Immunological Mechanisma Of Glucocorticoid Therapy In Acute Liver Failure

Background and aimsAcute liver failure(ALF)is a type of rapidly progressing clinical syndrome without specific treatment methods and has a high mortality rate.Liver transplantation is the most effective treatment for ALF currently,but the limition of the therapy is the lack of liver donors and more contraindications.Thus,finding effective medical treatment is of great significance to patients with ALF.Glucocorticoids can quickly suppress excessive immune and inflammatory responses.Previous clinical studies have shown that glucocorticoids have clinical benefits in treating ALF.However,the unclear mechanism of action,the indeterminate treatment population,and the inaccurate indication of treatment make its administration more controversial.The purpose of the study was to clarify the therapeutic effect of dexamethasone(Dex)on ALF mice induced by lipopolysaccharide/D-galactosamine(LPS/D-Ga IN)and to explore the specific immunological mechanisms.Methods1.C57BL/6 mice were given LPS/D-Ga IN by intraperitoneal injection to establish mouse ALF model.H&E staining of livers and serum liver enzymes ALT and AST test were performed 1-5h after modeling.CBA method was used to detected the changes of serum cytokines IL-6,TNF-?,CCL-2 and IL-10 at different time points during modeling.The specimens of liver and blood were taken for multicolor flow cytometry,to find the changes of immune cell subsets such as B cells,CD4 cells,CD8 cells,NK cells,NKT cells,Kupffer cells,monocytes,and neutrophils at different time points.2.LPS/D-Ga IN-induced ALF mice were injected intraperitoneally with Dex at 0h and 1 h to observe the therapeutic effect.A survival curve was drawn to analyze the effect of Dex at different time points on the survival rate of ALF mice.H&E staining and serum liver enzyme were detected to confirm the effect of Dex on the progression of liver injury in ALF mice.CBA method was used to detect cytokine changes.Multicolor flow cytometry was used to determine changes of immune cell subgroups in liver and blood.3.Kupffer cells of the mice from LPS/D-Ga IN and Dex 0h treatment group were separated by flow cytometry.Immunohistochemical staining F480 was used to confirm the results of flow cytometry.q PCR was used to detect the CD86 and CD163 m RNA levels of sorted Kupffer cells.RNA sequencing further confirmed the differences in Kupffer cells between the two groups.4.Clodronate Liposomes(CL),a liver macrophage scavenger,was used to clear liver macrophages.Then flow cytometry and immunohistochemistry were performed to clarify the clearance effect.ALF mice were treated with Dex or CL in the early stage,and H&E staining and serum liver enzyme detection were used to determine the role of macrophages in the treatment of ALF with Dex in the early stage.5.The early administration of Dex or GR inhibitor RU486 in ALF mice were used to bserve its effect on mouse survival.H&E staining and serum liver enzyme test were performed to confirm liver damage.q PCR was used to detect NR4A1 m RNA levels in Kupffer cells after RU486 treatment.Results1.LPS/D-Ga IN could successfully induce ALF in mice which showed symptom within 5 hours.There was a cytokine storm at early stage in ALF mice.Liver and blood innate immune cells such as Kupffer cells,neutrophils and monocytes changed significantly in the ALF progression.2.The use of Dex at 0h can significantly improve the progression of ALF induced by LPS/D-Ga IN in mice compared with the use of Dex at 1h.Dex treatment at 0h can significantly reduce the pro-inflammatory cytokines such as IL-6,TNF-? and CCL-2,and increase the anti-inflammatory factor IL-10.Innate immune cells,especially Kupffer cells and neutrophils,increased significantly in the Dex 0h treated group.3.Compared with the LPS/D-Ga IN group,the number of Kupffer cells in the liver of Dex treatment at 0h group increased significantly,and mainly was M2 type.RNA sequencing showed that m RNA levels of NR4A1 in Kupffer cells in the Dex treatment group increased significantly.4.CL applied 24 hours in advance could effectively clear liver macrophages.However,early clear of liver macrophages and intraperitoneal injection of LPS/D-Ga IN in mice did not show significant liver failure.5.The administration of RU486 supressed the effect of improving survival rate and relieving liver injury caused by the early use of Dex in ALF mice-induced by LPS/D-Ga IN.When GR were inhibited,the effect of early use of Dex to increase NR4A1 m RNA in liver Kupffer cells was also inhibited.Conclusion1.LPS/D-Ga IN could successfully induce mouse ALF model.2.Early use of Dex in LPS/D-Ga IN induced ALF could improve disease progression.3.Early use of Dex in ALF significantly increased liver Kupffer cells,mainly M2,and the m RNA level of NR4A1 in Kupffer cells significantly upgreluated.4.The direct removal of liver macrophages in the early stage of ALF could not explain the important role of Kupffer cells in the early treatment of ALF with Dex.5.Early use of Dex to improve LPS/D-Ga IN-induced ALF in mice mainly worked through the genomic mechanism of GR.The early use of Dex to increase the level of NR4A1 m RNA in liver Kupffer cells required GR to participate.