The Effect And Mechanism Of EGFR On Lipopolysaccharide Induced Acute Lung Injury

Part one Establish a mice model of the acute lung injury induced by lipopolysaccharide and detect the expression of EGFR and p-EGFRObjective: Establish a mice model of acute lung injury(ALI) induced by lipopolysaccharide(LPS), and detect the expression of epidermal growth factor receptor(EGFR) and phosphorylated epidermal growth factor receptor(p-EGFR) in lung tissues.Methods: C57BL/6 male mice aged from 6 to 8 weeks and weighted from 18 to 22 grams were enrolled in this study. The experiment group mice were received 2mg/kg LPS by intratracheal instillation, the control group without any treatment, 24 hours later, mice were sacrificed under anesthesia, lungs and bronchoalveolar lavage fluid(BALF) were collected.Hematoxylin-eosin(HE) staining was used for evaluating the lung injury score,immunofluorescent staining was used for testing the expression of EGFR and p-EGFR,Western Blot was used for detecting the protein levels of EGFR and p-EGFR.Results: HE staining showed that LPS could induce acute lung injury in mice, the lung injury score was significant higher than that of the control group. Immunofluorescence staining showed that the number of EGFR positive cells in LPS induced ALI group was not significantly changed, but the number of p-EGFR positive cells was significantly increased(p<0.05). There were no significant differences in the number of p-EGFR and EGFR positive cells in control group(p>0.05). Western Blot implied that the expression of EGFR was equal in experimental group and control group, but in LPS induced ALI group, the p-EGFR protein level was markedly increased(p<0.05).Conclusion: In the LPS-induced ALI murine model, the lung tissue injury degree was severe, and the expression of p-EGFR protein level was significantly increased.Part two Evaluate the effect of EGFR inhibitor Erlotinib on lipopolysaccharide induced acute lung injury in miceObjective: To detect the effect of EGFR inhibitor Erlotinib in LPS induced ALI models in mice.Methods: C57BL/6 male mice aged from 6 to 8 weeks and weighted from 18 to 22 grams were enrolled in this study. The mice were randomly divided into six groups, every group contains 6 mice. The blank control group without any treatment, the positive control group administrated 100mg/kg erlotinib gavage, LPS(2mg/kg) was used to induce the ALI model.In the other three drug treatment groups, mice respectively accept 10mg/kg, 25mg/kg,100mg/kg gavage, one hours later, those mice were intratracheal instillation of 2mg/kg LPS.24 hours later, mice were sacrificed under anesthesia. HE staining was performed to evaluate the degree of acute lung injury according to lung injury scoring system(LISS).The number of Gr-1 positive cells was observed by immunofluorescence staining. The total cells of BALF was counted by the microscope. Total protein level in BALF was tested by BCA method. The expression levels of IL-1? in BALF were examed by Enzyme linked immunosorbent assay(ELISA).Results: The LISS indicated that the lung injury score was significant higher in the ALI group than in the control group(p<0.05). After erlotinib treatment, the lung injury score decreased(p<0.05), and presented a dose-dependent manner. Gr-1 immunofluorescence showed that the LPS induced ALI group had obvious neutrophils aggregation(p<0.05), and the number of Gr-1 positive cells was decreased after treated by erlotinib(p<0.05). Total cell count showed that erlotinib treatment could inhibit LPS induced cell exudation(p<0.05). BCA assay showed that the total protein in BALF was significant higher in LPS induced ALI mice model, however, erlotinib treatment could inhibit protein exudation(p<0.05). As detected by ELISA, the inflammatory cytokine IL-1? in BALF was markedly increased in LPS induced ALI group, this expression was adversed by erlotinib treatment( p<0.05).Conclusion: In LPS induced ALI murine model, erlotinib could reduce the lung injury degree, decrease the recruitment of neutrophils and the exudation of protein and cells of lung tissue, block the secretion of IL-1?.Part three The mechanism of EGFR inhibitor Erlotinib on the acute lung injury induced by lipopolysaccharide in miceObjective: To explore the mechanism of Erlotinib in alleviating acute lung injury induced by lipopolysaccharide in mice.Methods: C57BL/6 male mice aged from 6 to 8 weeks and weighted from 18 to 22 grams were enrolled in this study. The mice were randomly divided into six groups, every group contains 6 mice. The blank control group without any treatment, the positive control group administrated 100mg/kg erlotinib gavage, LPS(2mg/kg) was used to induce the ALI model.In the other three drug treatment groups, mice received 10mg/kg, 25mg/kg, 100mg/kg gavage respectively, one hours later, those mice were intratracheal instillation of 2mg/kg LPS. 24 hours later, mice were sacrificed under anesthesia. The protein expression levels of TNF-?, IL-1?, TLR4, p65, p-p65, EGFR, p-EGFR, ERK, p-ERK, AKT, p-AKT were tested by Western Blot.Results: Compared with blank control group and positive control group, the protein levels of TNF-?, IL-1?, p-p65, p-EGFR, p-ERK, p-AKT were significantly increased in LPS induced ALI group(p<0.05), however, those increased expression levels were blocked by erlotinib lavage(p<0.05). But the expression of TLR4, p65, EGFR, ERK, AKT showed no obvious differences.Conclusion: Erlotinib inhibited the activation of EGFR signaling pathway in LPS-induced lung injury in murine model, and TLR4 signaling pathway was involved in this process.