The Effects And Mechanisms Of Glucosylceramide Synthase On The Outcome Of Immune Liver Injury

Background: Disorder of the immune system and excessive activation of invariant natural killer T(iNKT)cells can cause hepatocyte injury.Immune liver injury is associated with glucosylceramide synthase(GCS)activity--a key enzyme that catalyzes the glycosylation of ceramide(Cer)to glucosylceramide(Glu Cer),and its altered activity causes disturbances in intracellular sphingolipid metabolism.iNKT cells are enriched in liver,which can be activated by recognizing glycolipid antigens and participate in the regulation of innate and adaptive immune responses.Therefore,GCS can affect the function of iNKT cells by causing changes in glycosphingolipids,resulting in immune dysfunction and pathogenicity.In addition,lipid and autophagy pathways intersect each other.The liver is the central organ of lipid metabolism,and changes in its GCS activity inevitably affect lipid metabolism,which is closely related to autophagy.This study aimed to explore the role of GCS in iNKT cell-mediated immune liver injury and the related regulatory mechanisms.Objective: To study the effects and related mechanisms of GCS on the outcome of concanavalin A(Con A)-induced immune liver injury.Methods: The model of mice immune liver injury was established by using Con A,and then the mice were pre-administered with genetic and chemical drug interventions to suppress their GCS.The alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels,liver pathological changes,Th1/Th2/Th17 cytokine levels,GCS expression,Cer and Glu Cer levels,hepatocyte ultrastructural changes,and hepatocyte autophagy/apoptosis were observed in each group.Subsequently,the number and activation of lymphocyte subtypes such as T lymphocytes,NKT cells and iNKT cells in the mice liver were further analyzed.Next,iNKT cells were flow-sorted and cultured in vitro,and iNKT cells were stimulated with α-galactosylceramide(α-Gal Cer).On the basis of iNKT cells were activated,NF-κB signaling pathway or GCS or both were inhibited in advance,then,the activation of iNKT cells and nuclear factorkappa B(NF-κB)signaling pathway,the expression of GCS and cytokines were detected respectively.After iNKT cells co-cultured with primary hepatocytes,the levels of ALT,AST,lactate dehydrogenase(LDH),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)in the culture supernatant were detected.Then Genz-123346 or4-phenylbutyric acid(4-PBA)or both were applied to the co-cultured primary hepatocytes.The cell viability,cell injury indexes,the expression of glucose regulated protein 78(GRP78),p NF-κB p65/NF-κB p65 ratio,LC3BII/LC3 BI ratio,Cleaved caspase-3 and caspase-12 protein were detected in each group.Results: Compared to the control group,the serum levels of ALT,AST and Th1/Th2/Th17 cytokines(except IL-17A),GCS gene and protein,Glu Cer,and autophagy/apoptosis of the liver were significantly increased in the Con A group(all P value <0.05).Moreover,the results of histopathology and transmission electron microscopy(TEM)indicated that significant liver/hepatocyte injury occurred.After GCS was inhibited,the above indicators(except IL-17A)were improved(all P value<0.05),except for hepatocyte autophagy/apoptosis,which increased(all P value <0.05).As a complement,the GCS high expression group showed the completely opposite results to those after GCS inhibition.Flow cytometric analysis showed no difference in the total number of LMNC,lymphocytes,and T-lymphocytes in each group(all P value >0.05).Further analysis showed that the total number of NKT cells and actived iNKT cells in the Con A group were significantly increased than those in the control group(all P value <0.001),and the total number of NKT cells and actived iNKT cells were decreased after inhibition of GCS(all P value <0.01).After activating iNKT cells in vitro with α-Gal Cer,the p NF-κB p65/NF-κB p65 ratio and cytokines such as TNF-α,IFN-γ and IL-4 were at high levels.However,the activation of iNKT cells were reduced after inhibition of NF-κB signaling pathway or GCS or both(all P value<0.001),and the p NF-κB p65/NF-κB p65 ratio and the expression of cytokines such as tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ)and interleukin-4(IL-4)were reduced(all P value <0.01).Activated iNKT cells co-cultured with primary hepatocytes caused a significant increase in ALT,AST,LDH and MDA.After inhibiting the NF-κB or GCS or both in iNKT cells,the levels of ALT,AST,LDH and MDA in hepatocytes were significantly decreased(all P value <0.01).Next,cell counting kit-8(CCK-8)results showed that primary hepatocytes viability was significantly reduced after inhibition of GCS(all P value <0.001).In addition,inhibition of GCS in primary hepatocytes caused significant increase in GRP78,LC3BII/LC3 BI ratio,p NF-κB p65/NF-κB p65 ratio,Cleaved caspase-3 protein and caspase-12 protein(all P value<0.05).However,treatment with 4-PBA increased primary hepatocytes vialibity on the basis of GCS inhibition(all P value <0.001),and decreased GRP78 protein,LC3BII/LC3 BI ratio,p NF-κB p65/NF-κB p65 ratio,Cleaved caspase-3 protein and caspase-12 protein(all P value <0.05).Further studies showed that inhibition of GCS or treatment with 4-PBA in normal primary hepatocytes had no effect on ALT levels(all P value >0.05).However,inhibition of GCS decreased ALT levels in co-cultured primary hepatocytes and increased ALT levels after administration of 4-PBA(all P value <0.05).Conclusion: GCS impeded the activation of iNKT cells by inhibiting the NF-κB signaling pathway and triggered endoplasmic reticulum stress-mediated autophagy/apoptosis in hepatocytes,and thereby improving the outcome of iNKT cellmediated immune liver injury.