补肾活血方靶向Nrf2介导的铁死亡调控3T3-L1细胞成脂分化功能障碍的研究

洪耀南; 吴迪炯; 沈英英; 李杭超; 徐玲珑; 叶宝东; 邵科钉

doi:10.3969/j.issn.1006-2157.2023.04.010

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补肾活血方靶向Nrf2介导的铁死亡调控3T3-L1细胞成脂分化功能障碍的研究

实验研究 | 更新时间：2023-05-09

- 补肾活血方靶向Nrf2介导的铁死亡调控3T3-L1细胞成脂分化功能障碍的研究
- Study of Bushen Huoxue Formula modulate iron overload induced adipogenic differentiation dysfunction of 3T3-L1 cells by targeting Nrf2 mediated ferroptosis
- 北京中医药大学学报 2023年46卷第4期页码：509-519
- 作者机构：
  
  1.浙江中医药大学第一临床医学院　杭州　310053
  2.国家中医血液病临床研究基地
  3.台州市中心医院血液科
- 作者简介：
  
  洪耀南，男，在读本科生
  邵科钉，男，助理研究员，主治医师，主要研究方向：中西医结合治疗血液病，E-mail：skd@zcmu.edu.cn
- 基金信息：
  
  国家自然科学基金项目(82174138);浙江省自然科学基金项目(LY21H290003);浙江省中医药科学研究基金项目(2020ZB085;2023ZF099);浙江省卫生健康科技计划项目(2022RC216);台州市科技局资助项目(20ywa31)
- DOI：10.3969/j.issn.1006-2157.2023.04.010
  中图分类号： R285.5
- 收稿：2022-11-17，
  
  纸质出版：2023-04-30
- 稿件说明：
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洪耀南, 吴迪炯, 沈英英, 等. 补肾活血方靶向Nrf2介导的铁死亡调控3T3-L1细胞成脂分化功能障碍的研究[J]. 北京中医药大学学报, 2023,46(4):509-519.

HONG Yaonan, WU Dijiong, SHEN Yingying, et al. Study of Bushen Huoxue Formula modulate iron overload induced adipogenic differentiation dysfunction of 3T3-L1 cells by targeting Nrf2 mediated ferroptosis[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023, 46(4): 509-519. DOI: 10.3969/j.issn.1006-2157.2023.04.010.
洪耀南, 吴迪炯, 沈英英, 等. 补肾活血方靶向Nrf2介导的铁死亡调控3T3-L1细胞成脂分化功能障碍的研究[J]. 北京中医药大学学报, 2023,46(4):509-519. DOI： 10.3969/j.issn.1006-2157.2023.04.010.

HONG Yaonan, WU Dijiong, SHEN Yingying, et al. Study of Bushen Huoxue Formula modulate iron overload induced adipogenic differentiation dysfunction of 3T3-L1 cells by targeting Nrf2 mediated ferroptosis[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023, 46(4): 509-519. DOI: 10.3969/j.issn.1006-2157.2023.04.010. DOI：

摘要

目的

探讨补肾活血方对铁过载小鼠前脂肪细胞株(3T3-L1)成脂分化功能的影响及作用机制。

方法

连续7 d给45只SD大鼠灌胃补肾活血方(15 g/kg)以制备补肾活血方含药血清。将3T3-L1细胞分为正常对照组、空载体对照组、模型组、铁过载组、Nrf2基因沉默组。采用3T3-L1细胞，使用慢病毒颗粒转染构建核因子E2相关因子－2(Nrf2)基因沉默3T3-L1细胞(3T3-L1 shNrf2)与空载体对照3T3-L1细胞(3T3-L1 shCtrl)。使用柠檬酸铁铵(FAC)构建3T3-L1铁过载细胞模型，并筛选出最优浓度。正常对照组与空载体对照组不予干预，模型组给予FAC，铁过载组和Nrf2基因沉默组给予FAC＋补肾活血方含药血清。采用CCK-8法、油红O染色法分别检测各组细胞的存活率及脂质沉积情况。采用实时荧光PCR法检测过氧化物酶体增殖物激活受体(PPARγ)、激素敏感脂肪酶(HSL)、脂肪甘油三酯脂肪酶(ATGL)mRNA的表达量。采用酶联免疫吸附剂测定(ELISA)法检测细胞上清液中还原型谷胱甘肽(GSH)和脂质过氧化物(LPO)的含量。采用蛋白质印迹法检测转铁蛋白受体(TFR)、铁蛋白轻链(FTL)、铁蛋白重链(FTH)、铁调节蛋白(IRP)、谷胱甘肽过氧化物酶4(GPX4)、胱氨酸/谷氨酸反向转运体(XCT)、血红素氧合酶1(HO-1)、Nrf2的蛋白表达量。

