Sodium 4‑phenylbutyrate alleviates H9c2 cell apoptosis induced by high glucose by inhibiting the endoplasmic reticulum stress‑MAMs‑mitochondrial calcium overload axis

JI Yanwei; WEN Xinyu; TANG Hepeng; SU Wating; XIA Zhongyuan; LEI Shaoqing

doi:10.14188/j.1671-8852.2025.0026

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Sodium 4‑phenylbutyrate alleviates H9c2 cell apoptosis induced by high glucose by inhibiting the endoplasmic reticulum stress‑MAMs‑mitochondrial calcium overload axis

更新时间：2026-11-06

- Sodium 4‑phenylbutyrate alleviates H9c2 cell apoptosis induced by high glucose by inhibiting the endoplasmic reticulum stress‑MAMs‑mitochondrial calcium overload axis
- Medical Journal of Wuhan University Vol. 46, Issue 12, Pages: 1529-1536(2025)
- 作者机构：
  
  武汉大学人民医院麻醉科湖北武汉 430060
- 作者简介：
- 基金信息：
- DOI：10.14188/j.1671-8852.2025.0026
  CLC： R587.1
- Received：07 January 2025，
  
  Published：25 December 2025
- 稿件说明：
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纪延炜,文昕郁,唐赫鹏等.4‑苯基丁酸钠通过抑制内质网应激‑MAMs‑线粒体钙超载轴减轻高糖引起的H9c2细胞凋亡[J].武汉大学学报（医学版）,2025,46(12):1529-1536.

JI Yanwei,WEN Xinyu,TANG Hepeng,et al.Sodium 4‑phenylbutyrate alleviates H9c2 cell apoptosis induced by high glucose by inhibiting the endoplasmic reticulum stress‑MAMs‑mitochondrial calcium overload axis[J].Medical Journal of Wuhan University,2025,46(12):1529-1536.
纪延炜,文昕郁,唐赫鹏等.4‑苯基丁酸钠通过抑制内质网应激‑MAMs‑线粒体钙超载轴减轻高糖引起的H9c2细胞凋亡[J].武汉大学学报（医学版）,2025,46(12):1529-1536. DOI： 10.14188/j.1671-8852.2025.0026.

JI Yanwei,WEN Xinyu,TANG Hepeng,et al.Sodium 4‑phenylbutyrate alleviates H9c2 cell apoptosis induced by high glucose by inhibiting the endoplasmic reticulum stress‑MAMs‑mitochondrial calcium overload axis[J].Medical Journal of Wuhan University,2025,46(12):1529-1536. DOI： 10.14188/j.1671-8852.2025.0026.

摘要

目的

探讨内质网应激抑制剂4‑苯基丁酸钠（4‑PBA）对高糖条件下内质网应激‑线粒体相关内质网膜（MAMs）‑线粒体钙超载轴及心肌细胞凋亡的影响。

方法

体外培养H9c2细胞，按随机数字表法分为3组（每组6个复孔）：低糖组（LG组）、高糖组（HG组）、高糖+4‑PBA组（4‑PBA组）。LG组细胞以含5.5 mmol·L

-1

葡萄糖的培养基正常培养，HG组细胞以含35 mmol·L

-1

葡萄糖的培养基处理48 h，4‑PBA组细胞在行高糖处理前给予4‑PBA预处理2 h。细胞计数试剂盒（CCK‑8）检测细胞活性；酶联免疫吸附测定（ELISA）法检测细胞培养液中乳酸脱氢酶（LDH）水平；Rhod‑2细胞可渗透钙离子荧光探针法检测线粒体钙离子水平；2

7‑二氯荧光素二乙酸酯（DCFH‑DA）荧光探针法检测细胞内活性氧（ROS）水平；5

6'‑四氯‑1

3'‑四乙基苯并咪唑羰花青碘化物（JC‑1）法检测线粒体膜电位水平；线粒体红色荧光探针（Mito‑Tracker）法检测线粒体形态；透射电镜检测内质网应激、MAMs的激活水平及线粒体损伤水平；流式细胞仪检测细胞凋亡水平；免疫印迹法（Western Blot）检测

内质网应激标志蛋白BIP/GRP78、CHOP及MAMs标志蛋白IP3R、GRP75、VDAC的表达水平。

结果

与LG组相比：HG组细胞存活率明显降低（

0.05），LDH水平升高（

0.05），BIP/GRP78、CHOP、IP3R、GRP75、VDAC的表达水平升高（均

0.05），MAMs激活水平、线粒体内钙离子水平、线粒体破损比例、ROS水平及细胞凋亡率均升高（均

0.05）；与HG组相比：4‑PBA组细胞存活率升高（

0.05），LDH水平降低（

0.05），BIP/GRP78、CHOP、IP3R、GRP75、VDAC的表达水平降低（均

0.05），MAMs激活水平、线粒体内钙离子水平、线粒体破损比例、ROS水平及细胞凋亡率均降低（均

0.05）。

结论

4‑PBA可以通过抑制内质网应激‑MAMs‑线粒体钙超载轴抑制高糖引起的H9c2细胞凋亡。

Abstract

Objective

To investigate how the endoplasmic reticulum stress inhibitor 4‑phenylbutyric acid (4‑PBA) effectively reduces mitochondrial calcium overload by inhibiting endoplasmic reticulum stress

thereby preventing subsequent cellular damage and apoptosis.

Methods

H9c2 cells were cultured

in vitro

and divided into three groups (six replicate wells in each group) according to a random number table: the low glucose control group (LG group)

the high glucose injury group (HG group)

and the high glucose injury + 4‑PBA group (4‑PBA group). Cells in the LG group were cultured in a medium containing 5.5 mmol·L

-1

glucose

while cells in the HG group were treated with a medium containing 35 mmol·L

-1

glucose for 48 hours. Cells in the 4‑PBA group were pretreated with 4‑PBA for 2 hours prior to high glucose treatment. Cell viability was assessed using the Cell Counting Kit (CCK‑8)

and lactate dehydrogenase (LDH) levels in the cell culture fluid were measured using the enzyme‑linked immunosorbent assay (ELISA) method. The Rhod‑2 cell‑permeable calcium ion fluorescent probe method was employed to detect mitochondrial calcium ion levels

and the 2

7‑dichlorofluorescein diacetate (DCFH‑DA) fluorescent probe method was used to assess intracellular reactive ox

ygen species (ROS) levels. The mitochondrial membrane potential was evaluated using the 5

6'‑tetrachloro‑1

3'‑tetraethylbenzimidazolecarbocyanine iodide (JC‑1) method

and mitochondrial morphology was examined using a mitochondrial red fluorescent probe. The activation level of MAMs and the extent of mitochondrial damage were assessed using transmission electron microscopy. Flow cytometry was utilized to evaluate the level of cell apoptosis

while Western Blot analysis was conducted to determine the expression levels of endoplasmic reticulum stress marker proteins (BIP/GRP78 and CHOP) and mitochondrial‑associated endoplasmic reticulum membranes (MAMs) marker proteins (IP3R

GRP75

and VDAC).

Results

Compared to the LG group

the HG group showed a significantly decreased cell survival rate

increased LDH levels

and elevated expression of BIP/GRP78

CHOP

IP3R

GRP75

and VDAC (all

0.05). Additionally

HG increased MAM activation

mitochondrial calcium ion levels

mitochondrial damage ratio

ROS levels

and cell apoptosis rate (all

0.05). In contrast

the 4⁃PBA group exhibited increased cell survival rate

decreased LDH levels

and reduced expression of BIP/GRP78

CHOP

IP3R

GRP75

and VDAC compared to the HG group (all

0.05). Furthermore

4⁃PBA attenuated MAM activation

mitochondrial calcium ion levels

mitochondrial damage ratio

ROS levels

and cell apoptosis rate (all

0.05).

Conclusion

4⁃PBA can inhibit high glucose⁃induced apoptosis in H9c2 cells by targeting the endoplasmic reticulum stress⁃MAMs⁃mitochondrial calcium overload axis.

关键词

Keywords

references

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