Ursolic Acid Attenuates Lipopolysaccharide-induced Acute Lung Injury In Mice And Its Mechanism

Objective:1.To observe the protective effect of ursolic acid on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice.2.To investigate whether ursolic acid alleviates ALI by inhibiting endoplasmic reticulum stress.3.To investigate the effect of ursolic acid on the polarization of M1/M2 macrophages during ALI.Methods:Some ICR male mice were randomly divided into the following four groups: control group,model group,low-dose ursolic acid group(10 mg/kg)and high-dose ursolic acid group(20 mg/kg).The ALI mouse model was established by intratracheal instillation of LPS(3mg/kg).H&E staining was used to observed the pathological changes of the lungs,and pathological score was performed;the wet-dry weight ratio of lung tissue was calculated to assess the degree of pulmonary edema.Pulmonary capillary permeability was evaluated by Evans blue staining.The activity of myeloperoxidase(MPO)in lung tissue was detected by kits;the expression levels of TNF-α,IL-1β,IL-6 and IL-10 in lung tissue and bronchoalveolar lavage fluid(BALF)were detected by ELISA kits;the total protein concentration in BALF was measured by BCA method,and the number of white blood cells in BALF was counted.Western blot was used to detect the expression of endoplasmic reticulum stress marker proteins and inflammation-related proteins in cells.Mice alveolar macrophages(MH-S)induced by LPS and IFN-γ.MH-S cells were divided into the following five groups: control group,model group(LPS + IFN-γ),low does(1 μmol/L),medium dose(2 μmol/L)and high dose(4 μmol/L)ursolic acid group.CCK-8 assay was used to detect the survival rate of MH-S cells incubated with different concentrations of ursolic acid.The expression levels of M1/M2 polarization marker proteins and inflammatory pathway-related proteins in macrophages were detected by Western blot.Results:Animal experiments showed that compared with the control group,the lung water content,capillary permeability,MPO activity and pathological score of the model group were significantly increased.A large number of inflammatory cells infiltration,destruction of alveolar integrity,and thickening of alveolar septum were observed under light microscope.At the same time,the number of white blood cells and the total protein level in BALF of the model group were significantly increased,and the expression of proinflammatory cytokines TNF-α,IL-1β and IL-6 in BALF and tissues was significantly increased,while the expression of anti-inflammatory factor IL-10 was decreased.Ursolic acid pretreatment significantly inhibited LPS-induced lung pathological changes,decreased the total protein content and white blood cell number in BALF of ALI mice,reduced the expression of pro-inflammatory factors TNF-α,IL-1β and IL-6,decreased MPO activity,and increased the expression of anti-inflammatory factor IL-10.In addition,UA pretreatment significantly down-regulated the expression of GRP78,PERK,IRE1,ATF6 and TXNIP/NLRP3 in LPS-induced lung tissues.These results indicated that UA could inhibit the occurence of endoplasmic reticulum stress induced by LPS and the TXNIP/NLRP3 signaling pathway.Cell experiments showed that when cells were incubated with LPS and IFN-γ,the number of M1 macrophages increased significantly,while the number of M2 macrophages decreased significantly.Preincubation with ursolic acid reversed this result,that is,M1 macrophages were significantly decreased,while M2 macrophages were increased.Conclusions:Ursolic acid has a protective effect on LPS-induced ALI in mice,which may be related to inhibiting endoplasmic reticulum stress,down-regulating the expression of TXNIP/NLRP3 protein,and ultimately reducing the release of pro-inflammatory cytokines.At the same time,ursolic acid may alleviate lung injury by regulating macrophage polarization and inhibiting the expression of NF-κB and NLRP3 inflammatory signals.