[1]于洋,付晓燕,吴艳红,等.低剂量LPS通过上调巨噬细胞TLR4表达促进非酒精性脂肪性肝炎进展[J].陆军军医大学学报(原第三军医大学学报),2021,43(22):2423-2434.
　YU Yang,FU Xiaoyan,WU Yanhong,et al.Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages[J].J Amry Med Univ (J Third Mil Med Univ),2021,43(22):2423-2434.
点击复制

低剂量LPS通过上调巨噬细胞TLR4表达促进非酒精性脂肪性肝炎进展(/HTML )
分享到：

陆军军医大学学报（原第三军医大学学报）[ISSN:1000-5404/CN:51-1095/R]

卷:
43卷
期数:
2021年第22期
页码:
2423-2434
栏目:
基础医学
出版日期:
2021-11-30

文章信息/Info

Title:
Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages
作者:
于洋;  付晓燕;  吴艳红;  许虎;  杜宪红;  吴沛恩;  崔瑞翔;  程丽霞 ; 梁淑娟
潍坊医学院：山东省高校免疫学重点实验室，免疫学教研室，病原微生物教研室，第一附属医院内分泌科
Author(s):
YU Yang1;  FU Xiaoyan;  WU Yanhong;  XU Hu;  DU Xianhong;  WU Pei’en;  CUI Ruixiang;  CHENG Lixia;  LIANG Shujuan

Key Laboratory for Immunology in Universities of Shandong Province, 2Department of Immunology, 3Department of Pathogenic Microbiology, 4Department of Endocrinology, the First Affiliated Hospital of Weifang Medical University, Weifang Medical University, Weifang, Shandong Province, 261053, China

关键词:
非酒精性脂肪性肝炎; 高脂饲料; LPS; 趋化因子; 炎症因子; TLR4
Keywords:
non-alcoholic steatohepatitis;  high-fat diet;  lipopolysaccharide;  chemokine;  inflammatory cytokine;  Toll-like receptor 4
分类号:
R363.21; R392.3; R575.5
文献标志码:
A
摘要:

目的探讨LPS在促进非酒精性脂肪性肝炎（non-alcoholic steatohepatitis，NASH）中的作用及相关机制。方法6~8周龄B6.WT小鼠分别给予正常饲料（ND组）、高脂饲料（HFD组）和HFD联合LPS注射（HFD+LPS组）。HFD+LPS组每周1次腹腔注射LPS（0111∶B4，1.25 μg/g 体质量），其他两组注射等体积生理盐水，饲养10周。酶法检测血清和肝组织TG、TC、ALT、AST水平，油红O染色观察肝内脂质沉积；ELISA法、RT-qPCR检测血清和肝组织IL-1β、TNF-α、CCL5、CCL2、CCL4水平；HE染色观察肝组织病理变化和炎症细胞浸润；免疫荧光（immunofluorescence，IF）观察肝内巨噬细胞、中性粒细胞浸润及TLR4蛋白表达；流式细胞术（flow cytometry，FCM）观察肝脏浸润单个核细胞中巨噬细胞及中性粒细胞比例。结果HFD组血清TG、TC水平显著高于ND组（P均<0.05）。HFD+LPS组肝内脂质沉积以及TG、TC水平显著高于HFD组（P均<0.01），肝脏细胞空泡样变、炎症细胞浸润更为明显，NAS评分（P<0.05）和肝脏单个核细胞数（P<0.01）显著高于HFD组。同时，IF及FCM检测证实肝脏巨噬细胞、中性粒细胞浸润显著多于HFD组（P均<0.05），HFD+LPS组血清中IL-1β、TNF-α、CCL5、CCL2、CCL4水平显著高于HFD组（P<0.05）；肝组织中IL-1β、CCL5、CCL2的蛋白和mRNA水平均显著高于HFD组（P均<0.05）。免疫荧光结果提示LPS作用后肝组织中TLR4呈现明显的细胞膜转位，且TLR4与巨噬细胞共定位显示，HFD+LPS组巨噬细胞表面TLR4表达显著高于HFD组（P<0.01）。结论LPS通过诱导趋化因子及促炎因子释放，募集以巨噬细胞为主的炎性细胞浸润，从而加重HFD小鼠NASH进程；其机制可能与上调巨噬细胞表面TLR4表达有关。

Abstract:

