LPS induced HIF-1α expression of RAW264.7 cells under hypoxia by simultaneously activating p38/MAPK and NF-κB signal pathway

Fu Zhihui; Xu Ling; Huang Jiao; Yin Dongqing

doi:10.13406/j.cnki.cyxb.000985

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LPS induced HIF-1α expression of RAW264.7 cells under hypoxia by simultaneously activating p38/MAPK and NF-κB signal pathway

更新时间：2024-01-17

- LPS induced HIF-1α expression of RAW264.7 cells under hypoxia by simultaneously activating p38/MAPK and NF-κB signal pathway
- Journal of Chongqing Medical University Vol. 41, Issue 10, Pages: 1033-1039(2016)
- 作者机构：
  
  口腔疾病与生物医学重庆市重点实验室重庆医科大学附属口腔医院修复科重庆市高校市级口腔生物医学工程重点实验室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.000985
  CLC： R781.42
- Published：2016，
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[1]符智慧,徐凌,黄姣,尹东青.LPS通过p38/MAPK通路和NF-κB的活化诱导RAW264.7细胞HIF-1α的表达[J].重庆医科大学学报,2016,41(10):1033-1039.

Fu Zhihui, Xu Ling, Huang Jiao, et al. LPS induced HIF-1α expression of RAW264.7 cells under hypoxia by simultaneously activating p38/MAPK and NF-κB signal pathway. [J]. Journal of Chongqing Medical University 41(10):1033-1039(2016)
[1]符智慧,徐凌,黄姣,尹东青.LPS通过p38/MAPK通路和NF-κB的活化诱导RAW264.7细胞HIF-1α的表达[J].重庆医科大学学报,2016,41(10):1033-1039. DOI： 10.13406/j.cnki.cyxb.000985.

Fu Zhihui, Xu Ling, Huang Jiao, et al. LPS induced HIF-1α expression of RAW264.7 cells under hypoxia by simultaneously activating p38/MAPK and NF-κB signal pathway. [J]. Journal of Chongqing Medical University 41(10):1033-1039(2016) DOI： 10.13406/j.cnki.cyxb.000985.

摘要

目的:研究脂多糖（lipopolysaccharide

LPS）在低氧条件下对RAW264.7细胞低氧诱导因子-1α（hypoxia-inducible factor1α

HIF-1α）表达的影响及其可能的调控机制

探讨对牙周炎破骨细胞吸收的影响。方法:观察低氧条件下

用1μg/m L LPS刺激RAW264.7细胞

应用Western blot以及q RT-PCR检测HIF-1α的表达和P38/丝裂素活化蛋白激酶（mitogen-activated protein kinases

MAPK）和核转录因子-κB（nuclear transcription factor-κB

NF-κB）信号通路活化情况。干扰HIF-1α后在低氧中用LPS诱导RAW264.7细胞

ELISA检测炎症因子TNF-α、PGE2、IL-1β以及q RT-PCR检测破骨细胞分化标志基因肿瘤坏死因子受体相关因子6（tumor necrosis factor receptor associated factor 6

Traf6）、活化T细胞核因子c1（nuclear factor of activated Tcells cytoplasmic1

Nfatc1）、组织蛋白酶K（cathepsin K

Ctsk）、c Fos、抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase

Trap）的变化。结果:低氧条件下LPS诱导RAW264.7细胞3、6、9、12 h分别与1 h比较

HIF-1α表达量明显增加（P=0.000、P=0.000、P=0.000、P=0.000）。在低氧条件下

LPS刺激RAW264.7细胞HIF-1αm RNA的相对表达量水平（0.81±0.14）高于单纯的低氧组（0.21±0.05）

2组比较存在统计学差异（P=0.000）。低氧条件下LPS上调了p38的磷酸化水平（P=0.009）

抑制p38以及NF-κB的表达明显抑制HIF-1α的表达（P=0.011、P=0.039）。干扰HIF-1α的表达明显抑制了低氧条件下LPS诱导的RAW264.7细胞PGE2（94.46±8.89）和IL-1β（83.96±7.71）的生成

分别与si RNA-Control组的PGE2（134.66±21.11）和IL-1β（121.34±20.42）比较

2组比较存在统计学差异（P=0.008、P=0.008）;同时也抑制了低氧条件下LPS诱导的破骨细胞标志基因Traf6、Nfatc1的m RNA相对表达量（0.50±0.20

0.60±0.30）的表达

分别与si RNA-Control组（1.00±0.00、1.00±0.00）比较（P=0.000、P=0.000）。结论:LPS在低氧条件下通过p38/MAPK通路和NF-κB的活化诱导了RAW264.7细胞HIF-1α表达

干扰HIF-1α的表达能够抑制低氧条件下LPS诱导的炎症因子的生成以及破骨细胞的分化。

Abstract

Objective:To investigate the effect of lipopolysaccharide(LPS)on hypoxia-induced factor-1α(HIF-1α)expression in RAW264.7 cells under hypoxia condition and to discuss its effect on bone resorption of periodontitis associated osteoclast. Methods:RAW264.7 cells were treated with demanded concentration of LPS(1 μg/m L)under hypoxia condition. The expression of HIF-1α protein level was detected by Western blot and m RNA level was detected by q RT-PCR. Meanwhile

related signal molecules of p38/MAPK and NF- κB signaling pathways were determined. The changes of inflammatory cytokines(such as TNFα

PGE2

IL-1β)genereated by LPS-induced Raw264.7 cells and the expression of osteoclast-specific markers(such as Traf6

Nfatc1

Ctsk

c Fos

Trap) were measured by ELISA and fluorescence q RT-PCR after small inteferring RNA knocking-down HIF-1αexpression. Results:Under hypoxia condition

HIF-1α expression in cells induced by LPS for 3 h

6 h

9 h

12 h increased significantly compared with cells treated with LPS for 1 h(P=0.000

P=0.000

P=0.000). Under hopoxia condition

HIF-1αm RNA expressions in cells induced by LPS(0.81±0.14)were higher than those of pure hypoxia group(0.21±0.05)

with significant differences between the two groups(P=0.000). Under hypoxia condition

LPS caused up-regulation of the phosphorylation of p38/MAPK(P=0.009)

and reversely suppressed the expression of HIF- 1α by different specific inhibitors of p38/MAPK and NF-κB(P=0.011

P=0.039). After si RNA knocking down HIF-1α

LPS caused significant suppression in the level of PGE2(94.46±8.89)and IL-1β(83.96 ±7.71);there were significant differences in PGE2(134.66 ±21.11) and IL-1β(121.34 ±20.42) compared with those of si RNA-control group(P=0.008

P=0.008).Simutaneously

m RNA expressions of Traf6(0.50±0.20)and Nfatc1(0.60±0.30)were suppressed in si RNA-HIF-1α group compared with expressions of Traf6(1.00±0.00)and Nfatc1(1.00±0.00)in si RNA-control group(P=0.000

P=0.000). Conclusion:LPS can induce HIF-1α expression of RAW264.7 cells under hypoxia possibly by simultaneously activating the p38/MAPK and NF-κB signal pathway.The application of si RNA targeting HIF-1a could effectively suppress the expression of inflammatory cytokines and the osteoclast-differentiation of RAW264.7 cells induced with LPS under hypoxia condition.

关键词

脂多糖低氧低氧诱导因子-1αRAW264.7细胞

Keywords

lipopolysaccharidehypoxiahypoxia-induced factor-1αRAW264.7 cells

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