Mechanistic Study Of Liproxstatin-1 In The Inhibiting Ferroptosis To Attenuate Acute Lung Injury Caused By A Mouse Model Sepsis

Objectives: To observe the changes of ferroptosis levels and inflammatory indexes in the model of acute lung injury caused by cecum ligation perforation in mice.To observe the changes of ferroptosis levels and inflammatory indexes in mice in the model+ administration group after the intervention of the drug liproxstatin-1 with ferroptosis.Methods:Establishment and administration of acute lung injury mouse model: SPF-grade male C57BL/6 mice,8 weeks old,weighing 20-25 g.Mice were randomly divided into four groups:Control group,model group(CLP group),liproxstatin-1 group(Lip-1 group: 0.8 mg/kg),model+ liproxstatin-1 group(CLP + Lip-1 group: 0.8 mg/kg).The Control group and CLP group were injected with saline 16 h before surgery,and the Lip-1 group and CLP+Lip-1 group were injected with Lip-1 16 h before surgery.The Control group and Lip-1 group performed sham surgery.The CLP group and CLP+lip-1 group performed cecum ligation perforation surgery to replicate the acute lung injury model.Methods and indicators : After exposing the thoracic cavity,the right lung bronchus was ligated and the left lung was laved to obtain bronchoalveolar lavage fluid(BALF),which was used to detect the protein concentration and the content of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in BALF;the left lung was also taken from the same group of mice to determine the wet-to-dry weight ratio(W/D)of lung tissue;the upper and middle lobes of the right lung were fixed with 4% The upper and middle lobes of the right lung were fixed with 4% paraformaldehyde solution;the upper lobe of the right lung was used for pathological section staining to observe the histopathological conditions of the mice in each group;the middle lobe of the right lung was used for immunohistochemical determination of malondialdehyde(MDA)and4-hydroxynonenal(4-HNE)in lung tissues;the lower lobe of the right lung was used for the determination of glutathione peroxidase 4(GPX4),long-chain lipoyl coenzyme A Synthetase4(ACSL4),cyclooxygenase-2(COX-2)expression in lung tissue by Western blot,and biochemical analysis was used for the detection of ferroptosis.Results: Compared with the Control group: the W/D of mice in the CLP group increased,which was statistically significant(P<0.01);pathological sections showed that the CLP group had severe bronchial wall congestion,significant inflammatory cell infiltration,severe alveolar wall congestion,and significant fibrosis in the lungs,lung histopathological score increased,which was statistically significant(P<0.01);protein concentration,IL-6,TNF-α in alveolar lavage fluid increased,which was statistically significant(P < 0.01);Western blot results showed that compared with Compared with the Control group: GPX4 expression decreased in the CLP group,which was statistically significant(P<0.01);ACSL4 and COX-2 expression increased,which was statistically significant(P < 0.01);iron ion content increased,which was statistically significant(P < 0.01);In terms of MDA and 4-HNE,the number of positive staining cells in the Con and Lip-1 groups was normal and light yellow,while the number of positive staining cells in the CLP group was significantly increased and the color was brownish black.MDA optical density values increased with statistical significance(P<0.01)and 4-HNE optical density values increased with statistical significance(P<0.05)Compared with the CLP group: the W/D of lung tissue decreased in the CLP+LIP-1 group,which was statistically significant(P < 0.01);pathological sections showed that the CLP+Lip-1 group showed bronchial wall congestion with a small amount of obvious inflammatory cell infiltration,a few of which had alveolar wall congestion and mild fibrosis in the lungs,with a significant treatment effect compared to the CLP group,lung histopathological score decreased,which was statistically significant(P<0.01);protein concentration,IL-6,TNF-α in alveolar lavage fluid decreased,which was statistically significant(P < 0.01);Western blot results showed that compared with CLP+LIP-1 group compared with CLP group GPX4 expression increased,which was statistically significant(P < 0.01);ACSL4 and COX-2expression decreased,which was statistically significant(P<0.01);iron ion content decreased,which was statistically significant(P<0.01);In terms of MDA and 4-HNE,the CLP+Lip-1group showed a significant decrease in the number of positive stained cells and a change in color to brownish yellow.MDA optical density values increased with statistical significance(P<0.05)and 4-HNE optical density values increased with statistical significance(P <0.05).Conclusion: 1、Ferroptosis is involved in sepsis leading to acute lung injury.2、Inhibition of ferroptosis can play a therapeutic role in acute lung injury caused by sepsis.