ObjectiveTo investigate the role and underlying mechanisms of lipopolysaccharide (LPS) in the promotion of non-alcoholic steatohepatitis. MethodsWild type B6 (B6.WT) mice aged 6～8 weeks were randomly divided into 3 groups and fed with normal diet (ND), high-fat diet (HFD) and HFD combined LPS injection (HFD+LPS), respectively. LPS (0111∶B4, 1.25 μg/g body weight) was injected intraperitoneally to the mice of the HFD+LPS group once a week, and normal saline of same amount was given to the mice of the other 2 groups. After 10 weeks’ feeding, the levels of triglycerides (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate transaminase (AST) in the serum and liver tissue were detected by enzyme based assays. The lipid deposition in the liver was observed by oil red O staining. The levels of IL-1β, TNF-α, CCL5, CCL2 and CCL4 in the serum and liver tissue were measured by ELISA. The mRNA levels of IL-1β, TNF-α, CCL5, CCL2 and CCL4 in the liver tissue were detected by quantitative real time-PCR (RT-qPCR). The pathological changes of liver tissue and inflammatory cell infiltration were detected by HE staining. Infiltration of macrophages and neutrophils, and the expression of TLR4 in the liver tissue was analyzed by immunofluorescence (IF) assay. Proportion of macrophages and neutrophils in the liver was assessed by flow cytometry. ResultsThe serum levels of TG and TC were significantly higher in the HFD group than the ND group (P<0.05). Their contents in the liver were markedly increased in the HFD+LPS group than the HFD group (P<0.01). The mice from the HFD+LPS group had more obvious vacuolar degeneration of liver cells, infiltration of inflammatory cells, higher NAS score (P<0.05) and larger number of infiltrated mononuclear cells (P<0.01) when compared with the HFD group. IF assay and flow cytometry proved that HFD+LPS treatment induced significantly obvious infiltration of hepatic macrophages and neutrophil accumulation (P<0.05). ELISA proved that HFD+LPS treatment increased serum levels of IL-1β, TNF-α, CCL5 and CCL2 (P<0.05), elevated mRNA levels of IL-1β, CCL5 and CCL2 in the liver tissue (P<0.05). IF revealed that TLR4  showed obvious cell membrane translocation in liver tissue after LPS treatment, and the co localization of TLR4 and macrophages showed that the expression of TLR4 on the surface of macrophages in HFD + LPS group was significantly higher than that in HFD group(P<0.01).  ConclusionLPS mediates intrahepatic infiltration of macrophages and neutrophils and aggravates the process of NASH in mice fed with HFD through upregulating TLR4 expression on macrophages to induce chemokines and inflammatory cytokines. 

参考文献/References:

[1]FRIEDMAN S L, NEUSCHWANDER-TETRI B A, RINELLA M, et al. Mechanisms of NAFLD development and therapeutic strategies[J]. Nat Med, 2018, 24(7): 908-922. DOI: 10.1038/s41591-018-0104-9.
 
[2]RINELLA M E. Nonalcoholic fatty liver disease: a systematic review[J]. JAMA, 2015, 313(22): 2263-2273. DOI: 10.1001/jama.2015.5370.
 
[3]YOUNOSSI Z, ANSTEE Q M, MARIETTI M, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(1): 11-20. DOI: 10.1038/nrgastro.2017.109.
 
[4]YEH C F, CHEN Y H, LIU S F, et al. Mutual interplay of host immune system and gut microbiota in the immunopathology of atherosclerosis[J]. Int J Mol Sci, 2020, 21(22): E8729. DOI: 10.3390/ijms21228729.
 
[5]RATZIU V, HARRISON S A, FRANCQUE S, et al. Elafibranor, an agonist of the peroxisome proliferator-activated receptor-α and -δ, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening[J]. Gastroenterology, 2016, 150(5): 1147-1159.e5. DOI: 10.1053/j.gastro.2016.01.038.
 
[6]WIGG A J, ROBERTS-THOMSON I C, DYMOCK R B, et al. The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis[J]. Gut, 2001, 48(2): 206-211. DOI: 10.1136/gut.48.2.206.
 
[7]JINDAL A, BRUZZ S, SUTTI S, et al. Fat-laden macrophages modulate lobular inflammation in nonalcoholic steatohepatitis (NASH)[J]. Exp Mol Pathol, 2015, 99(1): 155-162. DOI: 10.1016/j.yexmp.2015.06.015.
 
