The Protective Effect And Mechanisms Of Lipoxin A4 On Acute Lung Injury Induced By Paraquat

Objective: After paraquat enters the human body,it can induce oxidative stress and inflammation,which can cause damage to multiple organs of the body,especially acute lung injury.At present,the pathogenesis of lung injury caused by paraquat poisoning has not been fully elucidated,and there is no effective treatment and specific antidote.Lipoxin A4 has anti-inflammatory,pro-inflammatory subsidence,damage repair and antioxidant effects,so it may have a certain protective effect on paraquat-induced acute lung injury.This experimental study used lipoxin A4 to treat paraquat poisoned rats to study its biological effect on paraquat poisoning acute lung injury and its potential mechanism,and through in vitro experiments to study the effect of lipoxin A4 on paraquat treatment of macrophages The biological functions of cells and their underlying biological mechanisms.Methods: In this study,male SD rats and RAW264.7 mouse macrophages were selected as experimental subjects.The poisoning model was established by administering paraquat,and lipoxin A4 was given as a therapeutic drug after poisoning.In terms of in vivo research,rats were randomly divided into the normal control group,the paraquat poisoning group,the paraquat poisoning + lipoxin A4 treatment group,and the lipoxin A4 administration group alone.A one-time intraperitoneal injection of paraquat(20mg/kg)was used to establish a rat poisoning model.After 1 hour of exposure,lipoxin A4(2ug/kg)was injected into the tail vein for therapeutic intervention.After 48 hours,the rats were sacrificed,BALF and lung samples were collected.H&E and the measurement of lung wet/dry weight ratio was performed to evaluated the severity of acute lung injury in each group of rats,the activity of superoxidase dismutase and contents of malondialdehyde in lung tissues were measured to assess the oxidative damage in lung tissues,the levels of pro-inflammatory factors TNF-α and IL-1β in rat alveolar lavage fluid was calculated by ELISA kits,RT-PCR technology was used to detect the m RNA levels of TLR4,MyD88,TRAM、TRIF、TNF-α and IL-1βin lung tissues,and Western-blot was performed to evaluate the protein expression levels of TLR4,MyD88,PI3K/P-PI3 K,AKT/P-AKT,NF-κB p65 and IκBα/P-IκBα,reflecting the level of inflammation in rat lung tissues and its possible mechanisms after paraquat poisoning.In terms of in vitro research,RAW264.7 mouse macrophages were randomly divided into normal control group,paraquat poisoning group,paraquat poisoning + lipoxin A4 treatment group,and lipoxin A4 group.After 24 hours of drug treatment,the cell supernatants and cell samples were collected.The CCK-8 method was used to determine the cell viability.The reactive oxygen species kit was performed to detect the level of reactive oxygen species in the cells to reflect the degree of cell oxidative damage,and the pro-inflammatory cytokines and related factors were assessed by ELISA,RT-PCR and Western-blot methods.In order to further verify the specific mechanism of LXA4 in treating PQ poisoning,we constructed the overexpression of TLR4 at the cellular level.We transfected the TLR4 plasmid constructed by GV417 vector into cells to achieve the overexpression of TLR4.After transfection,the expression level of TLR4 was evaluated by Western Blot and q RTPCR.Subsequently,the cells were transfected with TLR4 plasmid for 24 h,and RAW264.7 alveolar macrophages were treated with paraquat(100μM).After 1h,lipoxin A4(100 n M)was used to treat the infected RAW264.7 macrophages.After 24 h,ROS kit was used to detect the content of reactive oxygen species,TNF-α and IL-1βlevels in cell supernatants was assessed by ELISA method,RT-PCR method was performed to calculate the gene expression levels of TNF-α and IL-1β,and the related protein expression of MyD88,nuclear NF-κBp65,IκBα,p-IκBα,P-PI3 K,P-AKT was detected by Western Blot.Results: After paraquat poisoning,the structural integrity of rat lung tissue was destroyed,and the pulmonary edema was obvious.The activity of antioxidant enzyme superoxide dismutase SOD in lung tissue decreased,while the content of oxidative enzyme malondialdehyde MDA increased significantly.The secretion levels of proinflammatory cytokines TNF-α and IL-1β in the alveolar lavage fluid were significantly increased,and the gene levels of TLR4,MyD88,TRAM,TRIF,TNF-α and IL-1β in the lung tissues were significantly increased,and the Western Blot results showed that TLR4,MyD88,PIκBα,P-PI3 K,P-AKT protein expression increased significantly,NF-κBp65 nuclear metastasis increased,and IκBα protein expression decreased.After lipoxin A4 treatment,the acute lung injury of paraquat poisoning was reduced,the level of oxidative stress and inflammation was decreased,the activation of TLR4 and MyD88 was inhibited,but it had no effect on the activation of TRAM and TRIF.In addition,Lipoxin A4 inhibited the phosphorylation of PI3 K,AKT,and IκBα and the nuclear transfer of NF-κBp65.The experimental results also showed that LXA4 treatment reduced the toxic effect of PQ on cells,decreased the expression level of reactive oxygen species,and inhibited the activation of TLR4-mediated PI3K/AKT/NF-κB signaling pathway,thereby reducing inflammation damage.Conclusion: In conclusion,the in vitro and in vivo experiments of the present study demonstrated that LXA4 negatively regulated PQ-induced inflammation and oxidative stress through the PI3K/AKT/NF-κB pathway mediated by TLR4 and MyD88,exerting a protective effect on paraquat induced acute lung injury.