| Background: Acute Respiratory Distress Syndrome(ARDS)is a Respiratory disease caused by the inability of alveoli to conduct normal gas exchange in a short period of time,resulting in refractory hypoxemia and Respiratory failure.The treatment of ARDS has always been a medical focus and difficulty.After ARDS occurs,a series of inflammatory factors,such as Tumor Necrosis factor-alpha(TNF-α),are released,which activate the "death signal" pathway,may lead to dysfunction of liver,kidney,heart and other organs.It has been reported that ARDS can lead to acute liver injury,massive hepatocyte necrosis and severe liver function impairment in a short period of time,but the mechanism is not clear.Amphiregulin(Areg)is a ligand of Epidermal growth factor receptor(EGFR),which can regulate cell proliferation and differentiation to promote organ growth and development.Areg also has anti-apoptotic effect,which can alleviate injury of lung,liver,intestine and other organs and tissues and promote repair.Whether Areg also has a protective effect on acute liver injury secondary to ARDS and the mechanisms involved remain unclear.Therefore,we hypothesized that Areg can inhibit the activation of TNF-α mediated "death signal" pathway by activating EGFR signaling pathway,thereby reducing TNF-α damage to the liver and protecting the liver.Objective: To investigate the protective effect and mechanism of Areg on secondary acute liver injury caused by ARDS.Methods: In this study,male C57BL/6 mice aged 6-8 weeks,weighting 22-24 g were randomly assigned.(1)Mice were divided into Control group and LPS group.The Control group was intragastrically injected with the same volume of sterile PBS solution as LPS group,while the LPS group was intragastrically injected with Lipopolysaccharide(LPS)(3 mg/kg)to establish the ARDS model.After 24 h,mice were sacrificed.Lung and liver tissues were stained with HE for histological analysis and injury score.Bronchoalveolar lavage fluid(BALF)was used for Giemsa stain for neutrophil count and total protein content determination to evaluate the alveolar barrier function.Serum was used for Alanine aminotransferase(ALT),Aspartate aminotransferase(AST),and Lactate dehydrogenase(LDH)to assess liver function.(2)Mice were divided into 0 h group,3 h group,6 h group,12 h group and 24 h group,and ARDS model was established by intratracheal infusion of LPS(3 mg/kg).The expression levels of TNF-α in BALF,serum and liver of mice at different time were detected.(3)Mice were divided into control group(LPS+NS group)and inhibitor group(LPS+ETA group).The LPS+NS group was intraperitoneally injected with the same volume of normal saline(NS)as the LPS+ETA group,and the LPS+ETA group was intraperitoneally injected with the TNF-α inhibitor Etanercept(ETA)(10 mg/kg).LPS(3 mg/kg)was intraperitoneally injected 30 min later in both groups.Lung and liver histomorphology,alveolar barrier function and liver function were detected.(4)Mice were divided into 4 groups: Control group,Areg group,PBS+TNF-α group(TNF-α group)and Areg +TNF-α group.The Control group was intraperitoneally injected with 0.15% BSA solution equal to that of TNF-α group,and 30 min later,the Control group was intraperitoneally injected with the same volume of sterile PBS solution equal to that of TNF-α group.The Areg group was intraperitoneally injected with Areg(5 μg/ rat),and the same volume of sterile PBS solution as TNF-α group was intraperitoneally injected with Areg 30 min later.TNF-α group was intraperitoneally injected with 0.15% BSA solution of the same volume as Areg group,and TNF-α was intraperitoneally injected 30 min later(0.75 mg/kg).Areg+TNF-α group was intraperitoneally injected with Areg(5 μg/ mouse),and TNF-α was intraperitoneally injected 30 min later(0.75 