Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages

Yang YU; Xiaoyan FU; Yanhong WU; Hu XU; Xianhong DU; Pei'en WU; Ruixiang CUI; Lixia CHENG; Shujuan LIANG

doi:10.16016/j.1000-5404.202105117

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Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages

Basic Medicine | 更新时间：2026-01-09

- Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages
- Journal of Third Military Medical University Vol. 43, Issue 22, Pages: 2423-2434(2021)
- 作者机构：
  
  1.261053   山东   潍坊，潍坊医学院山东省高校免疫学重点实验室
  2.61053   山东   潍坊，潍坊医学院免疫学教研室
  3.61053   山东   潍坊，潍坊医学院病原微生物教研室
  4.61053   山东   潍坊，潍坊医学院第一附属医院内分泌科
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81873883);the National Natural Science Foundation for Young Scholars of China(82000525);the Project of Medical Science and Technology Development Plan of Shandong Province(2018WS065);the Project of Science and Technology Development Plan of Weifang City(2020YX011)
- DOI：10.16016/j.1000-5404.202105117
  CLC： R363.21;R392.3;R575.5
- Received：24 May 2021，
  
  Revised：2021-06-27，
  
  Published：30 November 2021
- 稿件说明：
移动端阅览
Yang YU, Xiaoyan FU, Yanhong WU, et al. Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages[J]. Journal of Third Military Medical University, 2021, 43(22): 2423-2434.
DOI：

Yang YU, Xiaoyan FU, Yanhong WU, et al. Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages[J]. Journal of Third Military Medical University, 2021, 43(22): 2423-2434. DOI： 10.16016/j.1000-5404.202105117.

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（

均

0.05）。HFD+LPS组肝内脂质沉积以及TG、TC水平显著高于HFD组（

均

0.01），肝脏细胞空泡样变、炎症细胞浸润更为明显，NAS评分（

0.05）和肝脏单个核细胞数（

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（

均

0.05），HFD+LPS组血清中IL-1β、TNF-α、CCL5、CCL2、CCL4水平显著高于HFD组（

0.05）；肝组织中IL-1β、CCL5、CCL2的蛋白和mRNA水平均显著高于HFD组（

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（

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

Objective

To investigate the role and underlying mechanisms of lipopolysaccharide (LPS) in the promotion of non-alcoholic steatohepatitis.

Methods

Wild 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.

Results

The serum levels of TG and TC were significantly higher in the HFD group than the ND group (

0.05). Their contents in the liver were markedly increased in the HFD+LPS group than the HFD group (

0.01). The mice from the HFD+LPS group had more obvious vacuolar degeneration of liver cells

infiltration of inflammatory cells

higher NAS score (

0.05) and larger number of infiltrated mononuclear cells (

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 (

0.05). ELISA proved that HFD+LPS treatment increased serum levels of IL-1β

TNF-α

CCL5 and CCL2 (

0.05)

elevated mRNA levels of IL-1β

CCL5 and CCL2 in the liver tissue (

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(

0.01).

Conclusion

LPS 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.

关键词

Keywords

references

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