Expression Of TREM-1 And Its Effect On Macrophages Autophagy In Acute Lung Injury

Objective:By observing the changes of TREM-1 expression in lung tissue during ALI and the effect of TREM-1 on the autophagy of macrophages,the role of TREM-1 in acute lung injury and the mechanism of amplification of inflammatory response were explored.Methods:(1)Kunming mice were divided into normal group and ALI group.ALI model was established by intraperitoneal injection of LPS 10mg/kg.Buxco method was used to observe the respiratory function of two groups,including respiratory rate,airway resistance,tidal volume and pulmonary compliance.HE staining method was used to observe the pathological changes of lung tissue in mice.The expression of TREM-1 in lung tissue was detected by real-time fluorescent quantitative PCR,and the expression of TREM-1 protein in lung tissue was detected by Western blot.(2)The logarithmic phase of macrophages(RAW264.7 cells)as the research object,with different concentrations of LPS(0,50,100,200,400,1000ng/mL)24h LPS 1000ng/mL;selection of different treatment time(0,6,12,24,36h).Detection of TREM-1 protein level in cells by Western blot.the macrophages(RAW264.7 cell line)were selected and divided into six groups:control group,LPS group,TREM-1 antagonist group,TREM-1 agonist group,TREM-1 antagonist+LPS group and TREM-1 agonist+LPS group.TREM-1 agonistlantagonist in advance into six well plates overnight,planting the logarithmic growth phase of macrophage,After 2h,LPS attached to join 1000ng/mL for 24h.The method of Western blot Determination of autophagy marker protein LC3 Ⅱ/Ⅰ and autophagy related gene expression of ATG7,The method of Real-time PCR determination mRNA of autophagy related genes(ATG5,7,12)and the expression of autophagy marker protein LC3 Ⅱ/Ⅰ.Detection of LC3 Ⅱ/Ⅰ protein expression in RAW264.7 macrophages by immunofluorescence staining.Result:1.Changes of TREM-1 expression in lung tissue at ALICompared with the normal group,the results of Buxco showed that the respiratory rate,tidal volume and lung compliance of the ALI group were decreased,while the airway resistance increased.HE staining was observed under light microscope,the pulmonary edema,pulmonary septal thickening and inflammatory cell infiltration in ALI group.The results of Real-time PCR and Western blot showed that the expression of TREM-1 in lung tissue of ALI group was significantly higher than that of normal group.2.Effects of TREM-1 on macrophage autophagyAt the cellular level,LPS could induce the expression of TREM-1 in macrophages,which was time and dose dependent.The results of Western blot showed that the expression of TREM-1 was significantly increased in macrophages treated 24h with 1000ng/mL LPS compared with the normal group.compared with LPS group,TREM-1 agonist+LPS group decreased the expression of autophagy marker protein LC3 Ⅱ/Ⅰ and autophagy related gene of ATG7.Real-time PCR results showed that compared with the LPS group,the TREM-1 agonist+LPS group of decreased expression of autophagy related gene mRNA(ATG5,ATG12 and ATG7);while the TREM-1 antagonist +LPS group increased the expression of autophagy related gene mRNA(ATG5,ATG7,ATG12).Immunofluorescence staining showed that LC3 Ⅱ/Ⅰ of macrophages showed strong positive expression in LPS group,the positive fluorescence expression of LC3 Ⅱ/Ⅰ of pure TREM-1 agonist group,TREM-1 agonist+LPS group of decreased,and the TREM-1 agonist+LPS group decreased significantly.Conclusion:1.LPS could induce the expression of TREM-1 and protein of ALI in lung tissue of ALI mice with mRNA.2.TREM-1 can inhibit macrophage autophagy.