结果

与空载体对照组比较，Nrf2基因沉默组中3T3-L1细胞的Nrf2的表达水平降低(

0.01)，表明3T3-L1 shNrf2构建成功。并确定15 μmol/L为FAC的最优浓度。与空载体对照组比较，模型组的细胞存活率降低(

0.01)，甘油三酯相对含量增加(

0.01)、细胞内脂质沉积增加，PPARγ、HSL和ATGL mRNA的表达量均降低(

0.01)，GSH和LPO含量均降低(

0.01)，Nrf2、IRP的蛋白表达量均增加(

0.05，

0.01)，TFR、FTL、GPX4、XCT、FTH、HO-1的蛋白表达量均降低(

0.01)。与模型组比较，铁过载组的细胞存活率增加(

0.05)，甘油三酯相对含量降低(

0.01)、细胞内脂质沉积降低，PPARγ、HSL和ATGL mRNA的表达量均增加(

0.05)，GSH和LPO含量增加(

0.05)，IRP蛋白表达量降低(

0.05)，Nrf2、TFR、FTL、GPX4、XCT、FTH、HO-1蛋白表达量均增加(

0.05，

0.01)。与铁过载组比较，Nrf2基因沉默组的细胞存活率降低(

0.05)，甘油三酯相对含量增加(

0.05)、细胞内脂质沉积增加，PPARγ、HSL和ATGL mRNA的表达量均降低(

0.05)，GSH和LPO含量均降低(

0.05)，IRP蛋白表达量增加(

0.05)，Nrf2、TFR、FTL、GPX4、XCT、FTH、HO-1蛋白表达量均降低(

0.05，

0.01)。

结论

补肾活血方可以有效保护3T3-L1细胞免受铁过载诱导的氧自由基损伤与细胞铁死亡，有利于脂肪细胞的增殖，并且可以抑制其终末成熟。

Abstract

Objective

We aimed to investigate the effects and the underlying mechanism of

Bushen Huoxue

Formula (BSHXF)on the adipogenic differentiation function of an iron overload (IO) mouse preadipocyte strain (3T3-L1 cells) and the underlying mechanism.

Methods

In total

45 Sprague-Dawley rats were given BSHXF(15 g/kg) by gavage for 7 days to prepare the medicated serum of BSHXF. 3T3-L1 cells were divided into the normal group

the shCtrl group

the model group

the BSHXF+ IO shCtrl group and the BSHXF+ IO shNrf2 group. 3T3-L1 cells were transfected with lentivirus particles to construct nuclear factor E2 related factor-2 (Nrf2) silenced 3T3-L1 cells (3T3-L1 shNrf2) and empty vector control 3T3-L1 cells (3T3-L1 shCtrl). Ferric ammonium citrate (FAC) was used to establish the 3T3-L1 IO cell model

and the optimal FAC concentration was determined. The normal group and the shCtrl group were not treated. The model group was given FAC

the BSHXF + IO shCtrl group and the BSHXF + IO shNrf2 group were given FAC+ BSHXF medicated serum. The cell survival rate and lipid deposition of each group were detected by the CCK-8 assay and Oil red O staining. The mRNA expression levels of peroxisome proliferator-activated receptor gamma (PPARγ)

hormone-sensitive triglyceride lipase (HSL) and adipose triglyceride lipase (ATGL) were detected by real-time PCR. The levels of glutathione reduced (GSH) and lipid peroxidation (LPO) in cell supernatant were detected by ELISA. The protein expression levels of transferrin receptor (TFR)

ferritin light chain (FTL)

ferritin heavy chain (FTH)

iron regulatory protein (IRP)

glutathione peroxidase 4 (GPX4)

cystine/glutamate antiporter system (XCT)

heme oxygenase 1 (HO-1) and Nrf2 were detected by Western blotting.

Results

Compared with the shCtrl group

the expression level of Nrf2 in the 3T3-L1 cells in the shNrf2 group was decreased (

0.01)

indicating that the establishment was successful. The optimal concentration of FAC was determined to be 15 μmol/L. Compared with the shCtrl group

the cell survival rate of the model group was decreased (

0.01)

the relative content of triglyceride was increased (

0.01)

intracellular lipid deposition was increased

the mRNA expression levels of PPARγ

HSL and ATGL were decreased (

0.01)

the levels of GSH and LPO were decreased (

0.01)

the protein expression levels of Nrf2 and IRP were increased (

0.05

0.01)

and the protein expression levels of TFR

FTL

GPX4

XCT

FTH and HO-1 were decreased (

0.01). Compared with the model group

the cell survival rate of the BSHXF+ IO shCtrl group was increased (

0.05)

the relative content of triglyceride was decreased (

0.01)

intracellular lipid deposition was decreased

the mRNA expression levels of PPARγ

HSL and ATGL were increased (

0.05)

the levels of GSH and LPO were increased (

0.05)

the protein expression level of IRP was decreased (

0.05) and the protein expression levels of Nrf2

TFR

FTL

GPX4

XCT

FTH and HO-1 were increased (

0.05

0.01). Compared with the BSHXF+ IO shCtrl group

the cell survival rate of the BSHXF+ IO shNrf2 group was decreased (

0.05)

the relative content of triglyceride was increased (

0.05)

intracellular lipid deposition was increased

the mRNA expression levels of PPARγ

HSL and ATGL were decreased (

0.05)

the levels of GSH and LPO were decreased (

0.05)

the protein expression level of IRP was increased (

0.05)

and the protein expression levels of Nrf2

TFR

FTL

GPX4

XCT

FTH and HO-1 were decreased (

0.05

0.01).

Conclusion

BSHXF can effectively protect 3T3-L1 cells from oxygen free radical damage and ferroptosis induced by IO

which benefits the proliferation of adipocytes

and can inhibit terminal maturation.

关键词

Keywords

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