Molecular Mechanism Of MiR-21 Regulating Endotoxic Shock Through A20

Background and Objective:Sepsis is a major clinical manifestation of septic shock.Due to the highly variable clinical characteristics of septicemia,it becomes more difficult to provide an accurate early diagnosis.Late diagnosis and delayed treatment can greatly increase mortality.Biomarkers are potentially valuable clinical tools.Although there have been many studies on early diagnosis of sepsis using some biomarkers,most have not been adopted or proven effective in clinical trials.In order to find new diagnostic and prognostic indicators in sepsis,a new genomic method has been applied to the study of sepsis,during which many miRNAs have been identified as key regulators of inflammation.As a regulator of immune response,miRNA has important implications for transcriptional regulation in septicemia.Although more and more expressions of miRNA have been studied,the pathological mechanism of these molecules in sepsis is not well understood.Some studies have shown that miR-21-3p is highly expressed in septicemia-related cardiac dysfunction induced by LPS.The pharmacological inhibition of miR-21-3p by antagomiR can improve survival rate in mice,however the mechanism remians unclear.Therefore,the role of miR-21 in endotoxic shock was studied and its regulatory mechanism was discussed.Methods: In vivo,miR-21 KO and WT mice were injected intraperitoneally with LPS to induce endotoxin-induced inflammatory shock model.The survival of mice within 48 hours was monitored and the survival curve was recorded.In addition,the levels of IL-1? and IL-18 in peripheral blood of mice were detected by ELISA 6h after LPS injection,and the peritoneal macrophages were obtained by centrifugation and the ratio of M1 and M2 macrophages was detected by flow cytometry.In order to observe the effect of miR-21 knockout on endotoxic shock,the infiltration of inflammatory cells was observed by H&E staining in liver,spleen and kidney of mice12 h after intraperitoneal injection of LPS.The activation of caspase-1 leads to the maturation and secretion of IL-1? and IL-18.In vitro,the activation of caspase-1 in the inflammatory body of NLRP3 was detected by western,and the secretion of IL-1? was detected by ELISA when caspase-1 was activated.miR-21 KO and WT BMDMs were pretreated with LPS for 4 h.Western was used to detect caspase-1activation trend after 0 min,10 min,20 min,30 min and 60 min stimulation by ATP or Nigeria.Meanwhile,the cell supernatant at the corresponding time point was used to detect IL-1? secretion and LDH content after caspase-1 activation.In order to exclude the effect of transfection reagent and siRNA on the activation of AIM2 inflammasome,we transferred dsDNAs to BMDM,detected the activation of caspase-1 by western and detected the contents of IL-1? and LDH in supernatants by ELISA.Studies have shown that A20 is a negative regulator of NLRP3 inflammatory bodies.In subsequent experiments,we detected the expression of NLRP3,caspase-1and ASC in miR-21 KO and WT BMDM by western and qRT-PCR.At the same time,luciferase reporter gene experiment and qRT-PCR experiment were used to verify that A20 is the target gene of miR-21.Then,the expression of NLRP3,caspse-1 and ASC was detected by western and qRT-PCR after transfection of si-A20 into miR-21 KO BMDMs,and the secretion of IL-1 ? in the supernatant was detected by ELISA.A large number of studies have shown that activation of NLRP3 inflammasome is associated with mitochondrial dysfunction.NLRP3 inflammasome can be activated by intracellular ROS produced by various cellular stressors.Mitochondrial damage was determined by detecting ROS in this study.Activation of caspase-1 can cleave GSDMDs and produce a GSDMD-N which is a direct fragment that causes pyroptosis.We transfect Flag-GSDMD lentivirus into BMDM cells,use anti-Flag to detect GSDMD cleavage by western,and use anti-GSDMD directly to detect GSDMD cleavage by western.Results:The results of survival curve in vivo showed that the survival of mice after miR-21 knockout was much better than that of the control group.The contents of IL-1? and IL-18 in serum and the percentage of peritoneal macrophages in the miR-21 KO group were lower than those in the control group.H&E staining in liver,spleen and kidney showed that the infiltration of inflammatory cells in miR-21 KO group was significantly less than that in control group.In vitro study,the activation of caspase-1 was promoted after activation of NLRP3,while the self-shearing of caspase-1 promoted the maturation and secretion of IL-1?.In miR-21 KO BMDMs,A20 is highly expressed,while NLRP3,caspase-1 and ASC are low expressed at mRNA level and protein level.A20 is a target gene for miR-21 and returns theexpression level of NLRP3,caspase-1 and ASC when the A20 is knocked down in miR-21 KO BMDMs.The release of ROS in miR-21 KO BMDM was significantly lower than that in control group.At the same time of caspase-1 activation,GSDMD-N induced pyroptosis of macrophages in miR-21 KO group was significantly lower than that in control group.Conclusion : These results suggest that the knockout of miR-21 can significantly alleviate the endotoxic shock induced by LPS in vivo as well as in vitro,and decrease the degree of inflammation.The mechanism is that miR-21 affects the occurrence of pyroptosis by regulating A20 and inflammasome,and ultimately affects the secretion of IL-1?.