Analysis Of Risk Factors For Poor Prognosis Of Biliary Atresia After Kasai Operation

Objective:To explore the risk factors for poor prognosis after Kasai operation,and to clarify the correlation between liver fibrosis and postoperative cholangitis and prognosis of children.To investigate the relationship between BMP-9 and biliary atresia liver fibrosis,to study the hepatic pathology of cholangitis after Kasai procedure with biliary atresia,and to explore the relationships between the expression of CD4~+,CD8~+and CD68~+cells and cholangitis,so as to provide theoretical basis for improving the prognosis of children with biliary atresia.Methods:1.Retrospective analysis was performed for 200 children with biliary atresia,who underwent LT with hepatic failure after KP.According to the interval between KP and LT,they were divided into three groups:G1(≤6-month),G2(6-month～2-year)and G3(>2-year).Gender,age of Kasai portoenterostomy,the degree of hepatic fibrosis during KP,jaundice-clearance,cholangitis after KP and liver function indexes before LT were compared among the three groups by Chi square test,analysis of variance and Kruskal Wallis H test.Multivariate logistic regression was used for multivariate analysis.2.Liver tissues were selected from 5 cases of choledochal cyst group(CBD group)and 14 cases of biliary atresia group(BA group).Immunohistochemical staining was used to compare the expression of BMP-9 and p-SMAD1/5,and the expression of BMP-9 and ID1 m RNA in liver tissues were detected by q RT-PCR.The difference between the two groups was analyzed by t test for statistical analysis.To detect the expression ofα-SMA in LX-2 cells by Western blot and q RT-PCR.LX-2 cells were treated with different concentrations of r BMP-9 solutions(0 ng/ml,10ng/ml,50ng/ml and 100ng/ml).The protein expressions ofα-SMA,Smad5,p-SMAD1/5 and ID1were detected by Western blot,and the m RNA expression of ACTA2 and ID1 was detected by q RT-PCR.3.27 children with biliary atresia who underwent liver transplantation after Kasai operation were divided into frequent cholangitis group,early cholangitis group and non-cholangitis group according to the previous outbreak of cholangitis.The hilar liver tissues of each group were stained with HE and immunohistochemical staining.The pathological changes of liver tissues and the expressions of CD4,CD8 and CD68were compared.Results:1.Among the 200 children with biliary atresia,there were 79 cases in group G1,40males and 39 females;91 cases in group G2,36 males and 55 females;30 cases in group G3,17 males and 13 females.There was significant difference in the proportion of children with operation age≤90 days(P=0.011),and the proportion in G1 was lower than G3(P=0.003).There was significant difference in the degree of liver fibrosis among the three groups(P=0.022).The rate of severe fibrosis in group G1 was higher than that in group G3(P=0.012).The clearance rate of jaundice in the three groups was statistically significant(P<0.001).From group G1 to group G3,the clearance rate of jaundice increased gradually.The incidence of early cholangitis was significantly lower in G3 than in G1(P=0.009).Kaplan-Meier survival analysis showed that the native liver survival rate in children with operation age>90 days was lower than that in children with operation age≤90 days(Log-rankX~2=9.221,P=0.002).The native liver survival rate in children with mild fibrosis was significantly higher than that in children with severe fibrosis(Log-rankX~2=14.265,P<0.001).The native liver survival rate in patients with jaundice clearance after Kasai operation was significantly higher than that in patients without jaundice clearance(Log-rankX~2=75.541,P<0.001).The native liver survival rate in children with early cholangitis was lower than that in children without early cholangitis(Log-rankX~2=4.931,P=0.026).Multivariate logistic regression analysis showed that uncleared jaundice after Kasai operation was an independent risk factor for native liver survival.2.Immunohistochemical staining showed that the expression levels of BMP-9 and p-Smad1/5 in BA liver tissue were higher than those in CBD group(P<0.05).The expression of BMP-9 in children with severe liver fibrosis was higher than that in children with mild liver fibrosis in BA subgroup(P<0.05).The expression of GDF2and ID1 m RNA in the BA group was higher than that in the CBD group(P<0.05),and the expression of GDF2 and ID1 m RNA in children with moderate to severe liver fibrosis in the BA subgroup was also higher than that in children with mild liver fibrosis(P<0.05).The expression levels of downstream signal proteins p-SMAD1/5,ID1 andα-SMA,ID1 and ACTA2 m RNA in LX-2 cells treated with different concentrations of r BMP-9 were gradually increased.3.HE staining showed that there were no significant differences in liver fibrosis,biliary duct hyperplasia and bile plug among the three groups,but there was significant difference in inflammatory cell infiltration.There were significant differences in CD8 and C68 protein expression among the three groups.The expression of CD8 and C68 protein in frequent cholangitis group was significantly higher than that in the non-cholangitis group.Conclusions:1.Kasai operation age>90 days,severe liver fibrosis during Kasai operation,jaundice-unclear after Kasai operation,early cholangitis after Kasai operation can reduce the survival time of postoperative autologous liver,which are the risk factors for poor prognosis after Kasai operation.Jaundice-unclear after Kasai operation is an independent risk factor for poor prognosis.2.The expression levels of BMP-9,p-SMAD1/5 and ID1 in liver tissue of children with biliary atresia were increased,and were positively correlated with the degree of liver fibrosis.BMP-9 can activate human hepatic stellate cells through SMAD/ID1signaling pathway and promote the progress of fibrosis.3.The main pathological changes of hepatic cholangitis after Kasai operation are inflammatory cell infiltration in portal area,which are an inflammatory reaction involving CD8~+T cells and CD68~+macrophages.Increased expression of CD8 and CD68 indicates the recurrence of cholangitis.