Protective Effect And Mechanism Of Quercetin On Lipopolysaccharide-Induced Acute Lung Injury

Research background and purpose:Acute lung injury(ALI)is a clinical syndrome caused by multiple factors.It is usually a progressive lung injury induced by inflammation,mainly manifested as diffuse alveolar injury,hypoxemia and respiratory distress.The mortality of ALI is as high as30%-40%.The pathogenesis of ALI is complex and has not been fully elucidated.Although the current treatment technology of ALI has made great progress,there is still a lack of specific treatment methods and drugs in addition to basic support treatment,and its treatment is still a difficult problem.The occurrence of ALI is often accompanied by strong inflammatory response and oxidative stress.A large number of inflammatory cells are activated to secrete various inflammatory mediators,which destroys the integrity of the alveolar-capillary endothelial barrier structure.At the same time,it induces mitochondrial damage and dysfunction of lung tissue cells,and accumulates a large amount of reactive oxygen species(ROS).When ROS exceeds the scavenging capacity of the body ’s antioxidant system,it presents an oxidative stress state.Studies have shown that potential anti-inflammatory and antioxidant pathways can be regulated by endogenous or exogenous compounds,which will provide new treatment methods for controlling the occurrence and development of acute lung injury.Many studies have confirmed that ferroptosis is closely related to the occurrence and development of diseases caused by inflammation and oxidative stress,and ferroptosis is an important cause of ALI.Ferroptosis is a new form of cell death,which is different from common death modes such as apoptosis and autophagy.It is mainly characterized by iron-dependent reactive oxygen species(ROS)accumulation,cell antioxidant inactivation and mitochondrial contraction.The significant feature of acute lung injury is uncontrollable inflammatory response.Therefore,inhibition of ferroptosis can reduce inflammatory response and reduce lung tissue damage,which is expected to become a new treatment for ALI.Quercetin is a common flavonoid compound found in a variety of fruits and vegetables.It has a wide range of pharmacological effects and is traditionally considered as an effective antioxidant and anti-inflammatory natural bioactive compound.At present,quercetin has been shown to effectively alleviate acute lung injury in animal models.However,the role and mechanism of ferroptosis in quercetin treatment of lipopolysaccharide-induced acute lung injury are still unclear.Therefore,this study explored the protective effect of quercetin on acute lung injury by constructing acute lung injury cells and animal models,and clarified that quercetin protected acute lung injury by regulating ferroptosis,in order to provide a new strategy for the prevention and treatment of acute lung injury.Materials and Methods:1.Protective effect of quercetin on lipopolysaccharide-induced acute lung injuryAnimal experiments: C57BL/6J mice were pretreated with different concentrations of quercetin(0,20,40,60 mg/kg).After pretreatment with quercetin for 1 h,acute lung injury model was induced by intratracheal instillation of LPS(5 mg/kg).HE staining,pathological score and expression of inflammatory factors(TNF-α,IL-6,IL-1β)in alveolar lavage fluid were used to evaluate the inflammatory infiltration of mice.The W/ D ratio of lung tissue was used to evaluate pulmonary edema.Neutrophil infiltration and alveolar-capillary barrier integrity were evaluated by detecting the number of cells in bronchoalveolar lavage fluid,protein concentration and myeloperoxidase(MPO)activity inlung tissue.Cell experiments: alveolar epithelial cells(AT2)were treated with different doses of LPS(0,5,10,20 μg/ml)to construct an acute cell injury model.After 6,12 and 24 hours of treatment,CCK-8 and LDH were used to detect the death of AT2 cells induced by LPS.In order to clarify the role of quercetin in acute lung epithelial cell injury,AT2 cells were pretreated with different concentrations of quercetin(0,5,10,20 μM).After pretreatment for 2h,LPS-induced acute cell injury model was established.CCK-8 and LDH release experiments were used to evaluate cell damage,and the