Correlation Between Bilirubin Metabolism And Hepatic Fibrosis Induced By Schistosomiasis Japonica

Background: Hepatic fibrosis of schistosomiasis is a serious pathological manifestation of the chronic development of schistosomiasis.Advanced schistosomiasis patients may have symptoms of hepatic fibrosis and decompensation of bilirubin.The Liver UGT1A1 enzyme is the only enzyme that catalyzes bilirubin metabolism.Whether bilirubin metabolism disorder caused by Schistosoma japonicum infection is mediated by UGT1A1 expression inhibition has not been reported.Meanwhile,the role of bilirubin in the body is controversial,and the correlation between bilirubin and liver fibrosis needs to be further investigated to better elucidate the mechanism of schistosome-induced liver fibrosis and to deepen the understanding of the disease process.In this study,descriptive analysis of serum total bilirubin and hepatic fibrosis indexes were conducted in a cohort study of advanced schistosomiasis patients in Jiangsu Province.The effects of Schistosoma japonicum infection on the expression of UGT1A1 and the metabolism of bilirubin in the liver were studied by cell and animal experiments.At the same time,the correlation between total bilirubin and schistosomiasis liver fibrosis was analyzed by epidemiological data,and the interaction between bilirubin and NF-κB pathway in liver cells was explored by combining cell experiments,and the mechanism of regulating the progression of liver fibrosis by bilirubin was initially clarified.From the perspective of metabolism,the inhibitory expression of liver metabolic enzyme UGT1A1 caused by Schistosoma japonicum infection and the role of abnormal bilirubin metabolism in the progression of schistosomiasis liver fibrosis were discussed.Methods:1.Correlation analysis of total bilirubin and liver fibrosis indexes in patients with advanced schistosomiasis in Jiangsu ProvinceThe clinical blood biochemical data were collected from a cohort study of advanced schistosomiasis patients in Jiangsu Province.The mean and variation levels of TBIL,liver damage(ALT,AST),and liver fibrosis(HA,LN,PⅢP,and CⅣ)were analyzed descriptively.χ~2 test to analyze the difference in the positive rate of liver injury and liver fibrosis indicators between patients with normal and abnormal TBIL;Spearman rank correlation analysis of the correlation between TBIL and liver injury and liver fibrosis index.2.To explore the relationship between activation of LX-2 cells and UGT1A1 expression using SEA in vitroLX-2 and L-O2 were treated with different concentrations of SEA(0,2.5,5,10,20,and 40 μg/m L)for 24 h and 48 h in vitro.The effects of SEA on the proliferation of LX-2 and L-O2 cells in vitro were detected by CCK-8.The concentration and time of SEA treatment for subsequent experiments were determined according to the results of the CCK-8 experiment.Annexin V-FITC/PI was used to detect the effect of SEA on the apoptosis of LX-2 and L-O2 cells,and q RT-PCR was used to detect the effect of SEA on the activation of LX-2.LX-2 and L-O2 cells were treated with SEA for 24 h and 48 h in vitro.The effect of SEA on UGT1A1 m RNA expression was detected by q RT-PCR.Effects of SEA in vitro to explore the relationship between liver UGT1A1 expression and activation of LX-2 cells.3.Effects of Schistosoma japonicum infection on hepatic UGT1A1 expression and bilirubin metabolism in miceBy establishing mouse models of Schistosoma japonicum infection at different times(6 w,8 w,and 10 w),liver pathological changes were observed by HE staining and Masson staining.q RT-PCR detected the expression levels of liver fibrosisrelated genes to reflect the changes of liver fibrosis in mice;meanwhile,q RT-PCR detected the expression of liver UGT1A1 and its upstream-related transcriptional regulators PXR/CAR,Western Blot for UGT1A1 protein expression;ELISA for unconjugated bilirubin and conjugated bilirubin levels in mice serum.To explore the effects of Schistosoma japonicum infection on the expression of UGT1A1 and the metabolism of bilirubin in the liver.4.Effect of bilirubin on L-O2 cells in vitro and NF-κB signaling pathwayL-O2 cells were treated with different concentrations