Xiao Yiyi,Zhao Ziyang,Tong Yanlu,et al.Experimental study of bile duct injury caused by cholesterol-activated CD177+ neutrophils in biliary atresia[J].Journal of Clinical Pediatric Surgery,,22():782-789.[doi:10.3760/cma.j.cn101785-202303064-016]
Experimental study of bile duct injury caused by cholesterol-activated CD177+ neutrophils in biliary atresia
- Keywords:
- Biliary Atresia; Cholesterol; CD177+ Neutrophils; Bile Duct Injury
- Abstract:
- Objective To explore the effect of total cholesterol (TC) on bile duct injury in children with biliary atresia (BA) and investigate the immunological mechanism.Methods Peripheral blood TC levels were retrospectively collected from 118 BA children diagnosed by intraoperative cholangiography, 80 infants of the same age with infantile hepatitis syndrome (IHS) and 165 normal control (NC) children of the same age without any evidence of hepatopathology and liver function abnormalities admitted to Guangzhou Women and Children’s Medical Center during the period of 2011-2020.The correlation between peripheral blood TC and various indicators of bile duct injury or multiple immune cells associated with BA formation was statistically analyzed respectively.Cholesterol was added into the co-culture system of CD177+ neutrophils and biliary epithelial cells (BECs) in vitro, and its effect on BEC apoptosis was observed.Simultaneously, the changes of related indicators in CD177+ neutrophils after cholesterol stimulation were observed.BA children were divided into high-cholesterol BA group and normal-cholesterol BA group according to the detection value of peripheral blood TC.Through RNA sequencing analysis, the differences in gene expression of CD177+ neutrophils between the high-cholesterol BA group and the normal-cholesterol BA group were compared.Results The peripheral blood TC level of[4.9(4.0, 6.1)]mmol/L in BA children was higher than that of[3.7(3.1, 4.1)]mmol/L in NC group and[4.2(3.3, 5.4)]mmol/L in IHS children, and the difference was statistically significant (P<0.05).The peripheral blood TC level of[4.9(4.0, 6.1)]mmol/L in BA children was positively correlated with gamma-glutamyltransferase (GGT) level of[497.0(298.5, 843.0)]U/L, an indicator of bile duct injury (r=0.401, P<0.05), as well as positively correlated with direct bilirubin (DBIL) and total bile acid (TBA) (r=0.338, P<0.05; r=0.235, P<0.05).In terms of the proportion of apoptotic cholangiocytes in liver tissue, the high-cholesterol BA group was 0.724±0.146, the normal-cholesterol BA group was 0.232±0.141, the NC group (children with portal vein cavernous transformation of similar age) was 0.242±0.228.There was a statistically significant difference between high-cholesterol BA group and the other two groups(P<0.05).Peripheral blood TC content[4.4(3.1, 5.5)]mmol/L in BA children was positively correlated with peripheral blood CD177+ neutrophil ratio (26.5±6.4)% (r=0.503, P<0.05), while there was no significant correlation between TC and peripheral blood monocytes[1.0(0.8, 1.2)]×109/L or neutrophils (3.2±1.4)×109/L (P>0.05).The peripheral blood TC level of BA children (4.7±1.6) mmol/L was significantly positively correlated with the number of peripheral blood T cells (4 952.1±1 985.0) cells/μL(r=0.488, P<0.05) and had no correlation with the number of peripheral blood B cells[971.0(664.8, 1 636.1)]cells/μL (P>0.05).Compared with the normal-cholesterol BA group, the expression of various mitochondrial oxidative phosphorylation-related genes (CYBB, CALR, MT-CYB, MT-ND5, MT-ND1) was significantly up-regulated in high-cholesterol BA group, and the difference was statistically significant (P<0.05).Under cholesterol stimulation, the mean fluorescence intensity (MFI) of TOMM20 and DNA 8-OHdG, the indicators of mitochondrial oxidative phosphorylation, were 73.47±6.36 and 159.07±10.17, respectively, and the differences were statistically significant as compared with those before stimulation (P<0.05).The CD177+ neutrophil extracellular trap indicators, MPO and H2B were 60.51±4.26 and 157.46±9.40 respectively, and the differences were statistically significant (P<0.05) as compared with those before stimulation.Meanwhile, in the co-culture system of CD177+ neutrophils and BECs, the viability of BECs after cholesterol stimulation was statistically significantly different from that before stimulation(P<0.05).
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Memo
收稿日期:2023-03-29。
基金项目:国家自然科学基金面上项目（81974056，82271750）；广州市科技计划项目（202206080002）
通讯作者:张锐忠,Email:cowboy2006@163.com