Protective Effects Of Fraxin On Acute Respiratory Distress Syndrome And Its Mechanisms

Objective:Acute respiratory distress syndrome is a common critical disease in clinical practice with the characteristics of high mortality,complicated mechanism and serious harmfulness。However,there is still a lack of effective drugs to cure it.Fraxin is one of the main effective components extracted from Cortex fraxini,a traditional Chinese medicine.So far,it has been shown that it has many pharmacological and biological activities.Therefore in this study,the effects and possible protective mechanisms of fraxin on a mouse model of bacterial endotoxin lipopolysaccharide(LPS)-induced ARDS were investigated.Methods:1.Healthy and aged 8-10 weeks C57BL/6 male mice were used as the research object of the experiment.All of the mice were randomly divided into five groups:control,LPS group,and LPS+fraxin groups at 10,20,or 40 mg/kg fraxin.The treated groups were intragastrically administered with fraxin based on their body weight(10,20,or 40 mg/kg),whereas the control and LPS groups were treated with the same volume of normal saline once a day.After the drugs were intragastrically administered for 7 consecutive days,the LPS+ fraxin groups and the LPS groups were intraperitoneally injected with LPS(20 mg/kg)to induce the ARDS mouse model.The control group was administered with the same volume of normal saline.After 6 h,the lung tissue,blood and alveolar lavage fluid of the mice were collected for the next experiment.Using the hematoxylin and eosin(HE)staining to observe the changes of mouse lung tissues in each group.Using the wet/dry(W/D)ratio of the mouse lung tissues to evaluate the degree of edema in the lung tissue of the mice in each group.Specific mouse ELISA kits were used to determination of the malondialdehyde(MDA)content and the superoxide dismutase(SOD)activity in the mouse lung tissues of each group and the contents of pro-inflammatory cytokines(IL-6,TNF-α,and IL-1β)in the bronchoalveolar lavage fluid(BALF)of the mice of each group.Furthermore,using a murine CD138 ELISA kit to detect the content of SDC-1 in the serum of each group of mice.Nonfluorescent DCFH-DA was used to determination of the ROS content in the mouse lung tissues of each group.Using the immunofluorescence techniques of mouse lung tissues to detect the change of the endothelial glycocalyx integrity and the expression of the glycocalyx-degrading enzyme MMP-9 in mice of each group.Changes in the pulmonary vascular permeability of the mice were determined using assays through EBD tissue extravasation and the leakage of FITC-albumin through lungs and blood vessel walls to assess pulmonary vascular permeability.Western blot were used to further analyze the two important inflammatory regulatory pathways,NF-κB and MAPKs in ARDS mice of each group.Results:Via observing the consequence of the hematoxylin and eosin staining,shows that fraxin might alleviate pathological changes in the lung tissues of mice with ARDS.The consequence of the mouse specific ELISA kit and Western blot results revealed that fraxin might inhibit the production of inflammatory factors,such as,IL-6,TNF-α,and IL-1β and the activation of NF-κB and MAPKs signaling pathways in the lungs of each group.Thus,the inflammatory responses were reduced.Furthermore,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.Conclusion:Thus,fraxin elicited protective effects on mice with LPS-induced ARDS by downregulating the inflammatory responses and oxidative damage and reducing pulmonary vascular permeability.