Protective Effect And Mechanism Of Glaucocalyxin A On Acute Respiratory Distress Syndrome Induced By Oleic Acid In Rats

Objective:To study the effects of Glaucocalyxin A(GLA)on acute respiratory distress syndrome(ARDS)in rats induced by oleic acid(OA),so as to provide theoretical guidance for the clinical treatment of ARDS.Methods:Male SD rats with body weight of 200±50g were randomly divided into five groups:control group,OA group,OA+GLA(5mg/kg)group,OA+GLA(lOmg/kg)group and OA+GLA(20mg/kg)group.Preparation of ARDS model:O.lml/kg OA femoral vein was rapidly injected into rats to establish the induction model.GLA group:immediately after O.lml/kg OA was injected,the prepared GLA solutions of different concentrations(5,10,20mg/kg)were successively injected.Control group:equal volume normal saline was injected into femoral vein.After 3h of injection,the rats were put to death.The left lung was taken and weighed immediately,is,”wet weight,,and then weighed again,is,"dry weight,after 48h of 60°C thermostat.The wet/dry ratio(W/D)was calculated.Bronchoalveolar lavage fluid(BALF)was extracted,and the total protein content in BALF was detected by BCA method.Count the total number of cells in BALF with Countstar cell count instrument.After BALF staining,use Giemsa staining.Count the neutrophils under high power microscope.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors in BALF.Using Western blot method to detect the lung tissue of p-PI3K,PI3K,and AKt,p-AKt,p-IKB,IKB,the NF-kB p65,NF-kB p-p65,p-AMPK,AMPK,Nrf2,HO-1 protein expression.Results:1.After the injection of OA,the rats experienced shortness of breath,agitation of the nasal wings,severe chest undulation,cyanosis of the lips and limbs.The lung tissues in the control group were pink without hyperemia and edema.In the OA group,the lung tissues became dark and bright in color,and their volume increased significantly,with obvious hyperemia and edema.Lung tissue volume decreased,congestion and edema decreased in GLA group and OA group,showing a dose-dependent relationship.2.Compared with the control group,the lung tissue W/D in OA group significantly increased {P<0.01).Compared with the OA group,the W/D of rat lung tissues in the OA+GLA(5mg/kg)group was reduced,but the difference was not statistically significant(P>0.05).Lung tissue W/D was significantly reduced in OA+GLA(10mg/kg)group and OA+GLA(20mg/kg)group(P<0.05).3.Compared with the control group,the alveolar walls of the OA group were thickened and ruptured,and the alveolar eavity effusion was increased,capillaries were congested and edema,and a large number of inflammatory cells were infiltrated,with transparent membrane formation visible.Lung injury score increased significantly(P<0.01).Compared with OA group,lung histopathological changes were reduced in low and medium dose GLA groups.The lung injury score was significantly reduced(P<0.05).Lung histopathological injury was significantly reduced in the high dose GLA group.Lung injury scores were significantly reduced(P<0.01).4.The results of cell count and total protein in alveolar lavage fluid(BALF)showed that,compared with the control group,the cell count and protein content in BALF of OA group were significantly increased(P<0.01).The cell count and protein content in BALF of medium and high dose GLA group were significantly reduced(P<0.01).5.ELISA results showed that GLA inhibited the release of TNF-a,IL-lp and IL-6 inflammatory factors in a dose-dependent manner(P<0.05).6.Westem blot results showed that GLA could inhibit the phosphorylation levels of PI3K,AKt and NF-?B p65 in lung tissues,especially at high doses(P<0.01).Phosphorylation of AMPK was up-regulated(P<0.01),and Nrf2 and HOI protein expression levels were significantly increased(P<0.01).Conclusions:1.Glaucocalyxin A(GLA)can reduce the degree of pulmonary edema induced by oleic acid in ARDS rats and inhibit the release of inflammatory factors.2.Glaucocalyxin A(GLA)may inhibit inflammation by inhibiting PI3K/Akt-NF-?B p65 pathway,and may play an antioxidant role through Ampk-Nrf2-Hol pathway,reducing oxidative stress.3.Glaucocalyxin A(GLA)has certain therapeutic effect on oleic acid-induced ARDS rats.