Mechanism Of Recombinant Protein EBI3 In Hepatobiliary Fibrosis Induced By Clonorchis Sinensis

Background:Clonorchiasis,also known as liver fluke disease,is a food borne parasitic disease,it is mainly prevalent in Asian countries and regions,among which China is the most seriously infected country.Studies have shown that C.sinensis infection is mainly prevalent in Northeast and South of China,especially in Guangxi.It is of great significance to study the C.sinensis infection in Guangxi.The pathogenic mechanism of C.sinensis is mainly because it can parasitize on the bile duct,long term parasitism can lead to chronic liver damage,and then lead to liver fibrosis and even hepatobiliary carcinoma.EBI3 is a subunit of anti-inflammatory cytokine IL-35 and contains a signal peptide and two fibronectin type III domains.In our preliminary experiment,we found that the expression of EBI3 protein was significantly increased in the liver of infected mice.Then,we reviewed the literatures and found that r EBI3 can significantly reduce bleomycin-induced skin fibrosis in scleroderma skin.Meanwhile,other studies also reported that r EBI3 can inhibit bleomycin-induced pulmonary fibrosis through the enrichment of STAT3 DNA,indicating that there is a potential relationship between EBI3 and fibrosis.Subsequently,we also found that EBI3 plays an important role in regulating the structural components of extracellular matrix and collagen catabolism by bioinformatics analysis.In present study,we used mice infected by C.sinensis as the model of liver fibrosis,and verified the role of r EBI3 in liver fibrosis caused by C.sinensis through in vivo and in vitro experiments,providing a new direction and strategy for the treatment of liver fibrosis caused by C.sinensis.Objective:1.To determine the optimal number of metacercaria infection in BALB/c mice with hepatobiliary fibrosis induced by C.sinensis through gavaged different numbers of metacercaria.2.To study the mechanism of r EBI3 in liver fibrosis induced by C.sinensis then provide a new direction and strategy for the follow-up treatment of liver fibrosis induced by C.sinensis.Methods:1.BALB/c mice were infected with 50,100 or 150metacercaria of C.sinensis respectively to determine the metacercaria infection number;2.Histopathological sections were used to observe the changes of liver fibrosis in mice infected with C.sinensis;3.ELISA,WB and RT-q PCR were used to detect the content of serum EBI3,the expression of liver EBI3 protein and m RNA transcription levels in mice infected with C.sinensis at different time points;4.Bioinformatics method was used to analyse the role of EBI3 in fibrotic liver;5.The in vitro experiment was carried out to verify the relevant mechanism of r EBI3 in p HSC;6.Animal experiments were conducted to verify the role of r EBI3 in liver fibrosis induced by C.sinensis;Results:1.After 14 days of C.sinensis infection,the liver and spleen of mice in the infected group were enlarged,and the liver showed obvious inflammatory lesions,especially the 150M group.On the 56th day of infection,pathological section staining and WB detection of liver tissue showed that liver fibrosis in 150M group was significantly increased compared with 50M and 100M groups（P<0.05）.Meanwhile,none of mice died due to the dose problem during the infection,indicating that it is feasible to infect BALB/c mice with 150 metacercariae by gavaged and BALB/c mice infected 150 metacercaria of C.sinensis can more quickly establish the model of hepatic bile duct fibrosis.Therefore,we infected the mice with 150 metacercaria of C.sinensis for the follow-up experiments.2.The liver tissue pathological section staining and liver hydroxyproline detection showed that the liver fibrosis of mice became worse with the prolongation of infection time.The liver inflammation increased significantly at the 1^stweek after infection,and then decreased gradually.At the 16^thweek after infection,the inflammation declined to the normal level and partial cirrhosis appeared.3.ELISA showed that the content of serum EBI3 in mice infected with C.sinensis was significantly higher than that in the control group at the 2^ndand 4^thweek after infection（P<0.001）and basically decreased to the normal level at the 8^thweek after infection.WB analysis showed that the expression of EBI3 in the liver of infected mice increased significantly at the 1^stand 2^ndweek after infection.Moreover,RT-q PCR results showed that the expression of EBI3 m RNA in the liver of infected mice decreased at the 4^thweek after infection,but increased significantly at the 16^thweek after infection（P<0.05）.4.Bioinformatics analysis showed that the level of EBI3 m RNA transcription in fibrotic liver was significantly higher than that in control group.GO and KEGG analysis showed that EBI3 may play an important role in regulating the structural components of extracellular matrix and collagen catabolism.5.The in vitro co-culture experiments showed that r EBI3 co-cultured with p HSC and Cs.ESP could significantly reduce the transcription levels ofα-SMA,COL1A1 and COL3A1 m RNA in Cs.ESP stimulated p HSC.Meanwhile,r EBI3,p HSC and Cs.ESP co-culture can promote the expression of gp130,JAK1,p-JAK1,STAT3 and p-STAT3 proteins in p HSC,suggesting that r EBI3 may reduce the expression ofα-SMA,COL1A1 and COL3A1 m RNA by inhibiting the activation of HSCs induced by Cs.ESP through JAK1/STAT3 pathway.6.The in vivo experiment showed that the liver fibrosis of mice in the infection group was significantly reduced after the tail vein injection of r EBI3.Through WB and RT-q PCR,we also found that r EBI3 can reduce liver fibrosis and inhibit the transcription of fibrosis related genes.Moreover,the vitro experients found that the expression of gp130,JAK1,p-JAK1,STAT3 and p-STAT3 protein in the liver of infected mice was significantly increased after the tail vein injection of r EBI3.These results suggested that r EBI3 may inhibit HSC activation through JAK1/STAT3pathway to alleviate liver fibrosis and related liver function damage induced by C.sinensis.Conclusions:1.The model of hepatobiliary fibrosis in BALB/c mice infected with C.sinensis was successfully established,which laid a foundation for the follow-up study on the mechanism of hepatobiliary fibrosis induced by C.sinensis.2.This study confirmed that r EBI3 can down regulate the liver fibrosis induced by C.sinensis through inhibiting the activation of HSC,indicating that r EBI3 may be a potential medicine for liver fibrosis,but the relevant mechanism still needs further research and exploration.