Protective Effect Of Oxyberberine On Acute Lung Injury In Mice

ObjectiveOxyberberine(OBB)is the oxidative metabolite of berberine.In recent years,studies have shown that OBB has a good activity of hypoglycemic,anti-inflammatory,antiarrhythmia and the treatment of ulcerative colitis.Acute lung injury(ALI)is a common critical and emergency disease in respiratory department,which can develop into acute respiratory distress syndrome(ARDS).Due to the high fatality rate and the lack of specific drugs at present,drug development for the prevention and treatment of ALI has been attracting much attention.Recent studies have found that the occurrence of ALI is closely related to the destruction of the alveolar epithelial barrier.The alveolar epithelial barrier is the physical barrier between the alveolar cavity and the outside world.It is mainly composed of epithelial cells and intercellular connections.The barrier function can be maintained stable according to its selection through and active transport of body fluid function.Acute lung injury leads to the damage of alveolar epithelial cells,the destruction of intercellular junction proteins,the occurrence of cytoskeleton migration,and the increase of permeability,which leads to the abnormality of alveolar epithelial barrier function,the imbalance of fluid transport,and finally the formation of pulmonary edema.Previous studies have shown that OBB can promote the expression of intercellular junction proteins in barrier cells and reduce the damage of epithelial barrier caused by diseases.At the same time,RhoA/ROCK is a signaling pathway that regulates various cellular functions and responses,and its activation can promote cell contraction and migration,thereby weakening the protective function of the alveolar epithelial barrier.Therefore,this study aims to explore the protective effect of oxyberberine on mice with acute lung injury and elaborate its related mechanism from the perspective of maintaining alveolar epithelial barrier.Methods1.Effect of oxyberberine on acute lung injury cell modelCCK-8 method was used to detect the effect of OBB on the survival rate of A549 cells.With IL-1β as the index,the ELISA reagent method was used to explore the dose of LPS modeling and OBB administration.TNF-α,IL-1β and IL-6 ELISA kit were used to detect the effect of OBB on acute lung injury cell model.2.Effects of oxyberberine on animal models of acute lung injuryBALB/c mice were randomly divided into normal group,model group,oxyberberine group(10,20,40 mg/kg),and berberine group(40 mg/kg).After prophylactic administration for 7 days and 1 hour after administration on the 7th day,20 mg/kg LPS was administered to the mice to establish the model through tracheal infusion.The survival of the mice was observed every 12 hours and the number of deaths was recorded for a total of 144 hours.BALB/c mice were randomly divided into normal group,model group,dexamethasone group(2 mg/kg),and oxyberberine group(10,20,40 mg/kg).The mice were given prophylactic medicine for 7 days,and 5 mg/kg LPS was used for endotracheal dripping modeling 1 hour after administration on the 7th day.After 24 hours of modeling,the left lung of some mice was taken for wet/dry lung weight weighing,while the other mice were lavaged with alveolar lavage and lavage fluid was collected.The pathological changes of lung tissue were observed by Hematoxylin-Eosin staining.The cell precipitation in the alveolar lavage fluid was observed and measured by Swiss-Gimsa staining,and the protein content in the alveolar lavage fluid was measured.The contents of TNF-α,IL-1β,IL-6 and MPO in lung tissues and alveolar lavage fluid were detected by ELISA reagent.3.The mechanism of protective effect of oxyberberine on acute lung injury in miceThe mRNA levels of Connexin-43 and ZO-1 were detected by ELISA reagent,and the mRNA levels of Connexin-43 and ZO-1 were analyzed by PCR.The expression levels of RhoA,ROCK,phosphorylated MLC and MLC were detected by Western-blot.Based on the view of maintaining alveolar epithelial barrier,the protective mechanism of oxyberberine against LPS-induced acute lung injury in mice was studied.4.Verification of the mechanism of oxyberberine in acute lung injury modelUsing U46619 as the RhoA/ROCK signaling pathway agonist,the cells were divided into Normal group,LPS group,LPS+U46619 group,LPS+OBB group and LPS+U46619+OBB group.The dose of U46619 was determined by RhoA ELISA kit,and the protein content of ROCK was also determined by ELISA method.The expressions of Connexin-43 and ZO-1 were detected by cell fluorescence assay,and RhoA and ROCK protein expressions were detected by Western-blot assay.The above experiments confirmed the mechanism of oxyberberine on acute lung injury model.Results1.Oxyberberine had no effect on the viability of A549 cells,and the dose of LPS modeling was determined as 100 ng/mL,and the dose of OBB was determined as 5,10,20μM.After prophylactic administration,the expression of inflammatory cytokines TNF-α,IL1β and IL-6 in A549 cells could be reduced,which proved that OBB had a protective effect on inflammatory lung epithelial cells.2.Preventive administration of oxyberberine and berberine improved survival in mice with acute lung injury,and oxyberberine was more effective at the same dose.Oxyberine can reduce the ratio of lung wet weight to lung dry weight,reduce the protein content of alveolar lavage fluid,alleviate pulmonary edema,alleviate pathological changes of lung tissue,reduce the number of neutrophils in alveolar lavage fluid,and reduce the contents of TNF-α,IL-1β,IL-6 and MPO in lung tissue and alveolar lavage fluid.3.The RhoA/ROCK signaling pathway was activated in the lung tissues of mice with acute lung injury,and the expression of Connexin-43 was increased while that of ZO-1 was decreased.The prophylactic administration of oxyberberine inhibited RhoA/ROCK signaling pathway activation,down-regulated RhoA,ROCK,p-MLC,and Connexin-43 protein expressions,and up-regulated ZO-1 expression.4.The dose of signaling pathway agonist U46619 was determined at 100 nM.After LPS stimulation,the protein content of ROCK was increased,and the expression of Connexin-43 protein in cell fluorescence was increased while the expression of ZO-1 protein was decreased.The protein expressions of RhoA and ROCK were increased in Western-blot assay,and the activation of RhoA/ROCK signaling pathway was inhibited after oxyberberine intervention.U46619 can counteract the inhibition effect of oxyberberine on RhoA/ROCK signaling pathway in LPS-induced A549 cell model.ConclusionIn conclusion,oxyberberine has a good protective effect on acute lung injury models,and its mechanism may be to inhibit the activation of RhoA/ROCK signaling pathway and enhance the alveolar epithelial barrier function,thus having a protective effect on acute lung injury diseases.