Role Of Necroptosis Mediated HMGB1 Release In Liver Injury Induced By Helicobacter Hepaticus Infection

Helicobacter hepaticus(H.hepaticus)is a pathogen that can infect a variety of rodents and causes liver injury,hepatitis,liver cancer and other diseases in some strains of mice.Necroptosis is a newly discovered programmed cell death,which plays an important role in liver diseases.Researches show that necroptotic cells induce liver inflammation by releasing damage-associated molecular patterns,including high mobility group box 1(HMGB1),which further aggravate liver injury.The previous work of our research group found that H.hepaticus infection can promote the release of HMGB1 from hepatocytes,but the exact mechanism remains unclear.The purpose of this study is to explore the mechanism of HMGB1 release from hepatocytes induced by H.hepaticus infection and its role in liver injury,so as to provide a theoretical basis for further revealing the pathogenic mechanism of liver injury induced by H.hepaticus infection.1.Effect of necroptosis of hepatocytes induced by Helicobacter hepaticus infection on the release of HMGB1To explore the mechanism of HMGB1 release from hepatocytes induced by H.hepaticus infection,in this study,AML12 cells were infected with H.hepaticus,ELISA,RT-qPCR,western blot and cell immunofluorescence were used to investigate.The results showed that compared with the control group,H.hepaticus infection group significantly increased the transcription levels of Ripk1,Ripk3,Mlkl and the protein expression levels of RIPK3,MLKL,p-RIPK1,p-RIPK3,p-MLKL and the levels of HMGB1 in cell supernatant(P<0.05 or P<0.01),and showed a dose-and time-dependent manner.HMGB1 is translocated from the nucleus to the cytoplasm(P<0.05 or P<0.01).After treatment with the MLKL inhibitor GW806742X,the levels of LDH and HMGB1 in the supernatant of H.hepaticus infection group were significantly decreased(P<0.05),the translocation of HMGB1 from the nucleus to the cytoplasm was alleviated(P<0.05),and the intracellular fluorescence intensity of Fluo-4 was decreased.These results suggest that H.hepaticus promote the translocation and release of HMGB1 by inducing necroptosis in hepatocytes.2.Preparation and functional identification of HMGBl-interfering adenovirusTo explore the role of HMGB1 in liver injury induced by H.hepaticus infection in BALB/c mice,three pairs of oligonucleotides coding for shRNAs targeting mouse HMGB1 gene were designed and synthesized,and were cloned into the shuttle vector pDC316-ZsGreen1-shRNA after annealing to obtain three recombinant shuttle plasmids.The recombinant adenoviruses Ad-shHMGB1-318,Ad-shHMGB1-376 and Ad-shHMGB1-772 were obtained by co-transfection with the recombinant shuttle plasmids and adenovirus backbone plasmid pBHGlox(delta)E1,3Cre into 293A cells.The recombinant adenovirus with the best interference effect was screened by infecting AML12 cells with the three adenoviruses separately or in combination,and its interference efficiency was verified in the liver tissue of BALB/c mice.The results showed that the recombinant shuttle plasmids were correct as verified by restriction enzyme digestion and sequencing.Compared with the control group,the gene transcription and protein expression levels of HMGB1 in the adenovirus infection group were significantly decreased(P<0.05),at the same time,the interference effect was better when the three adenoviruses were mixed.The recombinant adenovirus could reduce the transcription and protein expression levels of HMGB1 by 65%and 68%,respectively.It is suggested that HMGB1-interfering adenovirus prepared in this study can effectively inhibit the expression of HMGB1 in mice and can be used for subsequent experiments.3.Role of HMGB1 in liver injury induced by H.hepaticus infection in miceTwenty-eight male BALB/c mice were randomly divided into 4 groups with 7 mice in each group:Control group,H.hepaticus infection group,Ad-shHMGB1 group and Ad-shHMGB 1+H.hepaticus group.Recombinant adenovirus was injected through the tail vein to interfere with the expression of HMGB1.Two days later,H.hepaticus was injected intraperitoneally.Samples were collected on day 7 after H.hepaticus injection.The histopathological changes of liver were observed and the expressions of key regulatory proteins of necroptosis and inflammatory factors were detected.The transcription levels of Hmgb1,Ripk1,Ripk3,Mlkl,Il-6,Tnf-α,Il-1β,Inos and the protein expression levels of HMGB1,RIPK3,MLKL,p-RIPK1,p-RIPK3,p-MLKL were up-regulated(P<0.05 or P<0.01).Immunohistochemical results showed that HMGB1 could translocalize from the nucleus to the cytoplasm in H.hepaticus infection group,and the expression of MLKL in cytoplasm was increased.Interference on the expression of HMGB1 alleviates liver injury and the expression of inflammatory factors Il-6,Tnf-α,I-1β and Inos induced by H.hepaticus infection.The results showed that H.hepaticus infection could induce necroptosis of hepatocytes,release HMGB1,and interference on the expression of HMGB1 could alleviate liver injury.In conclusion,H.hepaticus infection can induce necroptosis of hepatocytes and promote the translocation and release of HMGB1.Interference on the expression of HMGB1 can reduce the release of HMGB1 and the expression of inflammatory factors,thus alleviating liver injury caused by H.hepaticus infection.