Effect Of MiR-221/222 Cluster On Klebsiella Pneumonia-induced Sepsis In Mice

BackgroundSepsis is currently considered to be a range of life-threatening multi-organ dysfunction caused by dysregulated host responses to infections.In recent years,the prevalence rate and in-hospital mortality of sepsis have shown an increasing trend,which seriously threatens the safety of human life and property.In respiratory and critical care medicine,klebsiella pneumonia（KP）is a common cause of sepsis.At present,the main treatment of sepsis is fluid resuscitation,anti-infection and other comprehensive treatment,and there is no specific therapeutic drug approved by the Food and Drug Administration for the treatment of sepsis.Therefore,it is a great challenge to find potential therapeutic targets for sepsis.micro ribonucleic acids（miRNAs）are a class of non-coding oligonucleotides that act on specific messenger RNAs and thus regulate biological functions.In recent 10 years of research,it has been found that miRNAs often occur disorders in various diseases such as cancer,infectious diseases and inflammatory diseases,and it has been found in a variety of animal experiments that miRNAs can be used as inhibitors or activators to regulate the occurrence and development of diseases,showing great prospects in the diagnosis,prognosis and treatment of diseases.Recent studies have found that miR-221/222 can be used as biomarkers to assist the diagnosis of lung cancer,asthma and chronic obstructive pulmonary disease,but its potential role and related mechanisms in Klebsiella pneumonia-induced sepsis models have not been reported.PurposeIn this study,we used Klebsiella pneumonia-induced sepsis mouse model to explore the role and possible mechanism of miR-221/222 in pneumonia-induced sepsis mice,providing theoretical basis for the search for novel biomarkers and potential therapeutic targets for sepsis.Method1.Bioinformatics analysis:Expression of miR-221 and miR-222 in the blood of patients with sepsis.2.Animal experiments:Male C57BL/6 mice were selected and wild-type（WT）mice and miR-221/222 cluster knockout（miR-221/222 KO）mice were randomly divided into 4 groups,as follows:WT Control group,miR-221/222 KO Control group,WT group,and miR-221/222 KO Sepsis group.The mice were infected with KP through the airway.48h after modeling,the mice were killed by CO₂,and the circulating blood,lung tissue,alveolar lavage fluid,liver and kidney were collected for clinical evaluation.（1）Lung tissue:real-time quantitative PCR（q RT-PCR）was used to detect the expression of miR-221 and miR-222 in the lung group of mice.Hematoxylin-eosin staining（HE）was used to evaluate the lung histopathological changes of mice.The wet and dry weight（W/D）ratio of lung tissue in each group was detected.The expression of MAPK and apoptosis-related proteins in mouse lung tissue was detected by Western Blot.The levels of pro-inflammatory factors IL-6,TNF-α,IL-1βand IL-18 and the activity of MPO in lung tissue were determined by enzyme-linked immunosorbent assay（ELISA）.（2）bronchoalveolar lavage solution（BALF）:The total number of BALF cells and total protein content of mice in each group were detected;Bacterial load in BALF was detected by solid medium coating method.（3）Plasma:The levels of IL-6 and TNF-αin plasma were detected by ELISA.（4）Blood:Bacterial load in blood of each group was detected by solid medium bacterial coating method.（5）Liver and kidney:histopathological changes of liver were evaluated by HE staining;Biochemical reagents were used to detect the related indexes of liver and kidney function:alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,serum creatinine（Cr）,urea nitrogen（BUN）;The levels of pro-inflammatory cytokines IL-6 and TNF-αwere determined by ELISA.Results1.Biological information analysis showed that the expression of miR-221 and miR-222 in peripheral blood of patients with sepsis decreased;Real-time quantitative PCR showed that the expression of miR-221 and miR-222 decreased in lung tissue of mice with sepsis caused by KP infection（P all<0.05）;2.Deletion of miR-221/222 alleviates the general status of Klebsiella pneumonia-induced sepsis in mice（P<0.05）,increased survival rate of mice（P<0.05）;3.HE pathological staining showed that miR-221/222 deletion alleviated lung histopathological changes in sepsis mice（P<0.05）;Total BALF protein concentration and lung W/D showed that miR-221/222 deletion reduced lung exudation and edema in mice with Klebsiella pneumonia-induced sepsis（P all<0.05）;BALF cell count showed that miR-221/222 deletion reduced the total number of BALF cells in sepsis mice（P<0.05）;4.ELISA results showed that miR-221/222 deletion decreased plasma IL-6 in mice with Klebsiella pneumonia-induced sepsis（P<0.05）,TNF-α（P=0.053）and decreased IL-6（P<0.01）,TNF-α（P<0.05）,IL-1β（P<0.01）level;miR-221/222 deletion decreased MPO activity in lung tissue of sepsis mice（P<0.05）;5.Bacterial load assessment showed that the deletion of miR-221/222 could enhance the KP clearance ability of immune cells in Klebsiella pneumonia-induced sepsis mice（P<0.05）;6.WB detection showed that miR-221/222 deletion may reduce acute lung injury in mice by inhibiting the activation of MAPK signaling pathway and regulating apoptosis pathway in lung tissue of sepsis induced by Klebsiella pneumoniae（P all<0.05）;7.Mouse liver function assay and ELISA results showed that miR-221/222 deletion could alleviate liver damage and decrease the levels of ALT and AST in mice with Klebsiella pneumonia-induced sepsis（P all<0.05）,decreased the levels of IL-6 and TNF-αin liver（P all<0.05）;8.Mouse renal function and ELISA results of mice showed that miR-221/222 deletion could alleviate kidney injury and decrease blood Cr（P<0.05）and renal IL-6（P<0.05）level.conclusionThe deletion of miR-221/222 can improve the survival rate of mice with sepsis,which may be caused by inhibiting the production of inflammatory cytokines,inhibiting the activation of MAPK pathway and regulating apoptosis pathway.These results suggest that miR-221/222 may be used as a potential biomarker to assist in the diagnosis of sepsis.It may be a potential therapeutic target for sepsis caused by Klebsiella pneumonia.