PEBP4 Attenuates LPS/D-GalN-induced Acute Liver Injury Via TLR4/NF-κB Signaling Pathway

Background and objective:Liver is an important metabolism and detoxification organ of human body.Liver damage caused by various types of liver diseases can eventually lead to liver failure,cirrhosis or liver cancer,which kills about 2 million people worldwide every year,according to statistics.Therefore,it is of great significance to further clarify the pathogenesis of liver injury and seek for more reasonable intervention targets and treatment schemes,so as to improve the prognosis of liver disease.Phosphatidyleth-anolamine binding proteins（PEBPs）are a family of ultra-conserved proteins with more than 400 members that are expressed in bacteria,plants,mammals and other species.Phosphatidylethanolamine binding protein4（PEBP4）is a member of the protein.Recent studies have found that PEBP4 is highly expressed in a variety of tumor tissues such as lung cancer and breast cancer,and plays an important role in tumor invasion and metastasis.More importantly,high expression of PEBP4 can also be detected in the cancer tissues of patients with primary liver cancer,and patients with high expression of PEBP4 are more likely to develop lymph node metastasis and distant metastasis than patients with low expression of PEBP4,and the 2-year survival rate is much lower than patients with low expression of PEBP4.These results suggest that the expression level of PEBP4 is closely related to the occurrence,progression and prognosis of liver cancer,and that PEBP4 may be involved in the occurrence and development of liver diseases.Studies have shown that PEBP4 is a secreted protein with a variety of biological functions,and can resist tumor necrosis factor-α（TNF-α）induced apoptosis.The purpose of this study was to investigate whether PEBP4 has an effect on acute liver injury（ALI）and to investigate its mechanism.Conclusion:To investigate whether PEBP4 has anti-ALI effect and its mechanism.Methods:1.To observe the association between PEBP4 and ALIAfter the successful establishment of the model,the expression level of PEBP4in normal liver tissues and liver tissues with acute injury induced by LPS/D-Gal N of WT mices was detected by Western Blot.2.Construct PEBP4 liver-conditional knockout（CKO）miceFirst,PEBP4^flox/-mices were constructed using CRISPR/Cas9 technology,and then PEBP4^flox/floxmices were obtained by self-crossbred PEBP4^flox/-mices.Alb-Cre mices were hybridized with PEBP4^flox/floxmices using the Cre-lox P recombinase system to obtain PEBP4^+/-;Alb-Cre.CKO mices could be obtained by PEBP4^+/-;Alb-Cregenotype self-crossing again.WT mices and CKO mices were randomly dissected,the genes and proteins of each organ were extracted and detected respectively.The gene expression and protein expression levels of PEBP4 in all organs of WT genotype mice were confirmed,at the same time,PEBP4 CKO mices were only knocked out PEBP4 in the liver.3.Model building and grouping processing（1）Healthy C57BL/6N mices（n=12）aged 6-8 weeks and weighing 18-22 g were randomly divided into normal control group and LPS/D-Gal N group.Model group was intraperitoneally injected with LPS（5μg/Kg）,30 minutes later,300 mg/Kg D-Gal N was intraperitoneally injected.The normal control group was intraperitoneally injected with the same dose of normal saline.Blood and liver were collected 6 h after administration.（2）Healthy C57BL/6N mices（n=24）and PEBP4 CKO mices（n=24）aged 6-8weeks and with body weight of 18-22 g were randomly divided into normal control group and LPS/D-Gal N group.The model was cloned by the same method.1.5 h after the end of administration,6 rats in each group were randomly selected for blood collection after anesthesia.Blood and liver samples were collected from the remaining6 mices in each group 6 h after administration.（3）Healthy C57BL/6N mices（n=36）and PEBP4 CKO mices（n=36）aged 6-8weeks and weighed 18-22 g were randomly divided into LPS/D-Gal N group,LPS/D-Gal N+PDTC group and LPS/D-Gal N+TAK-242 group,with 12 mices in each group.In the inhibitor group,60 mg/Kg PDTC or 3 mg/Kg TAK-242 were intraperitoneally injected 30 minutes before LPS injection,and D-Gal N was intraperitoneally injected 30 minutes after LPS injection.1.5 h after the end of administration,6 rats in each group were randomly selected for blood collection after anesthesia.Blood and liver samples were collected from the remaining 6 mice in each group 6 h after administration.4.Assess the extent of liver damage（1）He staining was used to observe and compare the liver injury in each group.（2）The activities of alanine aminotransferase（ALT）and aspartate aminotransf-erase（AST）in serums were detected by biochemical kit.5.Assess the level of inflammation（1）Biochemistry kit was used to detect the activity of myeloperoxidase（MPO）in liver homogenate.（2）The contents of IL-1β,TNF-αand IL-10 in serums were detected by enzyme-linked immunosorbent assay（ELISA）.（3）The expression level of cyclooxygenase-2（COX-2）was detected by Western Blot.6.Evaluate the degree of apoptosis（1）Cell apoptosis was observed by TUNEL staining.（2）The protein expression levels of Pro-caspase-3,Cleaved caspase-3,Pro-caspase-8,Pro-caspase-8,Pro-caspase-9,Pro-PARP,Cleaved PARP were detected by Western Blot.（3）The activities of Caspase-3,Caspase-8 and Caspase-9 were detected by the kits.（4）The expression levels of pro-apoptotic gene Bax and anti-apoptotic gene Bcl-2 were detected by Real-time PCR.7.To investigate the role of TLR4/NF-κB signaling pathway in the anti-liver injury effect of PEBP4The expression levels of TLR4,IκBα,P-IκBα,NF-κB p65,P-NF-κB p65 and NF-κB p65 in cytoplasm were detected by Western Blot.Result:1.Compared with normal mices,the expression level of PEBP4 protein in liver of mices in ALI model group decreased.2.The results of agarose gel electrophoresis and Western Blot showed that the gene and protein expressions of PEBP4 were found in all organs of WT mices.In CKO mices,the gene and protein expressions of PEBP4 were found in heart,lung,spleen,kidney and other organs,but not in liver.The results showed that PEBP4 CKO mice were successfully constructed.3.The results of HE staining and biochemical tests showed that LPS/D-Gal N could induce ALI.The liver tissues’structures of WT mices were disordered,with diffuse necrosis of liver cells,infiltration of a large number of red blood cells and inflammatory cells,and increased ALT/AST activities.Compared with WT mices,CKO mices had more severe liver injury and higher ALT/AST activities.It suggested that PEBP4 knockout could aggravate the severity of ALI.4.Compared with WT mices,CKO mices showed more severe inflammatory response.MPO activities increased;Increased inflammatory cell infiltration;The contents of IL-1β,TNF-αand IL-10 increased;The expressions of COX-2 protein increased.5.Compared with WT mices,CKO mices had more severe apoptotic response and TUNEL（+）cell rate increased;Increased expressions of Cleaved caspase-9,Cleaved caspase-8,and Cleaved caspase-3;The Cleaved PARP expression levels were increased.6.Compared with WT micess,TLR4/NF-κB pathway was significantly activated in CKO mices.TLR4 expressions were up-regulated.The phosphorylations of IκBαwere increased and a large amount of IκBαwas degraded.The phosphorylations of NF-κB p65 were increased and a large amount of NF-κB p65 was translocated into the nucleus.7.Both TLR4 blocker TAK-242 and NF-κB blocker PDTC partially reversed PEBP4-induced liver injury and TLR4/NF-κB signaling activation.Conclusions:PEBP4 attenuates LPS/D-Gal N-induced acute liver injury via inhibiting TLR4/NF-κB signaling pathway..