of bilirubin(0,20,50,100,and 200 μM)for 8 h,16 h,and 24 h in vitro to observe the morphological changes and cell proliferation.L-O2 cells were treated with 0,20(physiological concentration),and 50 μM(high concentration)bilirubin for 16 h.The m RNA expression of genes related to the NF-κB signaling pathway(p65,Ikk-β,and IκB-α)was detected by q RT-PCR.Western Blot was used to detect the expression of related proteins(p65,p-p65,Ikk-β,p-Ikk-β,IκB-α,and p-IκB-α).The regulatory effect of bilirubin on the NF-κB signaling pathway in hepatocytes was investigated in vitro.Results:1.Correlation analysis of total bilirubin and liver fibrosis indexes in patients with advanced schistosomiasis in Jiangsu ProvinceAbout 23.35% of the newly developed advanced schistosomiasis patients in Jiangsu Province had abnormal total bilirubin,and most of the patients had different degrees of liver injury and abnormal liver fiber four indexes.The positive rates of AST,LN,and CⅣ were different in patients with normal and abnormal total bilirubin(all P<0.001).In addition,TBIL was positively correlated with ALT,AST,LN,and CⅣ(all P<0.001).2.To explore the relationship between activation of LX-2 cells and UGT1A1 expression using SEA in vitroAfter treating LX-2 and L-O2 cells with different concentrations of SEA for 24 h and 48 h in vitro,the cells showed obvious morphological changes such as shrinkage,roundness,and swelling,and inhibited cell proliferation(PLX-2 48h<0.01)(PL-O2 48h<0.0001).SEA treated LX-2 and L-O2 cells for 48 h in vitro,induced apoptosis(all P<0.001),and inhibited the activation of LX-2 cells(PCollagen Ⅰ<0.01,PCollagen Ⅲ<0.0001,Pα-SMA<0.0001).SEA inhibited the expression of UGT1A1 m RNA in LX-2(P48h<0.001)and L-O2(P48h<0.0001)cells in vitro.3.Effects of Schistosoma japonicum infection on UGT1A1 expression and bilirubin metabolism in mouse liverAfter Schistosoma japonicum infection of mice(6 w,8 w,and 10 w),HE staining showed significant worm egg granulomas in the liver,Masson staining showed deposition of collagen fibers in the liver,and the expression of liver fibrosis-related factors Collagen Ⅰ,Collagen Ⅲ,and α-SMA also increased significantly(all P<0.001)and with the extension of time,and the area of worm egg granulomas and the degree of fibrosis increased with time.Meanwhile,the m RNA expression of UGT1A1,PXR,and CAR in the liver was continuously suppressed(all P<0.001)during the process of liver fibrosis,and the protein expression of UGT1A1 was also suppressed(P<0.0001).The bilirubin metabolism in the mice was disturbed,and the serum unconjugated bilirubin level increased(P<0.0001)and the conjugated bilirubin level decreased(P<0.0001).4.Effect of bilirubin on L-O2 cells in vitro and NF-κB signaling pathwayAfter treatment with different concentrations of bilirubin for 8 h,16 h,and 24 h,LO2 cells showed obvious morphological changes,such as roundness and shrinkage.It also inhibits cell proliferation(all P<0.001).The physiological concentration of bilirubin(20 μM)had no significant effect on the gene(P>0.05)and protein(P>0.05)expression of the NF-κB pathway in L-O2 cells.A high bilirubin concentration of 50 μM significantly increased the m RNA relative expression levels of p65(P<0.001),IκB-α(P<0.01),and IKK-β(P<0.001),and bilirubin concentration of 50 μM significantly increased the expression levels of phosphorylated proteins p-p65(P<0.01),p-IκB-α(P<0.001)and p-IKK-β(P<0.001)related to NF-κB signaling pathway.Conclusions: Some advanced schistosomiasis patients have abnormal bilirubin metabolism,which may be caused by the inhibition of liver UGT1A1 expression after Schistosoma japonicum infection.Soluble egg antigen(SEA)may be one of the important inducers of inhibiting liver UGT1A1 expression.In addition,there is a positive correlation between total bilirubin and some hepatic fibrosis indexes in advanced schistosomiasis patients.A high concentration of bilirubin can activate the NF-κB signaling pathway in hepatocytes.The inhibition of UGT1A1 expression in Schistosoma japonicum infection and the reduction of bilirubin metabolism may aggravate the progression of schistosomiasis liver fibrosis by activating the NF-κB signaling pathway.