Effect Of HNE And Its Inhibitor Ceverlastat Sodium On MUC5AC Expression In Intrahepatic Bile Duct Epithelial Cells

Objective(s)Intrahepatic bile duct stones(IBDS)are more common in the southwest of China,and it has a complex pathogenesis.Some patients have recurrence even after hepatectomy,resulting in the need for multiple surgeries,which causes great pain to patients.Chronic inflammation of the biliary tract is one of the important causes of IBDS,and some patients still have recurrence of stones after hepatectomy.We found that mucin(MUC)is one of the important factors in the pathogenesis of intrahepatic bile duct stones,and MUC can change bile viscosity and bile rheology to participate in the formation of bile stasis,which leads to the development of IBDS.It has been demonstrated that infection in the intrahepatic biliary epithelium,especially endotoxin from gram-negative bacilli,can lead to elevated MUC,but the exact mechanism is not known.After biliary tract infection,neutrophils are the first to make defense and a human neutrophil elastase(HNE)released by them plays a key role in regulating the inflammatory response and may be associated with the overexpression of MUC in the biliary tract.Controlling the regulation of MUC expression is of great significance to reduce the incidence of IBDS and the recurrence of stones after surgery.In this paper,we intend to explore whether HNE can regulate the expression of mucin 5AC(MUC5AC)in human intrahepatic bile duct epithelial cells and whether the HNE inhibitor civilex sodium can reduce the expression of MUC5 AC after HNE stimulation.In order to further explore the mechanism of stone recurrence after surgery in IBDS patients.Methods1.The gallstone gene data were downloaded from GEO database,screened to inflammatory factors such as EGFR and TLR4,and CHOL data were downloaded from TCGA database,which included 8 normal tissues and 38 tumor tissues,and the NE genes Elane and Muc5 AC were analyzed for correlation in the matrix using Limma package in R software.Further these factors P38 MAPK,TACE,TGF-α,TLR4,TNF-α,EGFR,IL,MUC5 AC,MUC2,ELANE were selected in the string data set for the interactions analysis.2.Immunohistochemistry(IHC)to detect the expression of bile duct HNE;3.Cellular experiments This experiment was validated at the cellular level.The original intrahepatic bile duct epithelial cell line was recovered,passaged and divided into five groups: Hi BEpi C cells blank control group,Hi BEpi C cells +(10n M)HNE group,Hi BEpi C cells +(10n M)HNE + Sivelastal sodium low dose group,Hi BEpi C cells +(10n M)HNE + Sivelastal sodium medium dose group,Hi BEpi C cells +(10n M)HNE + Sivelastal sodium high dose group.4.Cell proliferation activity assay was performed in Hi BEpi C cells after the addition of HNE and each dose of sevelamer sodium to evaluate the cytotoxicity and the effect on cell proliferation of each group of drugs.5.Western blot was performed to detect the expression of MUC5 AC in the cell mixture and supernatant of each group after 48 hours of culture.6.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the expression of MUC5 AC in the cell mixture and supernatant of each group after 48 hours of culture.Results1.Bioinformatic analysis revealed a reciprocal relationship between the HNE gene(Elane)and MUC5 AC,in which the expression of Elane and TLR4 factors were positively correlated.2.In the control group,only a small amount of HNE was expressed in the bile duct mucosa;while the HNE expression in the bile duct mucosa of the stone group was significantly higher than that of the control group.3.EDU cell proliferation experiments showed that the proliferation status of cells was good in the Hi BEpi C cell blank control group,Hi BEpi C cell +(10n M)HNE group,Hi BEpi C cell +(10n M)HNE + Sivelastal sodium low-dose group,Hi BEpi C cells +(10n M)HNE + Sivelastal sodium medium-dose group,Hi BEpi C cells +(10n M)HNE + Sivelastal sodium high-dose group.And according to the data of the percentage of EDU positive cells and the results of the analysis of differences between groups,there was no significant difference between the experimental group and the control group,and there was no significant promotion and inhibition of cell proliferation by drug treatment in the experimental group.4.Comparing the simple cell culture group and the group treated with HNE,WB and ELISA results showed that the expression of MUC5 AC was significantly higher after treatment with HNE;(p < 0.001)5.The expression of MUC5 AC was up-regulated in cells treated with HNE,WB and ELISA results showed that the expression of MUC5 AC was subsequently down-regulated after treatment of cells with HNE inhibitor Sivelastal sodium,and the down-regulation was more obvious with the increase of Sivelastal sodium concentration.(p < 0.001)Conclusion(s)1.Normal bile duct mucosa may express a small amount of HNE;stone bile duct mucosa HNE is overexpressed。2.HNE induces the expression of MUC5 AC in human intrahepatic bile duct epithelial cells,and its inhibitor,Sivelastal sodium,can effectively inhibit the HNE-induced MUC5 AC expression.3.This study indicates that HNE is a key mediator in regulating MUC5 AC expression,and it is expected to control MUC5 AC expression by inhibiting HNE activity,thus reducing the incidence of intrahepatic bile duct stones and the recurrence rate of stones after surgery.4.Further animal experiments can be performed to confirm the mechanism of postoperative stone recurrence in IBDS patients and how to reduce the rate of stone recurrence after surgery.