The Associations Between HBV Infection And Bilirubin Or Hepcidin And The Potential Mechanisms Of Dysregulated Hepcidin

Objective:The study aimed to probe into the effect of HBV infection and degree of liver injury on bilirubin and the main regulator of iron homeostasis,hepcidin,and the potential mechanisms that oxidative stress influences the expression of hepcidin in HBV replicating cells.Methods:1.A cross-sectional analysis of 28,500 Chinese participants which came from Dongfeng-tongji corhort study was performed to analyze the differences of bilirubin in terms of hepatitis B surface antigen?HBsAg?positive or negative,and using the logistics regression analysis to futher explore the correlations between bilirubin and severity of hepatic injury estimated by non-invasive indices.2.This was a hospital-based cross-sectional study.The participants who were HBV-infected patients admitted to the Department of Infectious Disease,Tongji Hospital of Tongji Medical College and diagnosed with CHB consecutively recruited.The diagnostic criteria used for mild,moderate and severe CHB were in accordance with the 2000 Xi'an Viral Hepatitis Management Guidelines recommended by the Chinese Society of Infectious Diseases and Parasitology,and the Chinese Society of Hepatology of the Chinese Medical Association?172 CHB patients,and 20 controls?.Based on the associations between hepcidin and severity of hepatitis using the logistics regression analysis,we searched for superior index to predict the severity of hepatitis in CHB.3.According to the results of previous section,this part futher explored the relationship between the status of oxidative stress and hepcidin.Targeting Bone morphogenetic protein 6?BMP6?/Smad family member 4?SMAD4?pathways,we used HepG2.2.15 which HBV was stably replicating to explore the potiential mechanisms of dysregulated hepcidin induced by oxidative stress with the methods of qRT-PCR,immunofluorescence technique and western blot.Results:1.This study included 28,500 paticipants and the average age of the HBsAg positive and negative groups were 62.8 ± 7.8 vs.64.4 ± 8.5,and the propotions of female were 56.6%vs.50.6%Compared with controls,higher bilirubin levels were detected in HBsAg carriers?total bilirubin:16.52±7.16 vs.14.70±6.07,P<0.001?.2.Higher bilirubin levels were consistently associated with elevated liver injury indices among HBsAg carriers.Compared with quartile 1 of total bilirubin?TBil?,the multivariable-adjusted ORs?95%CIs?for elevated fibrosis indices of quartile 4 were 2.24?95%CIs,1.57-3.21?estimated by FIB-4?fibrosis 4 score?and 2.22?95%CI,1.60-3.08?estimated by APRI?aspartate transaminase to platelet ratio index?.3.Direct bilirubin?DBil?had a stronger association with elevated liver injury indices than did indirect bilirubin?IBil?.And the associations between DBil and elevated FIB-4 and APRI were more robust among female,overweight or central fat distributed population.4.This section included 192 paticipants?172 CHB patients and 20 controls?.Compared with controls,the concentrations of hepcidin among CHB patients were lower.Hepcidin decreased along with the increase of severity in CHB.The levels of hepcidin?ng/mL?of each degree among CHB patients were 67.01?41.24,100.53?,38.84?29.61,58.84?and 27.54?10.99,47.11?.5.Among the CHB patients,plasma hepcidin,serum ferritin,et al were significantly associated with severity of hepatitis.The corresponding ORs?95%CIs?of hepcidin was 0.494?0.375,0.652?in univariate analysis.And in the multiple analysis,the decreased levels of hepcidin were associated with severity of hepatitis after adjusting related parameters,the corresponding ORs?95%CIs?comparing the highest quartile with the lowest quartile were 0.215?0.064,0.727?.6.Accoding to the results in logistic regression analysis,we calculated a combined parameter based on hepcidin and other parameters,H-score,with AUROCs of 0.823?0.755,0.879?,0.797?0.727,0.857?for more than moderate hepatitis and severe hepatitis respectively.7.Among the CHB patients,plasma MDA was significantly associated with hepcidin.The each concentrations of hepcidin?ng/mL?among tertils of MDA were 41.68?24.82,66.24?,48.77?29.74,70.61?,26.59?10.94,52.29?,P = 0.005.Further analyzing the relationship between plasma MDA and hepcidin,the negative association were detected,the corresponding rho =-0.243,P = 0.001.8.The negative association between MDA and hepcidin was only detected when MDA reached a certain concentrations,??95%CIs?=-2.80?-3.90,-1.70?.P<0.001,which suggesting that MDA's negative regulation on hepcidin existed when CHB patients' oxidative stress existed in relatively high level.9.Compared with control,H₂O₂ suppressed the expression of hepcidin in HepG2.2.15 while antioxidant?N-acetyl-L-cysteine,NAC?could antagonize the suppressive effect.H₂O₂ suppressed the expression of BMP6.After supplement of NAC,the suppression was alleviated.H₂O₂ decreased the nuclear expression of SMAD4.BMP6 inhibitor futher reduced the expression of SMAD4 while BMP6 activator relieved the suppression of SMAD4's expression in H₂O₂ treated cells.Conclusion:Bilirubin concentrations,especially direct bilirubin,were positively associated with elevated liver injury indices among HBsAg carriers.Hepdicin levels were significantly associated with severity of hepatitis among CHB patients.Additionally,MDA concentrations were negatively related with hepcidin's.Oxidative stress which suppressed hepcidin through BMP6/SMAD4 signaling pathway could be the potential mechanism of suppression of hepcidin expression in HBV replicating cell.