The Role And Underlying Mechanism Of IFIT1 In LPS-induced Acute Lung Injury

Background: Acute lung injury(ALI)is a serious inflammatory disease,and it is believed that uncontrolled inflammation is still the core link in the occurrence and progression of ALI.Therefore,exploring strategies to alleviate and reverse excessive inflammatory response is still a hot spot in the prevention and treatment of ALI.Inflammation occurs in the lungs,alveolar capillary permeability increases,edematous fluid enters the alveoli to affect the ventilation function of the lungs,and the alveolar surfactant is abnormal,further aggravating pulmonary edema and causing severe hypoxemia.Alveolar macrophages are the most important cells in the innate immune system,mainly distributed in the alveolar cavity,with phagocytosis,secretion function,alveolar macrophages account for about 90 %-95 % of the innate immune cells of the lung,participate in the entire pathogenesis of ALI,including regulating inflammatory response and repairing damaged lung tissue.Alveolar epithelial cells are the final effector cells of ALI,and apoptosis damage occurs in alveolar epithelial cells,which may lead to alveolar hydrops,loss of epithelial integrity,and loss of surfactant leading to decreased lung compliance.Apoptosis of alveolar epithelial cells due to lung inflammation is an important feature of ALI.In recent years,most of the literature has reported that interferon stimulated genes(IFITs)are involved in the occurrence,development and prognosis of pulmonary inflammatory diseases such as pulmonary bronchitis and pneumonia caused by Mycobacterium tuberculosis.Interferon-induced protein with tetratricopeptide repeats 1(IFIT1),a member of the interferon-induced gene family,is mainly distributed in the cytoplasm,and is expressed in low amounts in most cells under physiological conditions,but the IFN-JAK-STAT protein pathway is activated after IFN treatment or viral infection.IFIT1 protein can be briefly induced to express high levels.IFIT 1 have significant anti-inflammatory effects in pulmonary inflammatory diseases caused by viruses and bacteria.However,the effect and mechanism of IFIT1 on ALI have not been reported.Therefore,this project focuses on the regulatory effect of IFIT1 on macrophage secretion of inflammatory factors in acute lung injury,its mechanism and its effect on apoptosis of alveolar epithelial cells,in order to provide new research ideas for the prevention and treatment of acute lung injury.Objective:(1)To investigate the regulatory effect and mechanism of IFIT1 on the secretion of inflammatory factors in alveolar macrophages in lipopolysaccharide-induced acute lung injury;(2)To investigate the effect of IFIT1 on apoptosis of alveolar epithelial cells induced by alveolar macrophage inflammatory response.Methods: In vivo,C57BL/6J mice(male,weight 20 g-22 g)were divided into 4 groups(6 in each group),which were control group(PBS),model group(LPS),lentiviral control group(LV-empty+LPS)and lentiviral model group(LV-IFIT1+LPS).The model group of model group,lentiviral control group and lentiviral model group were instilled with LPS(5 mg/kg)by tracheal instillation of LPS(5 mg/kg),and the normal group was instilled with the same dose of PBS,and after 24 hours of modeling,the experimental mice were sacrificed and lung tissue and serum were collected.Bronchoalveolar lavage fluid was extracted and purified mouse primary alveolar macrophages were isolated,ELISA detected the expression of TNF-α,IL-1β and IL-6 in bronchoalveolar lavage supernatant,and RT-q PCR detected the expression of TNF-α,IL-1β and IL-6 in primary alveolar macrophages.Some tissues were taken to detect the expression of IFIT1 and related inflammatory factor m RNA and protein,and a small part of lung tissue was embedded and made into paraffin wax or frozen sections for histological and pathological observation such as fluorescent double staining and HE staining to detect lung damage.In vitro,MH-S cells were stimulated with LPS(200 ng/ml,8 h),the content of inflammatory factors in the cell supernatant was detected,and the IFIT1 protein and m RNA were extracted for expression analysis.Secondly,after the use of small interfering RNA,IFIT1-si RNA in vitro and the use of lentiviral overexpression of IFIT1,Western Blot and RT-q PCR were used to detect the changes of IFIT1 and inflammatory factors.At the same time,small interfering RNA was used to silence IFIT1 in MH-S cells,and the method of extracting MH-S cell RNA for RNA sequencing was observed,and the effect of IFIT1-si RNA on the expression of key proteins in the inflammatory signaling pathway in LPS-induced MH-S cells was observed.In addition,after overexpressing and silencing the supernatant of MH-S cells after IFIT1 was cultured MLE-12(mouse alveolar epithelial cell),the apoptosis rate of MLE-12 cells and the changes of related proteins were detected,so as to explore whether the inflammatory regulation of IFIT1 affected the apoptosis of alveolar epithelial cells.Result: In vivo,after the completion of modeling,the pathology and expression levels of inflammatory factors showed that LPS induced lung injury,indicating that the model induction was successful.Compared with the control group,the expression of m RNA and protein in the LPS-induced acute lung injury model was up-regulated,and the results of immunofluorescence double stain showed the co-location of IFIT1 to the macrophage marker CD68.After lentivirus were injected into mice for overexpression of IFIT1,the pathological section results showed that the degree of lung damage wasreduced and the apoptosis rate of lung tissue was significantly reduced.In vitro,compared with the control group,the expression levels of IFIT1 and various inflammatory factors showed an upward trend after treatment of MH-S cells with LPS.After overexpression of IFIT1,the level of a variety of inflammatory factors showed a downward trend,while after down-regulation of IFIT1 expression,the level of inflammatory factors showed an opposite trend,and further experiments found that IFIT1 regulated the expression of p65 and p-p65 proteins in CCL5 and NF-κB pathways.In addition,overexpression of IFIT1 in MH-S cells,centrifugation of MH-S cell supernatant cultured MLE-12 cells,apoptosis of MLE-12 cells decreased,while apoptosis of MLE-12 cells showed an upward trend after silencing IFIT1.Conclusion: From the results of in vivo and in vitro experimental results,it can be speculated that IFIT1 can inhibit the activation of LPS-induced alveolar macrophages,reduce the expression of pro-inflammatory factors and thereby reduce lung damage,this regulatory effect is achieved through the reverse regulation of CCL5 and NF-κB pathway,and this negative regulation of IFIT1 on inflammation greatly reduces the apoptosis of alveolar epithelial cells.