optimal concentration was selected.At the cellular level,the expression levels of inflammatory factors(TNF-α,IL-6,IL-1β)were detected to evaluate the anti-inflammatory effect of quercetin.Cell damage and cell proliferation were detected by Ed U and 7-AAD flow cytometry.2.The role of ferroptosis in quercetin treatment of acute lung injuryAcute lung injury cells and animal models were constructed by lipopolysaccharide induction.The concentrations of glutathione(GSH),malondialdehyde(MDA)and iron ion concentration were detected at the tissue level.Dihydroethidium(DHE)fluorescence staining was used to evaluate the release of ROS before and after quercetin treatment.The expression levels of GPX4 and 4HNE proteins were detected by immunohistochemistry.In addition,at the cellular level,the occurrence of oxidative stress was evaluated by detecting the expression levels of GSH,MDA and iron ion concentration.The accumulation of ROS was detected by flow cytometry,and the effects of quercetin on the expression of GPX4 and 4HNE were analyzed.Transmission electron microscopy was used to detect mitochondrial morphological changes and damage.3.The mechanism of quercetin inhibiting ferroptosis to protect acute lung injury by regulating SIRT1/NRF2/GPX4 pathwayIn order to further explore the specific mechanism of quercetin inhibiting ferroptosis,the expression levels of SIRT1,NRF2 and GPX4 were detected by western blot at the cellular and animal levels.C57 mice were divided into four groups(control group,LPS group,LPS + quercetin group,LPS + quercetin + EX527 group)for comparison.Collect alveolar lavage fluid and isolate lung tissue for subsequent experiments.HE staining,inflammatory injury score,total protein concentration and cell number in alveolar lavage fluid were used to evaluate lung tissue injury.The wet /dry weight ratio of lung was calculated to evaluate the degree of pulmonary edema.MPO activity and iron ion concentration in lung tissue were detected by kits.ROS level in lung tissue was detected by immunofluorescence.Western blot was used to detect the expression of ferroptosis-related protein.SIRT1 interference si RNA was purchased for effective sequence screening to construct SIRT1-silenced cells best interference sequence was screened by q RT-PCR and western blot experiments.The cell death after silencing SIRT1 was evaluated by CCK-8 assay and flow cytometry.Oxidative stress was assessed by detecting reactive oxygen species(ROS)levels and lipid peroxidation products(MDA)levels and iron ion concentrations.The mitochondrial damage induced by LPS was observed by transmission electron microscopy.Result:1.Protective effect of quercetin on lipopolysaccharide-induced acute lung injuryAt the animal level,ALI mouse model was successfully constructed,and different concentrations of quercetin(0,20,40,60mg/kg)were pretreated on this model.HE staining and lung injury score suggested that quercetin could significantly inhibit LPS-induced acute lung tissue injury and inflammatory cell infiltration.The results of lung dry / wet weight ratio showed that quercetin(40,60mg/kg)effectively alleviated lung edema.Compared with LPS group,MPO activity in lung tissue of LPS + quercetin group decreased significantly.At the same time,cell count,total protein concentration and inflammatory factors(TNF-α,IL-6,IL-1β)in alveolar lavage fluid were also significantly reduced.In addition,there was no significant difference between LPS +quercetin(40mg/kg)and LPS+quercetin(60mg/kg).Therefore,the optimal concentration of quercetin was 40mg/kg and used in subsequent experiments.At the cellular level,after alveolar epithelial cells were treated with LPS(0,5,10,20μM)for 6,12,24 h,CCK-8 and LDH assays showed that LPS treatment significantly inhibited cell proliferation and LDH release in a time-and dose-dependent manner.According to the above experimental results,we selected the LPS concentration of 10μg/ ml and 24 h model detection time point for subsequent experiments.In order to screen the effective concentration of quercetin,the experiment was divided into the following five groups: control group,LPS group and LPS + quercetin group(0,5,10,20 mg/kg).CCK-8 and LDH assay results showed that there was no significant difference in cell viability and LDH release between