受体相互作用蛋白3介导自身免疫性肝炎肝脏单核细胞来源巨噬细胞募集

目的:探索受体相互作用蛋白3(RIP3)对自身免疫性肝炎(AIH)肝脏单核细胞来源巨噬细胞浸润的调控作用。方法:纳入2018年1至6月于天津医科大学总医院消化科行肝穿刺活组织病理学检查的AIH患者10例，同期选择年龄和性别均匹配且无肝功能异常的5例肝囊肿患者作为对照，应用免疫荧光染色观察AIH患者和对照者肝组织单核细胞...

Receptor interaction protein 3 mediated the recruitment of hepatic monocytes/macrophages in autoimmune hepatitis

Objective To explore the role of receptor-interaction protein 3(RIP3)in regulating the infiltration of monocytes/macrophages into the liver in autoimmune hepatitis(AIH).Methods From January to June in 2018,at Department of Gastroenterology and Hepatology,Tianjin Medical University General Hospital,10 AIH patients who underwent liver biopsy were enrolled,and at the same time,5 age and gender matched individuals with normal liver function and hepatic cyst were selected as control.The infiltration of monocytes/macrophages in the liver tissues was observed by immunofluorescence detection in the patients with AIH and controls.Raw264.7 macrophages were divided into control group,lipopolysaccharide group and lipopolysaccharide+RIP3 inhibitor GSK872(GSK872)group.The expression of RIP3,mixed lineage kinase domain like pseudokinase(MLKL),tumor necrosis factor(TNF)-α,interleukin(IL)-6,IL-1β,nod-like receptor protein 3(NLRP3),CC motif chemokine ligand(CCL)2 and CCL5 at mRNA levels were detected by quantitative polymerase chain reaction(qPCR).Raw264.7 macrophages were also divided into control group,lipopolysaccharide group and lipopolysaccharide+dexamethasone group.The relative expression of TNF-α,NLRP3,RIP3 and MLKL at mRNA level in macrophage were detected by qPCR.Twenty-four 6-week-old female C57BL/6 mice were chosen to establish AIH mice model and were randomly divided into control group,concanavalin A(ConA)group,ConA+dexamethasone group and ConA+GSK872 group(6 mice in each group).After the mice were executed,the peripheral blood and liver tissues were collected.The histopathology of mice liver were observed and the serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured.The expression of CCL2 and CC motif chemokine receptor 2(CCR2)at mRNA level were detected by qPCR.The proportion of macrophages in mice livers were analyzed by flow cytometry.The independent sample t test and one-way analysis of variance were performed for statistical analysis.Results The percentages of CD68 positive macrophages and MAC387 positive infiltrated mononuclear macrophages in livers of AIH patients were both higher than those of controls((0.84±0.21)%vs.(0.09±0.03)%,(0.79±0.13)%vs.(0.03±0.01)%),and the differences were statistically significant(t=3.00 and 4.84;all P<0.05).The expression of RIP3,MLKL,TNF-α,IL-6,IL-1β,NLRP3,CCL2 and CCL5 at mRNA level of lipopolysaccharide group were all higher than those of control group and lipopolysaccharide+GSK872 group(1.64±0.16 vs.1.07±0.07 and 0.63±0.11;10.45±1.37 vs.1.10±0.33 and 1.51±0.63;5.43±0.59 vs.0.94±0.06 and 2.59±0.45;204.20±30.73 vs.1.26±0.19 and 111.40±11.62;20848.00±362.00 vs.1.09±0.26 and 10940.00±566.60;7.47±1.17 vs.1.09±0.09 and 3.79±0.89;68.03±5.15 vs.1.14±0.19 and 14.09±2.62;5935.12±96.20 vs.1.43±0.46 and 673.50±49.10),and the differences were all statistically significant(t=3.11,5.21,6.65,6.55,7.57,3.96,6.60,3.06,8.83,4.08,5.46,2.56,12.97,10.16,25.34 and 14.99;all P<0.05).The expression of TNF-α,NLRP3,RIP3 and MLKL at mRNA level of lipopolysaccharide group were all higher than those of control group and lipopolysaccharide+dexamethasone group(8.85±1.43 vs.1.44±0.43 and 3.63±0.63;6.42±0.86 vs.0.99±0.12 and 2.07±0.17;1.72±0.21 vs.0.93±0.09 and 0.43±0.07;6.87±0.85 vs.1.62±0.31 and 1.41±0.29),and the differences were all statistically significant(t=4.95,3.33,6.24,4.95,3.04,5.11,5.77 and 6.07,all P<0.05).The mice liver of ConA group showed obviously inflammatory cells infiltration and hepatocytes necrosis.The serum ALT and AST levels of ConA group were both higher than those of control group,ConA+dexamethasone group and ConA+GSK872 group((2569.00±45.44)U/L vs.(49.38±9.07),(103.00±14.07)and(759.30±34.99)U/L;(3335.00±88.79)U/L vs.(108.50±18.10),(460.00±97.40)and(1573.85±36.06)U/L),the serum ALT and AST levels of ConA+dexamethasone group were both lower than those of ConA+GSK872 group,and the differences were all statistically significant(t=5.54,5.42,3.90,4.63,4.16,3.79,6.70 and 2.71;all P<0.05).The expression of CCL2 and CCR2 at mRNA levels in mice liver of ConA group were both higher than those of control group,ConA+dexamethasone group and ConA+GSK872 group(92.64±10.57 vs.0.78±0.15,5.64±1.00 and 9.47±2.06;5.73±0.39 vs.0.98±0.22,2.18±0.22 and 2.98±0.33),and the differences were all statistically significant(t=7.66,7.24,5.87,8.71,8.58 and 5.45;all P<0.01).The proportion of CD45⁺CD11b⁺F4/80⁺total macrophages and CD45⁺CD11b^hiF4/80^lo infiltrated macrophages in mice livers of ConA group were both higher than those of control group,ConA+dexamethasone group and ConA+GSK872 group(0.86±0.02 vs.0.73±0.03,0.68±0.02 and 0.72±0.03;0.56±0.02 vs.0.08±0.02,0.11±0.01 and 0.08±0.01),however the proportion of CD45⁺CD11b^loF4/80^(hi )liver macrophages(Kupffer cells)was lower than those that of control group,ConA+dexamethasone group and ConA+GSK872 group(0.24±0.03 vs.0.58±0.04,0.52±0.07 and 0.56±0.07),and the differences were all statistically significant(t=4.27,5.90,3.89,18.70,19.87,20.52,7.35,3.82 and 3.87,all P<0.05).Conclusions The number of macrophages incread in the livers of AIH patients.RIP3 signaling mediates the migration of monocytes/macrophages infiltration in immune hepatitis,which may be a potential therapeutic target for AIH.