胆道闭锁中胆固醇活化CD177^(+)中性粒细胞致胆管损伤的实验研究

Experimental study of bile duct injury caused by cholesterol-activated CD177^(+)neutrophils in biliary atresia

目的通过实验研究胆道闭锁(biliary atresia,BA)患儿总胆固醇(total cholesterol,TC)对胆管损伤的影响,并探讨其免疫学机制。方法回顾性收集2011-2020年间广州市妇女儿童医疗中心收治的118例经术中胆管造影确诊的BA患儿、80例同龄婴儿肝炎综合征(infantile hepatitis syndrome,IHS)患儿以及165例无肝病学证据且无肝功能异常的同龄正常对照(normal control,NC)儿童外周血TC含量。分析TC与胆管损伤各指标及多种与BA形成相关免疫细胞之间的关联。在体外CD177^(+)中性粒细胞与胆道上皮细胞(biliary epithelial cell,BEC)共培养体系中加入胆固醇,观察其对BEC凋亡的影响,同时观察加入胆固醇刺激后CD177^(+)中性粒细胞内相关指标的变化;根据外周血TC检测数值将BA患儿分为高胆固醇BA组、正常胆固醇BA组,通过RNA测序分析,比较高胆固醇组、正常胆固醇组BA患儿CD177^(+)中性粒细胞基因表达差异。结果BA患儿外周血TC含量为4.9(4.0,6.1)mmol/L,高于NC组的3.7(3.1,4.1)mmol/L及IHS组的4.2(3.3,5.4)mmol/L,差异有统计学意义(P<0.05);BA患儿外周血TC含量[4.9(4.0,6.1)mmol/L]与胆管损伤指标γ-谷氨酰转移酶(gamma-glutamyltransferase,GGT)含量[497.0(298.5,843.0)U/L]呈正相关(r=0.401,P<0.05),同时与直接胆红素(direct bilirubin,DBIL)、总胆汁酸(total bile acid,TBA)含量呈正相关(r=0.338,P<0.05;r=0.235,P<0.05);在肝脏组织胆管细胞凋亡数量比例层面,高胆固醇BA组为0.724±0.146,正常胆固醇BA组为0.232±0.141,NC组(患儿年龄相近门静脉海棉样变儿童)为0.242±0.228,高胆固醇BA组与其余两组间差异有统计学意义(P<0.05);BA患儿外周血TC含量[4.4(3.1,5.5)mmol/L]与外周血CD177^(+)中性粒细胞比例[(26.5±6.4)%]呈正相关(r=0.503,P<0.05),与外周血单核细胞[1.0(0.8,1.2)×10^(9)/L]、中性粒细胞[(3.2±1.4)×10^(9)/L]无相关性(P>0.05);BA患儿外周血TC含量[(4.7±1.6)mmol/L]与外周血T细胞数量(4952.1±1985.0)个/μL呈正相关(r=0.488,P<0.05),与外周血B细胞971.0(664.8,1636.1)个/μL无相关性(P>0.05);与正常胆固醇BA组相比,高胆固醇BA组线粒体氧化磷酸化相关基因(CYBB、CALR、MT-CYB、MT-ND5、MT-ND1)表达显著上调,差异有统计学意义(P<0.05);在胆固醇刺激下,CD177^(+)中性粒细胞中代表线粒体氧化磷酸化的指标TOMM20及DNA 8-OHdG的平均荧光强度分别为73.47±6.36、159.07±10.17,较刺激前差异有统计学意义(P<0.05),而CD177^(+)中性粒细胞胞外诱捕网指标MPO和H2B分别为60.51±4.26、157.46±9.40,较刺激前差异有统计学意义(P<0.05);同时在CD177^(+)中性粒细胞与BEC共培养体系中,加入胆固醇刺激后BEC细胞活率较刺激前差异有统计学意义(P<0.05)。结论胆道闭锁中胆固醇可上调CD177^(+)中性粒细胞线粒体氧化磷酸化水平,促进胞外诱捕网的释放,引起胆管损伤。

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)]×10^(9)/L or neutrophils(3.2±1.4)×10^(9)/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(4952.1±1985.0)cells/μL(r=0.488,P<0.05)and had no correlation with the number of peripheral blood B cells[971.0(664.8,1636.1)]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).