the two groups after pretreatment with 10μM and20μM quercetin.Based on these data,we selected quercetin at a concentration of 10 μM and treated for 12 h for subsequent cell experiments.The results of Ed U cell proliferation assay and flow cytometry showed that quercetin treatment significantly inhibited the expression and secretion of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β),promoted the proliferation of lung epithelialcells and inhibited their death.It can be seen that quercetin treatment can alleviate the damage of lipopolysaccharide to alveolar epithelial cells and animal acute lung injury model.2.The role of ferroptosis in quercetin treatment of acute lung injuryC57 mice and AT2 cells were treated with LPS and quercetin,respectively.Compared with the control group,LPS-induced normal lung tissue structure loss,interstitial congestion and edema,thickening of the pulmonary septum,inflammatory cells and red blood cells in the alveolar space,and pneumonia injury score were also significantly increased.Quercetin treatment significantly improved the above pathological phenomena.Quercetin pretreatment significantly inhibited lipopolysaccharide-induced changes in oxidative stress-related protein levels in vivo and in vitro.Compared with the control group,the levels of ferroptosis-related indicators iron ion concentration,ROS and MDA increased significantly after LPS induction,and the level of GSH activity decreased,suggesting that with the increase of ferroptosis level,acute lung injury aggravated.Quercetin treatment significantly reversed the above phenomenon.In addition,quercetin effectively alleviated mitochondrial damage.In summary,acute lung injury is accompanied by obvious ferroptosis,and quercetin can effectively inhibit the occurrence of ferroptosis.3.The mechanism of quercetin inhibiting ferroptosis to protect acute lung injury by regulating SIRT1/NRF2/GPX4 pathwayLPS treatment down-regulated the expression of SIRT1,NRF2 and GPX4 proteins in lung tissue of mice.Quercetin treatment eliminated the inhibitory effect of LPS on SIRT1/NRF2/GPX4 signaling pathway-related proteins.After SIRT1 inhibitor EX527 intervention in mice,compared with LPS + quercetin group,EX527 treatment made lung tissue structural lesions more obvious,pulmonary interstitial thickening,alveolar hemorrhage and inflammatory cells,and lung injury score increased.In addition,the protein concentration in bronchoalveolar lavage fluid(BALF),lung W/D ratio and MPO activity were significantly increased.Meanwhile,the levels of ferroptosis-related indicators iron ion concentration,4HNE and ROS were significantly increased,and the expression of GPX4 protein was significantly decreased,suggesting that quercetin can inhibit acute lung injury and ferroptosis by regulating SIRT1 in vivo.Compared with the silent control group,in the alveolar epithelial cells stably silencing SIRT1,the cell proliferation ability was weakened,and the ferroptosis-related indicators iron ion concentration,ROS level and MDA content were significantly increased,and the mitochondrial ridge was reduced or disappeared.It can be seen that SIRT1 is a key target for quercetin to inhibit ferroptosis and exert its anti-inflammatory and antioxidant effects.Conclusion:Our study shows that quercetin can protect mice with acute lung injury by reducing lipopolysaccharide-induced alveolar epithelial cell injury and inhibiting inflammatory response and oxidative stress.The mechanism is closely related to the inhibition of SIRT1/NRF2/GPX4 signaling pathway and the inhibition of ferroptosis.Quercetin significantly improved histopathological changes,increased the proliferation of alveolar epithelial cells,inhibited the release of pro-inflammatory cytokines and the production of reactive oxygen species(ROS),and effectively improved lipopolysaccharide-induced tissue damage.Mechanistically,quercetin effectively up-regulated the SIRT1 / NRF2 /GPX4 signaling pathway,and selective inhibition of SIRT1 in vitro and in vivo significantly reversed the protective effect of quercetin on LPS-induced acute lung injury.In summary,quercetin inhibits lipopolysaccharide-induced ferroptosis by activating the SIRT1/NRF2/GPX4 pathway,thereby exerting its anti-inflammatory and antioxidant therapeutic effects.