MiRNA-145-5P Aggravates Sepsis-induced Inflammatory Lung Injury By Down-regulating Smad3

BackgroundSepsis is a common critical illness with high incidence,risky progress and high mortality in clinic,and it is one of the most common causes of death in intensive care unit.Excessive inflammatory response in sepsis can induce multiple organ dysfunction,with lung being the first and most susceptible organ to be damaged.Among the sepsis-associated inflammatory lung injuries,ARDS [Acute Respiratory Distress Syndrome] is the most common disease,which is often characterized by acute respiratory failure with progressive hypoxemia and diffuse pulmonary infiltration.ARDS is usually induced by diseases leading to systemic inflammatory response,with sepsis being the most common indirect cause.The mortality rate of ARDS is high.Even with the highly developed intensive care technology,the mortality rate of ARDS is still as high as 40%,indicating that it is still a major life-threatening disease.Compared with non-sepsis-induced ARDS,the ARDS induced by sepsis has more severe damage,worse prognosis,lower success rate of extubation,and higher mortality.Therefore,it is of great medical and social significance to further explore the occurrence mechanism of sepsis-induced lung injury,find the key process of disease progression and important targets,and then propose accurate molecular therapy.In our study,it was found that the anti-injury ability of Smad3 gene knockout mice was significantly reduced,which was manifested by significantly increased susceptibility of lung and other important organs to LPS,increased pulmonary edema and bleeding,and increased severity and mortality of sepsis-related lung injury.Through high-throughput sequencing of peripheral blood of patients with sepsis and healthy subjects,two types of miRNAs that are differentially expressed and may target Smad3,mir-145-5p and mir-744-5p,were selected step by step.To further explore the role and functional mechanism of miR-744-5p and miR-145-5p in sepsis-related lung injur,we studied them in vivo and in vitro and selected mice as experimental subjects.Part ? Construction of a model of sepsis-induced inflammatory lung injury in vivo and in vitroObjective (1)To construct a animal model of sepsis-induced inflammatory lung injury to simulate inflammatory injury in lung tissue of sepsis in vivo;(2)To construct a cell model of inflammatory injury of lung epithelial cells to simulate inflammatory lung injury in vitro.MethodsAn animal model of sepsis-induced inflammatory lung injury: C57 / BL mice was intraperitoneally injected with different concentration of LPS(lipopolysaccharide)and then observe and record gross morphology,HE staining and wet/dry weight of lung to detect tissue damage.RT-PCR is adopted to detect expression of IL-1(Interleukin 1)and TNF-a(Tumor Necrosis Factor alpha)of lung tissue.A cell model of inflammatory injury of lung epithelial cells: direct stimulation model of LPS-lung epithelial cells and indirect stimulation model of LPS-monocytes/macrophages-lung epithelial cells.In former model,LPS was directly added to medium of MLE-12,the mouse lung epithelial cell line,with different concentration.In latter model,LPS was added to the medium of RAW264.7(the mouse mononuclear/macrophage cell line)at first,and then the cell supernatant collected was added into MLE-12 medium.The expression levels of IL-1 and TNF-a in cells were detected by RT-PCR,cell proliferation was detected by CCK-8,and apoptosis was detected by Flow cytometry.Results(1)A model of sepsis-induced inflammatory lung injury in vivo: Compared with control group,hemorrhage and pulmonary edema,hemorrhage in lung tissue of HE staining,alveolar destruction,and migration of inflammatory cell are all increased in LPS group.The mRNA expression levels of il-1 and TNF-a were increased in lung tissue.(2)A model of inflammatory injury of lung epithelial cells in vitro: In LPS-lung epithelial cells direct stimulation model,LPS(1ug/ml-10 ug/ml)had no clear effect on inflammatory mediators production,proliferation and apoptosis of MLE-12.After stimulation of LPS to RAW264.7,the mRNA expression of the inflammatory mediators il-1 and tnf-a were significantly increased.After adding the supernatant of RAW264.7 cells after LPS stimulation into the MLE-12 medium,the proliferation rate of MLE-12 was significantly reduced,and the apoptosis rate was significantly increased,but the cell death rate was not significantly changed.Conclusion(1)Intraperitoneal injection of LPS can successfully construct a sepsis-induced inflammatory lung injury mode in vivo.