The Mechanism Of Amphiregulin Protect The Tight Junction Of Alveolar Epithelial Cells Damaged By Lipopolysaccharide

Objective: This study aimed to investigate the effect of Areg protect the tight junctions of the alveolar epithelial barrier by activating EGFR and AKT in acute lung injury to reduce pulmonary edema.Methods: There are two parts of the experiments.Experiments in vivo were conducted on male C57BL/6 mice of 6 to 8 weeks old,weighing 20 to 25 g.Mice were randomly divided into the control group,Areg group,PBS+LPS group,and Areg+LPS group.Mice in the control group were injected intraperitoneally with phosphate-buffered saline(PBS,the same volume as Areg).Mice in the Areg group were injected intraperitoneally with Areg(5 μg per mouse).Mice in the PBS+LPS group were injected intraperitoneally with PBS(the same volume as Areg),and 30 minutes later,they were treated intratracheally with lipopolysaccharide(3mg/kg).Mice in the Areg+LPS group were injected intraperitoneally with Areg(5 μg per mouse),and 30 minutes later,they were treated intratracheally with LPS(3mg/kg).After 24 h,taken bronchoalveolar lavage fluid(BALF)and lung tissue from mice.For lung tissue,the dry and wet weight of lung tissue in each group was then measured and statistical ratio.The HE staining of the paraffin section of lung tissue was observed by a microscope and the lung injury score was made.The content and distribution of ZO-1,occludin,and claudin-1 in lung tissue were detected by western blot and immunofluorescence.And detect phosphorylation levels of EGFR and AKT phosphorylation in mice lungs.For BALF: count the total cells in BALF;detect the total protein in BALF by BCA method;detect IgM,TNF-α and IL-6 in BALF by ELISA.Experiments in vitro were conducted on the murine lung epithelial cell line(MLE-12 cells).MLE-12 cells were randomly divided into the Control group,Areg group,PBS+LPS group,Areg+ LPS group,siRNA-Scramble group,and siRNA-EGFR group.Cells in the Control group were treated with PBS(the same volume as Areg).Cells in the Areg group were treated with Areg(100 ng/ml).Cells in PBS+ LPS group were treated with PBS(the same volume of Areg),and30 minutes later,they were treated with LPS(1 μg /ml).Cells in the Areg+LPS group were treated with Areg(100 ng/ml),and 30 minutes later,they were treated with LPS(1 μg /ml).Mixing the Opti-MEM with scrambled siRNA and the Opti-MEM with Lipofectamine 3000,incubating 13 minutes.Cells in the Scramble-siRNA group were treated with the mixture,culturing 6 hours,then replacing the medium to complete medium containing 10% fetal bovine serum.Continuing cultivating 24 hours,then cells were treated with Areg(100 ng/ ml),30 minutes later,treated with LPS(1μg/ml).Cells in the si-EGFR group were treated with a mixture containing EGFR-siRNA instead of scramble siRNA,and the remaining steps are the same as above.Assessment of epithelial monolayer permeability.Immunofluorescence and western blot were used to compare the expression of tight junction protein ZO-1,occluding,claudin-1 in alveolar epithelial cells in each group.And western blot experiments were used to compare the changes in phosphorylation levels of EGFR and AKT between groups.Then to detect the expressions of tight junction proteins ZO-1,occludin,and claudin-1 in alveolar epithelial cells after EGFR down-regulation in MLE-12.Results:1.Compared with PBS + LPS,the HE staining in lung tissue of mice in Areg + LPS showed almost normal alveolar structure,and there is also a significant decrease in lung injury score(P < 0.0001),a decrease in wet to dry ratio of lung(P < 0.05),a decrease in neutrophil count(P < 0.01),total protein(P< 0.01),IgM exudation(P < 0.0001),TNF-α(P < 0.05),and IL-6(P < 0.05)in BALF.After Areg intervention,the increase of MLE-12 monolayer cell permeability caused by LPS was relieved(P < 0.05).It can be concluded that Areg can reduce the pathological damage and edema of lung tissue induced by LPS,reduce the inflammatory exudation of lung tissue,and improve the function of the alveolar epithelial barrier.2.Compared with PBS + LPS,the levels of ZO-1(P < 0.01),occludin(P <0.0001),claudin-1(P < 0.01)in lung tissue of mice in Areg + LPS increased to different degrees,and the same results were obtained in MLE-12 alveolar cells.In the immunofluorescence experiment,the average fluorescence intensity of ZO-1,occludin,and Claudin-1 in PBS + LPS was lower than that in Control(P <0.0001),and the distribution was intermittent;the average fluorescence intensity of ZO-1(P < 0.05),occludin(P < 0.001)and claudin-1(P < 0.01)in Areg + LPS was increased,and the distribution was a continuous line or arc.In conclusion,Areg can alleviate the loss and abnormal distribution of ZO-1,occludin,and claudin-1,and protect the tight junction of the alveolar epithelial barrier.3.Compared with the Control,the EGFR(P < 0.0001)and Akt(P < 0.0001)in the lung tissues and MLE-12 cells of Areg and Areg + LPS were significantly phosphorylated;4.After siRNA downregulated EGFR in MLE-12 cells,the EGFR expression of MLE-12 cells in si-EGFR decreased and compared with other groups,the phosphorylation of EGFR(P < 0.001)and Akt(P < 0.05)by Areg decreased;5.The permeability of MLE-12 in si-EGFR was significantly higher than that in Control,Areg + LPS,and si-Scr(P < 0.0001).Western blot and immunofluorescence were used to detect the content and distribution of tight junction protein in MLE-12 cells.It was found that the protective effect of Areg on three kinds of tight junction protein disappeared,and the content of ZO-1,occludin,and claudin-1 decreased significantly(P < 0.0001),and normal linear or arc distribution could not be identified.These results suggest that inhibition of EGFR expression can inhibit the content and distribution of ZO-1,occludin,and claudin-1.Conclusion: Areg protects the tight junction of the alveolar epithelial barrier and reduces pulmonary edema by activating EGFR and Akt signaling pathways.