[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.
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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 )
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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. 

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更新日期/Last Update: 2021-11-23