[8]中华医学会肝病学分会脂肪肝和酒精性肝病学组, 中国医师协会脂肪性肝病专家委员会. 非酒精性脂肪性肝病防治指南(2018年更新版)[J]. 临床肝胆病杂志, 2018, 34(5): 947-957. DOI: 10.3969 /j.issn.1001-5256.2018.05.007.
 
National Group on Fatty Liver and Alcoholic Liver Disease of Chinese Society of Hepatology of Chinese Medical Association, Fatty Liver Expert Committee of Chinese Medical Doctor Association. Guidelines of prevention and treatment for nonalcoholic fatty liver disease: A 2018 update[J]. J Clin Hepatol, 2018, 34(5): 947-957. DOI: 10.3969 /j.issn.1001-5256.2018.05.007.
 
[9]BAECK C, WEI X, BARTNECK M, et al. Pharmacological inhibition of the chemokine C-C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly-6C(+) macrophage infiltration in mice[J]. Hepatology, 2014, 59(3): 1060-1072. DOI: 10.1002/hep.26783.
 
[10]吴剑平, 肖海英, 郑雪莲, 等. 白介素25及其相关因子在非酒精性脂肪肝患者血清及肝内的表达及其意义[J]. 第三军医大学学报, 2021, 43(01): 83-90. DOI: 10.16016/j.1000-5404.202005286.
 
WU J P, XIAO H Y, ZHENG X L, et al. Expression and significance of interleukin 25 and its related factors in serum and liver of patients with non-alcoholic fatty liver [J]. J Third Mil Med Univ, 2021, 43(01): 83-90. DOI: 10.16016/j.1000-5404.202005286.
 
[11]WESOLOWSKI S R, KASMI K C, JONSCHER K R, et al. Developmental origins of NAFLD: a womb with a clue[J]. Nat Rev Gastroenterol Hepatol, 2017, 14(2): 81-96. DOI: 10.1038/nrgastro.2016.160. 
 
[12]GRAVES D T, JIANG Y. Chemokines, a family of chemotactic cytokines[J]. Crit Rev Oral Biol Med, 1995, 6(2): 109-118. DOI: 10.1177/10454411950060020101.
 
[13]MARRA F, TACKE F. Roles for chemokines in liver disease[J]. Gastroenterology, 2014, 147(3): 577-594.e1. DOI: 10.1053/j.gastro.2014.06.043.
 
[14]PAN X F, CHIWANDA KAMINGA A, LIU A Z, et al. Chemokines in non-alcoholic fatty liver disease: a systematic review and network meta-analysis[J]. Front Immunol, 2020, 11: 1802. DOI: 10.3389/fimmu.2020.01802. 
 
[15]MIURA K, YANG L, VAN ROOIJEN N, et al. Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2[J]. Am J Physiol Gastrointest Liver Physiol, 2012, 302(11): G1310-G1321. DOI: 10.1152/ajpgi.00365.2011.
 
[16]LI B H, HE F P, YANG X, et al. Steatosis induced CCL5 contributes to early-stage liver fibrosis in nonalcoholic fatty liver disease progress[J]. Transl Res, 2017, 180: 103-117.e4. DOI: 10.1016/j.trsl.2016.08.006.
 
[17]BESSONE F, RAZORI M V, ROMA M G. Molecular pathways of nonalcoholic fatty liver disease development and progression[J]. Cell Mol Life Sci, 2019, 76(1): 99-128. DOI: 10.1007/s00018-018-2947-0.
 
[18]刘彦, 吴皓, 龚建平, 等. 阻断Kupffer细胞的TIM-4蛋白功能对小鼠肝脏缺血再灌注损伤的影响[J]. 第三军医大学学报, 2019, 041(001): 48-55. DOI: 10.16016 /j.1000-5404.201806145.
 
LIU Y, WU H, GONG J P, et al. Inhibition of TIM-4 in Kupffer cells alleviates hepatic ischemia-reperfusion injury in mice [J].J Third Mil Med Univ, 2019, 041(001): 48-55. DOI: 10.16016 /j.1000-5404.201806145.
 
[19]CARPINO G, DEL BEN M, PASTORI D, et al. Increased liver localization of lipopolysaccharides in human and experimental NAFLD[J]. Hepatology, 2020, 72(2): 470-485. DOI: 10.1002/hep.31056. 
 

更新日期/Last Update: 2021-11-23