Experimental Study On The Therapeutic Effect Of Aspernolide E On Acute Lung Injury After Fracture

Background and Objective:In recent decades,despite of the orthopedic surgical methods and postoperative supportive treatment have had significant development,the postoperative mortality of patients with fractures,especially those with multiple fractures and elderly patients,has not significantly decreased,with studies having shown that it is associated with subsequent MODS.In conclusion,uncontrolled inflammatory response syndrome after fracture can lead to Acute lung injury(ALI)or Acute respiratory distress syndrome(ARDS)after surgery,and if the injury develops to an irreversible degree,it can lead to pulmonary complications and eventually lead to respiratory failure and MODS.According to the "second blow" theory proposed by Moore et al.,as the first blow,fracture has made the body sensitized.And if it is subjected to another infectious factor(sepsis)or non-infectious factor(inappropriate resuscitation,surgical blow,etc.),it will induce more serious injury,causing a second blow.Then ALI/ARDS or even MODS will appear.However,endotoxemia caused by gram-negative bacteria infection is relatively common in clinical.Literature reports that 70%of clinical ALI is related to sepsis(40%)and aspiration(30%),which the latter may also be an infectious factor.For the main component of endotoxin is Lipopolysaccharide(LPS),the mouse endotracheal intubation infusion LPS is used as the second strike factor in this study,so as to better simulate ALI caused by clinical pulmonary infection.Marine microorganisms have been proved to be an important new drug source by many literatures and studies,and more and more reports have proved that the unique biological activity of Marine fungi.Aspernolide E is a small molecular compound extracted from Marine fungal soil aspergillus.In previous studies,it was found to have a certain anti-inflammatory effect.In this paper,its anti-inflammatory effect and mechanism in cell level and animal model of ALI were further studied.Objective:To explore the therapeutic effect and mechanism of Aspernolide E on inflammation in vivo and in vitro,and explore the possibility of using Aspernolide E in the treatment of ALI after clinical fracture.Methods:Part 1:The influence and mechanism of Aspernolide E on RAW264.7 cell inflammatory response model:choose the RAW264.7 cells grown in vitro culture as the research object,firstly using the MTT assay to explore the cytotoxicity of Aspernolide E on RAW 264.7 cells,then NO inhibition experiment and ELISA experiment were used to assess the inflammatory factor in supernatant of RAW264.7 cells.Further,Western Blot and IF were used to explore the pathway and effect of Aspemolide E’s antiinflammatory effect.Part 2:Male C57BL/6 mice(6-8 weeks old,20-25g)were used for LPS-induced ALI mouse model,which were divided into a blank control group,a LPS group,a low-dose Aspernolide E group,and a high-dose Aspernolide E group.In the intervention group,intraperitoneal injection was performed with Aspernolide E 24h in advance,followed by endotracheal intubation infusion LPS for modeling.And mice were sacrificed by neck removal 6 hours later,their lungs were collected for preservation.After part of the lungs fixation with formaldehyde,the inflammatory changes were observed by HE staining and the expression of NF-B subunit p65 was observed by IHC.The expression of iNOS and NF-B subunit p65 in the other lung was extracted and detected by Western Blot.Results:1.The levels of NO,IL-6 and TNF-α released by RAW264.7 cells treated with LPS were significantly increased compared with the control group,and all indexes were significantly decreased after the intervention of Aspernolide E.2.In RAW264.7 cells treated with LPS,the expression levels of iNOS and NF-B pathway activating related proteins p65 and p-p65 were significantly increased compared with the control group,and each indicator was significantly decreased after the intervention of Aspernolide E.3.Compared with the control group,the lung tissues of the ALI model induced by LPS group showed obvious inflammatory pathologic changes,and the inflammatory changes were significantly reduced by Aspernolide E,and the effect of the low-dose group was better than the high-dose group.4.Compared with the control group,the expression of iNOS in the lung tissue of LPS group was significantly increased,and the expression of iNOS in Aspernolide E group was significantly decreased,which the effect of low dose group was better than that of high dose group.5.Compared with the control group,the expression level of NF-κB subunit p65 in LPS group was significantly increased,and it decreased after the intervention of Aspemolide E,which the effect of low dose group was better than that of high dose group.Conclusions:Aspernolide E can reduce inflammation in RAW264.7 cells and ALI mice by inhibiting the phosphorylation of p65 in NF-κB pathway.Aspernolide E may be a potential treatment for post-fracture ALI.