Protective Mechanism Of DHQ On LPS-induced Acute Lung Injury

Acute lung injury(ALI)refers to acute lung inflammation or oxidative stress caused by a variety of factors.It is one of the most difficult Problems in clinical medicine around the world.In recent years,it has been threatening Peo Ple’s life and health.Recent studies have shown that li Po Polysaccharide(LPS)is the main cause of the induction of acute lung injury and Plays an im Portant role in the Pathogenesis of acute lung injury.However,there is currently no targeted and systematic treatment for LPS-mediated inflammatory res Ponse,es Pecially acute lung injury.Therefore,based on the excellent anti-inflammatory effects of dihydroquercetin(DHQ),the effect and mechanism of DHQ in LPS-induced ALI were fully studied in this study.Part 1: The Protective effect of DHQ on ALI induced by LPS in miceObjective: To clarify the Protective effect of DHQ on ALI induced by LPS in the body,and to ex Plore the a PPlication Pros Pect of DHQ in LPS-mediated inflammation.Methods: First,the mice were treated with DHQ to observe the effect of DHQ on the survival rate of mice after LPS induction,and to establish LPS-induced ALI model.DHQ was injected into mice by intra Peritoneal injection.After treatment with DHQ for 1 h,the mice were anesthetized with ether,and LPS was given intranasally to induce lung injury.After 12 h of LPS treatment,the animals were treated with CO2 as Phyxiation and collected lung tissue s Pecimens.Hematoxylin and eosin staining kit was used to Perform HE staining on mouse lung tissue.Collect all lungs and the same lung was dried in an oven at 80°C for 72 h to evaluate the wet-dry ratio of the mouse lungs after DHQ treatment.MDA content was detected in mouse lung tissue with li Pid Peroxidation MDA detection kit.The mRNA ex Pressions of Mir-132-3P and FOXO3 were detected by qPCR,the related Protein ex Pressions of P-P65,P-I κBαand FOXO3 were detected by Western blot,and the related Protein ex Pressions of TNF-α,IL-1β and IL-6 were detected by ELISA.Results: Test results show that DHQ can significantly im Prove the survival rate of ALI mice induced by LPS.Observed by HE staining,it was found that DHQ can significantly alleviate the Pathological changes such as inflammatory cell aggregation and alveolar hemorrhage in lung tissue caused by the inflammatory res Ponse induced by LPS.In addition,DHQ can also significantly reduce the wet-dry ratio of the lung tissue in mice with ALI induced by LPS.In addition,it can also significantly reduce the content of MDA.By analyzing the quantitative detection results of ELISA,qPCR and western blot,we found that DHQ can significantly increase the ex Pression of miR-132-3P m NRA,inhibit the ex Pression of FOXO3 mRNA,and inhibit the inflammatory Pathway P-P65,P-IκBα and inflammatory factors TNF-α,IL-1β,IL-6 ex Pression.Part 2: Study on the mechanism of DHQ ameliorating the damage of TC-1 cells induced by LPSObjective: To study the effect of DHQ on the inflammatory res Ponse of TC-1cells induced by LPS,and to ex Plore the Protective mechanism of DHQ.Methods:Use Cell counting kit-8 to measure the cell viability of TC-1 cells induced by LPS treated with DHQ.The Annexin V-FITC a Po Ptosis detection kit and flow cytometer were used to detect a Po Ptosis.qPCR,western blot and ELISA were used to detect the ex Pression levels of genes or Proteins such as miR-132-3P,FOXO3,P-P65,P-IκBα,TNF-α,IL-1β and IL-6 in TC-1 cells.The Potential binding site between miR-132-3P and FOXO3 was Predicted by Star Base bioinformatics software,and the dual luciferase ex Periment was used to verify it.qPCR and western blot were used to detect the ex Pression of FOXO3 in the Presence of miR-132-3P.Then construct the LPS-induced inflammatory res Ponse to ex Plore the inflammatory mitigation mechanism of DHQ.Results: The cell viability test results show that DHQ can significantly increase the viability of TC-1 cells induced by LPS.At the same time,the flow cytometry test also Points out that DHQ can also significantly inhibit the a Po Ptosis of TC-1 cells induced by LPS.ELISA,qPCR,Western blot results showed that DHQ treatment of TC-1 cells could significantly inhibit the ex Pression of Pro-a Po Ptotic Protein Bax and Promote the ex Pression of anti-a Po Ptotic Bcl-2,and significantly increase the ex Pression of Mir-132-3P mRNA.It also inhibited the ex Pression of FOXO3 mRNA,P-P65,P-I κBα,TNF-α,IL-1β,IL-6 mRNA and related Proteins in the inflammatory Pathway.The mechanism research results show that FOXO3 is the downstream target of miR-132-3P.miR-132-3P can significantly inhibit FOXO3 mRNA ex Pression and Protein ex Pression.DHQ can u P-regulate the ex Pression of miR-132-3P,inhibit the ex Pression of FOXO3,and inhibit the ex Pression of a Po Ptosis and inflammation Pathways P-P65,P-IκBα and inflammatory factors TNF-α,IL-1β,and IL-6.This effect can be reversed by inhibiting the ex Pression of miR-132-3P.In addition,overex Pression of FOXO3 can reverse the Protective effect of miR-132-3P on TC-1 cells and Promote LPS-induced inflammatory res Ponse.ConclusionDHQ has a good Protective mechanism against LPS-induced ALI in the body.By inducing the ex Pression of miR-132-3P,it can inhibit the ex Pression of Fox O3 and inflammatory factors TNF-α,IL-1β and IL-6,and inhibit the NF-κB Pathway,finally alleviate the inflammatory res Ponse of ALI caused by LPS,and im Prove the survival rate of animals.In addition,in the entire system,FOXO3 and miR-132-3P have a strong interaction relationshi P,FOXO3 is miR-132-3P downstream targets,miR-132-3P can alleviate inflammation by inhibiting FOXO3 and its associated inflammatory factors and inflammatory Pathways.Therefore,DHQ has a good Protective effect in LPS-induced ALI inflammatory res Ponse,which can Provide theoretical su PPort for its clinical a PPlication.