Study On The Recurrence Of Intrahepatic Stones Induced By Endogenous β-glucuronidase

Objectives: Primary intrahepatic stones refers to the stones located in the proximal part of the intrahepatic bile duct which are above the bifurcation of the common bile duct.At present,the mainstream view is that the occurrence of the disease is related to the infection of specific lithogenic bacteria,cholestasis,abnormal anatomy of bile duct and defect of bile metabolism.β-glucuronidase(β-G)plays an important role in the formation of stones.Lipopolysaccharide(LPS)produced by the lithogenic bacteria can induce the production of endogenous β-G in intrahepatic bile duct epithelial cells.In view of its pathogenesis,the therapeutic principles include surgical removal of all stones,establishment of the steady state of bile dynamic and resection of diseased liver were formed.However,due to the lack of study on the mechanism of the recurrence of intrahepatic stones,after treatment with the above principles,the stone recurrence rate is still high.The purpose of this study was to explore the long-term role and mechanism of endogenous β-G in stone recurrence by detecting and comparing the expression of endogenous β-G and its related signal pathways in human intrahepatic bile duct epithelial cells after LPS induction and LPS clearance.Provide a new idea for the study of the endogenous mechanism of recurrence of primary intrahepatic stones after the elimination of exogenous lithogenic factors.Methods: In this experiment,human intrahepatic bile duct epithelial cells were divided into 6 groups.Group A received LPS induction for 12 hours,group B received LPS induction for 24 hours,group C received LPS induction for 36 hours,group B1 eluted the LPS after receiving its induction for 24 hours,and then cultured for 24 hours.Group B2 received LPS induction again for 24 hours after the same intervention as group B1.And group O received no induction and cultured for 24 hours.The experimental LPS was produced from E.coli 055:B5.The induced concentration of LPS in the experiment was 1 μg/m L.After the proliferation of human intrahepatic bile duct epithelial cells,five independent media were designed for each experimental group to reduce the influence of accidental factors.The expression ofβ-G and key molecules in related signal pathways(TLR-4,NF-κB,c-myc)in each group was determined by Western-blot test.The ratio of gray value of target band to internal reference was used to reflect the expression level of target protein.The software SPSS 26.0 was used for statistical analysis.The experimental data of group A,B,C and O were compared to analyze the overexpression of endogenous β-G under different induction duration.The experimental data of group B,B1 and O were compared to analyze the effect of clearance of LPS on the overexpression of endogenous β-G.Compared with the experimental data of group B1,B2 and O,the response ability of intrahepatic bile duct epithelial cells to LPS re-induction was analyzed.Results: Compared with the control group,the expressions of TLR-4,NF-κB,c-myc and β-G were up-regulated in intrahepatic bile duct epithelial cells induced by LPS in experimental group.The average relative contents of target protein at 12 h,24h and 36 H were as follows: NF-κB was 0.538 vs.0.649 vs.0.736,c-myc was 0.510 vs.0.688 vs.0.881,TLR-4 was 0.425 vs.0.484 vs.0.611,β-G was 0.463 vs.0.623 vs.0.830.The expression level of each protein increased gradually with the prolongation of induction time.There was significant statistical difference in the data(P < 0.05).After scavenging LPS induction and conventional culture for 24 hours,the expression levels of NF-κB,c-myc,TLR-4 and β-G were still the same as those induced by LPS for 24 hours.The average relative contents of target protein expression induced by non-clearance and clearance were as follows: NF-κB was 0.649 vs.0.677,c-myc was0.688 vs.0.718,β-G was 0.623 vs.0.665.TLR-4 was 0.484 vs.0.558(except for the results of TLR-4 determination using Mann-Whitney U test due to variance,the other groups were analyzed by t-test,and the mean values of each group had no statistical difference).Some of the intrahepatic bile duct epithelial cells in group B2 died due to experimental stimulation,and the relative expression level of target protein in group B2 was significantly lower than that in group B1(P < 0.05).The relative expression of NF-κB,c-myc,TLR-4 and β-G were 0.677 vs.0.538,0.718 vs.0.506,0.558 vs.0.400 and 0.665 vs.0.543,respectively.However,it was still higher than that of the blank control(P < 0.05).Conclusion(s): 1.LPS can induce the opening of TLR-4/NF-κB/c-myc signal pathway in human intrahepatic bile duct epithelial cells,resulting in the overexpression of endogenous β-G.And its degree is positively correlated with the induction time.It suggests that the risk of formation of primary intrahepatic stones increases with the prolongation of the infection time of lithogenic bacteria.2.After LPS-induced clearance,the TLR-4/NF-κB/c-myc signal pathway remained open and endogenous β-G was continuously produced,which is an important reason for the high recurrence rate of primary hepatolithiasis after the removal of exogenous lithogenic factors.