Mechanisms Of Protective Effects Of MicroRNA-191 During Sepsis

Background:Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection.Sepsis is also the common clinical complication of severe burns,traumas and surgical operations,especially peritoneal infection.Complicated pathophysiological mechanism,rapid change and high fatality are the clinical hallmarks of sepsis,which is one of the most frequent cause of death in intensive care units.The primary cause of peritonitis to sepsis is the bacterial growth in the abdominal cavity.Neutrophils and macrophages are important immune cells,which play a crucial role in eliminating bacteria,limiting bacterial proliferation and controlling the process of sepsis.In particular,macrophages have a long life span and can continue to play a defensive role.MicroRNAs are small single-stranded non-coding RNAs,which are currently founded to be expressed in virus genome and eucaryon,with length of approximately18-22 nucleotides.Most microRNAs degrade or suppress the translation process by complementary binding to the 3'untranslated regions(3'UTR)of the target mRNA,thus regulating the growth and development,cell proliferation,differentiation,apoptosis,metabolism,immune reaction and other biological processes at the post-transcriptional levels.Abnormal expression of miR-191 has been reported recently in cancers,type 2diabetes,pulmonary arterial hypertension,alzheimer's and other diseases.Our previous experimental results confirmed that miR-191 also participated in the aseptic inflammatory response of liver IRI,and it was found that miR-191 promoted the inflammatory injury of liver IRI by promoting apoptosis and inhibiting cell cycle.So,is miR-191 involved in inflammatory injury of clinically critical severe sepsis?Bioinformatics analysis of the sequencing data from the GEO database showed that the expression of miR-191 in the peripheral blood of patients with systemicinflammatory response syndrome or sepsis was significantly decreased,suggesting that miR-191 was involved in sepsis,but the specific mechanism was unknown.Objective:Exploring the role and mechanism of miR-191 in sepsis through the study of septic mice and corresponding cell models.Methods:First,whether miR-191 is involved in mice sepsis was preliminarily analyzed.Cecal Ligation and Puncture(CLP)was used to simulate severe sepsis condition,and the expression of miR-191 in tissues,organs and peritoneal lavage fluid of C57BL/6(WT)mice were detected 18 hours after mouse modeling,which preliminarily verified whether mir-191 was involved in sepsis.To further explore the regulatory role of miR-191 in sepsis.miR-191 KO and WT mice were used as model animals to establish severe CLP models,respectively.Comparing the survival rates,bacteria load in peripheral blood and peritoneal lavage fluid,the proportion of different types of white blood cells between the two,in order to initially explore whether mir-191 regulates sepsis by affecting bacterial clearance and inflammatory cell infiltration.Based on the previous experimental data,further analysis was made on whether miR-191 regulates the clearance of sepsis bacteria by affecting the phagocytic function of neutrophils or macrophages.LPS stimulated RAW264.7 cells(mice mononuclear macrophages),WT mice peritoneal isolated primary macrophages or bone marrow isolated neutrophils for 4 hours,and the changes in the expression of miR-191 were detected,and the possible target cells of miR-191 were analyzed.On the basis of preliminary experiments,after the deletion of macrophages through intraperitoneal injection of Clodronate Liposomes,the survival rates of miR-191 KOand WT mice CLP models were compared,and the role of macrophages in the regulation of sepsis by miR-191 was discussed.Next,experiments were conducted to investigate the effect of miR-191 on the macrophage phagocytic function.We modulated miR-191 expression level of the peritoneal primitive macrophage and RAW264.7 cells,4 hours after LPS stimulated,incubate the cell with Enhanced Green Fluorescent Protein(EGFP)labeled Escherichia coli of 1 hours,confocal microscopy measures index,and combined with culture supernatant colonies count,analysis the role of miR-191 on macrophages bacteria clearance.At the same time,the differences in bacteria clearance ability of peritoneal primary macrophage of miR-191 KO and WT mice were compared,and the regulatory effect of miR-191 on the bacteria clearance of macrophage was confirmed.Finally,bioinformatics was utilized to predicted potential targets of miR-191,and the negative regulatory effect of miR-191 on the target was verified by Real-time PCR and luciferase reporter gene in the cell model,so as to preliminarily explore the possible molecular mechanism of miR-191 in the regulation of sepsis.Results:1.After CLP modeling for 18 hours,the expression levels of miR-191 in liver,intestine and peritoneal cavity cells of WT mice were significantly decreased.It suggested that miR-191 was involved in sepsis.2.After CLP modeling,miR-191 KO mice had a shorter survival time and higher bacterial load in peritoneal cavity and peripheral blood than WT mice.It suggested that miR-191 could alleviate sepsis.3.After CLP modeling,the recruitment of peritoneal neutrophils and macrophages of miR-191 KO and WT mice increased,but there was no difference in the proportion and absolute count between them.It suggested that both macrophages and neutrophils were involved in sepsis.4.In vitro,expression level of miR-191 in LPS stimulated RAW264.7 cells and peritoneal primary macrophages was decreased.However,there was no difference in the expression level of miR-191 in LPS stimulated neutrophils.The CLP model was repeated after further deletion of macrophages.The survival time of miR-191 KO and WT mice was shortened compared with that before the deletion of macrophages,and the more severe damage of miR-191 KO was eliminated.There was no statistical difference in the survival curve between the two groups.It suggested that miR-191 mainly alleviated sepsis by regulating macrophage function,but had no effect on neutrophil function.5.After regulating the expression of RAW264.7 cells and peritoneal macrophage miR-191,phagocytic tests and culture supernatant colonies count were performed.The high expression of miR-191 in RAW264.7 cells and peritoneal primary macrophages significantly increased the phagocytic ability and bacteria clearance of Escherichia coli.The macrophage phagocytic index and bacterial clearance of miR-191 with low expression were significantly reduced.Consistent with the above results,compared with WT mice,miR-191 KO mice significantly reduced the phagocytic index and bacterial clearance of peritoneal macrophages.6.bioinformatics analysis predicted that miR-191 in sepsis might target FOXP1,EGR1,CCND2,PHC2,ARPC4 and PLPP3 in macrophages.Real-time PCR and luciferase reporter gene verified that miR-191 negatively regulated EGR1 in macrophages.Conclusion:miR-191 perform the protective function through enhancing the bacteria clearance ability of macrophages by inhibiting EGR1 in mice,thereby prolonging survival time and alleviating sepsis.