Morris water maze and modified neurological severity scores (mNSS).. The data revealed that fraxin abated the OGD/R-mediated release of inflammatory and oxidative stress mediators, enhanced "M2″-like BV2 microglia polarization, and mitigated HT22 cell apoptosis. Mechanistically, fraxin boosted PPAR-γ expression, activated the Nrf2/HO-1 pathway, and suppressed NF-κB, IKK-β，p38 MAPK, ERK1/2 and Keap1 in a dose-dependent manner. Furthermore, attenuating PPAR-γ through pharmacological treatment with GW9662 (a PPAR-γ antagonist) mainly weakened the neuroprotective and anti-inflammatory functions of fraxin.. Fraxin could considerably ameliorate cerebral I/R damage by repressing oxidative stress, inflammatory response, and cell apoptosis through abrogating the PPARγ/ NF-κB pathway.

Topics: Animals; Anti-Inflammatory Agents; Brain Ischemia; Coumarins; Infarction, Middle Cerebral Artery; Inflammation; Kelch-Like ECH-Associated Protein 1; Neuroinflammatory Diseases; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; PPAR gamma; Rats; Reperfusion Injury; Signal Transduction

Fraxin, the effective component isolated from Cortex Fraxini, has been reported to have anti-inflammation effects. The aim of this study was to explore the effect of fraxin on lipopolysaccharide (LPS)-induced endotoxic shock in mice. We used Kunming male mice to establish the model, and we found that fraxin could improve the survival rate of the LPS-induced mice. Histopathological study showed that fraxin could mitigate the injuries in LPS-induced lung and liver tissues. The levels of tumour necrosis factor-α and interleukin-6 both in serum and lung, liver tissues, and the productions of nitric oxide (NO), aspartate transaminase and alanine transaminase in serum were decreased by fraxin. Western blot assay demonstrated that the pretreatment with fraxin could downregulate LPS-induced protein expressions of nuclear factor-kappa B (NF-κB) and NLRP3 inflammatory corpuscle signalling pathways. Overall, fraxin had protective effects on LPS-induced endotoxic shock mice and the possible mechanisms might activate through NF-κB and NLRP3 inflammatory corpuscle signalling pathways.

Topics: Animals; Anti-Inflammatory Agents; Coumarins; Cytokines; Disease Models, Animal; Inflammation; Lipopolysaccharides; Male; Mice; NF-kappa B; Nitric Oxide; NLR Family, Pyrin Domain-Containing 3 Protein; Shock, Septic; Signal Transduction; Survival Rate

Acute respiratory distress syndrome (ARDS) is a severe acute disease that threatens human health, and few drugs that can effectively treat this disease are available. Fraxin, one of the main active ingredients of Cortex Fraxini, a Chinese herbal medicine, has presented various pharmacological and biological activities. However, the effects of fraxin on ARDS have yet to be reported. In the present study, the protective effect of fraxin in lipopolysaccharide (LPS)-induced ARDS in a mouse model was analyzed. Results from the hematoxylin and eosin staining showed that fraxin might alleviate pathological changes in the lung tissues of mice with ARDS. ELISA and Western blot results revealed that fraxin might inhibit the production of inflammatory factors, namely, IL-6, TNF-α, and IL-1β, and the activation of NF-κB and MAPK signaling pathways in the lungs. Thus, the inflammatory responses were reduced. Fraxin might inhibit the increase in reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation in lung tissues. Fraxin might increase the superoxide dismutase (SOD) activity to avoid oxidative damage. Vascular permeability was also assessed through Evans blue dye tissue extravasation and fluorescein isothiocyanate-labeled albumin (FITC-albumin) leakage. Fraxin might inhibit the increase in pulmonary vascular permeability and relieve pulmonary edema. Fraxin was also related to the inhibition of the increase in matrix metalloproteinase-9, which is a glycocalyx-degrading enzyme, and the relief of damages to the endothelial glycocalyx. Thus, fraxin elicited protective effects on mice with LPS-induced ARDS and might be used as a drug to cure ARDS induced by Gram-negative bacterial infection.

Topics: Animals; Capillary Permeability; Coumarins; Down-Regulation; Glycocalyx; Inflammation; Lipopolysaccharides; Lung; MAP Kinase Signaling System; Mice; NF-kappa B; Oxidation-Reduction; Respiratory Distress Syndrome

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature