Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts

Xinyi HE; Ye MING; Hao TAN; Zhenxing ZHAO; Jing JING; Xuehuan MENG; Xiang LI; Leilei ZHENG

doi:10.16016/j.2097-0927.202204215

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Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts

Basic Medicine | 更新时间：2026-01-09

- Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts
- Journal of Army Medical University Vol. 44, Issue 18, Pages: 1809-1818(2022)
- 作者机构：
  
  401145 重庆，重庆医科大学附属口腔医院，口腔疾病与生物学重庆市重点实验室，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：
  
  the General Program of National Natural Science Foundation of China(81470772);the Innovative Leading Talents of Chongqing Talent Program(CQYC20210303384);the Natural Science Foundation of Chongqing(cstc2020jcyj-msxmX0307)
- DOI：10.16016/j.2097-0927.202204215
  CLC： R322.71;R329.25;R363.272
- Received：29 April 2022，
  
  Revised：2022-06-08，
  
  Published：30 September 2022
- 稿件说明：
移动端阅览
Xinyi HE, Ye MING, Hao TAN, et al. Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts[J]. Journal of Army Medical University, 2022, 44(18): 1809-1818.
DOI：

Xinyi HE, Ye MING, Hao TAN, et al. Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts[J]. Journal of Army Medical University, 2022, 44(18): 1809-1818. DOI： 10.16016/j.2097-0927.202204215.

摘要

目的

探讨在高糖环境下，与巨噬细胞Raw264.7共培养的成骨细胞MC3T3-E1氧化应激、自噬及细胞凋亡的变化。

方法

① 根据培养基的葡萄糖浓度将Raw264.7细胞分为25、30、35 mmol/L 3组，流式细胞术检测细胞CD86表达；RT-qPCR检测

-6、

TNF

、

iNOS

、

NOX

2、

phox

的mRNA表达水平，Western blot检测iNOS的蛋白表达水平，DCFH-DA探针检测ROS水平。②MC3T3-E1细胞分为正常糖单独培养组(5.5 mmol/L葡萄糖浓度，5.5-Mono组)、高糖单独培养组(35 mmol/L葡萄糖浓度，35-Mono组)和高糖共培养组(与Raw264.7细胞共培养24 h，35-Co组)3组，DCFH-DA探针检测MC3T3-E1细胞ROS水平；透射电镜、Western blot检测MC3T3-E1细胞自噬水平；流式细胞术检测MC3T3-E1细胞凋亡水平。

结果

高糖下Raw264.7细胞表面标志CD86表达增加，与25 mmol/L组相比，35 mmol/L组

-6、

TNF

、

iNOS

的mRNA表达水平显著增加(

0.05)，iNOS蛋白表达水平显著增加(

0.05)，ROS水平增加，

NOX

2、

phox

的mRNA表达水平显著增加(

0.05)；与5.5-Mono组相比，35-Mono、35-Co组MC3T3-E1细胞ROS均增加且35-Co组增加更明显，透射电镜显示35-Co组自噬溶酶体数量明显增加，35-Co组MC3T3-E1细胞中LC3-II及SQSTM1/p62的蛋白表达水平明显升高(

0.05)，35-Co组的MC3T3-E1细胞凋亡率较35-Mono组明显升高(

0.05)。

结论

与高糖下M1向极化的Raw264.7细胞共培养会诱导MC3T3-E1细胞凋亡，该作用可能与MC3T3-E1细胞胞内ROS增加和自噬过强有关。

Abstract

Objective

To evaluate the effects of high glucose on oxidative stress

autophagy

and apoptosis in MC3T3-E1 cells co-cultured with Raw264.7 cells.

Methods

① Raw264.7 cells were separated into 3 groups based on the glucose levels in the medium (25

and 35 mmol/L). Flow cytometry was used to detect CD86 expression. RT-qPCR was employed to detect the mRNA expression levels of

-6

TNF

iNOS

NOX

and

phox

in Raw264.7 cells. Western blotting was used to detect the protein expression level of iNOS. The DCFH-DA probe was used to measure the level of reactive oxygen species (ROS) in Raw264.7 cells. ②MC3T3-E1 cells were divided into 3 groups: normal glucose group (5.5 mmol/L glucose

5.5-Mono)

high glucose mono-culture group (35 mmol/L glucose

35-Mono) and high glucose co-culture group (co-cultured with Raw264.7 for 24 h

35-Co). The ROS level of MC3T3-E1 cells were detected by DCFH-DA probe. The autophagic level was assessed by transmission electron microscopy (TEM) and the expression of related proteins was detected with Western blotting. Flow cytometry was applied to determine the rate of apoptosis.

Results

Compared with the normal glucose group

the expression level of CD86 in Raw 264.7 cells was increased in other high glucose groups. Compared with the 25 mmol/L group

the mRNA levels of

-6

TNF

iNOS

NOX

and

phox

were increased (

0.05)

the protein level of iNOS was elevated (

0.05) and the ROS level in the Raw264.7 cells was raised in the 35mmol/L group as well. Compared with the normal glucose group

the ROS level of MC3T3-E1 cells in the high glucose groups was increased

especially in high glucose co-culture group. The number of autophagolysosomes in the MC3T3-E1 cells were increased sharply in the high glucose co-culture group. The protein levels of LC3-II and SQSTM1/p62 in MC3T3-E1 cells were increased significantly in the high glucose co-culture group (

0.05). The apoptotic rate of MC3T3-E1 cells was significantly higher in the high glucose co-culture group than the high glucose mono-culture group (

0.05).

Conclusion

Co-culture with M1-polarized Raw264.7 cells under high glucose induces apoptosis in MC3T3-E1 cells

which may be related to the production of ROS and over-enhancement of autophagy in MC3T3-E1 cells.

关键词

Keywords

references

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