Anti-inflammatory And Antioxidant Effects And Mechanisms Of Oxypeucedanin On Acute Lung Injury In Mice

BackgroundAcute lung injury(ALI)and respiratory distress syndrome(ARDS)is a clinical critical illness characterized by diffuse inflammation of the lung parenchyma and intractable hypoxemia.Since the COVID-19 pandemic,the number of ALI cases associated with it has increased.The lungs of ALI patients often exhibit a strong inflammatory response,with activated neutrophils and macrophages producing large amounts of pro-inflammatory mediators and disrupting the integrity of the barrier between alveolar epithelial cells and vascular endothelial cells.Therefore,suppression of the inflammatory response will be the key idea in the treatment of ALI.At the same time,ALI will cause different degrees of damage to mitochondria in cells,and the resulting mitochondrial dysfunction will produce a large amount of reactive oxygen species(ROS)accumulation,and when the scavenging ability of antioxidant system in the body is exceeded,the body will present oxidative stress.Nowadays,herbal monomers have been widely studied as a potential clinical drug,and oxypeucedanin(OPD),a furanocoumarin-like compound,have a wide range of anti-inflammatory and antioxidant effects.Therefore,in this study,we investigated the anti-inflammatory and antioxidant pathways of oxypeucedanin to reveal the protective effects of oxypeucedanin against ALI and related mechanisms.Methods1.Study on the anti-inflammatory effect of oxypeucedanin in ALI(1)LPS-induced ALI mouse model was constructed,and the lungs of mice were evaluated by HE staining of pathological sections between control and different treatment groups;(2)Myeloperoxidase(MPO)was used to detect the degree of neutrophil infiltration in lung tissue of mice in each group;(3)real-time fluorescence quantitative PCR and ELISA were used to examine the inflammatory factors IL-6,IL-1β,TNF-α m RNA transcript levels and secretion in lung tissue and alveolar lavage fluid(BALF),respectively.The levels of IL-6,IL-1β and TNF-αm RNA transcripts and their secretion in lung tissue and BALF were examined by real-time fluorescence PCR and ELISA,respectively;(4)the regulation of MAPK and AKT/NF-κB pathway proteins was examined by Western Blotting;the lung wet dry specific gravity of each group of mice was measured to assess the edema of lung tissue;(5)the total protein concentration in alveolar lavage fluid was measured by Evans Blue staining to detect pulmonary vascular permeability.The lung vascular permeability was measured by Evans Blue staining and the total protein concentration in the alveolar lavage fluid was measured by Western Blotting and immunofluorescence.(6)In in vitro cellular level assays: screening of appropriate cellular administration concentrations by CCK8,RT-PCR and ELISA methods to assess the m RNA transcript levels and secretion of inflammatory factors IL-1β,IL-6,TNF-α in each treatment group;(7)Western Blotting to detect the expression of MAPK and AKT/NF-κB proteins;immunofluorescence to observe the nuclear entry of P65.2.Oxypeucedanin exert a protective effect in ALI by improving mitochondrial function(1)In the ALI mouse model,the accumulation of ROS was measured by flow cytometry;the levels of antioxidant enzymes SOD,GSH,CAT and MDA were measured by ELISA;(2)the structural changes of mitochondria in mouse lung tissue were observed by electron microscopy;(3)the expression of HO-1 and the mitochondrial dynamics-related proteins Drp1,OPA1,Mfn2 and mitochondrial transcription factor A(TFAM)protein levels were measured by RT-PCR and Western Blotting.(4)In in vitro experiments,we used mouse macrophages(RAW267.4)and human alveolar macrophages(AM)to validate the above in vivo results.The optimal concentration of oxypeucedanin pre-protected alveolar macrophages was first determined by the CCK8 assay method;(5)the effect of oxypeucedanin on HO-1 expression was analyzed by cellular immunofluorescence assay;(6)cellular ROS production level was detected by cellular immunofluorescence;changes in cellular mitochondrial structure were observed by electron microscopy;mitochondrial membrane potential changes were detected by JC-1 staining and Mito-Tracker Red CMX Ros reagent.The expression of HO-1 and the mitochondrial dynamics-related proteins Drp1,OPA1,Mfn2 and TFAM were detected by Western Blotting.Results1.Oxypeucedanin s reduce LPS-induced lung injury in ALI mice by alleviating the inflammatory response(1)Oxypeucedanin significantly improved the extent of lung injury in ALI mouse model and inhibited the secretion of TNF-α,IL-6 and IL-1β in BALF.MPO activity,i NOS and COX-2 protein expression in lung tissues showed a decreasing trend,and MAPK and AKT/NF-κB protein phosphorylation levels were also significantly inhibited.(2)Meanwhile,oxypeucedanin effectively alleviated the edema of lung tissue in LPS-induced mice,and the dye leakage in alveolar fluid and total protein concentration in alveolar lavage fluid were significantly reduced,and the tight junction proteins Claudin 3 and Occludin also showed up-regulated expression.(3)In in vitro assays,oxidative prejuice inhibited LPS-induced i NOS,COX-2expression,cytokine IL-1β,IL-6,TNF-α secretion,MAPK and AKT/NF-κB protein phosphorylation levels,and P65 nucleation.2.Oxypeucedanin alleviates LPS-induced ALI/ARDS through HO-1 mediated mitochondrial kinetic homeostasis and TFAM(1)Oxypeucedanin reduced ROS levels,increased expression of antioxidant enzymes SOD,GSH and CAT,and decreased levels of lipid peroxidation products(MDA)in lung tissues of ALI mice.(2)Oxypeucedanin effectively alleviated mitochondrial damage in lung tissue cells,increased transcription and expression levels of HO-1,OPA1,Mfn2 and TFAM,and decreased transcription and expression of Drp1.(3)The above in vivo results were also verified in in vitro cellular assays,where oxypeucedanin promoted HO-1 expression,reduced ROS accumulation,restored mitochondrial membrane potential,and up-regulated expression of OPA1,Mfn2,and TFAM,and down-regulated expression of Drp1.After inhibition of HO-1,the regulatory ability of OPD on OPA1,Mfn2,TFAM and Drp1 proteins disappeared,ROS accumulation increased,mitochondrial damage reappeared,and mitochondrial membrane potential disappeared.Conclusion1.In in vivo and in vitro experiments,oxypeucedanin reduced inflammation in LPS-induced ALI mice by inhibiting the activation of MAPK and AKT/NF-κB signaling pathways.At the same time,oxypeucedanin significantly attenuated the degree of pulmonary edema and maintained the integrity of the lung air-blood barrier in the ALI mouse model.2.The present study demonstrated that oxypeucedanin alleviated LPS-induced mitochondrial dysfunction by promoting mitochondrial fusion and biogenesis,and alleviated oxidative stress caused by ROS excessive accumulation and mitochondrial damage.At the cellular level,we further demonstrated that the above phenomena were closely related to the regulation of HO-1 by oxypeucedanin.In summary,this study reveals the protective effects of oxypeucedanin on ALI mice at both anti-inflammatory and antioxidant levels.This study demonstrates the potential value of oxypeucedanin as a clinical alternative drug,and also provides a theoretical basis and pre-application basis for subsequent clinical treatment and prevention.