MiRNA-126 Inhibits LPS-induced Acute Respiratory Distress Syndrome By Targeting Endocan

Objective To investigate the effect and mechanism of mi RNA-126 targeting Endocan on lung injury in acute respiratory distress syndrome by intratracheal instillation of lipopolysaccharide(LPS)in animal model.Methods Animal experiment 1: Twelve 8-week old C57 BL male mice were randomly divided into control group(Control group)and lung injury group(LPS group)with 6 mice in each group.The model of lung injury in mice was induced by intraperitoneal injection of lipopolysaccharide(LPS,10 mg/kg),and the control group was injected with the same amount of normal saline.After the successful establishment of the mouse model of lung injury,the mice were killed with cervical dislocation method at a time point of 24 hours,lung tissue samples were collected,fixed with 4% paraformaldehyde,embedded in paraffin,sectioned,stained with HE,and scored for lung injury according to the sectioned.Levels of inflammatory factors in lung tissue of mice,such as TNF-α、 IL-6、IL-1 β And mi RNA-126 was detected by q RT PCR,and the expression level of Endocan was detected by Western blotting and immunohistochemistry.Animal experiment 2: Thirty six C57 BL male mice aged 8 weeks were randomly divided into control group(Control group),lung injury group(LPS group),simulant group(Agomir group),simulant+lung injury group(Agomir+LPS group),inhibitor group(Antagomir group)and inhibitor+lung injury group(Antagomir+LPS group).The lung injury mouse model and the control group were constructed according to the method of animal experiment 1.Mi RNA-126 simulant and inhibitors were injected into the tail vein of mice to increase and decrease mi RNA-126.The time point for collecting lung tissue samples is also the same as animal experiment 1,which uses 4% paraformaldehyde for fixation,paraffin embedding,sectioning,and HE staining.The levels of inflammatory factors in mouse lung tissue and mi RNA-126 were still detected by q RT PCR.The effect of mi RNA-126 on the expression of Endocan was detected by Western blotting and tissue immunofluorescence.Results Animal experiment 1:(1)24 hours after lps(10 mg/kg)was injected into the trachea,it was observed that the mice were slow in movement,listless,increased oral and nasal secretions,and increased respiratory rate;Open the chest,it can be seen that the appearance of the lungs of the mice in the control group is pink,and there are no abnormal changes such as congestion and edema in the lung tissue,while the lung volume of the mice in the ARDS group is significantly increased,the color of the lung surface is dark red,and spot or sheet bleeding can be seen under the capsule.(2)Compared with the control group,ARDS mice had disordered alveolar cavity structure,thickened alveolar wall,congestion of pulmonary interstitium,and a large number of inflammatory cells infiltration in blood vessels and alveolar cavity.The lung injury score increased significantly(P<0.0001).(3)Compared with the control group,the expression level of IL-1β、IL-6 and TNF-α in the lungs of ARDS mice was significantly increased(P<0.0001).(4)Compared with the control group,the expression level of mi RNA126 in the lungs of ARDS mice was significantly lower(P<0.001).(5)Compared with the control group,the expression level of endocan in the lungs of ARDS mice was significantly higher(P<0.001).Animal experiment 2:(1)Compared with the ARDS mice,the ARDS mice injected with the analog effectively increased the level of mi RNA126,and the ARDS mice injected with the inhibitor effectively decreased the level of mi RNA126.(2)In the control group,the structure of pulmonary alveoli and pulmonary interstitium was normal,and there was no obvious inflammatory cell infiltration.In simple ARDS mice,there was a large amount of bleeding in the alveolar cavity,the alveolar wall was thickened,and there was a large amount of inflammatory cell infiltration in the blood vessels and alveolar cavity.Compared with simple ARDS mice,the bleeding and inflammatory cell infiltration in the alveolar cavity of ARDS mice injected with the analog were reduced,and the lung injury of ARDS mice injected with mi RNA126 inhibitors was more serious.(3)Compared with ARDS mice,ARDS mice injected with mi RNA126 analog had significantly lower expression level of endocan in their lung tissues,while ARDS mice injected with mi RNA126 inhibitor had higher expression level of endocan in their lung tissues.(4)Compared with ARDS mice alone,ARDS mice injected with mi RNA126 analog had significantly lower expression level of IL-1β、IL-6and TNF-α in their lung tissues,while ARDS mice injected with mi RNA126 inhibitor had higher expression level of IL-1β、IL-6 and TNF-α in their lung tissues.Conclusion The expression of mi RNA126 decreases,and endocan increases in ARDS lung tissue.They play an important role in the process of ARDS,and may become biomarkers for the diagnosis of ARDS;Up regulation of mi RNA126 has protective effect in LPS induced ARDS mouse model;Moreover,mi RNA126 can inhibit the inflammatory response of ARDS and improve lung injury,possibly by negatively regulating the expression of endocan.