(2)LPS directly stimulates MLE-12 can't construct an inflammatory injury model of lung epithelial cells in vitro;(3)LPS induces RAW264.7 to secret inflammatory mediators,resulting in indirect damage of MLE-12.As a result,an inflammatory injury model of lung epithelial cells in vitro was constructed.Part ? verification of the correlation and study of causality between miRNA and sepsis-induced inflammatory lung injurySubjectiveTo verify the correlation between miR-744-5p/ miR-145-5p screened by high-throughput sequencing and sepsis-induced inflammatory lung injury,and then verify the causal relationship between them.MethodsRT-PCR was used to detect the expressions of mir-744-5p and mir-145-5p in the sepsis-induced inflammatory lung injury in vivo and in vitro.With transfection of miRNA mimic,the expression of miRNA in cells was increased;With transfection of miRNA inhibitor,the expression of miRNA in cells was decreased.The expression of IL-1 and TNF-a in cells were measured by RT-PCR,cell proliferation was detected by CCK-8,and apoptosis was detected by Flow cytometry.Results(1)the expression of mR-744-5p was decreased in vivo and in vitro for both types of inflammatory injury,but it did not show a clear concentration dependent of LPS.(2)the expression of mR-145-5p was increased in vivo and in vitro for both types of inflammatory injury,abd it was changed in a dose-dependent manner with LPS.(3)Transfection with miRNA mimic lead to high expression of miR-744-5p and miR-145-5p in cells.(4)Transfection with miRNA inhibitor down-regulate expression of miR-744-5p and miR-145-5p in cells.(5)Compared with RAW.264.7 cells transfected with negative control sequence,there was no significant difference in the increased expression of IL-1 and TNF-a induced by LPS of same dose in RAW 264.7 cells transfected with miR-744-5p mimic.After adding supernatant of RAW264.7 cells after LPS stimulation of same dose into the MLE-12 medium,there was no significant difference in the degree of inflammatory injury of MLE-12 between the two kinds of supernatants.(6)Compared with RAW.264.7 cells transfected with negative control sequence,the expression of IL-1 and TNF-a induced by LPS of same dose is high in RAW 264.7 cells transfected with miR-145-5p mimic.After adding supernatant of RAW264.7 cells after LPS stimulation of same dose into the MLE-12 medium,the degree of inflammatory injury of MLE-12 was more severe of RAW 264.7 cells transfected with miR-145-5p mimic than that of RAW.264.7 cells transfected with negative control sequence.(7)On the contrary,compared with RAW 264.7 cells transfected with negative control sequence,the expression of IL-1 and TNF-a induced by LPS of same dose is low in RAW 264.7 cells transfected with miR-145-5p inhibitor.Accordingly,the inflammatory injury degree of MLE-12 induced by cell supernatant was also reduced.Conclusion(1)Both miR-744-5p and miR-145-5p miRNAs are associated with sepsis-induced inflammatory lung injury;(2)overexpression of miRNA-145-5p aggravates LPS-induced inflammatory lung injury,suggesting that its hyperesponsiveness to LPS;down-regulation of miRNA-145-5p attenuated LPS-induced inflammatory lung injury,suggesting its hyporesponsiveness to LPS;miRNA-145-5p is more closely related to LPS-induced inflammatory lung injury,so it was selected as the research object for the next experiment.Part ? Down-regulation of Smad3 aggravates inflammatory injury of lung epithelial cells in vitroSubjectiveTo verify down-regulation of Smad3 aggravate the inflammatory injury effect of LPS on lung epithelial cells.MethodsTransfection with si-RNA inhibits the expression and activation of Smad3 in macrophages.The expression of IL-1 and TNF-a in cells were measured by RT-PCR,cell proliferation was detected by CCK-8,and apoptosis was detected by Flow cytometry.Results(1)Transfection with si-RNA inhibited mRNA expression,protein expression and phosphorylation of Smad3 in macrophages.(2)Compared with RAW 264.7 cells without transfection of si-RNA,the expression of IL-1 and TNF-a induced by LPS of same dose is high in RAW 264.7 cells transfected with si-RNA.(3)After adding supernatant of RAW264.7 cells induced by LPS of same dose into the MLE-12 medium,the degree of inflammatory injury of MLE-12 was more severe in RAW 264.7 cells transfected with si-RNA than that without transfection.ConclusionDown-regulation of Smad3 aggravates the inflammatory injury effect of LPS on lung epithelial cells.Part ? miRNA-145-5P aggravates sepsis-induced inflammatory lung injury by down-regulating Smad3Subjective(1)To verify that miR-145-5p down-regulates expression of Smad3;(2)To verify that the effect of miR-145-5p in aggravating sepsis-induced inflammatory lung injury was mediated by Smad3.MethodsWith transfection of miRNA mimic,the expression of miRNA in cells was increased;With transfection of miRNA inhibitor,the expression of miRNA in cells was decreased.Transfection with si-RNA inhibits the expression and activation of Smad3 in macrophages.The expression of IL-1 and TNF-a in cells were measured by RT-PCR,cell proliferation was detected by CCK-8,and apoptosis was detected by Flow cytometry.Results(1)Transfection of miRNA-145-5p inhibitor increased expression of mRNA,protein and phosphorylation of Smad3;(2)LPS hyperresponsiveness induced by transfection of miR-145-5p mimic and LPS hyporesponsiveness induced by transfection of miR-145-5p inhibitor was blocked by transfection with Smad3 si-RNA.Conclusion(1)miRNA-145-5p down-regulates expression of Smad3;(2)miRNA-145-5p aggravates the inflammatory injury effect of LPS on lung epithelial cells by down-regulating expression of Smad3;(3)down-regulation of miRNA-145-5p attenuated LPS-induced inflammatory lung injury by up-regulating expression of Smad3.