The Mechanism Of The Effect Of Cytolethal Distending Toxin B Subunit(CdtB)of Helicobacter Hepaticus On Mouse Hepatocytes

Helicobacter hepaticus(H.h),a common pathogen found in human and animals,is considered as an important factor in the pathogenesis of a variety of diseases such as chronic active hepatitis,cholecystitis,appendicitis,colitis,hepatic cancer,cholelithiasis,gallbladder cancer,breast cancer,and has a significance in public health.Helicobacter hepaticus has been classified as a kind of pathogen in developed countries of the world and ICLAS,which has to be eliminated on rodent laboratory animals.In our country,it has not been regarded seriously so that is holding huge potential threats nowadays.While Helicobacter hepaticus infection has been occurring frequently in rodents in China.Cytolethal distending toxin(CDT)is the only known virulence factor found in a wide range of gram-negative bacteria.It has been reported that CdtB could induce apoptosis in liver cells.However,whether CDT of H.h could induce inflammation and the underlying mechanisms of apoptosis induction are not clear.In this study,we are going to explain the underlying mechanism of the effect of cytolethal distending toxin B subunit(CdtB)of Helicobacter hepaticus on mouse primary liver cells.Firstly,AML12 cells were treated with CdtB at the indicated concentration,and the disruption and necrosis of cells morphology were observed at a dose-dependent manner.Cell viability assays(CCK-8)demonstrated that CdtB reduced cell viability in a dose-dependent manner compared with controls.Meanwhile,the results obtained by qRT-PCR showed that the expression of pro-inflammatory cytokine IL-6、IL-1β、TNF-α、IFN-α1、IFN-γ in AML12 cells was upregulated after CdtB treatment.Furthermore,the STAT3 was phosphorylated indicated that the cytokine production may be mediated by JAK-STAT signaling pathway.In addition,FACS assay demonstated that CdtB induced a dose-dependent apoptosis in AML12 cells.To investigate the mechanism of CdtB-induced apoptosis.The related protein in Caspase and MAPK pathways were detected,the results revealed that CdtB could activate Caspase-9,Caspase-3,PARP and p38 as well as Erk1/2 MAPK.Cells were pretreated with caspase inhibitor Z-VAD-FMK,Erkl/2 inhibitor U0126,and p38 inhibitor SB203580,then cells were treated with CdtB,the results showed that apoptosis were significantly inhibited,suggest that Caspase and p38 MAPK involved in CdtB-induced apoptosis.In combination with the results from qRT-PCR,we postulate that p38 MAPK not only play roles in CdtB-induced apoptosis but also cytokine production in AML12 cells.Autophagy is a vital cellular process shared by all eukaryotic organisms.In order to further investigate the function of CdtB-induced apoptosis.We evaluated the autophagy level in AML 12 and found that the basal autophagy level was high in AML 12.Intrestingly,CdtB could inhibit autophagy by PI3K/Akt/mTOR activation.To further study the function of autophagy in AML 12 cells.Cells were treated with authophagy inhibitor CQ piror to CdtB treatment.The results showed that CQ coud enhance CdtB-induced apoptosis in AML 12 cells,and the results were further verified by the observation of activation of Caspase-3 and PARP,suggesting that autophagy inhibition may augment the CdtB-mediated apoptosis in liver cells.Collectively,these findings provide new insights for explanation of the molecular pathogenesis of helicobacter hepaticus.