mg/kg).Lung and liver histopathology,alveolar barrier function,and liver function were detected.The apoptosis level of liver was detected by TUNEL,the expression of Cleaved-Caspase 3(Cl-Caspase 3)was detected by immunohistochemistry,and the protein expression of p-EGFR,EGFR,p-AKT,AKT,p-ERK1/2,AKT,Cl-Caspase 3,Bax,Bcl-2,and GAPDH was detected by Western Blot.Results:1.Compared with Control group,the alveolar structure changes and lung injury score were significantly increased in the LPS group(P﹤0.001);The function of alveolar barrier was impaired,and the number of neutrophils(P﹤0.05)and the protein content(P﹤0.01),which indicating the establishment of lung injury.Meanwhile,liver morphology and structure were changed in LPS group,and liver injury score was significantly increased(P < 0.01).ALT(P < 0.01),AST(P < 0.01)and LDH(P < 0.01)were significantly increased.These results indicated that acute liver injury could be secondary to ARDS in mice.2.In BALF,TNF-α in 3 h group was significantly increased compared with that in 0h group P﹤0.01),then gradually increased,and reached the peak at 6 h(P﹤0.001),and then showed a downward trend.Compared with 0 h group,the expression of TNF-α in serum of 3 h group was significantly increased(P﹤0.01),and then increased gradually,and reached the peak at 6 h(P﹤0.001),and then showed a downward trend.The concentration of TNF-α in serum and the concentration of TNF-α in alveolar lavage fluid were synchronized secretion.Compared with 0 h,the concentration of TNF-α in liver was significantly increased at 3 h(P﹤0.01),6 h(P﹤0.001),12 h(P﹤0.001),and 24 h(P﹤0.001)at each time point,and showed a step-by-step increase at each time point.These results suggested that TNF-α may be a key molecule in the secondary liver injury after ARDS.3.Compared with LPS+NS group,liver histomorphology and injury score(P﹤0.01),liver function ALT(P ﹤ 0.05),AST(P ﹤ 0.05)and LDH(P ﹤ 0.01)were significantly decreased in LPS+ETA group.The results showed that if the expression of TNF-α in liver was inhibited,then the secondary liver injury was significantly reduced.4.Compared with the Control group,liver tissue structure was significantly changed,liver injury score was significantly increased(P﹤0.001),liver function ALT(P﹤0.001),AST(P﹤0.001),LDH(P﹤0.01)were significantly increased in TNF-α group;Compared with TNF-α group,liver tissue structure was significantly improved,liver injury score was significantly decreased(P﹤0.01),liver function ALT(P﹤0.001),AST(P﹤0.001),LDH(P﹤0.05)were significantly decreased.These results indicated that TNF-α can mediate secondary liver injury after ARDS,and Areg can alleviate secondary liver injury mediated by TNF-α.5.Compared with Control group,the expression of p-EGFR(P﹤0.001),p-AKT(P﹤0.05),p-ERK1/2(P﹤0.05)was significantly increased in Areg group,even the expression of p-EGFR(P ﹤ 0.001),p-AKT(P ﹤ 0.05),p-ERK1/2(P ﹤ 0.01)and Cl-Caspase 3(P ﹤ 0.001)and hepatocyte apoptosis rate(P ﹤ 0.001)was also significantly increased in TNF-α group.Compared with TNF-α group,the expression of p-EGFR(P﹤0.05),p-AKT(P﹤0.01),p-ERK1/2(P﹤0.05)was further increased in Areg+ TNF-α group,and the express of Cl-Caspase 3(P﹤0.01)and hepatocyte apoptosis rate(P﹤0.001)was significantly decreased in Areg+TNF-α group.These results indicated that both Areg and TNF-α could prompt the phosphorylation level of EGFR,AKT and ERK1/2.Areg can alleviate TNF-α induced apoptosis and secondary liver injury.Conclusion: ARDS can lead to acute liver injury,in which TNF-α plays an important role.Areg not only has a protective effect on lung injury after ARDS,but also promotes liver repair and protects liver by activating EGFR pathway and down-regulating TNF-α mediated "death signal" pathway. |
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