Epidemiological Investigation Of Helicobacter Hepaticus And Study On CdtB-induced Liver Injury

Helicobacter hepaticus(H.hepaticus)is a kind of micro-aerobic,curved Gram-negative bacteria belonging to the genus Helicobacter,which would colonize the lower digestive tract of rodents.H.hepaticus infection are subclinical in laboratory animals,which has posed a serious threat to the health of laboratory animals and public health.In recent years,it has been shown that H.hepaticus infection is closely related to chronic hepatitis,cirrhosis,liver cancer and colon cancer,and the pathological features of mouse liver fibrosis/cirrhosis induced by H.hepaticus infection are similar to those of human hepatocellular carcinoma,but the underlying molecular mechanism has not been fully elucidated.Cytolethal distending toxin(CDT)is one of the main virulence factors of H.hepaticus with highly conservative property.In current study,H.hepaticus infection prevalence in laboratory rats and mice of Jiangsu province was investigated by a self-established quantative PCR method targeting CdtB,providing technical method and data support for further control of the H.hepaticus infection in Chinese laboratory animal facilities.By using animals models infected by wild type H.hepaticus and CdtB-deficient H.hepaticus(ΔCdtB)and CdtB overexpressed cell models,the underlying mechanism of liver injury caused by H.hepaticus infection are going to be explored from the view of histopathology,immunology,molecular cytology.1.Establishment of TaqMan-MGB PCR for Helicobacter hepaticus detection and epidemiological investigation of laboratory rats and mice in JiangsuTo develop a TaqMan-MGB probe quantitative PCR method for Helicobacter hepaticus,we designed a set of specific primers targeting CdtB gene of H.hepaticus ATCC 51449 strain.The results showed that the established TaqMan-MGB probe quantitative PCR method can detect H.hepaticus as low as 10 copies/μL,and the detection sensitivity is 100 times higher than that of conventional PCR with high specificity.1506 samples of mouse feces from different facilities inf Jiangsu province were investigated by the established mehtod.The results showed that the H.hepaticus infection rate in 2019 and 2020 in Jiangsu Province was 34.18%and 14.90%respectively.Among them,the infection rate in clean rats was 27.50%and 6.15%respectively in 2019 and 2020,while the infection rate of SPF rats was 18.33%in 2019,and there were no H.hepaticus infection was detected in 2020.And the infection rates of clean mice in 2019 and 2020 were 43.46%and 21.37%,respectively,and those of SPF mice were 33.33%and 15.61%.Importantly,BALB/c,ICR,SCID,KM,Knock out mice represents higher infection rates,BALB/c mice is a highly sensitive strain with 71.13%、46.34%incidence in 2019 and 2020.2.The study of inflammatory signaling pathways activated by CdtB of Helicobacter hepaticus infection induced liver injuryStudies have indicated that Helicobacter hepaticus infection can cause liver injury diseases such as hepatitis,liver fibrosis and so on.As one of the important virulence factors,the mechanism of CdtB remains unclear.In order to explore the role of CdtB in H.hepaticus infection induced liver damage.The Wild type H.hepaticus and CdtB-deficient H.hepaticus(ΔCdtB)were used to infect 6-week-old male BALB/c mice.Samples were collected at 3,6 and 10 months post infection(MPI),and histopathology observation and H.hepaticus colonization level in liver and colon detection as well as key inflammatory factors and key regulatory proteins in inflammatory signaling pathways were performed.The results showed that during the infection period,there was no significant difference of colonization level both in colon and liver between the wild type infection group and the ΔCdtB infection group(P>0.05).At 6 MPI and 10 MPI,compared with WT infection group,the degree of hepatitis and fibrosis of mice in ΔCdtB infection group was weaker,the transcription levels of IL-6、IL-1β、TNF-α、IL-10、TGF-β in liver were decreased,and the expression levels of stat3,nuclear factor-κB(p65),α-SMA and yH2AX were decreased.Which suggesting that CdtB activates the inflammatory pathway leading to liver injury during Helicobacter hepaticus infection.3.The exploration of CdtB in Helicobacter hepaticus long-term infection induced hepatocellular carcinomaAlthough there is growing evidence that H.hepaticus infection induces chronic active hepatitis and hepatocellular carcinoma in susceptible strains and genetically engineered mice,the effect of CdtB of H.hepaticus on the progression of hepatocellular carcinoma remains unclear.In order to investigate the mechanism of CdtB in H.hepaticus-induced liver cancer in mice,wild type H.hepaticus(WT)and CdtB deletion strain(ΔCdtB)were used to infect BALB/c male mice for long time.The results showed that,at 12 and 18 months post infection,there was no significant difference in liver colonization level between the wild-type H.hepaticus and CdtB deletion strains(P>0.05),and three stages of lymphoid nodules and cirrhosis in the wild-type H.hepaticus group were observed.The liver pathological scores were significantly higher than those in the CdtB mutant group.In addition,AFP and Ki67 were positive in the liver of wild type stain infected group but not in the CdtB mutant infected group.Western blot analysis showed that during 12 and 18 months post infection,γH2AX expression in the wild-type H.hepaticus infected group was suppressed,while γH2AX expression in the CdtB mutant infected group was continuously activated,suggesting that after long-term exposure to CdtB in mouse liver,Hepatocytes are damaged and the conventional structure were replaced by fibrous connective tissue,leading repair ability lost and the ability to inhibit tumor occurrence reduction.4.In vitro study of hepatocyte injury induced by CdtB of Helicobacter hepaticusCdtB has DNase activity and can induce DNA damage in cells,but its mechanism has not been fully elucidated.To further elucidate the mechanism of CdtB-induced hepatocyte injury in mice,we constructed a lentivirus expressing CdtB,a Hepa1C1C7 cell line which over-expressed CdtB gene was constructed.Cytological experiments were carried out from the perspective of DNA damage.The results showed that the expression of γH2AX and apoptotic necrotic cells both in wild type H.hepaticus and H.hepaticus(ΔCdtB)infected groups had been upregulated at 72 h post infection.When CdtB expression initiated by Dox based on the Tet-On system,ATM-dependent DNA damage,DNA repair activation,G1/G2 cell cycle arrest and cell death occurred in cells,suggesting that CdtB could induce liver cell damage by inducing ATM-dependent cell cycle arrest.In conclusion,the study has revealed that CdtB may promote CLD development in the course of H.hepaticus persistent infection both in vivo and in vitro,and found the DNA damage and impairment of cellular control responses as critical events in the process.Furthermore,regarding CdtB-displayed cancerogenic activities in vivo,our results underline chronic exposure to CdtB and the repair mechanisms that may be involved with malignant transformation for the disease,ultimately providing a new objective model for H.hepaticus infection induced liver injury.