Helicobacter Hepaticus Infection Induces Liver Fibrosis In BALB/c Mice And The Preliminary Study Of Its Pathogenic Mechanism

Liver fibrosis is a pathological process from the chronic liver injury to cirrhosis.In our country,the incidence of liver fibrosis is high,which is harmful for human health.At present,the research on animal models of liver fibrosis mainly induced by chemical drugs whereas few models are available for the study of pathogenic microorganism infection.It has been reported that Helicobacter hepaticus(H.hepaticus)infection can induce chronic active hepatitis,liver cancer,and hepatobiliary tumors in mice,however,there are fewer reports of liver fibrosis which belongs to the middle process.This project is based on an animal model of liver fibrosis in BALB/c mice infected with H.hepaticus,and focuses on the preliminary exploration of its pathological characteristics and pathogenic mechanisms.The mechanism of liver fibrosis infected with H.hepaticus remains unclear.To investigate the mechanism of IL-33 in hepatic fibrosis in mice,an animal model of liver fibrosis was established and the gene silencing effect of plasmid vector on IL-33 in hepatocytes was constructed in this study.The mechanism of liver damage was studied from multiple angles such as immunology,histopathology and molecular biology.1.Research on liver fibrosis in BALB/c mice infected by Helicobacter hepaticusIt has been documented that H.hepaticus infection is linked to chronic hepatitis and liver cancer.However,our understanding of the molecular mechanisms underlying progression of the H.hepaticus-induced hepatic inflammation to cellular hepatocarcinoma is still limited.In our study,male BALB/c mice were infected by H.hepaticus for 8,12,16,20 and 24 weeks.Histopathology,H.hepaticus colonization,select signaling pathways,and expression of key inflammatory cytokines in the liver were examined.The results showed that H.hepaticus was detectible in feces of mice at 7 days post infection(DPI)by PCR,but it was not detected in the livers by PCR until 8 weeks post infection(WPI).H.hepaticus-induced hepatic inflammation and fibrosis were aggravated over the infection duration,and necrosis or cirrhosis developed in the infected liver at 24 WPI.H.hepaticus infection increased levels of alanine aminotransferase and aspartate aminotransferase.Moreover,mRNA levels of IL-6,IL-1β,TGF-β and TNF-αwere significantly elevated in the livers of H.hepaticus-infected mice compared to uninfected control from 8 WPI to 24 WPI.Furthermore,STAT3,nuclear factor-κB(P65),MAPK(Erk1/2,and p38)and α-SMA were activated by H.hepaticus infection.These data demonstrated that male BALB/c mice can be used as a new model of H.hepaticus-induced liver diseases.2.Construction of a recombinant adenovirus vector that interferes with the expression of mouse IL-33 geneIn order to investigate the effect of interleukin 33 on mice liver fibrosis infected by H.hepaticus,recombinant adenovirus containing shRNA targeting IL-33 was constructed.The mouse IL-33(mIL-33)gene sequence was obtained from the NCBI gene library and shRNA was designed.The adenoviral vector pDC316-ZsGreen1-shRNA was digested with PstⅠ and BamHI and ligated with the interfering RNA fragment shIL-33 to construct an adenovirus vector.It was co-transfected with adenovirus backbone plasmid pBHGlox(delta)E1,3Cre into 293 A cells.After 10 days,the cell supernatant was collected to obtain the first-generation virus,and was purified and concentrated to achieve a virus titer of 1×1010 PFU/mL.The adenovirus was injected into male BALB/c mice via tail vein.Livers were collected after 24 weeks,one part for protein extraction and one for immunohistochemistry to verify the gene silence efficiency by adenovirus injection.The results showed that the recombinant adenoviral vector pDC316-ZsGreen1-shIL-33 was successfully constructed by verification of enzyme digestion and the sequencing results.The adenovirus titer was calculated by concentration to meet experimental requirements.The results of Western Blot and immunohistochemistry revealed that the expression level of IL-33 in the adenovirus injection group was significantly lower than that in the control group,and the expression of IL-33 in liver cells decreased accordingly.The results proved that the adenovirus vector was successfully constructed and could interfere with the expression of IL-33 in mouse hepatocytes.These achievements provided a foundation for the further study on the mechanism of IL-33 in liver fibrosis.3.Helicobacter hepaticus infection-induced IL-33 promotes hepatic inflammation and fibrosis through ST2 signaling pathways in BALB/c miceIt has been documented that H.hepaticus infection is linked to hepatic inflammation and fibrosis.IL-33 is a cytokine involved in inflammatory and fibrotic diseases,but its relevance to H.hepaticus infection-induced liver inflammation and fibrosis is unknown.Mice were infected by H.hepaticus for 8 weeks before additional IL-33 recombinant adenovirus treatment.All mice were sacrificed at 24 WPI,the sera and liver tissues from all of the mice were collected.Histopathology,H.hepaticus colonization,select signaling pathways,and expression of key inflammatory cytokines in the liver were examined.The location and expression levels of IL-33 and ST2 were detected by immunohistochemistry.In this study,we found that the expression of IL-33 in mice liver was significantly induced by H.hepaticus infection at 24 weeks post infection(WPI).Immunohistochemistry analysis revealed that IL-33 was transferred from the nucleus to the cytoplasm due to infection.The quantitation of inflammatory cytokine and histopathology evaluation showed that IL-33 knockdown attenuated the H.hepaticus-induced hepatic inflammation and fibrosis.More importantly,H.hepaticus promoted the expression of the IL-33 receptor ST2 on cell surfaces,and the expression of ST2 then activated the expression nuclear factor-κB(P65),α-SMA,and Erk1/2.These observations provided novel insights into the pathogenic mechanism of hepatic inflammation and fibrosis during H.hepaticus infection.